| Reference Number | First/Senior Author | Year | Author Final Product Nomenclature. Prior to Analysis | Correct Final Product Nomenclature. Prior to Analysis | Manual or Automated | Species | Fat Source | Fat Isolation Technique | Mincing | Volume Fat Processed | Pre-Enzyme Processing | Enzyme (s) | Enzyme Source | Digestion Solutions | Enzyme Concentration | Digestion Vessel | Temperature (C) | Time | Agitation | Collagenase Neutralization/Removal | Separation | Wash (s) | Secondary Digestion | Filtration Selection | RBC Removal | FACS Separation | Characterization | Cell Counting Method | Yield | Viability | Microbiology | Endotoxin | Author Final Product Terminology (Before Analysis) | Application | Cell Characterization | Reference | Methods Cited | Notes | Isolation Method | Senior Author address/email | |
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| 1 | Lasfargues | 1957 | Epithelial Cells | Adipocytes and SVF | Manual | Mouse C57 | Mammary | Surgical | Scissors | Washed in Simm’s solution | Collagenase | Innes Mandl | 0.2 mg/ml | round-bottom centrifuge tube | 37 | 1.5 hours | wrist action shaker with glass beads | Centrifugation 1200 rpm 10 minutes | 3x in Simm’s solution. 1200 RPM 10 minutes | Cell Culture | Morphology Stains | LASFARGUES EY. Cultivation and behavior in vitro of the normal mammary epithelium of the adult mouse. Anat Rec. 1957 Jan;127(1):117-29. doi: 10.1002/ar.1091270111. PMID: 13411582. | MATERIALS AND METHODS Tissue. To provide the best experimental conditions, mammary glands are taken from C-57 mice before the 10th day of pregnancy, although an epithelial growth can be obtained from glands taken prior to or at the end of pregnancy. They are washed in Simms’ solution and the lymph nodes eliminated. Collagenase The collagenase used is isolated from Clostridium histolyticum according to the technique described by I. Mandl, J. MacLennan et al. ( ’53). This enzyme hydrolyzes 25 mg of collagen in 18 to 24 hours when its concentration is of 0.02 mg per milliliter. For tissue culture purposes it is desirable to have a short but highly effective enzymatic action. After preliminary tests (table 1) a concentration of 0.2 mg of collagenase per milliliter of Simms ’ solution appeared as most favorable. Technique. The two abdominal mammary glands are collected in the concavity of a Maximow slide containing 1 ml of collagenase solution and are finely minced with scissors. This suspension is transferred into a centrifuge tube (round bottom) containing 3 4 ml of the same collagenase solution and a few glass beads. The tube, stoppered, is mounted on a shaker in an incubator regulated to 37°C. A wrist-shaking action of medium strength is maintained from 1 to 2 hours according to the original size of the fragments. About 10ml of Simms’ solution is then added. After 10 minute centrifugation at 1200 rpm most of the fat, collected on top of the supernatent, can easily be discarded. A complete drainage leaves the epithelial cells at the bottom of the tube. Two additional washings in plain saline solution are made before the tissue is finally suspended in 4 to 5ml of culture nutrient. This suspension is used for slides and roller tube cultures. *This enzyme was kindly supplied by Doctors I. Mandl and J. D. MacLennm. | Etienne Yves “Steve” Lasfargues. Etienne Lasfargues died 2013. Was at Pasteur Institute in Paris, Associate Professor of Microbiology at Columbia University in New York until 1966 and then moved to Coriel Institute in Camden New Jersey. | |||||||||||||||||
| 2 | Rodbell | 1964 | Adipocytes and Stromal Vascular Cells | Adipocytes and SVF | Manual | Rat | Rat Parametrial / Uterine fat pads | Surgical Excision | 3 pieces | 1.9 gm | 40% ammonium sulfate fraction from Clostridium histolyticum, Worthington Biochemical Corporation | Worthington | 3 ml of bicarbonate Krebs-Ringer buffer, pH 7.4, containing 4% dialyzed bovine albumin (Armour’s, Fraction V), 10 mg of collagenase (40% ammonium sulfate fraction from Clostridium histolyticum, Worthington Biochemical Corporation), and 0.5 mg of glucose per ml | 10 mg/ml | 25 ml Erlenmeyer | 37 | ~ 60 | Shaking Dubnoff Water Bath | Centrifugation 400 x g 1 minute | Bouyant fat cells and SVF pellet washed separately. 3x, centrifugation, 400 x g 1 minute. | SVF – 140ug DNA per 1.9 gm fat. Fat Cells 92 ug DNA per gm fat. 1.9 gms fat contained 346 ug DNA | Fat Cells and Stromal Vascular Cells | Fat Cells Biochemical Evaluation | Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucos-e metabolism and lipolysis. J. Biol. Chem. 239: 375-380. | Primary Reference Isolation Adipocytes & Stromal Vascular Cells | Assuming 7.1. pg DNA per cell the SVF cell yield is 10.4 million SVF per gm fat processed. | Rodbell 1964 EXPERIMENTAL PROCEDURE Preparation of Adipose Cells-Adipose tissue was obtained from female Sprague-Dawley rats (170 to 200 g) fed a high fat diet (6). The animals were killed by decapitation. The parametrial fat pads were removed, rinsed in 0.9% NaCl, blotted, and weighed. One fat pad was cut into three pieces and incubated at 37°, with shaking, in 3 ml of bicarbonate Krebs-Ringer buffer, pH 7.4, containing 4% dialyzed bovine albumin (Armour’s, Fraction V), 10 mg of collagenase (40% ammonium sulfate fraction from Clostridium histolyticum, Worthington Biochemical Corporation), and 0.5 mg of glucose per ml. The contralateral fat pad, which served as the intact tissue control, was incubated in the buffer in the absence of collagenase. Incubations were performed in 25-ml siliconized Erlenmeyer flasks. Tissues incubated with collagenase were dispersed into fragments within an hour. Gentle stirring resulted in the liberation of cells, which was manifested by an increased turbidity in the medium. The fragments of undispersed tissue were removed, rinsed in buffer, and saved for further analyses (see below). Centrifugation of the cell suspension for 1 minute at 400 X g caused the fat cells to float and the stromal-vascular cells to sediment. After separation, the fat cells and stromal-vascular cells were washed by suspending them in warm (37°) albumin buffer and centrifuging at 400 X g for 1 minute. After three washings, the final fat cell suspension was devoid of stromal-vascular cells as determined by histological examination. | Martin “Marty” Rodbell died in 1998. Perrformed research at National Institute of Health Duke University University of North Carolina at Chapel Hill | |||||||||||||
| 3 | Fain | 1965 | Adipocytes and Stromal Vascular Cells | Adipocytes and SVF | Manual | Rat | Rat Parametrial / Uterine fat pads | Surgical | albumin-bicarbonate buffer solution | 37 | 60 | Fain JN, Kovacev VP, Scow RO. Effect of growth hormone and dexamethasone on lipolysis and metabolism in isolated fat cells of the rat. J Biol Chem. 1965 Sep;240(9):3522-9. PMID: 5835934. | “Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J. Biol. Chem. 239: 375-380.” | Isolated fat cells were prepared by a modification of the procedure of Rodbell in which parametrial adipose tissue was incubated for only 1 hour in albumin-bicarbonate buffer solution containing bacterial collagenase. Glucose was not present in the buffer solution which contained collagenase or in the solu- tion which was used to wash the cells. A 30% solution of albumin (bovine, Fraction V, hrmour) was dialyzed overnight against 9 volumes of the bicarbonate buffer and then diluted to a 4y0 solution. | Robert O Scow National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Bethesda, United States | ||||||||||||||||||||||||||
| 4 | Ng/Poznanski | 1971 | Sedimented Cells | SVF | Manual | Human | SubQ | Surgical | Collagenase | Sedimented cells | In Vitro Studies | Oil Red O | Ng, C. W., Poznanski, W. J., Borowecki, M., and Reimer, G. Differences in growth in uitro of adipose cells from normal and obese patients. Nature (Land.) 231: 445, 1971 | Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucos-e metabolism and lipolysis. J. Biol. Chem. 239: 375-380. | Subcutaneous adipose tissue was obtained from patients, aged 35-50, undergoing abdominal surgery. Cells were dispersed.with collagenase according to Rodbell and centrifuged at low speed. The sedimented cells were cultured on glass cover slips in Leighton tubes containing 1.5 mi. of lactalbumin hydrolysate growth medium supplemented with 20% foetal calf serum, in an atmosphere of 5% C0 -95% air at 37° C. | Wojciech J Poznanski. Metabolism Research Laboratory, Department of Metabolism, Ottawa Civic Hospital, 1053 Carling Ave, Ottawa 3, Canada | |||||||||||||||||||||||||
| 5 | Wagner | 1972 | Capillaries | Microvascular Fragments | Manual | Rat | Epididymal | Surgical | Worthington Type II Collagenase | Worthington | 37 | 45 | Gentle stirring | 25 micron Nylon Mesh Filter Centrifugation 200 g 10 mmin | Capillaries | Biochemical | Wagner RC, Kreiner P, Barrnett RJ, Bitensky MW. Biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase in isolated capillary endothelium. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3175-9. doi: 10.1073/pnas.69.11.3175. PMID: 4564206; PMCID: PMC389729. | Primary Reference Isolation Microvascular Fragments | Wagner 1972 Isolation of Capillaries. The distal portion of epididymal fat pads from Sprague-Dawley rats (250-300 g) were removed and placed in Tyrode’s basal salt solution (pH 7.2) at 0oC, thoroughly minced, and placed in siliconized vials (three fat pads per vial) each containing 40 mg of crude collagenase (Worthington Biochemicals), 6 mg of bovine-serum albumin, and 6 ml of Tyrode’s balanced salt solution. The mixture was incubated at 37oC for 45 min on a wrist action shaker and cen-trifuged in siliconized conical tubes (30 X g for 2 min). The resulting pellet contained larger blood vessels, stromal cells, and attached fat cells, and was discarded. The supernatant containing the fat cake was decanted and centrifuged (800 X g for 2 min). The vascular pellets from the second centrifugation were twice pooled and washed in Tyrode’s solution, and collected by centrifugation at 800 X g for 2 min. The final pellet was pink; it contained fragments of intact capillary networks with minimal stromal elements, without fat, as monitored by phase-contrast and electron microscopy. | Roger C Wagner. Emeritus Professor. University of Delaware Newark, DE. Email: Rags@Udel.com | |||||||||||||||||||||
| 6 | Poznanski | 1973 | Stromal Cells | Stromal Vascular Fraction (SVF) | Manual | Human | Abdominal | Surgocal | Fat cut into small pieces | Collagenase | 37 | 30 | Constant Stirring | Centrifugation 700 x g | Poznanski WJ, Waheed I, Van R. Human fat cell precursors. Morphologic and metabolic differentiation in culture. Lab Invest. 1973 Nov;29(5):570-6. PMID: 4753021. | Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucos-e metabolism and lipolysis. J. Biol. Chem. 239: 375-380. | Fat tissue samples were obtained from patients undergoing abdominal surgery. All patients were between 25 and 35 years of age and of normal weight. Adipose tissue was dispersed with collagenase according to the technique of Rodbell and the cells were prepared for culture according to the methods in our previous publication. Adult method human skin tissue was obtained from normal subjects by biopsy. Tissues were cut into small pieces in Hanks’ balanced salt solution then and incubated with collagenase at 37oC for 30 minutes with constant stirring. The resulting suspension was centrifuged at 700 x g and the stromal elements were resuspended in growth media for cultures. | Wojciech J Poznanski. Metabolism Research Laboratory, Department of Metabolism, Ottawa Civic Hospital, 1053 Carling Ave, Ottawa 3, Canada | |||||||||||||||||||||||
| 7 | Wagner | 1975 | Capillary Endothelium | Adipose Microvascular Fragments (MVF) | Manual | Rat | Epididymal Fat | Surgical | Worthington Type II Collagenase | Worthington | Dulbecco’s PBS | 7.5 mg/ml | 50 ml Erlenmeyer | 37 | 45 | gentle stirring | Centrifuge 100 g 3 min. Pelelt resuspended then Capillaries captured on top of a 200 micron stainless steel screen | 1x. 100 g 3min | Capillaries | Culture Endocytosis Studies | WAGNER, R. C., AND MATTHEWS, M. A. (1975). The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10, 286-297. | Primary Reference Culture of Microvascular Fragments | Capillary Isolation The distal two-thirds of epididymal fat pads from adult (350-400 g) rats (Flow Labs. Inc.) were aseptically removed and pinned out flat on beeswax in the bottom of a Petri dish containing lo-ml sterile Dulbecco’s PBS (pH 7.4). Portions of fat free of large blood vessels (greater than 60-pm diam) were removed from the pads under a dissection microscope. This microvascularized fat was placed in 50-ml screwcap Erlenmeyer flasks (2 pads/flask) containing lo-ml Dulbecco’s PBS (pH 7.4), Worthington Type II Collagenase (7.5 mglml), bovine serum albumin (5 mg/ml), and a magnetic stirring bar. The fat was incubated with gentle stirring at 37” for 45 min or until the pieces of fat were digested into a slurry. The collagenase digest was centrifuged at 100 g for 3 min, and the floating cake of adipocytes and the supernatant were decanted. The vascular pellet was washed with 5 ml of Delbecco’s PBS and recentrifuged at 100 g for 3 min. The washed pellet (0.2 ml packed pellet/fat pad) was resuspended in 5 ml of Medium 199 (Earle’s salts plus bicarbonate), fetal calf serum (20x), gentamycin (50 pg/ml), and amphotercin B (2.5 ,ug/ml). The vascular suspension was then filtered through a stainless steel screen (200 microns) attached to a suction flask. Tufts of capillary networks and smaller fragments were retained by the screen and free cells in suspension were filtered through the holes. The capillaries were washed from the screen with a jet of culture medium (15 ml) from a 30-ml (20-gauge needle) into a plastic Petri dish, transferred to centrifuge tubes, and repelleted at 100 xg for 3 min. The entire isolation procedure was performed in a laminar flow hood, and sterile apparatus and solutions were used in all operations. | Roger C Wagner. Emeritus Professor. University of Delaware Newark, DE. Email: Rags@Udel.com | |||||||||||||||||
| 8 | Fain | 1975 | Fat Cells | Adipocytes | Manual | Fat cut inot pieces | Crude Collagenase | Worthington | Krebs-Ringer phosphate buffer containing 4% albumin | 0.2-0.7 mg/ml | Erlenmeyer Flask | 37 | water bath shakers equipped for reciprocal mot!on. Gliemann 12 has shown that shaking fat cells in 22 ml plastic vials at up to 120 cpm did not affect the hormonal response, but in cells isolated by shaking at 140 cpm there was an increase in basal glucose metabolism which suggests cell damage | Filtration. Centrifugation | Stromal Vascular Cells | Fain JN. Isolation of free brown and white fat cells. Methods Enzymol. 1975;35:555-61. doi: 10.1016/0076-6879(75)35184-7. PMID: 164607. | The major problem in the isolation of hormonally sensitive fat cells is the variability seen in commercially available collagenase preparations. These problems cannot be circumvented as yet by the use of more highly purified preparations of collagenase since Kono 4 found that digestion of adipose tissue requires the presence of a proteolytic enzyme(s) as well as collagenase. | For the isolation of white fat cells, white adipose tissue is obtained from the appropriate source and kept at 37 ° C. The tissue is rinsed, cut into pieces weighing about 200 mg, and any connective tissue present is removed. Isolation of white fat cells from most animal species is readily accomplished except with ruminants where the cells often clump. This may result from the difficulty in keeping the triglycerides of ruminant fat cells in the liquid state. The pieces of adipose tissue are rinsed with buffer containing albumin and added to 1 ounce polyethylene bottles containing buffer plus albumin and eollagenase. Plastic 25 ml Erlenmeyer flasks are also suitable for digestion of adipose tissue. Two milliliters of Krebs-Ringer phosphate buffer containing 4% albumin and 0.2-0.7 mg/ml of eollagenase are added per gram of adipose tissue. The tissue is ordinarily incubated for about 45 minutes at 37 ° in a shaking water bath. Our experience with rat white adipose tissue has not indicated that shorter periods of digestion using greater amounts of collagenase resulted in superior results. After 45-60 minutes of incubation with collagenase in buffer containing albumin, the cells are filtered through nylon chiffon to remove undigested adipose and connective tissue. This is readily accomplished by cutting the top out of 1 ounce bottle caps, placing a piece of chiffon on top of the bottle, and then screwing on the top. The filtration of the cells is facilitated by squeezing the side of the polyethylene bottle. The suspension of fat cells is added to plastic centrifuge tubes (12 ml) and centrifuged briefly. The time and force of centrifugation should be just sufficient to float the fat cells to the surface. Generally 10-15 seconds of centrifugation in an International clinical centrifuge with a horizontal head to hold 15 ml tubes is used. With large fat cells centrifugation can even be omitted. The stromal-vascular cells, erythrocytes, and other sedimented material are removed by aspiration using a Pasteur pipette attached to a water aspirator. | John N Fain. Died 2016 Research associate, Emory University, Atlanta, 1960-1961; National Science Foundation fellow, National Institutes of Health, Bethesda, Maryland., 1961-1962; postdoctoral fellow United States Public Health Service, National Institutes of Health, Bethesda, Maryland., 1962-1963; biochemist, National Institutes of Health and National Institute Arthritis and Metabolic Diseases, Bethesda, 1963-1965; assistant professor, Brown U., Providence, 1965-1968; associate professor, Brown U., Providence, 1968-1971; professor, Brown U., Providence, 1971-1985; chairman biochemistry, Brown U., Providence, 1975-1985; Van Vleet professor, chairman, U. Tennessee, Memphis, since 1985 email: jfain@utmem.edu | ||||||||||||||||||||||
| 9 | Adebonojo | 1975 | Dissociated Adipose Cells | Adipocytes and Stromal Vascular Fraction | Manual | Human 1 day to 11 years | anterior abdominal walls | Surgical Excision | Sliced | 30 to 50 mg | Collagenase B Grade | Calbiochem | of MIcCoy’s 5a medium (Microbiological Associates, Bethesda, Md | 10 mg/gm tissue | 25 ml Erlenmeyer Falsk | 37 | 60 min | slow con- stant gyratory rotation (50 to 100 | Centrifugation 400 x g | Morphology | Adebonojo FO. Monolayer cultures of disaggregated human adipocytes. In Vitro. 1975 Jan-Feb;11(1):50-4. doi: 10.1007/BF02615322. PMID: 1126738. | Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucos-e metabolism and lipolysis. J. Biol. Chem. 239: 375-380. | Methods not clear whether adipocytes or SVF or both are being cultured | Adipose tissue was disaggrrgatcd by a modifiication of the methods described by Rodbell (6). At abdominal laparotomies, 1 to 2 g of white adipose tissue were obtained from anterior abdominal walls of 31 human infants and children aged 1 day to 11 )’ears. The tissue was sliced into pieces weighing 30 to 50 mg, and placed in 10 ml of :\[rCoy’s .5a medium (Microbiological Associates, Bethesda, Md.) in a 25-ml siliconized Erlenyer flask. Ten milligrams of collagenase (B Grade Calbiochem) per g of tissue were added. The mixture was incubated in slow constant gyratory rotation (50 to 100 rpm) for 1 hr at 37°C. After incubation, the contents of the flask were gently stirred with a plastic spatula and the cells were further dispersed by gentle pipet-ting (plastic pipets). Undisperscd pieces of tissue wrre remowd with sterile forceps and the suspPnsion of cells was centrifuged (LE.C. UV Centrifugp) in sterile 10-ml nlastic centrifmre ubes at 400 X g for 1 min. Fat droplets from ruptured cells and intact fat cells floated to the surface, whereas stromal vascular cells formed a pellet on the bottom of the centrifuge tube. The cells were gently pipetted and resuspended in 10 ml of fresh McCoy’s medium and recentrifuged as before. This step was repeated once more, af-ter which the cells were plated in monolayers in 25-ml plastic culture flasks (Falcon Plastics). The cells were incubated at 37°C for 4 hr during which time they adhered to the bottom of the flask. To each flask was then added 2 ml of McCoy’s medium supplemented with 20% fetal calf serum, penicillin (60 units per ml), and streptomycin (60 units per ml). The culture flasks were incubated at 37°C in a gas phase of 95% air and 5% CO•. The medium was changed every 5 to 7 days and the culture pH kept be-tween 7.2 and 7.4. Cells were subcultured by removing all of the medium with a sterile pipet. The cells were al-lowed to remain in contact for 1 min with 3 ml of Puck’s solution (7) containing 200 mg of EDTA per liter. The Puck’s solution was re-moved and replaced with 1.5 ml of trypsin in Puck’s solution mixture (trypsin (B Grade, Calbiochem) final concentration of 0.25%). Un-der microscopic examination it was possible to establish when the cells became loosened from the culture flask. The trypsin in Puck’s solution mixture was then removed and replaced with 1.0 ml of culture medium (McCoy’s and 20% fetal calf serum) and the cells were gently dispersed with a pipet and divided equally into new cul-ture flasks. Additional medium was added to each bottle to bring the volume to 2.0 ml. | |||||||||||||||||
| 10 | Van | 1977 | Stromal Fraction | Stromal Vascular Fraction (SVF) | Manual | Van RL, Roncari DA. Isolation of fat cell precursors from adult rat adipose tissue. Cell Tissue Res. 1977 Jul 11;181(2):197-203 | Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucos-e metabolism and lipolysis. J. Biol. Chem. 239: 375-380. | Tissue was obtained from male Wistar rats which weighed 480-580 g (Canadian Breeding Laboratories); they had been fed ad libitum. Epididymal adipose tissue and abdominal skin fibroblast were re ected immediately upon sacrifice and immersed in Hanks’ balanced salt solution (Flow Laboratories Inc.) at 0-4°C, pH 7.4. The adipose and skin tissues were minced and treated with collagenase (I mg/ml, Worthington Biochemical Corp.) at 37°C for 30 and 60 min, respectively (Rodbell, 1964). After centrifugation (700 xg, 15 min, 22° C), the digested stromal fraction of adipose tissue and the skin fibroblast preparations were suspended in Medium 199 (Flow Laboratories Inc.), supplemented with 20% fetal calf serum {Flow Laboratories Inc.), and with penicillin (100 units/ml) and gentamicin (50 μg/ml). | Rien L. Van. Department of Adult Mental Health Care, Arkin Mental Health Care, Amsterdam, Netherlands. And. Daniel A. K. Roncari, MD. emigrated to Canada from Italy when he was 14. He received his MD from Queen’s University in Kingston in 1961 and his MSc in experimental medi- cine under Dr Charles H. Hollenberg at McGill Univer- sity in Montreal in 1965. At Washington University in St Louis with Dr I’. Roy Vagelos, he received his PhD in biological chemistry. After becoming a member of the faculty of medicine at the University of Toronto, he went on to become the third director of the Institute of Medical Science from 1980 to 1983. He then moved to the University of Calgary to become the first Julie McFarlane Professor of Diabetes Research. Dr Roncari returned to Toronto in 1988 to become Physician-in-Chief at Sunnybrook Health Science Centre. Most recently he was Professor of Investigative Medicine at Sunnybrook. He served on the editorial boards of sev | ||||||||||||||||||||||||||||||||
| 11 | Van | 1978 | Stromal Fraction | Stromal Vascular Fraction (SVF) | Manual | Human | Omental Fat | Surgical Excision | Minced | Collagenase | Worthington | Hanks Balanced Salt Solution | 1 mg/ml | 37 | 15 | Centrifugation | Van RL, Roncari DA. Complete differentiation of adipocyte precursors. A culture system for studying the cellular nature of adipose tissue. Cell Tissue Res. 1978 Dec 28;195(2):317-29. | Van RLR, Bayliss CE, Roncari DAK 1976 Cytological and enzymological characterization of adult human adipocyte precursors in culture. J Clin Invest 58:699 | Source and Preparation of Cells Adipocyte precursor cells were obtained and isolated from the stromal fraction of omen taI adipose tissue from 6 adult humans (30-40 years of age) undergoing elective abdominal surgery, and from tbe epididymal adipose tissue of 8 adult male Wistar rats (450-500 g) which had been fed ad libitum. In each case, fibroblasts were obtained from the abdominal skin of the same patients and animals. Isolated adipocyte precursors and mature adipocytes were prepared as previously reported (Yan et al., 1976; Van and Roncari, 1977). | Rien L. Van. Department of Adult Mental Health Care, Arkin Mental Health Care, Amsterdam, Netherlands. And. Daniel A. K. Roncari, MD. emigrated to Canada from Italy when he was 14. He received his MD from Queen’s University in Kingston in 1961 and his MSc in experimental medi- cine under Dr Charles H. Hollenberg at McGill Univer- sity in Montreal in 1965. At Washington University in St Louis with Dr I’. Roy Vagelos, he received his PhD in biological chemistry. After becoming a member of the faculty of medicine at the University of Toronto, he went on to become the third director of the Institute of Medical Science from 1980 to 1983. He then moved to the University of Calgary to become the first Julie McFarlane Professor of Diabetes Research. Dr Roncari returned to Toronto in 1988 to become Physician-in-Chief at Sunnybrook Health Science Centre. Most recently he was Professor of Investigative Medicine at Sunnybrook. He served on the editorial boards of sev | |||||||||||||||||||||
| 12 | Bjorntorp | 1978 | Preadipocytes/ adipoblasts | Adipose Stromal Cells (ASC) | Manual | Rat | Epididymal Fat | Surgical Excision | None | Collagenase Worthington CLS 46D112X | Worthington | 0.1 M HEPES (hydroxyethylpiperazine- ethane-sulfonic acid, Sigma, St. Louis, MO) buffer, 0.12 M NaCl, 0.05 M KCI, 0,001 M CaCI2, 0.005 M glucose, and 1.5% (w/v) bovine serum albumin (Bovine serum albumin Fraction V, batch WB 1370, Armour, Eastbourne, England). | 0.2 % (w/v) Collagenase | 50 ml Erlenmeyer | 37 | 30 | Water bath 120 RPM | 250 micro filter after digestion, 1 g flotation of adipocytes – 15 min – 25 micron filter centrifugation. The density gradient system with colloidal silica and polyvinylpyrrolidone described by Pertoft and Laurent was utilized | 250 micron post digest 25 micron post adipocyte float | Morphology | preadipocytes/ adipoblasts | Adipocyte differentiation | Oil Red O | P. Bjorntorp, M. Karlsson, H. Pertoft, P. Pettersson, L. Sj€ostr€om, U. Smith, Isolation and characterization of cells from rat adipose tissue developing into adipocytes, J. Lipid Res. 19 (1978) 316–324. | Primary Reference Isolation of Adipose Stromal Cells | Bjorntorp 1978 Two fat pads from the small rats or one from the larger rats were incubated in a 50-ml siliconized Erlenmeyer flask containing 10 ml of a solution with final concentrations of 0.1 M HEPES (hydroxyethylpiperazine- ethane-sulfonic acid, Sigma, St. Louis, MO) buffer, 0.12 M NaCl, 0.05 M KCI, 0,001 M CaCI2, 0.005 M glucose, and 1.5% (w/v) bovine serum albumin (Bovine serum albumin Fraction V, batch WB 1370, Armour, Eastbourne, England). This solution contained 0.2% (w/v) collagenase (Worthington, Freehold, NJ, batch CLS 46D112X). The gas phase was air; the pH was 7.4; and the temperature was 37°C. Incubation time was 30 min if not noted otherwise. Incubations were performed in a water bath with rotating flasks (120 rpm). After incubation the tissue remnants were removed by filtration through a nylon screen with a pore size of 250 microns into a siliconized test tube. The fat cells were then allowed to float to the surface for 15 min whereafter the infranatant was aspirated through a siliconized injection needle. The infranatant was then filtered through a nylon screen with a pore size of 25 microns to remove cell aggregates. The cells passing the filter were pelleted by centrifugation and then resuspended in the appropriate buffer or medium for further processing. | Per Bjorntorp, MD, PhD. Died 2003 Research fellow, National Institutes of Health, 1961-1962; research fellow, Medical Research Council, Sweden, 1964-1970; clinical teacher, U. Goteborg, Sweden, 1970-1974; professor, 1976-1996 | ||||||||||||||
| 13 | Williams | 1979 | Capillaries | Stromal Vascular Fraction (SVF) | Manual | Rat | Epididymal fat | Surgical | Collagenase Worthington Type II | Worthington | Dulbecco’s Cation Free PBS | 7.5 mg/ml | 50 ml Erlenmeyer | 37 | 45 | Dubnoff Water Bath Shaking | Centrifugation 100g 3min | 1 x 100g 3min. Final 50 g 1 min | Capillary Endothelium / Rat Fat Capillary Endothelium | Endocytosis Studies | Williams SK, Matthews MA, Wagner RC. Metabolic studies on the micropinocytic process in endothelial cells. Microvasc Res. 1979 Sep;18(2):175-84. doi: 10.1016/0026-2862(79)90027-x. PMID: 491985 | “WAGNER, R. C., AND MATTHEWS, M. A. (1975). The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10, 286-297.” | Isolation and incubation. Capillaries were isolated from rat epididymal fat pads essentially according to the method of Wagner and Matthews (1975). However, following collagenase treatment the following procedure was used. The collagenase digest was centrifuged at 100 xg for 3 min and the floating cake of adipocytes and supernatant were decanted. The vascular pellet was washed with 2 ml Dulbecco’s PBS and recentrifuged at 100xg for 3 min. This washing procedure was repeated two times; however, the final centrifugation was performed at 50xg for only 1 min. The final vascular pellet upon resuspension in Dulbecco’s PBS contained essentially tufts of capillary networks and smaller fragments. | Stuart K Williams PhD Univesity Delaware, Yale,, TJU, UA, UL. Bioficial Organs. Scottsdale AZ. cvregen@gmail.comRoger C Wagner. Emeritus Professor. University of Delaware Newark, DE. Email: Rags@Udel.com | |||||||||||||||||
| 14 | Plaas/Cryer | 1980 | Stromal Cells | Adipose Stromal Cells (ASC) | Manual | Bovine | SubQ | Surgical | 1 mm slices | Collagenase Sigma Type II | Sigma (London) | Krebs Ringers Bicarbonate | 1 ml/100 mg tissue | 42 | 40 to 50 | Digest Filtered through 130 micron Nylon mesh. Floatation removal of adipocytes. Centrifugation 800 g 5 min. | 3x M199 | 130 micron Nylon mesh | Stromal Cells | Plaas HA, Cryer A. The isolation and characterization of a proposed adipocyte precursor cell type from bovine subcutaneous white adipose tissue. J Dev Physiol. 1980 Oct;2(5):275-89. PMID: 7252095. | VAN, adipose R.L.R. & tissue. RCell and Tissue .K. Research, (1977) 181, Isolation of fat cell precursors from adult rat adipose tissue Cell&Tissue Research 181; 197=203 | Preparation and culture of stromal cells isolated from bovine adipose tissue Within 40 min of collection, I mm thick slices of adipose tissue were prepared without tissue cooling. The slices were incubated in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 4% (w/v) bovine serum albumin and 1 mg of collagen ase/ml (1 ml/100 mg tissue) at 42°C for between 40 and SO min. Subsequent!, the digested tissue suspension was filtered through nylon mesh (0.13 1_nm diameter). Intact adipocytes were removed from the suspension by floata hn (Rodbell, 1964). The adipocyte free cell suspension was centrifuged f?r 5 mm at 800 gav to sediment the stromal cells. The cells were washed three time s by resuspension in serum-free ti ssue culture medium (Medium 199). The final p elle of wa shed stromal cells wa s then suspended in Medium 199 supplemen ed with 20% (v/v) fetal calf serum, penicillin (200 units/ml) and strepto y cn (50 μ/ml). Two ml aliquots of the cell suspension ( I 0 5 cells/ml) were :tbut ed mto plastic tissue _ culture flask s (25 cm 2 growth area). The fht/rent cell s were fed by medrnm changes three times per week. At 5-8 day rvals the cells were subcultured. For this, cells were detached from the 0;1h surface with 0.25% (w/v) trypsin in 1 mM EDTA and redi stributed into P cat e flasks at the original cell density. For the preparation of primary ‘tiures approximately 1 g of adipose tissue yielded sufficient stromal cells to ate two flasks. In all the e • d. xpenments | |||||||||||||||||||
| 15 | Williams | 1981 | Capillary Endothelium | Stromal Vascular Fraction (SVF) | Manual | Rat | Epididymal Fat | Surgical | Minced Scissors | Collageanse Type II Sigma | Sigma | Dulbecco’s Cation Free PBS | 2 mg/ml | 50 ml Erlenmeyer with Teflon magneitic spin bar | 37 | 40 | Dubnoff Water Bath Shaking | Centrifuge 100g 7 min. Percoll 23K 15 min washed twice in DCF-PBS | 2 x DCF PBS | Morphology | Capillary Endothelium / Rat Fat Capillary Endothelium | Endocytosis Studies | Phase and EM | Williams SK, Wagner RC. Regulation of micropinocytosis in capillary endothelium by multivalent cations. Microvasc Res. 1981 Mar;21(2):175-82. doi: 10.1016/0026-2862(81)90030-3. PMID: 6783813. | WAGNER, R. C., ANDREWS, S. B., AND MATTHEWS, M. A. (1977). A fluorescence assay for micropinocytosis in isolated capillary endothelium. Microvasc. Res. 14, 67-80. | Isolation of Capillary Endothelium. Microvessels were isolated by a modification of the method of Wagner and Matthews (1975). The distal two-thirds of epididymal fat pads from (-300- g) male Sprague-Dawley rats were dissected, minced, and suspended (two pads per Erlenmeyer flask) in 15 ml of Dulbecco’s divalent cation-free, phosphate-buffered saline containing collagenase (type II, Sigma) at 2 mg/ml, bovine serum albumin (fatty acid-free, Sigma) at 2 mg/ml, and a Teflon coated magnetic stir bar. The flasks were incubated for 40 min at 37°C with gentle stirring. The slurry was then centrifuged (100 x g, 7 min, 4°C) and adipocytes and supernatant were decanted. The vascular pellet was resuspended with 5.0 ml of DCF-PBS and centrifuged at 100 x g for 5 min. The pellet was resuspended in 45% (vol/vol) Percoll (Pharmacia) in DCF PBS NaCl and centrifuged (23,000 x g, 15 min, 4°C). This Percoll gradient centrifugation results in the separation of microvessels, contaminating blood cells, and large blood vessels. The band of microvessels was removed and washed twice with DCF PBS NaCl. The final vascular pellet was resuspended in Dulbecco’s phosphate-buffered saline and contained predominantly capillary endothelium as observed by phrase-contrast and electron microscopy. | Stuart K Williams PhD University Delaware, Yale,, TJU, UA, UL. Bioficial Organs. Scottsdale AZ. cvregen@gmail.com. Roger C Wagner. Emeritus Professor. University of Delaware Newark, DE. Email: Rags@Udel.com | ||||||||||||||
| 16 | Williams | 1981 | Microvessels | Microvascular Fragments | Manual | Rat | Epididymal Fat | Surgical | Minced Scissors | Collageanse Type II Sigma | Sigma | Dulbecco’s Cation Free PBS | 2 mg/ml | 50 ml Erlenmeyer with Teflon magneitic spin bar | 37 | 40 | Dubnoff Water Bath Shaking | Centrifuge 100g 7 min. Pellet resuspended and centrifuged 100xg 5 min. 45% Percoll 23K 15 min washed twice in DCF-PBS | 2 x DCF PBS | Morphology | Endocytosis Studies | Phase Mmicroscopy | Williams SK, Devenny JJ, Bitensky MW. Micropinocytic ingestion of glycosylated albumin by isolated microvessels: possible role in pathogenesis of diabetic microangiopathy. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2393-7. doi: 10.1073/pnas.78.4.2393. PMID: 6941299; PMCID: PMC319352. | “WAGNER, R. C., AND MATTHEWS, M. A. (1975). The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10, 286-297.” | Isolation of Capillary Endothelium. Microvessels were isolated by a modification of the method of Wagner and \1atthews (18). The distal two-thirds of epididymal fat pads from (=300- g) male Sprague-Dawley rats were dissected, minced, and suspended (two pads per Erlenmeyer flask) in 15 ml of Dulbecco’s (19) divalent cation-free, phosphate-buffered saline (CF-P,/ NaCl) containing collagenase (type II, Sigma) at 2 mg/ ml bovine serum albumin (fatty acid-free, Sigma) at 2 mg/ml, and a Teflon-coated magnetic stir bar. The flasks were incubated for 40 min at 37°C with gentle stirring. The slurry was then centrifuged (100 x g, 7 min, 4°C) and adipocytes and supernatant were decanted. The vascular pellet was resuspended with 5.0 ml of CF-P/l’\’aCI and centrifuged at 100 X g for 5 min. The pellet was resuspended in 45% (vol/vol) Percoll (Pharmacia) in CF-P,/ NaCl and centrifuged (23,000 X g, 15 min, 4°C). This Percoll gradient centrifugation results in the separation of microvessels, contaminating blood cells, and large blood vessels. The band of microvessels was removed and washed twice with CF-P,/ NaCl. The final vascular pellet was resuspended in Dulbecco’s phosphate-buffered saline (19) and contained predominantly capillary endothelium as observed by phrase-contrast and electron microscopy (18). | ||||||||||||||||
| 17 | Madri/Williams | 1983 | Capillary Endothelial Cells | Stromal Vascular Fraction (SVF) | Manual | Rat | Epididymal Fat | Surgical | Minced | 2 pads per flask | Collagenase | Sigma | 10 ml of HEPES buffer. bovine serum albumin (BSA), 5 mg/ml | 5 mg/ml | bovine serum albumin (BSA), 5 mg/ml | 37 | 30 | gentle agitation | bovine serum albumin (BSA), 5 mg/ml BSA content adjusted to 10%. Centrifuged 200 g 7 min bovine serum albumin (BSA), “The pellet is resuspended in HEPES buffer containing 10% BSA and spun down, as described above, two times. The resultant pellet is resuspended in 45% Percoll (Pharmacia Fine Chemicals, Piscataway, NJ) (9 ml of Percoll, 1 ml of !Ox HEPES buffer and 10 ml of HEPES buffer containing 10% BSA), and centrifuged at 15,000 g for 20 min at 4°C in a SS 34 fixed-angle rotor (Sorvall).” | “tufts of capillary endothelial cells were washed twice by pelleting them by centrifugation in HEPES-BSA buffer 200 g for 3 min.” | Growth on Collagen | Capillary Endothelial Cells | 2D In Vitro Angiogenesis | Madri, JB, and Williams SK. THE JOURNAL OF CELL BIOLOGY • VOLUME 97 JULY 1983 153-165 | “WAGNER, R. C., AND MATTHEWS, M. A. (1975). The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10, 286-297.” | “Rat capillary endothelial cells were isolated from epididymal fat pads as described by Wagner and Matthews (1975), with several modifications. Briefly, the epididymal fat pads from adult rats (400 g} were removed and pinned out flat on dental Byte wax sheets in petri dishes containing sterile HEPES Buffer (pH 7.4; containing 140 mM NaCl, 10 mM HEPES, 10 mM KCI, 0.1 mM CaCI,, 0.2 mM MgCl2, with 11 g/liter NaHCO3 and 5.0 g/liter glucose). Fat free of large vessels was removed, minced, and placed in sterile 50-ml screw cap Erlenmayer flasks (2 pads/flask} containing 10 ml of HEPES buffer and collagenase (Sigma Chemical Co., St. Louis, MO; type II} 5 mg/ml and bovine serum albumin (BSA}, 5 mg/ml. The flasks were incubated for 30 min at 37°C with gentle agitation. At this time the contents of the flask have the appearance of a pinkish slurry. This slurry is made 10% with respect to BSA (Sigma Chemical Co. Fx 5, fatty acid free} and centrifuged at 200 xg for 7 min in I5-cm3 disposable, sterile conical tubes (Corning Glass Works, Corning, NY}. The pellet is resuspended in HEPES buffer containing 10% BSA and spun down, as described above, two times. The resultant pellet is resuspended in 45% Percoll (Pharmacia Fine Chemicals, Piscataway, NJ} (9 ml of Percoll, I ml of lOx HEPES buffer and 10 ml of HEPES buffer containing 10% BSA}, and centrifuged at 15,000 xg for 20 min at 4°C in a SS 34 fixed-angle rotor (Sorvall). The tufts of capillary endothelial cells are in a milky off-white layer beneath the lymphocytes and above a translucent layer containing larger cells and above a translucent layer containing larger vessel fragments. The tufts of capillary endothelial cells were washed twice by pelleting them by centrifugation in HEPES-BSA buffer 200 xg for 3 min. The tufts were then plated on 60-mm petri dishes coated with type I/III collagen ([100 µg/dish] by the methods of Elsdale and Bark in media (Medium 199 E [Gibco, Grand Island, NY]) containing 20% heat-inactivated fetal bovine serum, penicillin (100 U/ml}, streptomycin (100 U/ml), and glutamine mixed one to one with the same media conditioned for 48 h by incubation with confluent bovine aortic endothelial cell cultures. Cultures were incubated in a 5% CO2, 95% air humidified atmosphere at 37°C. After passage number 10, the cells also grew well in nonconditioned media. | Joseph Madri. Yale University. Stuart K Williams PhD Univesity Delaware, Yale,, TJU, UA, UL. Bioficial Organs. Scottsdale AZ. cvregen@gmail.com. | ||||||||||||||
| 18 | Bjorntorp | 1983 | Capillaries | Mesothelium | Manual | Rat | Epididymal Fat | Surgical | Mince 25 mg pieces | 1 .0 mg/ml of collagenase (Worthington, Freehold, NJ, batch CLS 46 N 026) | Worthington | 10 ml of a solution, pH 7.4, with final concentrations of 0.1 M HEPES buffer (hydroxyethylpiperazineethanesulfonic acid, Sigma, St. Louis, MO), 0.12 M NaCI, 0.05 M KCI, 0.001 M CaCI2, 0.005 M glucose, 1.5% .:w/v) bovine serum albumin (Fraction V, batch WB 13’70, Armour, Eastbourne, England), and 1 .O mg/ml of collagenase (Worthington, Freehold, NJ, batch CLS 46 N 026) | 1 mg/ml | 50 ml siliconized Erlenmeyer Flask | 37 | 15 min | Water Bath 120 cycles/min. After 10 and 15 min, respectively, the flasks were vigorously mixed for 10 sec in a vibrating device (Super-mixer, Lab-Line Instruments, Melrose Park, IL) | After incubation the contents of the flasks were fil- tered through a nylon screen (250 pm pore size) to col- lect any remaining, nondisintegrated tissue. Thereafter the cell suspension was centrifuged at about 300 g for 3 min in a quick start and stop centrifuge. Fat cells and infranatant were now removed with a siliconized Pas- teur pipette. The sediment was resuspended in 1.5 ml of the same HEPES-buffer with additions (except col- lagenase) as above, and the suspension was filtered through a 25-pm pore size nylon screen. This nylon screen was cut to fit into a 5-cm diameter Petri dish. After rinsing the filter with another 1 ml of buffer, the filter was placed upside down in the Petri dish con- taining 2.5 ml culture medium. Next the filter was rinsed, with the aid of mechanical scraping with a plastic spatula, with another 1.5 ml of medium. The nylon net was then removed and the cell suspension was distrib- uted into a Multidish (Flow, Rockville, MD) for cell cul- ture. Cells corresponding to at least one-fourth of the fat pad were used in each culture in a total of 1 ml of culture medium. | Cells were removed from culture vessels by incubat- ing them for 10 min at 37°C with collagenase (1.0 mg/ ml) in buffer as described above. Cells were counted in a Fuchs-Rosenthal blood cell counting chamber with 0.2 mm depth and 1/16 mm2 area. | Bjorntorp, P., G. K. Hansson, L. Jonasson, P. Pettersson, and G. Sypniewska. Isolation and characterization of endo- thelial cells from the epididymal fat pad of the rat. J. Lipid Res. 24: 105-1 12. | Four epididymal fat pads were cut into pieces of about 25 mg and incubated to liberate the cells in 50- ml siliconized Erlenmeyer flasks containing 10 ml of a solution, pH 7.4, with final concentrations of 0.1 M HEPES buffer (hydroxyethylpiperazineethanesulfonic acid, Sigma, St. Louis, MO), 0.12 M NaCI, 0.05 M KCI, 0.001 M CaCI2, 0.005 M glucose, 1.5% .:w/v) bovine serum albumin (Fraction V, batch WB 13’70, Armour, Eastbourne, England), and 1.0 mg/ml of collagenase (Worthington, Freehold, NJ, batch CLS 46 N 026). Incubations were performed for 15 min at 37°C in a water bath where the flasks were shaken at a speed of 120 cycles/min. After 10 and 15 min, respectively, the flasks were vigorously mixed for 10 sec in a vibrating device (Super-mixer, Lab-Line Instruments, Melrose Park, IL). After incubation the contents of the flasks were filtered through a nylon screen (250 micron pore size) to col- lect any remaining, nondisintegrated tissue. Thereafter the cell suspension was centrifuged at about 300 g for 3 min in a quick start and stop centrifuge. Fat cells and infranatant were now removed with a siliconized Pasteur pipette. The sediment was resuspended in 1.5 ml of the same HEPES-buffer with additions (except collagenase) as above, and the suspension was filtered through a 25 micron pore size nylon screen. This nylon screen was cut to fit into a 5-cm diameter Petri dish. After rinsing the filter with another 1 ml of buffer, the filter was placed upside down in the Petri dish con- taining 2.5 ml culture medium. Next the filter was rinsed, with the aid of mechanical scraping with a plastic spatula, with another 1.5 ml of medium. The nylon net was then removed and the cell suspension was distributed into a Multidish (Flow, Rockville, MD) for cell culture. Cells corresponding to at least one-fourth of the fat pad were used in each culture in a total of 1 ml of culture medium. The culture medium consisted of medium 199 with 5 mM glucose, 20% fresh human serum, 40 mU/ml of insulin (Vitrum, Stockholm, Sweden), and 0.1 mg/ml of sodium cefalotin (Keflin, Eli Lilly, Indianapolis, IN). | Per Bjorntorp, MD, PhD. Died 2003 Research fellow, National Institutes of Health, 1961-1962; research fellow, Medical Research Council, Sweden, 1964-1970; clinical teacher, U. Goteborg, Sweden, 1970-1974; professor, 1976-1996 | |||||||||||||||||||
| 19 | Nechad | 1983 | Stromal Vascular Fraction | Adipose Stromal Cells (ASC) | Manual | Rat | Cervvical Brown Fat. Epididymal White Fat | Surgical | cut into small pieces | Crude Collagenase | Sigma | The isolation buffer consisted of 123 mM Na+, 5 mM K+, 1.3 mM Ca*+, 131 mM Cl-, 5 mM glucose, 1.5% (w/v) crude bovine serum albumin and 100 mM N-2-hydroxyethylpiperizine-N’-2- ethanesulfonic acid (HEPES) (pH-adjusted with NaOH to 7.4). When used for cell isolation from animals, this buffer was sterile filtered (Millipore 0.45 urn-HA; 0.22 urn Millex-GS) before use, after the addition of collagenase. | 0.2 % (w/v) | siliconized scintillation vials | 37 | 30 | shaking water bath, with vortexing of the vials every 3-5 min | After incubation, the tissue remnants were removed by filtration through a 250~urn nylon screen into plastic test tubes. The mature adipocytes and the fat droplets resulting from broken cells were then allowed to Boat to the surface of the cell suspension for about 30 min, whereafter the infranatants were collected through a siliconized needle and filtered through a 25urn nylon screen to remove cell aggregates. The cells passing the filter were finally pelleted in test tubes by centrifugation at 700 g,,, for 10 min. | 250 micron Nylon then 25 micron nylon. | hypoosmotic shock (1 ml Eagle’s medium (see below) + 2 ml HZ0 for 20 set at 4°C) | Btirker chamber | average of 1- 1.5~ lo6 such cells were obtained from the epididymal fat (both pads) | SVF | Cell Culture | MYRIAM NfiCHAD,1,2, * PERTTI KUUSELA,’ CLAES CARNEHEIM,’ Development of Brown Fat Cells in Monolayer Culture I. Morphological and Biochemical Distinction from White Fat Cells in Culture. Experimental Cell Research 149 (1983) 105-l 18 | “P. Bjorntorp, M. Karlsson, H. Pertoft, P. Pettersson, L. Sj€ostr€om, U. Smith, Isolation and characterization of cells from rat adipose tissue developing into adipocytes, J. Lipid Res. 19 (1978) 316–324.” | Nylon screens (250 and 25 pm) were obtained from Nytal (Thai, Switzerland). Crude collagenase was purchased from Sigma (St Louis, MO), and fatty acid-free bovine serum albumin from Miles Laboratories (Kankakee, Ill.). FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) was from Pierce Eurochemie (Rotterdam, Holland). Other chemicals were pro analysi. | From each rat, interscapular plus cervical brown fat, and epididymal white fat, were carefully dissected out under sterile conditions. The tissues were then cut into small pieces and treated for isolation of the stromal-vascular fraction essentially as described by Bjomtorp et al. [14]. Each tissue from each rat was firstly incubated in the presence of 0.2 % (w/v) collagenase in 4-ml isolation buffer (see below) in siliconized scintillation vials at 37°C for 30 min in a shaking water bath, with vortexing of the vials every 3-5 min. After incubation, the tissue remnants were removed by filtration through a 250~urn nylon screen into plastic test tubes. The mature adipocytes and the fat droplets resulting from broken cells were then allowed to Boat to the surface of the cell suspension for about 30 min, whereafter the infranatants were collected through a siliconized needle and filtered through a 25urn nylon screen to remove cell aggregates. The cells passing the filter were finally pelleted in test tubes by centrifugation at 700 g,,, for 10 min. The concentrated cells were then resuspended in 500 ~1 culture medium (see below). Numerous red cells were contained in the final brown fat cell suspension. These were eliminated by a hypoosmotic shock (1 ml Eagle’s medium (see below) + 2 ml HZ0 for 20 set at 4°C) [38]. After addition of 5 ml Eagle’s medium, the remaining cells were pelleted and washed once with Eagle’s medium. The cells were finally resuspended in 500 ul culture medium (see below). (This treatment was not performed on the white fat cell suspensions used here, but is now routinely carried out with no effects on any parameters measured.) | MYRIAM NeCHAD The Wenner-Gren Institute, University of Stockholm, S-106 91 Stockholm, Sweden; and Laboratoire de Physiologie Cornparke, Centre National de la Recherche Scientifique UA 307, Universiti Pierre et h4arie Curie, F-75252 Park Cedex, France | ||||||||||||
| 20 | Kern | 1983 | Endothelial Cells | MicroVascular Fragments (MVF) | Manual | Human | Omental and SubQ | Surgical | Cut 2 to 3 pieces, minced | Collagenase | Worthington | Krebs Ringers Bicarbonate, 3 mM Glucose, 4% BSA | 2 mg/ml | Plastic flask | 37 | 15 min | gyratory water bath | 10% FCS | 250 micron nylon mesh (Nitex) , Filtrate 1 g sedimentation, media below adipocytes centrifuged 300 x g 10 min. Pellet resuspended and endothelium captured on a 30 micron nylon mesh. Captured endothlium layered on 5 % BSA, 1 x g for 10 min and lower cell population removed and centrifuged. | Product filtered through 250 micron filter and capturd on 30 micron filter. | FVII rAg | Kern PA, Knedler A, Eckel RH. Isolation and culture of microvascular endothelium from human adipose tissue. J Clin Invest. 1983 Jun;71(6):1822-9. doi: 10.1172/jci110937. PMID: 6306056; PMCID: PMC370387. | Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucos-e metabolism and lipolysis. J. Biol. Chem. 239: 375-380. | Isolation of human microvascular endothelial cells. Af- ter informed consent was obtained, -10 g of omental and/ or subcutaneous adipose tissue was removed during elective surgery for nonmalignant conditions from patients who were in overall good health. The tissue was immediately placed into sterile, iced phosphate-buffered saline (PBS) with 1.0 mM Ca”2 and processed within 2 h. Using a modification of the technique of Rodbell (18), the tissue was divided into 2-3-g pieces that were minced and transferred into a plastic flask containing 7 ml of 2 mg/ml collagenase (Worthington Biochemicals, Freehold, NJ) in Kreb’s-Ringer bicarbonate buffer with 3 mM glucose and 4% bovine serum albumin (BSA). Digestion proceeded at 37°C in a gyratory water bath shaker for 15 min (19). The suspension was then passed through 250-,tm pore nylon mesh (Nitex, Tetko, Inc., Elms- ford, NY) to remove the undigested pieces. To inactivate the collagenase, Medium 199 (Gibco Laboratories, Grand Island, NY) containing 10% heat-inactivated fetal calf serum (Re- heis Chemical Co., New York), 100 U/ml penicillin, and 100 ,gg/ml streptomycin (both from Gibco Laboratories) was added. In the filtrate, the adipocytes floated to the surface and the medium below the adipocytes, containing endothe- lium and stromal cells, was removed and centrifuged at 300 g for 10 min. The cell pellet was resuspended in 5 ml of serum-containing medium, and this in turn was passed through 30-am nylon mesh (Nitex). Clumps of endothelium, containing 4-15 cells, were retained by the mesh, while sin- gle cells, including most of the nonendothelial stromal cells, passed through. The cell aggregates were then washed into a plastic centrifuge tube, recentrifuged, and the pellet was resuspended in 5 ml of serum-containing medium. This sus- pension was then gently layered on 40 ml of PBS containing 1.0 mM Ca”2 and 5% BSA, to allow separation of the larger endothelial cell aggregates from the few remaining single cells. After 10 min at room temperature and at unit gravity, the top 10 ml of the PBS-albumin was removed and the remaining 30 ml was centrifuged. The pellet containing – 1,000 cells, was resuspended in 4 ml of Medium 199 con- taining 10% serum, and plated in a 25-cm2 flask (Corning Glass Corp., Corning, NY). Cultures were incubated at 37°C in a humid 5% CO2, 95% air environment. The culture me- dium was changed every 2-3 d. Serial passage was accom- plished by a brief exposure to 0.25% trypsin with 0.01% EDTA, with the flasks split three for one. | PHILIP A. KERN, ANN KNEDLER, and ROBERT H. ECKEL, Department of Medicine, Endocrinology Division, University of Colorado Health Sciences Center, Denver, Colorado 80262; Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309. Now Professor of Internal Medicine in the Division of Endocrinology at the University of Kentucky. philipkern@uky.edu | ||||||||||||||||
| 21 | Djian | 1983 | Adipocyte Precursors | Adipose Stromal Cells (ASC) | Manual | Rat | epididymal and perirenal | surgical | Collagenase Type II | Sigma | Hanks’ balanced salt solution, pH 7.4, and in the presence of 1.5 mg/ml bovine serum albumin (Fraction V, Sigma Chemical Co.) | 1.5 mg/ml | 37 | 30 to 45 min (until complete disaggregation of tissue occurred | After filtration through a 25-micron pore nylon sieve (Nitex CH325, Thomson Band SM, Montreal, Canada) the cell suspension was centrifuged at 800 g for 10 min. The cells contained in the pellet were then plated on 100-mm Petri dishes. | 25 micron | Culture. Differentiation to adipocytes | Djian P, Roncari AK, Hollenberg CH. Influence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture. J Clin Invest. 1983 Oct;72(4):1200-8. doi: 10.1172/JCI111075. PMID: 6630508; PMCID: PMC370403. | Van, R. L. R., C. E. Bayliss, and D. A. K. Roncari. 1976. Cytological and enzymological characterization of adult human adipocyte precursors in culture. J. Clin. Invest. 58:699-704. Van, R. L. R., and D. A. K. Roncari. 1978. Complete differentiation of adipocyte precursors. A culture system for studying the cellular nature of adipose tissue. Cell Tissue Res. 195:317-329. | Isolation of adipocyte precursors. Adipocyte precursors were isolated from epididymal and perirenal adipose tissue and cultured by methods similar to those previously reported in references 6 and 7. To summarize the procedure and specifying the modifications, the fat pads were digested at 37°C with a 1.5 mg/ml collagenase preparation Type II, Sigma Chemical Co., St. Louis, MO), in Hanks’ balanced salt solution, pH 7.4, and in the presence of 1.5 mg/ml bovine serum albumin (Fraction V, Sigma Chemical Co.) for 30-45 min (until complete disaggregation of tissue occurred). After filtration through a 25-gm pore nylon sieve (Nitex CH325, Thomson Band SM, Montreal, Canada) the cell suspension was centrifuged at 800 g for 10 min. The cells contained in the pellet were then plated on 100-mm Petri dishes. | Daniel A. K. Roncari, MD. emigrated to Canada from Italy when he was 14. He received his MD from Queen’s University in Kingston in 1961 and his MSc in experimental medi- cine under Dr Charles H. Hollenberg at McGill Univer- sity in Montreal in 1965. At Washington University in St Louis with Dr I’. Roy Vagelos, he received his PhD in biological chemistry. After becoming a member of the faculty of medicine at the University of Toronto, he went on to become the third director of the Institute of Medical Science from 1980 to 1983. He then moved to the University of Calgary to become the first Julie McFarlane Professor of Diabetes Research. Dr Roncari returned to Toronto in 1988 to become Physician-in-Chief at Sunnybrook Health Science Centre. Most recently he was Professor of Investigative Medicine at Sunnybrook. | ||||||||||||||||||||
| 22 | Kern | 1984 | Adipose Stromal Cells. Stromal Vascular Fraction | SVF | Manual | Human | Omental SubQ | Surgical | Minced | 2 to 3 gm | Collagenase II Collagenase Type R | Worthington. Calbiochem-Behring | Krebs Ringers Bicarbonate 3mM glucose. 4% BSA | 2 mg/ml | Plastic Flask | 37 | 60 to 90 min. 15 min | Filtered 250 micron nylon mesh. | Stromal Vascular Fraction | Biochemistry Lipo Protein Lipase | Kern PA, Eckel RH. Absence of lipoprotein lipase in cultured human adipose stromal cells. Arteriosclerosis. 1984 May-Jun;4(3):232-7. doi: 10.1161/01.atv.4.3.232. PMID: 6712537. | Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucos-e metabolism and lipolysis. J. Biol. Chem. 239: 375-380. | Omental and/or subcutaneous adipose tissue was excised; was immediately placed into sterile, iced, PBS containing 1.0 mM CaCI2; and was processed within 2 hours. With the method of Rodbell,17 the tissue was divided into 2 to 3 g pieces, which were minced and transferred to a plastic flask containing 7 ml of 2 mg/ml collagenase II (Worthington Biochemicals, Freehold, New Jersey) and Krebs Ringer-bicarbonate buffer with 3 mM glucose and 4% BSA. In some experiments (Subjects 2 and 3), a more gentle digestion was accomplished using 2 mg/ml of collagenase type R (Calbiochem-Behring Corporation, La Jolla, California). In most instances, the tissue was digested for 60 to 90 minutes at 37°C to liberate most of the stromal cells. The stromal cells from Subjects 12 and 14, however, were obtained after a 15- minute collagenase digestion. The suspension was then passed through a 250^ pore nylon mesh, and Medium 199 (Gibco Laboratories, Grand Island, New York) containing 10% heat-inactivated whole fetal bovine serum (Reheis Company, New York, New York), penicillin (100 units/ml, Gibco), and streptomycin (100 /xg/ml, Gibco) was added to the filtrate. The medium below the adipocytes, containing the putative adipocyte precursors, was removed and centrifuged. The cell pellet was then washed once and resuspended in 4 ml of the above medium containing either penicillin-streptomycin or cephalothin (100 /ig/ml, Squibb, Princeton, New Jersey) and plated in 25 cm2 flasks (Corning Glass Corporation, Corning, New York). The size of the cell inoculum varied depending on the degree of digestion and the amount and cellularity of the adipose tissue. In some instances, cells were plated at near confluent density. The cultures were incubated at 37°C in a humid 5% CO2, 95% air environment. The medium was changed every 2 to 3 days, and serial passage was accomplished by a brief exposure to 0.25% trypsin with 0.01% EDTA, with the flasks split three from one. The counting of trypsinized cells was performed with a hemocytometer. The staining of cells in the monolayer was accomplished using oil red 0 and hematoxylin after fixing cells in Bouin’s fixative. | PHILIP A. KERN, ANN KNEDLER, and ROBERT H. ECKEL, Department of Medicine, Endocrinology Division, University of Colorado Health Sciences Center, Denver, Colorado 80262; Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309. Now Professor of Internal Medicine in the Division of Endocrinology at the University of Kentucky. philipkern@uky.edu | |||||||||||||||||
| 23 | Williams US Patent 4,820,626 | 1985 | Microvascular Endothelial Cells | SVF | Manual | Human | Abdominal | Surgical | Scissors | Crude Clostridial Collagenase | PBS | 37 | 15 to 30 | Centrifugation 700 g 5 min. Can use Percoll | Microvascular Endothelial Cells | Charaterization Studies | US Patent 4,820,626 | an amount of human microvascularized fat (A) may be procured from a number of sources. Although less preferred, it is possible to obtain human perinephric fat from brain-dead but heart beating cadaver donors, or from donors other than the patient during the donor’s surgery. In any event, the donated tissue is then immediately transferred to ice cold buffered saline (pH 7.4) wherein the buffering agent is preferably a phosphate, i.e., a phosphate buffered saline (PBS). The tissue is minced (Step B) with fine scissors and the buffer decanted. The proteolytic enzyme collagenase, containing caseanase and trypsin, is added to the tissue and incubated at 37° C. until the tissue mass disperses. This digestion occurs within thirty (30) minutes, and generally should be less than fifteen (15) minutes. The digest is transferred to a sterile test tube and centrifuged (Step C) at low speed (700 X g) in a table top centrifuge for five (5) minutes at room temperature. The pellet of cells thus formed consists of greater than ninety-five percent (95%) endothelial cells. These endothelial cells are described herein as microvascular endothelial cells (MEC) since they originate from the arterioles, capillaries and venules, all elements of the microvasculature. | |||||||||||||||||||||||
| 24 | Hauner | 1986 | Rat Adipocyte Precursor Cells | SVF | Manual | Rat | epididymal fat | Surgical | Mechanically minced | Collagenase CLS type I Worthington | Worthington | Media 199 10% FCS | 1 mg/ml | 37 | 40 to 60 | Sedimentation | 1x | 250 micron Nylon | Adipose Derived Stromal Cells | In Vitro Studies | Hauner H, Schmid P, Pfeiffer EF. Differential effect of insulin on the synthesis of lipoprotein lipase and glycerophosphate dehydrogenase activity in developing rat adipocyte precursor cells. Horm Metab Res. 1986 Oct;18(10):719-21. doi: 10.1055/s-2007-1012417. PMID: 3536708. | Bjomtorp, P., M. Karlsson, H. Pertoft, P. Pettersson, L. Sjostrom, and U. Smith. 1978. Isolation and characterization ofcells from rat adipose tissue developing into adipocytes. J. Lipid Res. 19:316- 324. | Primary cultures of rat adipocyte precursor cells were established using a modification of a method described previously (Bfornrorp. Karlsson, Pertoft, Pettersson, Sjostrom and Smith 1978). Thccpididymal fat pads of young male Wistar rats weighing between 150 and 180 g were removed under sterile conditions, washed in phosphate buffered saline and were mechanically minced. The tissue pieces were incubated for 40 to 60 min. in medium 199 supplemented with 10% fetal calf serum (Seromed, Munich) and 1 mgJml collagenase (CLS Type I, Worthington). After filtration through a nylon mesh of 250 um pore size and centrifugation the floating fat cells were aspirated. The sedimented cells were washed, resuspended in medium 199 supplemented with 10% fetal calf serum and inoculated in 35 mm culture dishes (Greiner, Niirtingen, Germany). | Hans Hauner Diabetes Research Institute, D-40225 Düsseldorf, Germany. Head of Department at Technical University of Munich Germany. hans.hauner(at)tum.de | |||||||||||||||||
| 25 | Hauner | 1987 | Stromal Vascular Cells | SVF | Manual | Human | SubQ | Surgical | Cut small pieces | Collagenase CLS type I Worthington | Worthington | Krebs Ringers HEPES | 2 mg/ml | 37 | 50 | Intermittent Agitation | Centrifugation 500g 10 min | Digest through 250 micron Nylon mesh | Morphology | Adipose Stromal Vascular Cells | In Vitro Studies | Hauner, H., P. Schmid, and E. F. Pfeiffer. 1987. Glucocorticoids and insulin promote the differentiation of human adipocyte precursor cells into fat cells. J. Clin. Endocrinol. Metab. 64:832-835. | Van RLR, Bayliss CE, Roncari DAK 1976 Cytological and enzymological characterization of adult human adipocyte precursors in culture. J Clin Invest 58:699 | Tissue samples from the individual donors were put in phosphate- buffered saline containing 20 mg/mL BSA. Blood vessels and fibrous material were cut off and discarded, and the tissues were repeatedly washed to remove any remaining blood. Then, the tissues were cut into small pieces and digested by a modification of the method described by Van et al. (4) using 2 mg/ mL collagenase (CLS type I, Worthington Biochemical Corp., Freehold, NJ) in Krebs-Ringer-HEPES (25 mmol/L), pH 7.4, containing 20 mg/mL BSA (Sigma, Munich, West Germany; fraction V powder) for approximately 50 min with intermittent agitation. The ratio of adipose tissue mass to incubation solution was approximately 1 g/3 mL. After dispersion, the cells were filtered through a nylon mesh (pore size, 250 nm) and centrifuged at 500 X g for 10 min. Collagenase digestion of adipose tissue results in the formation of two distinct fractions: the floating mature fat cells and the sedimented stromal-vascular cells. The latter is the source of the adipocyte precursor cells, and it also contains fibroblasts, endothelial cells, and a high proportion of blood cells (~80-90%). After the floating fat cells were removed by aspiration, the sedimented cells were washed twice with phosphate-buffered saline and suspended in medium 199 supplemented with 15% fetal calf serum (Seromed, Berlin, West Germany), penicillin (100 U/mL), and streptomycin (0.1 mg/mL). This standard medium contained cortisol at a final concentration of approximately 10~9 mol/L. | Hans Hauner Diabetes Research Institute, D-40225 Düsseldorf, Germany. Head of Department at Technical University of Munich Germany. hans.hauner(at)tum.de | ||||||||||||||||
| 26 | Knedler | 1987 | Human Microvascular Endothelial Cells | Human | Omentum | Knedler A, Ham RG. Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum. In Vitro Cell Dev Biol. 1987 Jul;23(7):481-91. doi: 10.1007/BF02628418. PMID: 3301790. | Kern PA, Knedler A, Eckel RH. Isolation and culture of microvascular endothelium from human adipose tissue. J Clin Invest. 1983 Jun;71(6):1822-9. doi: 10.1172/jci110937. PMID: 6306056; PMCID: PMC370387. | Initially, primary cultures of HMVEC were prepared as previously described (34 | ANN KNEDLER’ AND RICHARD G. HAM’ Department of Molecular, Cellular and Developmental Biology. Campus Box 347, University of Colorado, Boulder, Colorado 80309 | ||||||||||||||||||||||||||||||||
| 27 | Hauner | 1988 | Stromal Vascular Cells | SVF | Manual | Human | SubQ Abdominal | Surgical | dissected into small pieces | 0.7 to 3.5 gm | Collagenase Type I | Worthington CLS Type I | Krebs Ringer HEPES | 2 mg/ml | 37 | 40 to 60 | shaking water bath | Filtration through a nylon mesh (pore size 250 μm) After repeated washing and centrifugation at 800 x g a fracton of stromal-vascular cells was obtamed | repeated | 250 micron | Hauner H, Wabitsch M, Pfeiffer EF. Differentiation of adipocyte precursor cells from obese and nonobese adult women and from different adipose tissue sites. Horm Metab Res Suppl. 1988;19:35-9. PMID: 3235057. | “Bjomtorp, P., M. Karlsson, H. Pertoft, P. Pettersson, L. Sjostrom, and U. Smith. 1978. Isolation and characterization ofcells from rat adipose tissue developing into adipocytes. J. Lipid Res. 19:316- 324.” | Adipose tissue samples of 0.7 to 3.5 g were immers ed into Krebs-Ringer-Hepes ( 1 0 mmol/1) buffe supplemented with 20 mg/ml bovine serum albumin, pH 7.4. The samples were repeatedly washed, dis_sected into small pieces and incubated with Krebs-Rmger-Hepes. buffer containing 2 mg/ml collagenase (CLS type I, Worthington Biochemical Corp., Freehold, NJ)}or 40 to 60 minutes in a shaking water bath at 37 Celsius Before filtration through a nylon mesh (por e size 250 μm) aliquots of the floating fat_cell lay er were removed for fat cell sizing accordmg to Br_ay (1 970). More than 90 % of the adip?cytes wre mble as assessed by the acridin orange dymg techmq (Lorch and Rentsch 1969). After re?eated wa mg and centrifugation at 800 x g a fracton of se!IDentable stromal-vascular cells was obtamed, w;:s d din medium 199 supplemented wit o }:e;al; serum (Gibco, Berlin), 0, 1 mg/ml strept? my- . d 100 U/ml penicillin (Boehringer, Mannheim) cm an . db (B” . . torp Karlsson Per- as originally de scnbe .. y 1 0m “th , 1 978) The s··· tram and Sm1 toft, Pettersson, 10s . lated at a density of | Hans Hauner Diabetes Research Institute, D-40225 Düsseldorf, Germany. Head of Department at Technical University of Munich Germany. hans.hauner(at)tum.de | |||||||||||||||||
| 28 | Nougues | 1988 | Stromal Vascular Cells in Pellet. Rabbit Adipocyte Precursor Cells | Adipose Stromal Cells (ASC) | Manual | Rabbit | Perirenal | Surgical | Minced | 1.8 mg/ml collagenase, 1.4 mg/ml dispase 0.3 mg/ml hyaluronidase | Collagenase, dispase, hyaluronidase, were obtained from Boehringer Mannheim (FRG) | 1.8 mg/ml collagenase, 1.4 mg/ml dispase 0.3 mg/ml hyaluronidase | 37 | 90 | Controlled Agitation | 25 micron Nylon Mesh Filter Centrifugation 200 g 10 mmin | 25 micron Nylon Mesh | Morphology. Oil Red O | Stromal Vascular Cells in Pellet. Rabbit Adipocyte Precursor Cells | Cell Culture Characterization | Jean NOUGUES, Yves REYNE and Jean-Paul DULOR Jntemational Joumal of Obesity (1988) 12, 321-333. | Perirenal adipose tissue was removed from 28-day-old rabbit fetuses under sterile conditions and stromal-vascular cells were separated by enzymatic digestion of the minced tissues. The digestion was performed at 37°C for 90 min under controlled agitation in Hank’s buffer (pH 7.3) containing 1.8 mg/ml collagenase, 1.4 mg/ml dispase and 0.3 mg/ml hyaluronidase. Subsequently the digested tissue suspension was filtered through a 25-µm nylon mesh filter and centrifuged at 200 g for JO min in order to sediment the stromal-vascular cells. These cells were washed three-times by resuspension in Dulbecco’s modified Eagle’s medium (DME medium) + 10 per cent rabbit serum. The final pellet of washed stromal cells was then suspended in a DME medium containing HEPES buffer (pH 7.4), biotin, pantothenate, penicillin and JO per cent rabbit serum or JO per cent rabbit plasma as indicated below. The cells were seeded into 35-mm culture dishes at a density of 1.4 X J04 cells/cm2; seeding efficiency was routinely 50 per cent. Cultures were kept at 37°C in an atmosphere of 95 per cent air- 5 per cent CO2 and culture media were changed every 2 days. The volume of medium used per dish was I ml. At confluence (4-5 days after plating), the medium was changed to one supplemented with 17 nM insulin, 2 nM triiodothyronine, lymph or chylomicrons, JO per cent rabbit plasma or 10 per cent rabbit serum as indicated below. | Jean NOUGUES | ||||||||||||||||||
| 29 | Hauner | 1989 | Stromal-Vascular Cells | SVF | Manual | Human | SubQ | Surgical | Cut small pieces 10-20 mg | 2 – 15 gm | Collagenase CLS type I Worthington | Worthington | HEPES Buffer 20mg/ml BSA | 1.5 mg/ml | 37 | First Digestion 30 – 45 min. Filtered 250 micron Nylon Mesh. Seccond digestion 30 min | Intermittant Shaking | Centrifugation 200g 10 min | Centrifugation | 250 mircron | Lysis Buffer | Trypan Blue | Stromal Vascular Fraction. | SVF differentiation to adipocytes in Tissue Culture | Hauner et al., “Promoting Effect of Glucocorticoids on the Differentiation of Human Adipocyte Precursor Cells Cultured in a Chemically Defined Medium,” J Clin Invest,(1989), vol. 84, pp. 1663-1670. | Bjomtorp, P., M. Karlsson, H. Pertoft, P. Pettersson, L. Sjostrom, and U. Smith. 1978. Isolation and characterization ofcells from rat adipose tissue developing into adipocytes. J. Lipid Res. 19:316- 324. | Adipose tissue samples were obtained from the subcutaneous abdominal depot of 17 male and 10 female adults in the age range between 20 and 83 yr undergoing elective abdominal surgery. Patients suffering from an inflammatory or malignant disease were excluded. The operations were carried out for the following reasons: 10 for herniotomy, 7 for cholecystectomy, 3 for abdominal vascular surgery, for transabdominal colon or Sigma polypectomy, 2 for selective proximal vagotomy, and 1 for tubal ligation. All subjects were of normal weight as defined as a body mass index under 27.8 kg/m2 for male and 27.3 kg/M2 for females (14). All patients had a normal physical examination and routine laboratory tests were within normal limits. None of the subjects was under a reduction diet at the time ofexamination. The data on age and body mass index ofthe individual subjects are given in Table I. The procedure followed in this investigation has been approved by the Ethical Committee of the University of Ulm. Isolation and culture of stromal-vascular cells. The isolation of stromal-vascular cells was performed according to the method originally described by Bjorntorp et al. (15) with some modifications. Tissue samples (2-15 g) from the individual donors were kept at room temperature in PBS containing 20 mg/ml BSA (Sigma Chemical Co., Munich, West Germany), pH 7.4, and immediately transferred to the laboratory. The samples were repeatedly rinsed in PBS to remove any blood. Fibrous material and blood vessels were carefully dissected and discarded. The remaining tissue cut into small pieces (10-20 mg) was digested in Krebs-Ringer buffered with 25 mM Hepes containing 1.5 mg/ml collagenase (CLS type I; Worthington Biochemical Corp., Freehold, NJ) and 20 mg/ml BSA in a two-step procedure. The ratio between adipose tissue mass to incubation solution was 1 g/4 ml. The first digestion was carried out for 30-45 min at 370C under intermittent shaking. The dispersed tissue was filtered through a nylon mesh (pore size 250 Mm). The remaining tissue was again digested for another 30 min and then filtered as above. More than 95% of the tissue was disaggregated by this procedure and the remaining fibrous material was discarded. Both cell suspensions were mixed and centrifuged for 10 min at 200 g. The main contaminating cells of the cell suspension were erythrocytes, which constituted more than 90% oftotal cell number at this stage. The high contamination with red blood cells was found to markedly decrease cell adherence and proliferation. To eliminate red blood cells, the stromal-vascular cell fraction was incubated with a erythrocyte lysing buffer consisting of 0.154 M NH4CI, 10 mM KHCO3, and 0.1 mM EDTA for 10 min at room temperature. Under these conditions, > 95% of red blood cells were lysed without damaging the nucleus-containing cells as assessed by Trypan blue exclusion. The application of the erythrocyte lysing buffer did not interfere with cell attachment, growth or differentiation of nucleus-containing cells in the stromal-vascular fraction. (Control experiments showed that treatment with the erythrocyte lysing buffer reduced dramatically the number ofred blood cells previously mixed with 3T3-LI cells, with no change in growth and differentiation of the latter.) After additional washing and centrifugation steps, the floating mature adipocytes were aspirated and the sedimented stromal-vascular fraction resuspended in DME/Ham’s F-12 medium (1:1, vol/vol) supplemented with 10% FCS (Seromed, Berlin, West Germany), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Boehringer, Mannheim, West Germany). Aliquots of the cell suspension were counted with a hemocytometer to determine the number of nucleus-containing cells. Cells were inoculated at various densities as indicated into 12-well plates, each well representing – 4.5 cm2 (Linbro, Flow Laboratories, McLean, VA). After 16-20 h routinely used for cell attachment, cells were carefully washed with PBS to remove nonadhering material, mainly white blood cells and cell debris. Unless otherwise stated cells were inoculated at a density of 30,000/cm2. | ||||||||||||||
| 30 | Curti | 1989 | Human Adipose Endothelial Cells. HAEC | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Surgical | Mechanically Minced | Sigma Type I | Sigma | Dulbeccos PBS 0.1 % BSA | 2 mg/ml | 25 ml Erlenmeyer | 37 | 30 | Constant Agitation | Percoll | 1. 200xg 10 min 2. 120 micron screen 3. 200 x g 10 min. 4. Percoll 1500 g 20 min 5. two washes 200 xg 10 min | 120 micron Nylon Screen | Percoll | Hemocytometer Trypan Blue Dye Exclusion. Electron Microscopy. Actin Desmin. Vimentin. Factor VIII. Gpiib IIIa. UEA1 Lectin Alkaline Phosphatase | Human Adipose Endothelial Cells HAEC | Vascular Graft Seeding | Curti T, Pasquinelli G, Preda P, Freyrie A, Laschi R, D’Addato M. An ultrastructural and immunocytochemical analysis of human endothelial cell adhesion on coated vascular grafts. Ann Vasc Surg. 1989 Oct;3(4):351-63. doi: 10.1016/S0890-5096(06)60159-3. PMID: 2532036. | “Human subcutaneous adipose tissue was ob tained from patients (n = 20) undergoing abdominal””surgery. The tissue was immediately placed in cold Dulbecco’s phosphate buffered saline (PBS). Using a modification of the Jarrel method [13] the fat was mechanically minced and transferred to an Erlenmeyer flask containing 50 ml of PBS, pH 7.4, with collagenase* 2 mg/ml and bovine serum albu min (BSA) (Sigma type V) 2 mg/ml. The flask was incubated for 30 minutes at 37°C with constant agitation. The suspension, containing HAEC collected from below the adipocytes floating on the surface, was then centrifuged at 200 x g for 10 minutes. The resulting pellet was resuspended in PBS containing 0.1% BSA, and the suspension was filtered through a 120 µ,m nylon screen to remove undigested fragments of tissue. After being centrifuged again, the pellet was resuspended in 2 ml of PBS-BSA and gently layered on 8 ml of 45% Percollt in PBS (final osmolarity 280 mOSM) and centrifuged at 1500 x g for 20 minutes at room temperature. HAEC were recovered in a milky-white layer at the top of the Percoll cushion while red cells, white cells, and cellular debris were found in the pellet. HAEC were washed twice by centrifugating in PBS-BSA at 200 x g for 10 minutes and finally resuspended in Medium 199 containing 20% heat inactivated fetal calf serum.§ Cells were counted in a hemocytometer. Cell viability, as determined by Trypan Blue exclusion, resulted in higher than 90% of isolated HAEC.” | https://www.linkedin.com/in/tiziano-curti-b1086065?lipi=urn%3Ali%3Apage%3Ad_flagship3_profile_view_base_contact_details%3BG7sZ0c2rQNuLuhpAvdfhhg%3D%3D | ||||||||||||||
| 31 | Rehnmark | 1989 | Brown Fat Precursor Cells | Adipose Stromal Cells (ASC) | Manual | Rat | Cervvical Brown Fat | Surgical | cut into small pieces | Crude Collagenase | Sigma | The isolation buffer consisted of 123 mM Na+, 5 mM K+, 1.3 mM Ca*+, 131 mM Cl-, 5 mM glucose, 1.5% (w/v) crude bovine serum albumin and 100 mM N-2-hydroxyethylpiperizine-N’-2- ethanesulfonic acid (HEPES) (pH-adjusted with NaOH to 7.4). When used for cell isolation from animals, this buffer was sterile filtered (Millipore 0.45 urn-HA; 0.22 urn Millex-GS) before use, after the addition of collagenase. | 0.2 % (w/v) | siliconized scintillation vials | 37 | 30 | shaking water bath, with vortexing of the vials every 3-5 min | After incubation, the tissue remnants were removed by filtration through a 250~urn nylon screen into plastic test tubes. The mature adipocytes and the fat droplets resulting from broken cells were then allowed to Boat to the surface of the cell suspension for about 30 min, whereafter the infranatants were collected through a siliconized needle and filtered through a 25urn nylon screen to remove cell aggregates. The cells passing the filter were finally pelleted in test tubes by centrifugation at 700 g,,, for 10 min. | 250 micron Nylon then 25 micron nylon. | hypoosmotic shock (1 ml Eagle’s medium (see below) + 2 ml HZ0 for 20 set at 4°C) | Btirker chamber | Rehnmark S, Kopecký J, Jacobsson A, Néchad M, Herron D, Nelson BD, Obregon MJ, Nedergaard J, Cannon B. Brown adipocytes differentiated in vitro can express the gene for the uncoupling protein thermogenin: effects of hypothyroidism and norepinephrine. Exp Cell Res. 1989 May;182(1):75-83. doi: 10.1016/0014-4827(89)90280-2. PMID: 2497023. | Nechad, M., Kuusela, P., Cameheim, C., Bjotntorp, P., Nedergaard, J., and Cannon, B. (1983) Exp. Cell Res. 149, 105-118. | Cell isolatjon and culture. The brown-fatp recursocr ellsw erei solatedfr omt he interscapulabrr own fat of theS -week-olmd ice,e xactly asd escribe-fodr rat cells[ 2], but without a hypoosmotisch ock.F or each experiment, brown fat from a full litter from an untreated or from an MM&treated dam was pooled,w hereaftere qual amountso f cell suspensiown ere inoculatedin to 25-cm*c ulture flasks (NunclonD elta).A cellq uantity correspondintgo thec ellsi solatedfr om 1.5a nimalsw asu sedf or one flask,c ontaining5 mlo f culturem ediumc onsistingof DMEM (Dulbecco’sm od&d Eagle’sm edium) supplementewdi th 10% newborn-calsf erum,4 nM insulin, and 10 m&4H epes,a nd with 50 IU penicillina nd5 0 pg streptomycinp er milliliter( i.e., a mediump racticallyi denticalt o that usedb y N&had et al. [3], but without the Intralipid). | Jan Nedergaard. Professor of Physiology at the Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University. Jan Nedergaard e-mail: jan@metabol.su.se | ||||||||||||||||
| 32 | Rehnmark | 1990 | Brown Fat Precursor Cells | Adipose Stromal Cells (ASC) | Manual | Rat | Cervvical Axillary and interscapular Brown Fat | Surgical | cut into small pieces | Crude Collagenase | Sigma | The isolation buffer consisted of 123 mM Na+, 5 mM K+, 1.3 mM Ca*+, 131 mM Cl-, 5 mM glucose, 1.5% (w/v) crude bovine serum albumin and 100 mM N-2-hydroxyethylpiperizine-N’-2- ethanesulfonic acid (HEPES) (pH-adjusted with NaOH to 7.4). When used for cell isolation from animals, this buffer was sterile filtered (Millipore 0.45 urn-HA; 0.22 urn Millex-GS) before use, after the addition of collagenase. | 0.2 % (w/v) | siliconized scintillation vials | 37 | 30 | shaking water bath, with vortexing of the vials every 3-5 min | After incubation, the tissue remnants were removed by filtration through a 250~urn nylon screen into plastic test tubes. The mature adipocytes and the fat droplets resulting from broken cells were then allowed to Boat to the surface of the cell suspension for about 30 min, whereafter the infranatants were collected through a siliconized needle and filtered through a 25urn nylon screen to remove cell aggregates. The cells passing the filter were finally pelleted in test tubes by centrifugation at 700 g,,, for 10 min. | 250 micron Nylon then 25 micron nylon. | hypoosmotic shock (1 ml Eagle’s medium (see below) + 2 ml HZ0 for 20 set at 4°C) | Btirker chamber | Rehnmark S, Néchad M, Herron D, Cannon B, Nedergaard J. Alpha- and beta-adrenergic induction of the expression of the uncoupling protein thermogenin in brown adipocytes differentiated in culture. J Biol Chem. 1990 Sep 25;265(27):16464-71. PMID: 1697859. | Rehnmark S, Kopecký J, Jacobsson A, Néchad M, Herron D, Nelson BD, Obregon MJ, Nedergaard J, Cannon B. Brown adipocytes differentiated in vitro can express the gene for the uncoupling protein thermogenin: effects of hypothyroidism and norepinephrine. Exp Cell Res. 1989 May;182(1):75-83. doi: 10.1016/0014-4827(89)90280-2. PMID: 2497023. | Brown fat precursor cells were isolated from cervical, axillary, and interscapular brown adipose tissue of 4- week-old male mice, as described in Rehnmark et al. (20) (identical to the procedure used for rat cells by N&had et al. (12), except for the hypoosmotic shock). A cell quantity corresponding to the cells isolated from one animal was inoculated per culture flask (Bibby, Stockholm, Sweden, 25 cm’), containing 5 ml of culture medium consisting of Dulbecco’s modified Eagle’s medium supplemented with 10% newborn calf serum, 4 nM insulin, 10 mM Hepes, and 50 IU of penicillin, 50 pg of streptomycin, and 25 Kg of sodium ascorbate per ml. The cells were grown at 37 “C in an atmosphere of 8% CO, in air (as recommended for Dulbecco’s modified Eagle’s medium by the suppliers) with 80% humidity. The medium was changed on days 1 and 3 after plating. Unless otherwise specified, the cultures were analyzed on day 6 after plating (i.e. around confluency). The cells were exposed to norepinephrine (freshly made aqueous solution for each experiment) and/or other agents for 4 h (if not otherwise stated) and harvested. Cell Harvesting, | Jan Nedergaard. Professor of Physiology at the Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University. Jan Nedergaard e-mail: jan@metabol.su.se | ||||||||||||||||
| 33 | Park | 1990 | Human Adipose Endothelial Cells | SVF | Manual | Human | SubQ | Surgical | Minced | Collagenase | Worthington | Dulbeccos PBS 0.1 % BSA | 4mg/ml | 50 ml Erlenmeyer | 37 | 50 | Dubnoff Shaking water bath | Centrifugation 700g 4 minutes Filtered 500 micron filter, Centrifuged and resuspended. | 1x | Digest 500 micron filter | Endothelial Cells | Human Vascular Graft Sodding | Park PK, Jarrell BE, WILLIAMS SK, Carter TL, Rose DG, Martinez-Hernandez A and Carabasi RA: 1990. Thrombus-free, Human Endothelial Surface in the Mid-region of a Dacron Vascular Graft in a Splanchnic Venous Circuit-observations after Nine Months of Implantation. Journal of Vascular Surgery. 11(3):468-475. | First in Man report | Fifty grams of subcutaneous fat was excised and taken to a sterile back table within the operating room. The fat was then minced, mixed with collagenase (W orthington Biochemicals, Freehold, N.J.) 4 mg/ml, diluted in Dulbecco’s cation-free physiologic saline solution containing 4 mg/ml human serum albumin, and incubated at 37′ C for a total of50 minutes. The resulting slurry was filtered and centrifuged at 700 g for 4 minutes at room temperature. The endothelial cell pellet was washed in media 199£ with 1 % human scrum albumin (M-199 /HSA), centrifuged, and resuspended for subsequent graft incubation. | Stu Williams | |||||||||||||||
| 34 | Gregoire | 1990 | stroma-vascular fraction | Adipose Stromal Cells (ASC) | Manual | Rat | peri-epididymal and subcutaneous inguinal fat | Surgical | Minced | Collagenase | Boehringer Mannheim | 5 mg/ml of control medium per g of tissue | 37 | 45 | Agitation | The resulting cell suspension was homogenized by mechanical dissociation with 10 ml serological plastic pipette, filtered through a 100 ~m nylon filter and centrifuged at 400 g for 5 min. | The floating mature adipocytes were washed and centrifuged again in order to obtain a second pellet of stroma-vascular cells that-was pooled with the first pellet and filtered through a 25 ~m nylon filter to eliminate residual aggregates. | 100 micron 25 micron | Grégoire F, Todoroff G, Hauser N, Remacle C. The stroma-vascular fraction of rat inguinal and epididymal adipose tissue and the adipoconversion of fat cell precursors in primary culture. Biol Cell. 1990;69(3):215-22. doi: 10.1016/0248-4900(90)90348-7. PMID: 2097004. | The stroma-vascular cells were obtained by collagenase digestion (Clostridium histolyticum – specific activity 0,44 U/mg, Boehringer Mannheim, FRG) of the minced tissue. The collagenase concentration was adjusted to the tissue weight in a ratio : 5 mg/ml of control medium per g of tissue. The digestion was performed for 45 rain at 37°C under agitation. The resulting cell suspension was homogenized by mechanical dissociation with 10 ml serological plastic pipette, filtered through a 100 ~m nylon filter (Tripette & Renaud, Combles, France) and centrifuged at 400 g for 5 min. The floating mature adipocytes were washed and centrifuged again in order to obtain a second pellet of stroma-vascular cells that-was pooled with the first pellet and filtered through a 25 ~m nylon filter to eliminate residual aggregates. Half of this stroma-vascular fraction was then treated for transmission electron microscopy (TEM), the other half being inoculated in primary culture. | C;aude Remacle. Université Catholique de Louvain, Life Sciences Institute, Louvain-la-Neuve, Belgium. claude.remacle@uclouvain.be | ||||||||||||||||||||
| 35 | Kirkland | 1990 | Stromal Vascular Cells | Insufficient Data To Classify | Manual | Rat | epididymal fat | surgical | Minced | Collagenase | Hanks’ balanced salt solution (HBSS) | 1 mg/ml | 37 | centrifugation at 800 g for 10 min | Adipose Stromal Vascular Cells | Cell Culture. Growth and Differentiation to adipocytes | Kirkland JL, Hollenberg CH, Gillon WS. Age, anatomic site, and the replication and differentiation of adipocyte precursors. Am J Physiol. 1990 Feb;258(2 Pt 1):C206-10. doi: 10.1152/ajpcell.1990.258.2.C206. PMID: 2305864. | DJIAN, P., D. A. K. RONCARIA, NDC . H. HOLLENBERGI.n fluence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture. J. Clin. lnuest. 72: 1200-1208, 1983. 9. DJIAN, | Charles Hollenberg started the Banting and Best Institute at Univ Toronto. Died in 2003 | fragments by mincing and digestion in 1 mg collagenase/ ml Hanks’ balanced salt solution (8)(Djian 1983) . Stromal vascular cells were isolated by centrifugation at 800 g for 10 min and were plated in loo-mm Petri dishes in a basal medium (a-minimal essential Eagle’s medium with 10% fetal bovine serum). After 12 h, a period over which no cell replication occurs | kirkland.james@mayo.edu C. H. Hollenberg, Institute of Medical Science, Rm. 7238, Medical Sciences Bldg. Univ. of Toronto, Toronto, Ontario M5S lA8, Canada. | ||||||||||||||||||||
| 36 | Klaus | 1991 | Stromal Vascular Fraction SVF | SVF | Manual | Hamster | Brown Fat – axillary, interscapular, dorsal-cervical, and parts of subscapular depot | Surgical | Cut in small pieces | Collagenase | 100 mM Hepes (pH 7.4), 123 mM NaCl, 5 mM KCI, 1 .3 mM CaC12, 5 mM glucose, 1 .5% (wt/vol) BSA, | 1 mg/ml (10 ml/gm tissue) | 37 | 30 to 40 | Vortexed every 5 min | Mixture filtered over a 250 micron Nylon filter 1 g separation at RT for 5 to 10 min, Buoyant adipocytes discradrd and lower phase centrifuged 700g for 10 min RT. | 1 wash culture media centrifuged 700 g 10 min RT | cells counted in a Malassez counting chamber. | 1.1 x 106/g BAT | Stromal Vascular Fraction Name changed to preadipocytes | Culture – Culture media 50% MEM (GibcoBRL, Cergy Pontoise, France) and 50% F12 Hams F12 medium (GibcoBRL) supplemented with Na HC03 (1 .2 g/1), biotine (4 mg/1), Capanthotenate (2 mg/1), glutamine (5 mM), glucose (4 .5 g/1) and Hepes (pH 7.4, 15 mM), penicillin G (6.25 mg/1), and streptomycin (5 mg/1) . Until appearance of differentiated adipocytes the medium was supplemented with 10% FCS | Klaus S, Cassard-Doulcier AM, Ricquier D. Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP. J Cell Biol. 1991 Dec;115(6):1783-90. doi: 10.1083/jcb.115.6.1783. PMID: 1684582; PMCID: PMC2289199. | Rehnmark S, Kopecký J, Jacobsson A, Néchad M, Herron D, Nelson BD, Obregon MJ, Nedergaard J, Cannon B. Brown adipocytes differentiated in vitro can express the gene for the uncoupling protein thermogenin: effects of hypothyroidism and norepinephrine. Exp Cell Res. 1989 May;182(1):75-83. doi: 10.1016/0014-4827(89)90280-2. PMID: 2497023.. | Isolation and culture of brown preadipocytes was adapted from the protocol used in reference 32 Rehmark et al 1989 . Animals aged between 4 and 6 wk were anaesthetized with chloroform and killed by cardiac puncture . The following depots of BAT were excised : axillary, interscapular, dorsal-cervical, and parts of subscapular depot . Tissue was cut in small pieces and digested at 37°C for 30-40 min in a buffer containing 100 mM Hepes (pH 7.4), 123 mM NaCl, 5 mM KCI, 1 .3 mM CaC12, 5 mM glucose, 1 .5% (wt/vol) BSA, and 1 mg/ml collagenase (10 ml/g tissue) . Digestion mixture was vortexed every 5 min . All this as well as following procedures were performed under sterile conditions . After digestion, mixture was filtered over a 250-pm nylon filter and left at room temperature for 5-10 min, until a discrete fat layer on top had formed . This layer, which contains mature adipocytes, was discarded and the lower phase centrifuged at 700 g for 10 min at room temperature. The pellet containing the stromal-vascular fraction was washed with 20-40 ml cell culture medium (preheated to 37°C) and repelleted. This pellet was resuspended in 20-40 ml culture medium and cells counted in a Malassez counting chamber. | Department of Physiology of Energy Metabolism, German Institute of Human Nutrition Potsdam-Rehbrücke, Arthur-Scheunert-Allee 114-116, 14458, Nuthetal, Germany. klaus@dife.de. | ||||||||||||||||
| 37 | Hewett | 1993 | Microvessel Endothelial Cells | MVF | Manual | Human | Mammary Adipose | Surgical | Fat was dissected free of connective tissue and large (visible) blood vessels, and chopped fine | type II collagenase | Sigma | PBS/A containing 0.5% BSA | 0.25%. 10 ml collagenase per 20 to 30 gm fat | 37 | 60 | rotary shaker | Trypsin neutralized with HBSS and 5% FBS | Centrifugation 400 g 10 min , Lower layer 200 g 10 min, washed 2 x 10% BSA in PBS. | Crude microvessel pellet incubated in 0.25 % trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA) solution for 15 min with occasional agitation | UEA1 Lectin Dynabead selecction | Microvessel Endothelial Cells | Characterization. 2D Culture | Morphology Cytochemistry | Hewett PW, Murray JC, Price EA, Watts ME, Woodcock M. Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue. In Vitro Cell Dev Biol Anim. 1993 Apr;29A(4):325-31. doi: 10.1007/BF02633961. PMID: 7686548. | Isolation and culture of microvessel endothelial cells. Human mammary adipose tissue obtained from breast reduction surgery was washed in 2% antibiotic-antimycotic solution (Sigma) in PBS/A, and either processed im- mediately or stored for up to 48 h at 40 C before use. The fat was dissected free of connective tissue and large (visible) blood vessels, and chopped finely before incubation with 0.25% type II collagenase (Sigma) in PBS/A containing 0.5% BSA on a rotary stirrer at 370 C for 1 h (approximately 10 ml collagenase solution per 20 to 30 g fat). Adipocytes and free oil were then separated by centrifugation (400 g, 10 min, room temperature), from the aqueous lower layer containing microvessels. The lower layer was col- lected and centrifuged with 10% BSA in PBS/A (200 g, 10 min), the pellet was washed twice with 10% BSA in PBS/A, and finally in PBS/A. The crude microvessel pellet obtained was then incubated in 0.25% trypsin (Sigma) and 1 mM ethylenediamine tetraacetic acid (EDTA) solution for 15 min with occasional agitation. Trypsin was neutralized by the addition of Hanks’ balanced salt solution (HBSS) containing 5% fetal bovine serum (FBS). Large fragments of connective tissue were then removed by washing the digest above 100-jim nylon mesh (Lockertex, Warrington, Cheshire, UK). The filtrate was then centrifuged and the pellet resuspended in 0.5 to 1 ml of HBSS + 5% FBS and incubated with 30 #l of UEA-1-coated Dynabeads (1 to 2 X 107 beads) for 10 min at 40 C with occasional agitation to facilitate attachment of the beads. HBSS + 5% FBS was then added to give a final volume of 10 to 12 ml, and the microvessel fragments selected using a magnetic particle concentrator (MPC-1, Dynal) for 3 min. Purified microvessel fragments were then washed a further 3 to 5 times with 12 ml of HBSS + 5% FBS, suspended in growth medium of M199 with 0.014 M N-[2-Hydroxyethyl] piperazine-N’-[2-hydroxy-propane sulphonic acid] (HEPES, Sigma), 0.15% sodium hydrogen carbonate, 2 mM L-gluta- mine, 100 U/ml penicillin, 100 zg/ml streptomycin (Sigma), 90 zg/ml heparin (Sigma), 30% FBS (GIBCO BRL, Paisley, UK), and 15 eg/ml endothelial cell growth supplement (Boehringer Mannheim, Mannheim, Germany). Selected cells were plated onto 25-cm2 tissue culture dishes freshly coated for 1 h with 5 tg/cm2 bovine fibronectin (Sigma). Cells were maintained at 370 C in an atmosphere of 5% CO2. The medium | ||||||||||||||||
| 38 | Vici | 1993 | Microvessel Endothelial Cells | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Surgical | Minced with Scissors | Collagenase | 0.20% | Erlenmeyer | 37 | 30 | constant | Floating adipocytes discharged. Remaining solution centrifuged 200g 10 minutes. 120 micron filte, centrifugation wshese. Percoll. Centrifuge 1500g 20 min | Several | 120 micron for digestate. | 2.5 to 8 x 105 cells/g of fat | >90% | Microvessel Endothelial Cells | In Vitro Studies | QB-End/10 mAb for CD34. | Vici M, Pasquinelli G, Preda P, Martinelli GN, Gibellini D, Freyrie A, Curti T, D’Addato M. Electron microscopic and immunocytochemical profiles of human subcutaneous fat tissue microvascular endothelial cells. Ann Vasc Surg. 1993 Nov;7(6):541-8. doi: 10.1007/BF02000148. PMID: 8123456. | 5. Curti T, Pasquinelli G, Preda P, et al. An ultrastructural and immunocytochemical analysis of human endothelial cell adhesion on coated vascular grafts. Ann Vase Surg 1989;3:351- 363. 6. Pasquinelli G, Preda P, Vici M, et al. Development of a rotation device for endothelial cell seeding. Cells Materials I 992;4:29 I -297. | Isolation of MECs MECs were enzymatically harvested as previously described.5-6 Ten to 15 g of subcutaneous fat was removed by simple sterile excision from the abdominal wall of patients undergoing surgery for abdominal aneurysms. The fat tissue was minced with iris scissors, transferred to an Erlenmeyer flask, and incubated in a 0.2% collagenase solution for 30 minutes at 37° C under constant agitation. After collagenase digestion the floating adipocytes were discharged and the remaining solution was centrifuged at 200 g for 10 minutes. The resulting pellet was resuspended in phosphate- buffered saline (PBS) containing 0.1 % pyrogen- free bovine serum albumin (BSA)- The suspension was then filtered through a 120 μm nylon filter. After repeat washing and centrifugation the pellet was resuspended in 2 ml of PBSBSA, gently layered on 8 ml of sterile 45% Percoll (Sigma) in PBS (280 mOsm), and centrifuged at 1500 g for 20 minutes at room temperature. Microvascular cells were recovered in a milky white layer at the top of the Percoll cushion. After an additional washing in PBS-BSA the microvascular cells, the viability of which was estimated to be greater than 90% by trypan blue exclusion, were counted in a hemocytometer. A variable number of cells ranging from 2.5 to 8 x 105 cells/g of fat tissue were recovered. These differences were related to the age and sex of the donor patients. | Gianandrea Pasquinelli, MD, Istituto di Microscopia Elettronica Clinica, Policlinico S. Orsola, via Massarenti 9, 40138 Bologna, Italy. | ||||||||||||||
| 39 | Williams | 1994 | Microvessel Endothelial Cells | SVF | Manual | Human | SubQ | Liposuction | Collagenase | Worthington | 37 | 9 and 20 min | Centrifugation 700 g 4 minutes | vWF. EN4. alpha SMC actin. Cytokeratin | Microvessel Endothelial Cells | Immuno and EM Characterization | Williams SK, Wang TF, Castrillo R, Jarrell BE. Liposuction-derived human fat used for vascular graft sodding contains endothelial cells and not mesothelial cells as the major cell type. J Vasc Surg. 1994 May;19(5):916-23. doi: 10.1016/s0741-5214(94)70019-2. PMID: 8170048. | Williams SK, Jarrell BE, Rose DG, et al. Human microvessel endothelial cell isolation and vascular grafr sodding in the operating room. Ann Vasc Surg 1989;3:146-52. Williams SK. Isolation and culture of microvessel and large vessel endothelial cells: their use in transport and clinical studies. In: McDonagh PF, ed. Microvascular perfusion and transport in health and disease. Vienna, Karger; 1986:204-5. | Microvessel endothelial cell isolation. Tengram aliquots of fat were used for cell isolation. The cell isolation procedures were identical to our previously published methods.ll,19 Briefly, the fat was mixed with an equal volume of crude clostridial collagenase and digested for 20 minutes at 37° C in an Erlenmeyer flask. The fat-collagenase mixture was transferred to centrifuge tubes and centrifuged at 700 g for 4 minutes, and the vascular pellet was suspended in media. | ||||||||||||||||||||||
| 40 | Williams | 1995 | Microvessel Endothelial Cells | SVF | Manual | Human | SubQ | Liposuction | 10 gm each test | 8 different lots of crude collagenase. Purified Collagenase Trypsin | Boehringer Mannheim and Worthington | 50 ml Erllenmeyer with Spin Bar | 37 | Dubnoff Water Bath max shaking | Centrifugation 700 x g 7 min. | 1 wash 700 x g. 7 min | 0.8 to 1.5 10E6 cells/gm fat | Microvessel Endothelial Cells | Testing Collagenase Lots | Williams SK, McKenney S, Jarrell BE. Collagenase lot selection and purification for adipose tissue digestion. Cell Transplant. 1995 May-Jun;4(3):281-9. doi: 10.1177/096368979500400306. PMID: 7640867. | All samples of collagenase were tested for their ability to dissociate microvascularized fat tissue. 10 g samples of liposuction derived human SC fat was placed in 50 mL Erlenmeyer flasks with 10 mL samples of specific collagenase solutions. Fat samples from three different donors was evaluated. A magnetic spin bar was placed in the Erlenmeyer flask and the collagenase-fat solution was incubated at 37°C with vigorous shaking in a Dubenoff water bath. Following digestion, the fat slurry was centrifuged in two 15 mL conical centrifuge tubes at 700 x g for 7 min. Following this centrifugation the floating cake of adipocytes was decanted with the supernatant and the vascular pellet was suspended in divalent cation free DCF-PBS. The suspension was centrifuged at 700 x g for 4 min and the pellet was subsequently suspended for cell counting. The suspended microvascular endothelial cells were counted electronically using a Coulter counter (Coulter Electronics, Inc. Hialeah, FL). Fat samples were digested in duplicate flasks and each cell isolate was counted five times. | Stuart K Williams PhD University Delaware, Yale,, TJU, UA, UL. Bioficial Organs. Scottsdale AZ. cvregen@gmail.com. Roger C Wagner. Emeritus Professor. University of Delaware Newark, DE. Email: Rags@Udel.com | |||||||||||||||||||
| 41 | Hauner | 1995 | Adipose Stromal Cells (ASC) | Adipose Stromal Cells (ASC) | Manual | Human | Mammary | Surgical | minced into pieces of approximately 10 mg weight | 1.5 mg/ml collagenase | PBS 20 mg/ml BSA | 1.5 mg/ml | 37 | After short centrifugation at 200 x g, the floating fat cells and the incubation solution were removed by aspiration. The sedimented cells were resuspended and incubated with an erythrocyte-lysing buffer consisting of 154 mmol/1 NH4C1, 5.7 retool/1 K2HPO4, and 0.1 mmol/1 EDTA for 10 min to remove contaminating erythrocytes. The dispersed material was filtered through a nylon mesh with a pore size of 150 ~tm. After additional washing and centrifugation steps, the sedimented cells were resuspended in Dulbecco’s Modified Eagle’s/Ham’s F-12 medium (v/v, 1 : 1) supplemented with 10 % fetal bovine serum | erythrocyte-lysing buffer consisting of 154 mmol/1 NH4C1, 5.7 retool/1 K2HPO4, and 0.1 mmol/1 EDTA for 10 min | Culture TNF effects | Hauner H, Petruschke T, Russ M, Röhrig K, Eckel J. Effects of tumour necrosis factor alpha (TNF alpha) on glucose transport and lipid metabolism of newly-differentiated human fat cells in cell culture. Diabetologia. 1995 Jul;38(7):764-71. doi: 10.1007/s001250050350. PMID: 7556976. | Hauner et al., “Promoting Effect of Glucocorticoids on the Differentiation of Human Adipocyte Precursor Cells Cultured in a Chemically Defined Medium,” J Clin Invest,(1989), vol. 84, pp. 1663-1670. | Cell preparation and culture. Adipose tissue samples (20 to 80 g) were obtained from the mammary adipose of young normal- weight women (body mass index < 26 kg/m 2, age between 18 and 37 years) undergoing surgical mammary reduction. All subjects were otherwise healthy, and had no evidence of diabetes mellitus according to routine laboratory tests. The procedure for obtaining adipose tissue has been approved by the Ethical Committee of the University of Diisseldorf. Stromal cells from human adipose tissue samples were isolated as described recently [24]. Briefly, the samples were carefully dissected from other tissues, all visible fibrous material and blood vessels were discarded. The remaining adipose tissue was minced into pieces of approximately 10 mg weight and digested in 10 mmol/1 phosphate buffered saline (PBS) containing 1.5 mg/ml coHagenase and 20 mg/ml bovine serum albumin, pH 7.4. To obtain full disaggregation the cells were isolated in a two-step procedure. After short centrifugation at 200 x g, the floating fat cells and the incubation solution were removed by aspiration. The sedimented cells were resuspended and incubated with an erythrocyte-lysing buffer consisting of 154 mmol/1 NH4C1, 5.7 retool/1 K2HPO4, and 0.1 mmol/1 EDTA for 10 min to remove contaminating erythrocytes. The dispersed material was filtered through a nylon mesh with a pore size of 150 ~ microns. After additional washing and centrifugation steps, the sedimented cells were resuspended in Dulbecco’s Modified Eagle’s/Ham’s F-12 medium (v/v, 1 : 1) supplemented with 10 % fetal bovine serum and inoculated into 35- mm or 100-ram dishes at a density of 30,000 to 50,000 cells per cm 2. | ||||||||||||||||||||||
| 42 | Hoying | 1996 | Microvessel Fragments | MVF | Manual | Rat | Epididymal Fat Pad | Surgical | Fine scissors | Crude Clostridial Collagenase | Boehringer | Crude Clostridial collagenase (Boehringer Mannheim Biochemicals, Indianapolis, IN), 2 mg/ml bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, MO Dulbecco’s cation free phosphate-buffered saline (DCF PBS; pH 7.4) | 2 mg/ml | 50 ml Erllenmeyer with Spin Bar | 37 | 12 | Dubnoff Water Bath max shaking | Centrifugation. Pellete washed DCF-PBS, Centrifugation, DCF-PBS, Passed through 400 micron mesh Nylon screen, Filtrate captured on a 30 micron mesh Nylon screen. | 400 micron then 30 micron Nylon | Morphology | Hemocytometer | Approxi mately 5000 fragments per two fat pads. | Microvessel Fragments | Characterization/ 3D Culture | Hoying JB, Boswell CA, Williams SK. Angiogenic potential of microvessel fragments established in three-dimensional collagen gels. In Vitro Cell Dev Biol Anim. 1996 Jul-Aug;32(7):409-19. doi: 10.1007/BF02723003. PMID: 8856341. | “WAGNER, R. C., AND MATTHEWS, M. A. (1975). The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10, 286-297.” | Rat fat microvessel fragment (RFMF) isolation. RFMF were isolated from epididymal fat pads of retired breeder Sprague-Dawley rats (> 400 g) using modifications of the collagenase digestion methods previously reported by Wagner and Matthews (38). The major deviation from the methods reported by these authors, and used extensively by other investigators for the isolation and culture of rat fat microvascular endothelial cells, was the use of a limited collagenase digestion due to a reduction in collagenase concentration and duration of digestion. Epididymal fat pads were removed from euthanized animals, minced with fine scissors, and incubated with a solution containing: 2 mg/ml crude Clostridial collagenase (Boehringer Mannheim Biochemicals, Indianapolis, IN), 2 mg/ml bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, MO) and Dulbecco’s cation free phosphate-buffered saline (DCF PBS; pH 7.4) for 12 min at 37? C with vigorous shaking. The resulting slurry was centrifuged to remove adipocytes, and the vascular pellet was washed once with DCF-PBS containing 0.1% BSA (DCF-PBS + BSA). Following further centrifugation, the pellet was suspended in 12 ml of DCF-PBS + BSA and passed through a sterile 400 pm mesh nylon screen (Tetko, Elms ford, NY). The filtrate was then placed on a sterile 30 pm mesh nylon screen to remove single cells. The retained RFMFs were collected from the screen by flushing with DCF-PBS + BSA and an aliquot was removed for counting of the RFMF with a hemocytometer. The | ||||||||||||||
| 43 | Chung-Welch | 1997 | Human Omental Microvascular Endothelial Cells HOME | MVF | Manual | Human | Residual Omental Tissue Mesothelium Removed | Surgical | 1 gm pieces minced in Mcilwain Tissue Chopper | Collagenase CLSII Worthington | Worthington | 0.20% | Digest filtered then adipocytes allowed to float at 1g. Supernatant filtered through 20 micron Nylon filter catching fragments | 215 then a 100 micron filter | MicroVascular Endothelial Cell | Characterization | Chung-Welch N, Patton WF, Shepro D, Cambria RP. Two-stage isolation procedure for obtaining homogenous populations of microvascular endothelial and mesothelial cells from human omentum. Microvasc Res. 1997 Sep;54(2):121-34. doi: 10.1006/mvre.1997.2039. PMID: 9327383. | Isolation of HOME cells. HOME cells were isolated plates were rinsed 21 with serum-free medium and from the residual omental tissue remaining after MESO incubated in serum-free medium (M199 supplemented cell isolation by enzymatic dissociation and differential with 2.5 mg/ml BSA) for 24 hr at 377C in a humidified filtration (Chung-Welch et al., 1997). Briefly, residual 5% CO2/95% air incubator. After 24 hr, fresh serumomental tissue was cut into small pieces (1 cm2) and free medium containing human recombinant PDGF AA finely minced in a McIlwain tissue chopper (Brinkman or BB (Pepro Tech) at varying concentrations (0–30 ng/ Instruments). The minced tissue was enzymatically di- ml) was added. After 24 hr exposure to PDGF, HOME gested with 0.2% collagenase and subsequently filtered and MESO cells were labeled with [3H]thymidine (1 through a succession of graded nylon meshes (Tetko) mCi/ml, 6.7 Ci/mmole, New England Nuclear) for 2 hr of 215 and 100 mm to remove undigested tissue frag- at 377. [3H]Thymidine incorporation into the DNA of ments. The recovered filtrate was allowed to settle at HOME and MESO cells was measured in the trichlororoom temperature to allow the adipocytes to float to acetic acid (TCA)-precipitable cell pellet as described the surface. The cell suspension beneath the layer of by Vale et al. (88). Briefly, labeled medium was aspifloating adipocytes containing the microvascular frag- rated from the culture dishes and the cells were rinsed ments and single cells (i.e., stromal cells and red blood 21 with ice-cold PBS. Cells were precipitated with icecells) was retrieved and filtered through a 20-mm nylon cold 5% TCA for 1 hr on ice. Precipitable material in the mesh to remove erythrocytes and single cells. The mi- wells were rinsed 21 with ice-cold 5% TCA followed crovascular fragments retained by the mesh were sepa- by two washes in ice-cold methanol. TCA-precipitable rated from single cells through a 5% BSA cushion at material was scraped from the wells and solubilized in unit gravity. The microvascular fragments were col- 0.5 N NaOH. The TCA-precipitable material was neulected, resuspended in complete medium, and seeded tralized with 12 N HCl and transferred to vials cononto tissue culture plates coated with 1% gelatin. | Chung-Welch. To whom correspondence should be addressed at current address: gan sites (Chung-Welch et al., 1988; Dodge et al., 1989; Oncogene Research Products, 84 Rogers Street, Cambridge, MA 02142. Patton To whom reprint requests should be addressed at Microvascular Research Laboratory, Biological Sciences, Room 207, Boston Univer- late and culture endothelial cells from the microvascusity, 5 Cummington Street, Boston, MA 02215. | ||||||||||||||||||||||
| 44 | Halverson Patent | 1998 | Adipose Tissue-Derived Stromal Cell | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | Collagenase Type I Worthington | Worthington | PBS | 2 mg/ml | 37 | 30 to 35 minutes | intermittant shaking | Centrifugation 500xg 5 min | Centrifugation 500g 5 min | Adipose Stromal Cells | In Vitro conversion of ASCs to Osteoblasts | Such assays include, but are not limited to those that assess morphological or biochemical characteristics ( e.g., secreted osteocalcin or other osteoblast-specific proteins or RNA). | US Patent 6,391,297 | Rodbell M. Localization of Lipoprotein Lipase in Fat Cells of Rat Adipose Tissue. J Biol Chem. 1964;239:753-5. PubMed PMID: 14154450. Hauner et al., “Promoting Effect of Glucocorticoids on the Differentiation of Human Adipocyte Precursor Cells Cultured in a Chemically Defined Medium,” J Clin Invest,(1989), vol. 84, pp. 1663-1670. | SVF to Stromal Cells coversion by change of terminology | Human stromal cells were isolated from adipose tissue according to the procedures described by Rodbell (1964) J Biol Chem 239:375 and Hanner et al. (1989) J Clin Invest 84:1663-1670. Briefly, human adipose tissue from subcutaneous depots was removed by liposuction surgery. The adipose tissue was then transferred from the liposuction cup into a 500 ml sterile beaker and allowed to settle for about 10 minutes. Precipitated blood was removed by suction. A 125 ml volume ( or less) of the tissue was transferred to a 250 ml centrifuge tube, and the tube was then filled with KrebsRinger Buffer. The tissue and buffer were allowed to settle for about three minutes or until a clear separation was chieved, and then the buffer was removed by aspiration. The tissue was washed with Krebs-Ringer Buffer an additional four to five times or until it was orange-yellow in color and the buffer was light tan in color. The cells of the adipose tissue were dissociated by collagenase treatment. Briefly, the buffer was removed from the tissue and replaced with a 2 mg collagenase/ml Krebs Biffer (Worthington, Me., USA, type I) solution at a ratio of 1 ml collagenase solution/ml tissue. The tubes were incubated in a 37 degree water bath with intemittent shaking for 30 to 35 minutes. Stromal cells were isolated from other components of the adipose tissue by centrifugation for 5 minutes at 500x g at room temperature. The oil and adipocyte layer was removed by aspiration. The remaining stromal-vascular fraction was resuspended in approximately 100 ml of phosphate buffered saline (PBS) by vigorous swirling, divided into 50 ml tubes and centrifuged for five minutes at 500xg. The buffer was carefully removed by aspiration, leaving the stromal cells. The stromal cells were then resuspended in stromal cell medium (DMEM (Morton (1970) In Vitro 6:89-108; Dulbecco (1959) Virology 8:396)/Ham’s F-10 medium (Ham (1963) Exp Cell Res 29:515) (1:1, v/v); 10% (v/v) fetal calf serum; 15 mM HEPES, pH 7.4; 60U/ml penicillin; 60U/ml 25 streptomycin; 15 µg/ml amphotericin B), plated at an appro priate cell density and incubated at 37° C. in 5% CO2 overnight. Once attached to the tissue culture dish or flask, the cultured stromal cells may be used immediately or maintained in culture for up to 5 passages before being | |||||||||||||||||
| 45 | Williams | 1999 | MicroVascular Endothelial Cells. MVEC | SVF | Automated. Becton Dickinson | Human | SubQ | Liposuction | 50 gm | Partially Purified Type II Crude Collagenase | Purified from Crude obtained from Worthington | 4 mg/ml | 37 | 35 | Sterile Air Agitation | Centrifugation 100 x g 4 min | 1 x | Proprietary removal | Endothelial Cells | Vascular Graft Sodding | WILLIAMS SK: 1999. Human Clinical Trials of Microvascular Endothelial Cell Sodding. In: Tissue Engineering of Prosthetic Vascular Grafts, P Zilla and HP Greisler (eds), R.G. Landes Company pp. 143-147. | The abdomen and the site of graft placement was prepped and draped. To obtain adipose tissue for microvessel endothelial cell isolation, liposuction of subcutaneous fat from the abdominal wall was performed with a liposuction cannula and specially designed 60 cc syringe. Approximately 50 g of fat was removed from each patient and processed for MVEC isolation. The completely assembled kit, being used during a human endothelial cell sodrung procedure, is shown in Figure 12.3. The isolation procedure as outlined above resultedi n ePTFEg raftsw itha pproximately2 x 105 cells/cm2 graft lumenal surface area. Grafts were immediately implanted in patients | |||||||||||||||||||
| 46 | Cousin/Castella | 1999 | Stromal Cells | Adipose Stromal Cells (ASC) | Manual | Mouse Rat | Inguinal Fat | Surgical | Type II Collagenase Sigma | Sigma | phosphate buffer PBS containing 0.2% BSA | 2 mg/ml | 37 | 45 | Centrifugation | 25 micron | Stromal Vascular Fraction. SVF | In Vitro Studies | MOMA2. ED2 Mac1 F480 | Cousin B, Munoz O, Andre M, Fontanilles AM, Dani C, Cousin JL, Laharrague P, Casteilla L, Penicaud L. A role for preadipocytes as macrophage-like cells. FASEB J 1999; 13: 305 – 312. | “P. Bj€orntorp, M. Karlsson, H. Pertoft, P. Pettersson, L. Sj€ostr€om, U. Smith, Isolation and characterization of cells from rat adipose tissue developing into adipocytes, J. Lipid Res. 19 (1978) 316–324.” | Method according to Bjornthorp: Two fat pads from the small rats or one from the larger rats were incubated in a 50-ml siliconized Erlenmeyer flask containing 10 ml of a solution with final concentrations of 0.1 M HEPES (hydroxyethylpiperazine- ethane-sulfonic acid, Sigma, St. Louis, MO) buffer, 0.12 M NaCl, 0.05 M KCI, 0,001 M CaCI2, 0.005 M glucose, and 1.5% (w/v) bovine serum albumin (Bovine serum albumin Fraction V, batch WB 1370, Armour, Eastbourne, England). This solution contained 0.2% (w/v) collagenase (Worthington, Freehold, NJ, batch CLS 46D112X). The gas phase was air; the pH was 7.4; and the temperature was 37°C. Incubation time was 30 min if not noted otherwise. Incubations were performed in a water bath with rotating flasks (120 rpm). After incubation the tissue remnants were removed by filtration through a nylon screen with a pore size of 250 pm into a siliconized test tube. The fat cells were then allowed to float to the surface for 15 min whereafter the infranatant was aspirated through a siliconized injection needle. The infranatant was then filtered through a nylon screen with a pore size of 25 p m to remove cell aggregates. The cells passing the filter were pelleted by centrifugation and then resuspended in the appropriate buffer or medium for further processing. | E-mail addresses: cousinb@toulouse.inserm.fr (B. Cousin), casteil@ toulouse.inserm.fr (L. Casteilla). | ||||||||||||||||||
| 47 | Halvorsen | 2000 | Stromal-Vascular (SV) Fraction | SVF | Manual | Human | Collagenase Type I | centrifugation | Stromal-Vascular (SV) Fraction | Halvorsen, Y.-D.C., Wilkison, W.O., and Gimble, J.M. Adipose-derived stromal cells—their utility and potential in bone formation. Int. J. Obes. Relat. Metab. Disord. 24, Suppl. S41, 2000. | Van RL, Roncari DA. Complete differentiation of adipocyte precursors. A culture system for studying the cellular nature of adipose tissue. Cell Tissue Res 1978; 195: 317 ± 329. | Review. State that endothelial cells do not adhere to plastic without pre-coating and without endothelial cell growth hormones | The most abundant source of accessible stroma, however, is from the adipose tissue. The surgical procedure of obtaining subcutaneous adipose is relatively noninvasive and can be performed in outpatient clinics. The isolation of adipose stromal cells was ®rst described by Van and Roncari8 using type I collagenase to digest the extracellular matrices. The ¯oating primary adipocytes could be separated by centrifugal force from the cells that settle down, the stromal ± vascular (SV) fraction. The SV fraction includes the stromal cells, the blood cells and the blood vessel cells. Without pre-coating the culture wear and supplementing endothelial growth hormones, the capillary endothelial cells do not attach or grow well. After overnight culture to allow adhering, between 10000 and 25000 stromal cells per gram of adipose tissue can be isolated. | YC Halvorsen, Zen-Bio Inc., 3200 Chapel Hill- Nelson Blvd, Research Triangle Park, NC 27709, USA. E-mail: yuan-di@zen-bio.com | |||||||||||||||||||||||||||
| 48 | Gronthos/ Gimble | 2001 | Stromal Vascular Fraction Preadipocytes Adipose Tissue Derived Stromal Cells | SVF | Manual | Human | SubQ | Liposuction | Collagenase Type I | Krebs with 1% BSA | 1mg/ml | 37 | 60 | Intermittant Shaking | Centrifugation 300 g 5 min | Gronthos S, Franklin DM, Leddy HA, Robey PG, Storms RW, Gimble JM. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol. 2001 Oct;189(1):54-63. doi: 10.1002/jcp.1138. PMID: 11573204. | The subcutaneous adipose was acquired from elective surgeries with the patient’s consent as approved by the Institution Review Boards. The tissues used were from female patients of ages between 23 and 58 with a mean ( SEM) of 39.4 3.1 years. The patients displayed a mean body mass index (kg/m2) ( SEM) of 32.2 4.6 (summarized in Table 2). Liposuction tissues were transported to the laboratory in saline solution within 2 h post-surgery. The tissue was washed at least three times with 2 volumes of Krebs±Ringer-bicarbonate (KRB; Sigma) to remove blood. The tissue was then digested with one volume of collagenase type I (1 g/liter of KRB with 1% BSA) for 60 min at 378C with intermittent shaking. The ¯oating adipocytes were separated from the stromal±vascular fraction by centrifugal force (300g) for 5 min. The preadipocytes in the stromal±vascular fraction were plated in tissue culture flasks at 3,500 cells/cm2 in Dulbecco’s modi®ed Eagle’s± Ham’s F-10 medium (vol/vol, 1:1) supplemented with 10% FBS, 15 mM HEPES (pH 7.4), 100 U/ml penicillin, 100 mg/ml streptomycin, and 25 ng/ml amphotericin B (pre-adipocyte medium). | ||||||||||||||||||||||||
| 49 | Hauner | 2001 | Human Adipose Precursor Cells | Adipose Stromal Cells (ASC) | Manual | Human | Visceral/SubQ | Liposuction/ Surgical | Mince | Crude collagenase (e.g., Worthington CLS type 1, specific activity 172 U/mg) at a final concentration of approx 200 U/mL, pH 7.4, is dissolved in phosphate-buffered saline (PBS): 10 mMKH2PO4, 10 mMNa2HPO4, 2.7 mM KCl, 0.137 M NaCl, pH 7.4, supplemented with 2% bovine serum albumin (fraction V, according to Cohn). | Worthington | phosphate-buffered saline (PBS): 10 mMKH2PO4, 10 mMNa2HPO4, 2.7 mM KCl, 0.137 M NaCl, pH 7.4, supplemented with 2% bovine serum albumin (fraction V, according to Cohn). | 200 U/ml | 37 | 60 to 90 min | shaking water bath | centrifuged at 200gmax for 10 min. suspension is filtered through a 150 μm nylon mesh, and centrifuged once again. Filtration using a 70 micron filter Filtration using a 25 micron filter | 150 micron. 70 micron. 25 micron | Erythrocyte lysing buffer: 155 mM NH4Cl, 5.7 mM K2HPO4, 0.1 mM EDTA at pH 7.3. | Determination of Total Cell Number 1. For determination of the total cell number, a 50-μL aliquot of the cell fraction, obtained after resuspension in basal medium, is taken, and diluted with 100 μL medium and 50 μL trypan blue (0.4% in H2O). 10 μL of this solution is transferred to a Neubauer chamber and counted under the microscope. This procedure is repeated and the mean value used to calculate total stromal cell number. The yield of isolated stromal cells/g wet AT is in the range of 100,000–350,000. In samples from severely obese subjects, the yield of stromal cells is usually lower than in samples from lean persons. 2. After cell counting, the still-concentrated stromal cell solution is diluted with inoculation medium to a final concentration of 150,000 cells/mL for subsequent seeding of the cells in a 4.5-cm2 well. Thus, 1 mL corresponds to a seeding density of approx 33,000 cells/cm2 | Protocol | Hauner, H., Skurk, T., Wabitsch, M. (2001). Cultures of Human Adipose Precursor Cells. In: Ailhaud, G. (eds) Adipose Tissue Protocols. Methods in Molecular Biology™, vol 155. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-231-7:239 | Hauner, H., Röhrig, K., Spelleken, M., Lin, L. S., and Eikel, J. (1998) Development of insulin-responsive glucose uptake and GLUT4 expression in differentiating human adipocyte precursor cells. Int. J. Obesity 22, 448–453. Björntorp, P., Karlsson, M., Pertoft, H., Pettersson, P., Sjöström, L., and Smith, U. (1978) Isolation and characterization of cells from rat adipose tissue developing into adipocytes. J. Lipid Res. 19, 316–324. | Cell Isolation 1. After transportation to the laboratory, the crudely prepared fat pads are carefully liberated from remaining connective tissue and blood vessels. This step is followed by collagenase digestion. Different types of collagenases are offered by several companies, and batches may differ substantially in their quality and activity. This problem renders it difficult, if not impossible, to standardize the conditions for collagenase digestion. However, variation in collagenase activity can be compensated by the adaption of concentration and duration of incubation. For the standard digestion procedure, a crude collagenase preparation at a concentration of 200 U/mL and 3 mL/g tissue is used. The incubation time is approx 60–90 min in a shaking water bath at 37°C. 2. To collect the disaggregated cells, samples will be centrifuged at 200gmax for 10 min. The supernatant with the adipocytes is discarded, or can be used for other purposes. 3. The pellet is resuspended in erythrocyte-lysing buffer, and incubated for not more than 10 min. This suspension is filtered through a 150 μm nylon mesh, and centrifuged once again. The resulting pellet is resuspended in an appropriate volume of basal medium. The cells are optionally filtered through a nylon mesh with a pore size of 70 μm (see Note 2: 2. AT samples from the intra-abdominal depots are densely vascularized. For this reason, there is usually a variable but significant contamination of the stromal cell fraction by endothelial cells (EC). In contrast to the stromal cell fraction from subcutaneous AT, which is essentially free of ECs, this cell type may cause several problems. The proportion of ECs should be reduced to less than 5% of total cell number. Therefore, after a first filtration through a 150-μm mesh in erythrocyte-lysing buffer, a second filtration is recommended, using a nylon mesh with a pore size of 25-μm to reduce this contamination (2). ECs reaggregate rapidly, and are, for this reason, quantitatively retained by this filter size, but some contamination by ECs cannot be fully prevented. If precursor cells from various depots are compared, all samples must undergo the same filtration procedure. | |||||||||||||||||
| 50 | Zuk | 2001 | Stromal Vascular Fraction. SVF | Adipose Stromal Cells (ASC) | Manual | Human | Human Sub Q | Liposuction epinephrine | 300 ml | 0.075% Collagenase | 0.08% | 37 | 30 | Centrifugation 1200 xg 10 min | Centrifugation | 100 micron Nylon Screen | NH4Cl | SVF Pellet | SVF | Cell Culture Characterization | “Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228.” | Hauner, H., Schmid, P., and Pfeiffer, E.F. Glucocorticoids and insulin promote the differentiation of human adipocyte precursor cells into fat cells. J. Clin. Endocrinol. Metabol. 64, 832, 1987 | Human adipose tissue was obtained from elective liposuction procedures under local anesthesia (HSPC #98-08 011-02). In this procedure, a hollow blunt-tipped cannula was introduced into the subcutaneous space through small (, 1 cm) incisions. The cannula was attached to gentle suction and moved through the adipose compartment, mechanically disrupting the fat tissue. A solution of saline and the vasoconstrictor epinephrine was infused into the adipose compartment to minimize blood loss and contamination of the tissue by peripheral blood cells. The raw lipoaspirate (, 300 cc) was processed according to established methodologies to obtain a stromal vascular fraction (SVF).33,34 To isolate the SVF, lipoaspirates were washed extensively with equal volumes of phosphate-buffered saline (PBS), and the ECM was digested at 37°C for 30 min with 0.075% collagenase. Enzyme activity was neutralized with Dulbecco’s modified Eagle’s medium (DMEM), containing 10% FBS and centrifuged at 1200 3 g for 10 min to obtain a high-density SVF pellet. The pellet was resuspended in 160 mM NH4Cl and incubated at room temperature for 10 min to lyse contaminating red blood cells. The SVF was collected by centrifugation, as detailed above, filtered through a 100-micron nylon mesh to remove cellular debris and incubated overnight at 37°C/5% CO2 in control medium (DMEM, 10% FBS, 1% antibiotic/antimycotic solution). Following incubation, the plates were washed extensively with PBS to remove residual nonadherent red blood cells. The resulting cell population was termed a processed lipoaspirate (PLA), to distinguish it from the SVF obtained from excised adipose tissue. PLA cells were maintained at 37°C/5% CO2 in noninductive control medium. Cells did not require specific FBS sera lots for expansion and differentiation (data not shown). For immunofluorescence studies, a population of MSCs was obtained from human bone marrow aspirates according to the protocol of Rickard et al.17 and maintained in control medium. To prevent spontaneous differentiation, cells were maintained at subconfluent levels. | ||||||||||||||||||
| 51 | Halvorsen | 2001 | Adipose Tissue-Derived Stromal Cell Adipose Tissue-Derived Stem Cell. Preadipocyte Stromal Vascular Fraction. Stromal Vascular Fraction | Adipose Tissue-Derived Stromal Cell Adipose Tissue-Derived Stem Cell. Preadipocyte Stromal Vascular Fraction. Stromal Vascular Fraction | Manual | Human | SubQ | Liposuction | Collagenase Type I | Krebs-Ringer-Bicarbonate (KRB) | 1 mg/ml | 37 | 60 | 1 min vigorous shake evry 5 to 10 min | Centrifuge 300 x g 5min | 8.6 x 10E4/gm fat | Preadipocytes in the Stromal Vascular Fraction | In Vitro Studies | Oil Red O | Halvorsen Y-DC, Bond A, Sen A et al . Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: biochemical, cellular and molecular analysis. Metabolism 2001;50:407 /13. | Hauner H, Entenmann G, Wabitsch M, et al: Promoting effect of glucocorticoids on the differentiation of human adipocyte precursor cells cultured in a chemically defined medium. J Clin Invest 84:1663- 1670, 1989 Rodbell papers 1964 | Primary isolate is SVF. They state the preadipocytes from SVF are plated but do not disclose separation – probably no separation. “Here, we describe the isolation of human adipose tissuederived stromal cells in large quantities from liposuction and abdominoplasty specimens. The method is a modification of previously reported techniques.” | Subcutaneous adipose tissue was acquired from elective liposuction or abdominoplasty surgeries. The tissues used were from patients of ages 28 to 46 with an average of 36.8 5.4. The patients displayed a mean body mass index (BMI; kg/m2) of 25.5 7.3, with a range between 19 and 40. Other than obesity (defined as a BMI 25) in some instances, the patients were in good health; no diabetes or other complications were reported. The abdominoplasty tissues were minced with scissors into pieces with diameters between 1 mm and 5 mm; liposuction tissues were used without further processing. The minced tissue was washed at least 3 times with 2 vol of Krebs-Ringer-Bicarbonate (KRB) to remove contaminating blood. The tissue was then digested with 1 vol of collagenase type I (1 g/L of KRB with 1% BSA) for 60 minutes at 37°C with intermittent shaking. The floating adipocytes were separated from the stromal-vascular fraction by centrifugal force (300 g) for 5 minutes. The preadipocytes in the stromalvascular fraction were plated in tissue culture flasks without precoating of extracellular matrices. The initial plating density was approximately 3,500 cells/cm2. The primary cells were cultured for 4 to 5 days until they reached confluence; these cells were defined as “passage 0.” | ||||||||||||||||||
| 52 | Frye | 2002 | Microvascular endothelial Cells (MECS). Microvascular Fragments (MVF) Adipose Stromal Cells (ASC) | Microvascular endothelial Cells (MECS). Microvascular Fragments (MVF) Adipose Stromal Cells (ASC) | Manual | Rat | Epididymal Fat | Surgical | scissors | 3% collagenase (catalog # C2139, Sigma | Sigma | PBS 3% BSA | 3% | 37 | 30 | orbital shaker for 30 min at 37 ~ C | The digested tissue was serially filtered through a 250-p~m screen mesh (Sigma) and a 40-p,m nylon mesh cell strainer (Falcon, Franklin Lakes, NJ). material remaining in the 40-1xm nylon mesh cell strainer, the filtrate, was rinsed with media and layered on top of a 45% Percoll solution in a 50-ml conical tube (Falcon). The 45% Percoll solution was prepared with 10 ml of 10% BSA in 1x PBS, 9 ml Percoll (Sigma), and 1 ml 10X PBS. The tube was centrifuged (Juoau, Winchester, VA) at 400 x g for 30 min at 4 ~ C. This results in three distinct layers and a cell pellet. The top layer was discarded, and the second layer containing the MECs and microvascular fragments (MVFs) was transferred to a 15-ml conical tube with fresh EGM-2-MV media (Clonetics, San Diego, CA) to fill the tube. The tube containing MECs-MVFs was centrifuged for 5 rain at 200 X g. EGM-2-MV is a standardized proprietary media containing human recombinant epiden~ml growth factor, human fibroblast growth factor-basic with heparin, vascular endothelial growth factor, ascm-bic acid, hydrocortisone, human recombinant insulin-like growth factor, heparin, 5% fetal bovine serum, gentamicin, and amphotercin. Separation via differential cell attachment. The pellet was resuspended in media, plated in a 100-ram tissue culture dish (Falcon), and incubated at 37 ~ C with 5% C02 for 1.5 h. This allowed contaminating fibroblasts (FBs) and preadipocytes (PAs) to attach. The medium (containing suspended MECs-MVFs) was carefully removed from the flask without disturbing the attached cells and was transferred to another tube. | 250 then 40 micron | Morphology CD31 | Coulter Z1 particle counter with an aperture of 100 tzm, an upper threshold of 16.00 | The average numbers of MECs/MVFs per rat (two fat pads) recovered after sieving, the Percoll density gradient separation, the attachment phase, and the immunomagnetic separation were 3.6 • 106 ~ 6.3 X 105, 1.1 X 106 –+ 2.7 X 105, 3.5 • 105 -4- 1.5 X 10 z, and 1.7 • 1@ -+ 1.5 • 103, respectively. | Frye CA, Patrick CW Jr. Isolation and culture of rat microvascular endothelial cells. In Vitro Cell Dev Biol Anim. 2002 Apr;38(4):208-12. doi: 10.1290/1071-2690(2002)038<0208:IACORM>2.0.CO;2. PMID: 12197772. | “WAGNER, R. C., AND MATTHEWS, M. A. (1975). The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10, 286-297.” | Fat pad digestion and cell straining. The minced tissue was enzymatically digested in PBS supplemented with 3% collagenase (catalog # C2139, Sigma, St. Louis, MO) and 3% bovine serum albumin (BSA, Sigma) using an orbital shaker for 30 min at 37 ~ C. The digested tissue was serially filtered through a 250-p~m screen mesh (Sigma) and a 40-p,m nylon mesh cell strainer (Falcon, Franklin Lakes, NJ), rinsing each time with Dulbecco modified Eagle L with 10% fetal bovine serum (Sigma). Density separation. The material remaining in the 40-1xm nylon mesh cell strainer, the filtrate, was rinsed with media and layered on top of a 45% Percoll solution in a 50-ml conical tube (Falcon). The 45% Percoll solution was prepared with 10 ml of 10% BSA in 1x PBS, 9 ml Percoll (Sigma), and 1 ml 10X PBS. The tube was centrifuged (Juoau, Winchester, VA) at 400 x g for 30 min at 4 ~ C. This results in three distinct layers and a cell pellet. The top layer was discarded, and the second layer containing the MECs and microvascular fragments (MVFs) was transferred to a 15-ml conical tube with fresh EGM-2-MV media (Clonetics, San Diego, CA) to fill the tube. The tube containing MECs-MVFs was centrifuged for 5 rain at 200 X g. EGM-2-MV is a standardized proprietary media containing human recombinant epiden~ml growth factor, human fibroblast growth factor-basic with heparin, vascular endothelial growth factor, ascm-bic acid, hydrocortisone, human recombinant insulin-like growth factor, heparin, 5% fetal bovine serum, gentamicin, and amphotercin. Separation via differential cell attachment. The pellet was resuspended in media, plated in a 100-ram tissue culture dish (Falcon), and incubated at 37 ~ C with 5% C02 for 1.5 h. This allowed contaminating fibroblasts (FBs) and preadipocytes (PAs) to attach. The medium (containing suspended MECs-MVFs) was carefully removed from the flask without disturbing the attached cells and was transferred to another tube. | |||||||||||||||||
| 53 | van Harmelen | 2003 | Stromal cells (i.e. preadipocytes) | Adipose Stromal Cells (ASC) | Manual | Human | Mammary Fat | Surgical | Dissected of fibrous tissue and blood vessels Minced Small Pieces | Collagenase | PBS | 250U/ml | 37 | 90 | Centrifuge 200g 10 min 250 micron mesh, lysis, centrifuged 200 g 10 minutes. 70 micron filter | 250 micron polypropelene mesh, 70 micron | Lysis buffer | Percentage of differentiated cells (standard conditions) 67 7711 0–49 Percentage of differentiated cells (+IBMX) 67 23719 0–76 Percentage of differentiated cells (+troglitazone) 67 34723 0–86 Percentage of differentiated cells (+trog + IBMX) 67 46721 1–87 | Adipose Derived Stromal Cells | In Vitro Studies | van Harmelen V, Skurk T, Rohrig K, Lee YM, Halbleib M, Aprath-Husmann I, Hauner H. 2003. Effect of BMI and age on adipose tissue cellularity and differentiation capacity in women. Int J Obes Relat Metab Disord 27:889– 895. | Papers by Hauner cited – but not for isolation methods | readipocyte isolation from mammary adipose tissue and primary culture of the cells Adipose tissue was dissected from fibrous material and visible blood vessels, weighed, minced into small pieces and digested in PBS containing 20 mg/ml BSA and 250 U/ml collagenase for 90min at 371C. The completely disaggre- gated material was centrifuged for 10 min at 200 g and the pellet of stromal cells (ie preadipocytes) was resuspended in an erythrocyte lysis buffer (consisting of 0.154 mol/l NH4Cl, 10 mmol/l KHCO3 and 100 mmol/l ethylenediamine tetra- acetate) to remove contaminating red blood cells, filtered through a polypropylene mesh (pore size 250mm) and centrifuged for 10 min at 200 g. The cell pellet was resuspended in DMEM/F-12 medium, filtered (pore size 70mm) and the total cell number was determined micro- scopically using a Neubauer chamber. The cells were seeded in 12-well plates in a density of 33000 cells/cm2 and incubated in DMEM/F-12 medium, containing 50 mg/ml gentamycin and 10% fetal calf serum (FCS) for 20 h at 371C in 5% CO2 to become attached. Then, cells were washed twice with PBS and differentiation was started using a chemically defined, serum-free medium.6,12 This medium consisted of DMEM/F-12 (50:50, v:v) with 10 mg/ml transfer- rin, 100 nmol/l cortisol, 66 nmol/l insulin, 200 pmol/l T3 and 50 mg/ml gentamycin. Three wells each were also incubated with 500 mmol/l 3-isobutyl-1-methylxanthine (IBMX), 1 mg/ ml troglitazone or a combination of both substances. The cells were cultured for 16 days and the medium was renewed every 3 days. IBMX and/or troglitazone were added to the medium only for the first 3 days | *Correspondence: Dr H Hauner, Deutsches Diabetes-Forschungsinstitut an der Heinrich-Heine-Universita¨t, Auf’m Hennekamp 65, 40225 Du¨sseldorf, Germany. E-mail: hauner@ddfi.uni-duesseldorf.de. Leiden University Medical Center, Department of Human Genetics, Einthovenweg 20, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail address: V.J.A.van_Harmelen@lumc.nl (V. van Harmelen). | |||||||||||||||||
| 54 | Cousin/Casteilla | 2003 | Stromal Vascular Fraction. SVF | Adipose Stromal Cells (ASC) | Manual | Mouse | Inguinal Fat | Surgical | Type II Collagenase Sigma | Sigma | phosphate buffer PBS containing 0.2% BSA | 2 mg/ml | 37 | 45 | Centrifugation | 25 micron | FACS CD34 CD45 | Stromal Vascular Fraction. SVF | transplantaion | Cousin B, Andre M, Arnaud E, Penicaud L, Casteilla L. Reconstitution of lethally irradiated mice by cells isolated from adipose tissue. Biochem Biophys Res Commun. 2003;301(4):1016-22. doi: 10.1016/s0006-291x(03)00061-5. PubMed PMID: 12589814. | P. Bj€orntorp, M. Karlsson, H. Pertoft, P. Pettersson, L. Sj€ostr€om, U. Smith, Isolation and characterization of cells from rat adipose tissue developing into adipocytes, J. Lipid Res. 19 (1978) 316–324. | In contrast to BM cells, intravenous injections of the same number of SVF cells induced immediate death of mice by heart and/or brain failure (data not shown). Attempts to inject a smaller number of cells were performed, but mice did not survive (data not shown). Thus, all presented results were obtained after intraperitoneal SVF or BM cell transplantations | Cells were isolated according to Bj€orntorp et al. with minor modifications. Inguinal adipose tissue was excised from C57Bl/6 6-week-old male mice and was digested at 37 C in phosphate buffer PBS containing 0.2% BSA and 2 mg/ml collagenase for 45 min (type II collagenase, Sigma Aldrich, St. Quentin Fallavier, France). After filtration through 25 lm filters and centrifugation, isolated SVF cells were suspended in PBS, counted, and used for transplantation, FACS analyses or methylcellulose cultures. | ||||||||||||||||||
| 55 | Harmon Patent | 2003 | Adipose-Tissue Derived Stem Cells | SVF | Manual | Mammal | Multiple Sites | Surgical Excision Liposuction | Mince | Sigma Adlrich Cat # 2674. Serva NB4 | Sigma. Serva | PBS | 0.01 to 10 mg/ml | 37 | 20 to 60 min | Centrifugation 400g 5minutes | Centrifugation 400g 5minutes | Adipose-Tissue Derived Stem Cells | Transplantation | US Patent 11,129,855 | Rodbell, M., “Metabolism of Isolated Fat Cells: I. Effects of Hormones on Glucose Metabolism and Lipolysis,” Journal of Biological Chemistry, 239:2, 1964, 375-380. Rodbell, M., “Metabolism of Isolated Fat Cells: V. Preparation of “Ghosts” and Their Properties; Adenyl Cyclase and Other Enzymes,” Journal of Biological Chemistry, 242:24, 1667, 5744-5750. “Van et al., “”Complete Differentiation of Adipocyte Precursors. A Culture System for Studying the Cellular Nature of Adipose Tis sue,”” Cell and Tissue Research 195(2):317-329, 1978.” | Example 1 Preparation of Adipose-Tissue Derived Stem Cells Using Enzymatic Degradation Stem cells from adipose tissue obtained from four equines were prepared according to the following procedure, and the number and viability of purified cells was determined. a. Label processing tubes (50 ml conicals). Determine the weight of each conical, record on the label. b. Transfer fat sample to the inside surface of a lid from a Petri dish. Allow to drain. c. Place fat sample in pre-weighed conical tube. d. Record weight of fat sample and tube. e. Add 30-40 ml of sterile PBS to the conical, cap and gently invert several times. f. Carefully pour off liquid by using sterile forceps to retain the fat. g. Transfer fat to the bottom portion of a sterile petri dish and mince with scissors and forceps. h. Return minced sample to original 50 ml conical by using sterile scoop. i. Rinse the petri dish with 15 ml of sterile PBS to dislodge any remaining fat particles and pour liquid/particles into the conical. j. Carefully aspirate PBS from the tube until approximately 5 ml of PBS remains. k. Prepare the required amount of collagenase solution by determining the volume of the fat present in the conical. Make sufficient volume of collagenase to equal 1.lx the volume of fat to be treated. Collagenase stock is 0.075% in PBS (i.e., 75 mg per 100 ml). Filter sterilize the collagenase stock solution by using a 0.22 μm sterile filter. Place collagenase stock solution in a 37° C. water bath. I. Add enough sterile PBS to bring level to 40-45 ml mark, then cap. m. Mix by inverting several times. n. Aspirate the aqueous layer present, leaving 5 ml of volume. o. Repeat Steps 21-2n until aqueous layer is relatively clear of debris/blood (e.g., 2x or 3x, depending on how bloody the sample is). In one specific embodiment, a method of treatment comprises injecting a composition comprising stem cells isolated from an adipose tissue sample obtained from the tail head region of a horse and prepared according to a method of the invention into the same horse at a site of actual or potential injury, such as a tendon or ligament. p. After final aspiration, add collagenase enzyme solution (warmed to 37° C.). Mix well. Add a volume of collagenase stock solution equal to the total volume of the minced fat sample (fat and residual PBS). q. Incubate tubes in 37° C. water bath with agitation for 20-60 minutes, depending on the rate of fat degradation. r. Dry tubes and spray outside of tubes with 70% Isopropyl alcohol. s. Add enzyme-neutralizing solution (optional, based on adding DMEM with FCS). t. Centrifuge tube at 400xg for 5 minutes, swinging bucket rotor ( set at 4 ° C. ). u. Aspirate/pour off supernatant, removing fat layer. v. Resuspend pellet by gently “flicking” the bottom of the tube with a finger. w. Add 10 ml of PBS and mix gently by inversion. to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety. x. Pour resuspended cells through tissue filter ( e.g., 70 μm) into a labeled 50 ml conical tube to remove any tissue matrix and debris. After the suspension has drained into the conical, gently rinse the tissue filter with approximately 20 ml of sterile PBS, collecting the rinsate in the conical (including the underside of the filter unit itself). y. Centrifuge the tube at 400xg for 5 minutes, swinging bucket rotor. z. Aspirate/pour off supernatant. aa. Resuspend cell pellet in a small volume of media. The invention claimed is: 1. A method of treating an injury or disease in a mammal, comprising providing to the mammal a cell population comprising adipose-tissue derived stem cells, wherein said cell population was prepared by a cell processing method comprising: (a) processing adipose tissue obtained from a mammal to release cells therein, wherein said processing releases cells and a fat layer; and (b) separating cells released in (a) from the fat layer; thereby preparing the cell population comprising adipose tissue-derived stem cells; wherein said cell processing method does not include isolating stem cells separated in (b) from other cells separated in (b), thereby treating the injury or disease in the mammal. 2. The method of claim 1, wherein the processing of (a) comprises one or more procedures selected from the group consisting of: physical cutting or mincing of the adipose tissue; treating the adipose tissue with an enzyme that facilitates the release of cells; exposing the adipose tissue to ultrasonic energy; and treating the adipose tissue with perfluorocarbons. (a) preparing a cell population comprising adipose tissue derived stem cells for introducing into a mammal by a method comprising: (i) processing adipose tissue obtained from a mammal to release cells therein, wherein the adipose tissue comprises stem cells, wherein said processing releases cells and a fat layer; and (ii) separating the cells released in (i) from the fat layer, wherein the preparing does not include isolating stem cells separated in ( a )(ii) from other cells separated in (a)(ii), thereby preparing the cell population comprising adipose tissue-derived stem cells; and (b) providing the cell population of ( a )(ii) to the mammal, thereby treating the disease of disorder in the mammal. 3. The method of claim 1, wherein the processing of (a) separating comprises one or more procedures selected from comprises physical cutting or mincing of the adipose tissue. 4. The method of claim 1, wherein the separating of (b) comprises one or more procedures selected from the group consisting of: use of a density gradient; centrifugation; and filtration. 5. The method of claim 1, wherein: the processing of (a) comprises one or more procedures selected from the group consisting of: physical cutting or mincing of the adipose tissue; treating the adipose tissue with an enzyme that facilitates the release of cells; exposing the adipose tissue to ultrasonic energy; and treating the adipose tissue with perfluorocarbons; and the separating of (b) comprises one or more procedures selected from the group consisting of: use of a density gradient; centrifugation; and filtration. 6. The method of claim 1, wherein the adipose tissuederived stem cells are autologous to the mammal being treated. 7. The method of claim 1, wherein said cell population further comprises one or more cells selected from the group consisting of: red blood cells, white blood cells, fibroblasts, neutrophils, monocyte/macrophages, and basophils. 8. The method of claim 1, wherein said cell population further comprises one or more other tissue components. 9. The method of claim 8, wherein the one or more other tissue components comprise: extracellular matrix polypeptides, proteoglycans, lipids, cytokines, or growth factors. 10. The method of claim 1, wherein the disease or disorder is selected from the group consisting of: inflammatory diseases or disorders, cardiovascular diseases, nervous system diseases, tumors, demyelinating diseases, digestive system diseases, endocrine system diseases, reproductive system diseases, hemic and lymphatic diseases, immunological diseases, mental disorders, musculoskeletal diseases, neuromuscular diseases, metabolic diseases, skin and connective tissue diseases, and urological diseases. 11. The method of claim 1, wherein the injury is a wound selected from the group consisting of: abrasions, avulsions, blowing wounds, incised wounds, bums, contusions, puncture wounds, surgical wounds and subcutaneous wounds. except as by the appended claims. 12. A method of treating a disease or disorder in a mammal, comprising: (a) preparing a cell population comprising adipose tissue derived stem cells for introducing into a mammal by a method comprising: (i) processing adipose tissue obtained from a mammal to release cells therein, wherein the adipose tissue comprises stem cells, wherein said processing releases cells and a fat layer; and (ii) separating the cells released in (i) from the fat layer, wherein the preparing does not include isolating stem cells separated in ( a )(ii) from other cells separated in (a)(ii), thereby preparing the cell population comprising adipose tissue-derived stem cells; and (b) providing the cell population of ( a )(ii) to the mammal, thereby treating the disease of disorder in the mammal. 13. The method of claim 12, wherein the processing comprises one or more procedures selected from the group consisting of: physical cutting or mincing of the adipose tissue; treating the adipose tissue with an enzyme that facilitates the release of cells; exposing the adipose tissue to ultrasonic energy; and treating the adipose tissue with perfluorocarbons. 14. The method of claim 12, wherein the wherein the separating comprises one or more procedures selected from the group consisting of: use of a density gradient; centrifugation; and filtration. 15. The method of claim 12, wherein the adipose tissue- derived stem cells are autologous to the mammal being treated. 16. The method of claim 12, wherein the cell population further comprises one or more cells selected from the group consisting of: red blood cells, white blood cells, fibroblasts, neutrophils, monocyte/macrophages, and basophils. 17. The method of claim 12, wherein the cell population further comprises one or more other tissue components. 18. The method of claim 17, wherein the one or more other tissue components comprise: extracellular matrix polypeptides, proteoglycans, lipids, cytokines, or growth factors. 19. The method of claim 12, further comprising: (c) freezing the cell population in a physiologically compatible buffer before step (b). 20. The method of claim 12, further comprising: ( c) suspending the cell population in a physiologically compatible buffer before step (b ). 21. The method of claim 12, wherein the disease or disorder is selected from the group consisting of: inflammatory diseases or disorders, cardiovascular diseases, nervous system diseases, tumors, demyelinating diseases, digestive system diseases, endocrine system diseases, repro ductive system diseases, hemic and lymphatic diseases, immunological diseases, mental disorders, musculoskeletal diseases, neuromuscular diseases, metabolic diseases, skin and connective tissue diseases, and urological diseases. 22. The method of claim 12, wherein the injury is a wound selected from the group consisting of: abrasions, avulsions, blowing wounds, incised wounds, bums, contusions, puncture wounds, surgical wounds and subcutaneous wounds. 23. The method of claim 12, wherein the disease or disorder is a musculoskeletal injury or disease. 24. The method of claim 23, wherein the musculoskeletal injury or disease is a sprain, strain, dislocation, bruising, tear, microtear, contusion, bursitis, tendonitis, articular cartilage injury or fracture in a tendon, ligament, cartilage, bone, or hoof laminae. | |||||||||||||||||||
| 56 | Aust | 2004 | Stromal Vascular Fraction Cultured Cells Adipose Derived Adult Stem Cells | SVF | Manual | Human | SubQ | Liposuction | Collagenase Type I Worthington | Worthington | Krebs Ringers Buffer | 0.1 % Collagenase | 37 | 45 | Internittent Shaking | Centrifugation 300g 5min | The undifferentiated ADAS cells were homogeneously positive for the cell-surface markers CD10, CD13, CD29, CD44, CD49e, CD59, CD90, and HLA-ABC, and homogeneously negative for the cell surface markers CD11b, CD45, and HLA-DR. | 4.0410E5 cells/ml fat | > 85 % | Stromal Vascular Fraction | Culture derivation of stem cells | Aust L, Devlin B, Foster SJ, Halvorsen YD, Hicok K, du Laney T, Sen A, Willingmyre GD, Gimble JM. Yield of human adipose-derived adult stem cells from liposuction aspirates. Cytotherapy. 2004;6(1):7-14. doi: 10.1080/14653240310004539. PMID: 14985162. | Halvorsen Y-DC, Bond A, Sen A et al . Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: biochemical, cellular and molecular analysis. Metabolism 2001;50:407 /13. | Liposuction procedures were performed using a liposuction needle with an internal diameter of either 2 or 3 mm. Liposuction tissue samples were digested for 45 min at 37oC with intermittent shaking in Krebs Ringers buffer (KRB) (Sigma Chemical Co., St Louis, MO, USA) containing 1% BSA (Type V) (Sigma) and 0.1% collagenase (Type I) (Worthington Biochemical, Lakewood, NJ, USA). The floating adipocytes were separated from the stromal-vascular fraction by centrifugation (300 g) for 5 min. The stromal-vascular fraction was plated at a density equivalent to 0.12 mL of liposuction tissue per cm2 in multi-deck tissue culture flasks (Nunc, Napierville, IL, USA), fed with Stromal Medium composed of DMEMHam’s F-12 medium (vol/vol, 1:1) supplemented with 10% FBS (HyClone, Logan, UT, USA) and 1% antibiotic/ antimycotic solution (Invitrogen, Carlsbad, CA, USA), and maintained for 24 /72 h. | |||||||||||||||||
| 57 | Planat-Benard /Casteilla | 2004 | SVF | Adipose Stromal Cells (ASC) | Manual | Human | dermolipectomy | dermolipectomy | Collagenase | Sigma | PBS 2% BSA | 2mg/ml | 37 | 45 | 25 micron filter then Centrifugation. 600g. 10 minutes | 25 micron | VEGF. Angiogenic Assay. CD 34 31 13 45 14 144 | SVF | In Vitro. In Vivo Angiogenesis | VEGF. Angiogenic Assay. CD 34 31 13 45 14 144 | Planat-Benard V, Silvestre JS, Cousin B, André M, Nibbelink M, Tamarat R, Clergue M, Manneville C, Saillan-Barreau C, Duriez M, Tedgui A, Levy B, Pénicaud L, Casteilla L. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation. 2004 Feb 10;109(5):656-63. doi: 10.1161/01.CIR.0000114522.38265.61. Epub 2004 Jan 20. PMID: 14734516. | Bjorntorp P, Karlsson M, Pertoft H, et al. Isolation and characterization of cells from rat adipose tissue developing into adipocytes. J Lipid Res. 1978;19:316 –324. | Abstract: cultured human SVF cells differentiate into endothelial cells, incorporate into vessels, and promote both postischemic neovascularization in nude mice and vessel-like structure formation in Matrigel plug | Cells were isolated from adipose tissues according to Bjorntorp et al17 with minor modifications. Inguinal adipose tissue excised from mice and human adipose tissue from dermolipectomy were digested at 37°C in PBS containing 2% BSA and 2 mg/mL collagenase (Sigma) for 45 minutes. After elimination by filtration through 25 m filters of undigested fragments, mature adipocytes were separated from pellets of SVF cells by centrifugation (600g, 10 minutes). SVF cells were plated (30 000 cells/cm2) in DMEM-F12 10% newborn calf serum (NCS) medium. Six hours after plating, all nonadherent cells were removed by washing. SVF cells were then cultured for 3 days in the same medium before use, as previously described.17 The mature adipocyte fraction was also recovered, washed gently in DMEM-F12 10% NCS medium, and prepared as a 106 cells/mL suspension. A 100- L aliquot of the cell suspension was layered onto a 25-mm Thermanox coverslip (Nunc) placed in a 35-mm culture dish. The first coverslip was covered by a second one, and after 15 minutes at room temperature, 1.5 mL of DMEM-F12 10% NCS was added. After 4 to 5 days, adherent cells with small lipid droplets (preadipocyte-like cells) appeared and developed a fibroblast-like morphology free of lipids. | |||||||||||||||||
| 58 | Curat | 2004 | SVF | Adipose Stromal Cells (ASC) | Manual | Human | SubQ and Visceral | Liposuction and Surgical | collagenase solution (300 units/ml in PBS and 2% BSA), | PBS | 300 Units/ml | 37 | 200g centrifugation. resuspended in PBS/2% FCS and sequentially filtered through 100-, 70-, and 40- micron sieves | resuspended in PBS/2% FCS and sequentially filtered through 100-, 70-, and 40- m sieves | 10 min in an erythrocyte-lysing buffer (155 mmol/l NH4Cl, 5.7 mmol/l K2HPO4, and 0.1 mmol/l EDTA) | In Vitro culture characterization | Curat CA, Miranville A, Sengenès C, Diehl M, Tonus C, Busse R, Bouloumié A. From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes. Diabetes. 2004 May;53(5):1285-92. doi: 10.2337/diabetes.53.5.1285. PMID: 15111498. | Isolation of the SVF of human AT. Human subcutaneous AT was obtained from patients undergoing plastic surgery by lipoaspiration of the gluteal regions or lipectomy of the abdominal region (mean BMI 25 1 and 25.3 0.5 kg/m2, respectively). Human visceral AT was obtained from patients undergoing abdominal tumor surgery (colorectal carcinoma, pancreas, and stomach) that was not associated with obvious inflammatory disease (mean BMI 26.3 0.8 kg/m2). The study was approved by the ethics committee of the University Hospital of Frankfurt am Main. After digestion of the AT in a collagenase solution (300 units/ml in PBS and 2% BSA), followed by a 200g centrifugation, the pellet containing the SVF was incubated for 10 min in an erythrocyte-lysing buffer (155 mmol/l NH4Cl, 5.7 mmol/l K2HPO4, and 0.1 mmol/l EDTA) and finally resuspended in PBS/2% FCS and sequentially filtered through 100-, 70-, and 40- micron sieves. | |||||||||||||||||||||||
| 59 | Ariff/Williams. US patent 9144583 | 2004 | Cells | SVF | Automated | Mammal | Collagenase | Digestion and Separation Chamber | 37 | Centrifugation | 200 micron | Cell Transplantatiom | Ariff/Williams. US patent 9144583 | 39.A methodforseparatingcelscomprising: adding an amount oftisuetothe innerbowl ofthe aparatusofclaim13;heatingthetisuetoatempera tureofabout37°C.; addinganefectiveamountofcollagenasetotheinner bowl; digestingthetisuebyoscilatingtheinnerbowlforan efectiveamountoftime; removingfibroustisuefromthedigestedtisue; spinningtheinnerbowlatabout3100RPM forabout5 minutes; displacingfatcelstothecenteroftheinerbowlby addingfluidtotheinerbowl; directingthefatcelsthroughatleastoneapertureinthe inerbowlintotheouterbowlbycentrifugalaction; rinsingtheinnerbowlwithfluid; directingatleastonecelpeletfromatleastonelobeof theinnerbowltothebottomoftheinnerbowl; optionaly,addingfluidtotheinerbowlandspinningthe innerbowlatabout3100RPM forabout5minutes;and colectingatleastonecelpelet. | |||||||||||||||||||||||||||
| 60 | Ariff/Williams. US Patent 9683210 | 2004 | Cell Pellet | SVF | Automated | Mammal | Collagenase | Oscillating Bowl | 37 | Centrifugation | 200 micron | Endothelial Cells | Cell Transplantation | Ariff/Williams. US Patent 9683210 | |||||||||||||||||||||||||||
| 62 | Sengenes | 2005 | Stromal Vascular Fraction SVF | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | Collagenase | Biochrom AG | phosphate buffered saline (PBS),2%bovine serum albumin (BSA),pH7.4 | 300U/ml | 45 | Constant Shaking | Floatin adipocytes removed, Centrifugation 200g 10 minutes, pellet resuspended in erythrocyte lysis buffer (155 mmol/L NH4Cl; 5.7 mmol/L K2HPO4; 0.1 mmol/L EDTA, pH 7.3) for 10 min. After successive filtrations through 100, 70, and 40 micron sieves, the cells were resuspended in PBS/2% fetal calf serum (FCS). | Successive filtrations through 100, 70, and 40 micron sieves. | pellet resuspended in erythrocyte lysis buffer (155 mmol/L NH4Cl; 5.7 mmol/L K2HPO4; 0.1 mmol/L EDTA, pH 7.3) for 10 min. | adipocyte progenitor cells, i.e., the preadipocytes, are included in the CD34þ/CD31 cell fraction | Sengenès C, Lolmède K, Zakaroff-Girard A, Busse R, Bouloumié A. Preadipocytes in the human subcutaneous adipose tissue display distinct features from the adult mesenchymal and hematopoietic stem cells. J Cell Physiol. 2005 Oct;205(1):114-22. doi: 10.1002/jcp.20381. PMID: 15880450. | Human adipose tissue was obtained from individuals undergoing plastic surgery by lipoaspiration. The adipose tissue was digested using collagenase (300 U/ml in phosphatebuffered saline (PBS),2%bovine serum albumin (BSA),pH7.4) for 45 min under constant shaking. Following removal of the floating mature adipocytes, the lower layer containing the SVF was centrifuged (200g, 10 min) and the pellet resuspended in erythrocyte lysis buffer (155 mmol/L NH4Cl; 5.7 mmol/L K2HPO4; 0.1 mmol/L EDTA, pH 7.3) for 10 min. After successive filtrations through 100, 70, and 40 mm sieves, the cells were resuspended in PBS/2% fetal calf serum (FCS). | |||||||||||||||||||||
| 63 | Rodriguez | 2005 | human multipotent adipose-derived stem (hMADS) cell | Insufficient Data to Classify | Manual | Human | Surgical | collagenase | DMEM containing antibiotics (100 U/ml of penicillin and 100 g/ ml of streptomycin),, and 20 mg/ml bovine serumalbumin. | 2 mg /ml | low speed centrifugation (200 g, 10 min) | Rodriguez AM, Pisani D, Dechesne CA, Turc-Carel C, Kurzenne JY, Wdziekonski B, Villageois A, Bagnis C, Breittmayer JP, Groux H, Ailhaud G, Dani C. Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J Exp Med. 2005 May 2;201(9):1397-405. doi: 10.1084/jem.20042224. PMID: 15867092; PMCID: PMC2213197. | Hauner, H., T. Skurk, and M. Wabitsch. 2001. Culture of human adipose precursor cells. In Adipose Tissue Protocols (Methods in Molecular Biology). G. Ailhaud, editor. Humana Press Inc., Totowa, NJ. 239–247. | Isolation of hMADS cells. For the isolation of hMADS cells from young donors, adipose tissue was obtained with the informed consent of the parents as surgical scraps from surgical specimen of various surgeries, as approved by the Centre Hospitalier Universitaire de Nice Review Board. We modified a previous published protocol used to isolate adipocyte precursors from adipose tissue (23). In brief, 200 mg/ml adipose tissue was dissociated for 5–10 min in DMEM containing antibiotics (100 U/ml of penicillin and 100 g/ ml of streptomycin), 2 mg/ml collagenase, and 20 mg/ml bovine serumalbumin. The crude SVF was separated from the adipocyte fraction by low speed centrifugation (200 g, 10 min). The adipocyte fraction was discarded and cells from the pelleted SVF were seeded onto uncoated tissue culture plates (Greiner) at 1,000–3,500 cells/cm2 in low glucose DMEM (Invitrogen | |||||||||||||||||||||||||||
| 64 | Tchkonia | 2005 | Preadipocytes | Insufficient Data to Classify | Manual | Human | Abdominal SubQ, Mesenteric, Omental | Surgical | Minced | collagenase | Hank’s balanced salt solution (HBSS) | 1 mg/ml | 37 | until fragments were no longer visible and the digest had a milky appearance. | shaking water bath | Digests were filtered and centrifuged at 800 g for 10 min. | filtered | The digests were treated with an erythrocyte lysis buffer | Digest | Culture Differentiation to adipocytes | Tchkonia T, Tchoukalova YD, Giorgadze N, Pirtskhalava T, Karagiannides I, Forse RA, Koo A, Stevenson M, Chinnappan D, Cartwright A, Jensen MD, Kirkland JL. Abundance of two human preadipocyte subtypes with distinct capacities for replication, adipogenesis, and apoptosis varies among fat depots. Am J Physiol Endocrinol Metab. 2005 Jan;288(1):E267-77. doi: 10.1152/ajpendo.00265.2004. Epub 2004 Sep 21. PMID: 15383371. | regional variation in abundance of preadipocyte subtypes likely contributes to the distinct characteristics of different fat depots | Preadipocyte culture. Fat tissue was minced and then digested in Hank’s balanced salt solution (HBSS) containing 1 mg/ml collagenase in a 37°C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800 g for 10 min. The digests were treated with an erythrocyte lysis buffer (37, 81). The cells were then plated using a low-serum plating medium [1:1 Dulbecco’s modified Eagle’s medium (DMEM)-Ham’s F-12 that contained 0.5% bovine serum and antibiotics]. After 12 h, a period during which no replication occurs (21), the adherent preadipocytes were washed extensively, trypsinized, and replated at a density of 4 104 cells/cm2 in plating medium. Replating helps to eliminate mesothelial cell and macrophage contamination. Linearity of preadipocyte recovery has been shown previously using this approach (51). Macrophages were rare ( 5 106 cells as assessed by phase contrast microscopy) in the replated cultures, irrespective of depot origin. In previous studies (79), we cloned cells from similarly prepared replated primary cultures and treated the resulting colonies with differentiation-inducing medium. The proportion of clones that contained cells capable of more extensive lipid accumulation than lung fibroblasts or other nonpreadipocyte cell types was determined. Essentially 100% of the clones prepared from the replated primary cultures accumulated lipid more extensively than nonpreadipocyte cell types, confirming the purity of our preadipocyte preparations. Plating medium was changed every 2 days until confluence. | ||||||||||||||||||
| 65 | Katz | 2005 | human adipose- derived adherent stromal (hADAS) | Adipose Stromal Cells (ASC) | Manual | Human | intraoperative suction lipectomy or from laboratory liposuction of panniculectomy specimens. | surgical | enzyme | Filtered | Contaminating erythrocytes were lysed with an osmotic buffer | Katz AJ, Tholpady A, Tholpady SS, Shang H, Ogle RC. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells. Stem Cells. 2005 Mar;23(3):412-23. doi: 10.1634/stemcells.2004-0021. PMID: 15749936. | Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001Katz AJ. Mesenchymal cell culture: adipose tissue. In: Atala A, Lanza RP, eds. Methods of Tissue Engineering. Academic Press, NY;2002:277–286.;7:211–228. | Cells were isolated from adipose tissue using methods previously described [1, 19]. Briefly, harvested tissue was washed several times and then enzymatically dissociated. The dissociated tissue was then filtered to remove debris, and the resulting cell suspension was centrifuged. Pelleted stromal cells were then recovered and washed several times. Contaminating erythrocytes were lysed with an osmotic buffer, and the stromal cells were plated onto tissue culture plastic (10-cm dishes). Cultures were washed with buffer 24–48 hours after plating to remove unattached cells, and then refed with fresh medium. Plating and expansion medium consisted of DMEM/F12 with 10% FBS and antibiotic-antimycotic. Cultures were maintained at 37°C with 5% CO2 and fed three times per week. | |||||||||||||||||||||||||||
| 66 | Boquest | 2005 | SVF | Adipose Stromal Cells (ASC) | manual | Human | SubQ. Abdomen Hip and Thigh | Liposuction | Collagenase | Possibly Sigma | HBSS | 0.20% | 37 | 120 | shaker | Centrifugation 400 x g. 10 min, Pellet resuspended in HBSS containin 2% FBS and allowed to setle for 1 min. Suspended cells passed through a 100 micron and then a 40 micron sieve. Final separation used Histopaue-1077 gradietns. Final isolate passed through a 30 micron mesh | 100 then 40 then 30 micron filters/meshes.5 | red blood cell lysis buffer (2.06 g/l Tris base, 7.49 g/l NH4Cl, pH 7.2) for 10 min at room temperature | Boquest AC, Shahdadfar A, Frønsdal K, Sigurjonsson O, Tunheim SH, Collas P, Brinchmann JE. Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture. Mol Biol Cell. 2005 Mar;16(3):1131-41. doi: 10.1091/mbc.e04-10-0949. Epub 2005 Jan 5. PMID: 15635089; PMCID: PMC551479. | “Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228.” | Adipose tissue was obtained by liposuction from abdominal, hip, and thigh regions of healthy female donors aged 18–39. The stromal vascular fraction (SVF) was separated from adipose tissue using a procedure modified from Zuk and colleagues (Zuk et al., 2001). Briefly, lipoaspirate (300–400 ml) was washed with Hanks’ balanced salt solution (HBSS; Life Technologies-BRL, Paisley, UK) containing antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin, Life Technologies-BRL) and 2.5 g/ml amphotericin B (Life Technologies-BRL). Washed adipose tissue was digested for 2 h on a shaker at 37°C in HBSS containing 0.2% collagenase. Floating adipocytes were aspi- rated from pelleted SVF cells after centrifugation at 400 g for 10 min. Pellets were resuspended in red blood cell lysis buffer (2.06 g/l Tris base, 7.49 g/l NH4Cl, pH 7.2) for 10 min at room temperature. After resuspending SVF cells in HBSS containing 2% fetal bovine serum (FBS), tissue clumps were allowed to settle for 1 min. Suspended cells were passed through 100- m and then 40- m cell sieves (Becton Dickinson, San Jose, CA). Cell suspensions (15 ml) were applied to Histopaque-1077 gradients (15 ml) in 50-ml tubes. After centrifugation (400 g, 30 min), cells at the gradient interface were collected, washed in HBSS, and passed through a 30- m mesh. Cell counts and viability assessment were performed. | Address correspondence to: Jan E. Brinchmann (j.e.brinchmann@ labmed.uio.no) or Philippe Collas (philippe.collas@medisin.uio.no). | |||||||||||||||||||
| 67 | Dicker | 2005 | MSC | Insufficient Data for Classification | MSC Culture | Dicker A, Le Blanc K, Aström G, van Harmelen V, Götherström C, Blomqvist L, Arner P, Rydén M. Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res. 2005 Aug 15;308(2):283-90. doi: 10.1016/j.yexcr.2005.04.029. PMID: 15925364. | M. Rodbell, Metabolism of isolated fat cells: I. Effects of hormones on glucose metabolism and lipolysis, J. Biol. Chem. 239 (1964) 375– 380. M. Ryden, A. Dicker, C. Gotherstrom, G. Astrom, C. Tammik, P. Arner, K. Le Blanc, Functional characterization of human mesenchymal stem cell-derived adipocytes, Biochem. Biophys. Res. Commun. 311 (2003) 391– 397. | Mature adipocytes were separated out according to Rodbell et al. . The remaining stromal– vascular portion was used for subsequent isolation of MSC as described previously [Ryden]. | E-mail address: mikael.ryden@medhs.ki.se (M. Ryde´n). | ||||||||||||||||||||||||||||||||
| 68 | Cao | 2005 | ADAS | Insufficient Data to Classify | Manual | Human | Liposuction | Collagenase II | Sigma | 37 | 30 | 2 x | ADAS | Cao Y, Sun Z, Liao L, Meng Y, Han Q, Zhao RC. Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem Biophys Res Commun. 2005 Jul 1;332(2):370-9. doi: 10.1016/j.bbrc.2005.04.135. PMID: 15896706. | Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228.””” | Preparation of human ADAS cells from the adult human fat. Human raw lipoaspirates from patients undergoing selective suction-assisted lipectomy were collected after obtaining informed consent from the patients according to procedures approved by the Ethics Committee at Chinese Academy of Medical Sciences and Peking Union Medical College. The procedure was described by Zuk et al. [15] with some modifications. The raw liposuctioned aspirate was extensively washed with D-Hanks solution to remove contaminating blood cells and local anesthetics. The extracellular matrix was digested with 0.2% collagenase II (Sigma) at 37 C for 30 min to release the cellular fractions. The cells were washed two times and plated in T-75 tissue culture flasks at a density of 2 · 106/ml. Expansion medium contained 57% DMEM/F-12 (Gibco Life Technologies, Paisley, UK), 40% MCDB-201 (Sigma, St. Louis, MO, USA), 2% fetal calf serum (FCS; Gibco), 1· insulin transferrin selenium (ITS; Gibco), 10 8 M dexamethasone (Sigma), 10 4 M ascorbic acid 2-phosphate (Sigma), 10 ng/ml epidermal growth factor (EGF; Sigma), 10 ng/ml platelet-derived growth factor BB (PDGF-BB; Sigma), 100 U/ml penicillin, and 100 lg/ml streptomycin (Gibco). Once adherent cells were more than 70% confluent, they were detached with 0.125% trypsin and 0.01% EDTA, and replated at a 1:3 dilution under the same culture conditions. All the experiments were done with the 5th passage and the 20th passage, and the results present in the article are all 5th passage. | E-mail address: chunhuaz@public.tpt.tj.cn (R.C. Zhao). | ||||||||||||||||||||||||
| 69 | Boquest | 2005 | Stromal Vascular Fraction ( SVF) | Adipose Stromal Cells (ASC) | Manual | Human | abdominal, hip, and thigh regions of healthy female donors aged 18–39 | Liposuction | lipoaspirate (300–400 ml) | collagenase | HBSS | 0.20% | 37 | 120 min | shaker | Floating adipocytes were aspirated from pelleted SVF cells after centrifugation at 400 g for 10 min. Pellets were resuspended in red blood cell lysis buffer (2.06 g/l Tris base, 7.49 g/l NH4Cl, pH 7.2) for 10 min at room temperature. After resuspending SVF cells in HBSS containing 2% fetal bovine serum (FBS), tissue clumps were allowed to settle for 1 min. Suspended cells were passed through 100- m and then 40- m cell sieves (Becton Dickinson, San Jose, CA). Cell suspensions (15 ml) were applied to Histopaque-1077 gradients (15 ml) in 50-ml tubes. After centrifugation (400 g, 30 min), cells at the gradient interface were collected, washed in HBSS, and passed through a 30- micron mesh. Cell counts and viability assessment were performed. | 100 micro. 40 micron. 30 micron | red blood cell lysis buffer (2.06 g/l Tris base, 7.49 g/l NH4Cl, pH 7.2) for 10 min at room temperature | Microarray Analysis. CD31 + and CD31 – cells isolated from ASCs and compared | Boquest AC, Shahdadfar A, Frønsdal K, Sigurjonsson O, Tunheim SH, Collas P, Brinchmann JE. Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture. Mol Biol Cell. 2005 Mar;16(3):1131-41. doi: 10.1091/mbc.e04-10-0949. Epub 2005 Jan 5. PMID: 15635089; PMCID: PMC551479. | Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228.””” | Surprisingly von Willebrand factor, a molecule with a very restricted expression profile including predominantly endothelial cells, was expressed by all CD31 cells, but also by most of the CD31 cells. CD31 cells also overexpressed transcripts related to bone, cartilage and muscle biology. | Isolation of Stromal Vascular Cells from Human Adipose Tissue Adipose tissue was obtained by liposuction from abdominal, hip, and thigh regions of healthy female donors aged 18–39. The stromal vascular fraction (SVF) was separated from adipose tissue using a procedure modified from Zuk and colleagues (Zuk et al., 2001). Briefly, lipoaspirate (300–400 ml) was washed with Hanks’ balanced salt solution (HBSS; Life Technologies-BRL, Paisley, UK) containing antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin, Life Technologies-BRL) and 2.5 g/ml amphotericin B (Life Technologies-BRL). Washed adipose tissue was digested for 2 h on a shaker at 37°C in HBSS containing 0.2% collagenase. Floating adipocytes were aspirated from pelleted SVF cells after centrifugation at 400 g for 10 min. Pellets were resuspended in red blood cell lysis buffer (2.06 g/l Tris base, 7.49 g/l NH4Cl, pH 7.2) for 10 min at room temperature. After resuspending SVF cells in HBSS containing 2% fetal bovine serum (FBS), tissue clumps were allowed to settle for 1 min. Suspended cells were passed through 100- m and then 40- m cell sieves (Becton Dickinson, San Jose, CA). Cell suspensions (15 ml) were applied to Histopaque-1077 gradients (15 ml) in 50-ml tubes. After centrifugation (400 g, 30 min), cells at the gradient interface were collected, washed in HBSS, and passed through a 30- m mesh. Cell counts and viability assessment were performed. | Address correspondence to: Jan E. Brinchmann (j.e.brinchmann@ labmed.uio.no) or Philippe Collas (philippe.collas@medisin.uio.no). | |||||||||||||||||
| 70 | van Harmelon | 2005 | Preadipocytes | Adipose Stromal Cells (ASC) | Manual | Human | various | elective or laparoscopic abdominal surgery | collagenase | 3 mL solution/mL or gram adipose tissue | 50 ml tubes | 37 | 90 min | shaking water bath | After the digestion, the tubes are centrifuged at 200g for 10 min at room temperature; thereafter the supernatant containing mature adipocytes and collagenase solution is removed from the pellet that contains the preadipocytes. The pellet is resuspended (pellets from several tubes can now be pooled) in erythrocyte lysis buffer (1 9, v v) (see Note 4) and incubated for not more than 10 min. The suspension is filtered through a 150-μm filter into another 50-mL tube and centrifuged again as above (see Note 5). The supernatant is discarded and the pellet is resuspended in an appropriate volume of basal medium (10–20 mL, depending on the expected yield of preadipocytes). The suspension is filtered through a 70-μm pore size filter into another 50-mL tube. An aliquot of the suspension is taken for cell counting (see below) and the rest of the suspension is centrifuged again as described above. | 150 micron. 70 micron | erythrocyte lysis buffer (1 9, v v) | Neubauer chamber | Trypan Blue | Methodology | van Harmelen V, Skurk T, Hauner H. Primary culture and differentiation of human adipocyte precursor cells. Methods Mol Med. 2005;107:125-35. doi: 10.1385/1-59259-861-7:125. PMID: 15492368. | Adipose tissue samples from the visceral region are densely vascularized. Therefore, it is very likely that there is a contamination by endothelial cells in the stromal cell fraction. The proportion of endothelial cells should be reduced to less than 5% of the total cell number in order to avoid major effects of the endothelial cells on preadipocyte functioning. Therefore, it is recommended, after the first filtration through 150 μm to do a second filtration immediately using a filter with pore size 30 μm. Endothelial cells reaggregate rapidly and are therefore retained by this filter (10). However, some contamination by endothelial cells cannot be avoided. If precursor cells from different depots are compared, all samples must undergo the same filtration procedure. | Cell Isolation 1. The adipose tissue specimens are carefully liberated from remaining visible connective tissue and blood vessels in Petri dishes. Readily prepared pieces are kept in PBS in Petri dishes until the collagenase digestion procedure. 2. The adipose tissue pieces are put into 50-mL tubes, cut gently into very fine pieces using surgical scissors, and incubated in collagenase solution (3 mL solution/mL or gram adipose tissue) (see Note 2). For the incubation, the tubes are closed tightly and connected horizontally under water in a 37°C shaking water bath for 90 min. The tubes are never filled more than 45 mL in order to let the solution move freely in the tube and to allow the collagenase to reach all adipose tissue pieces. 3. After the digestion, the tubes are centrifuged at 200g for 10 min at room temperature; thereafter the supernatant containing mature adipocytes and collagenase solution is removed from the pellet that contains the preadipocytes (see Fig. 1). The pellet should not dry, so 1 mL solution should be kept at the bottom. 4. The pellet is resuspended (pellets from several tubes can now be pooled) in erythrocyte lysis buffer (1 9, v v) (see Note 4) and incubated for not more than 10 min. 5. The suspension is filtered through a 150-μm filter into another 50-mL tube and centrifuged again as above (see Note 5). The supernatant is discarded and the pellet is resuspended in an appropriate volume of basal medium (10–20 mL, depending on the expected yield of preadipocytes). 6. The suspension is filtered through a 70-μm pore size filter into another 50-mL tube. An aliquot of the suspension is taken for cell counting (see below) and the rest of the suspension is centrifuged again as described above. | *Correspondence: Dr H Hauner, Deutsches Diabetes-Forschungsinstitut an der Heinrich-Heine-Universita¨t, Auf’m Hennekamp 65, 40225 Du¨sseldorf, Germany. E-mail: hauner@ddfi.uni-duesseldorf.de. Leiden University Medical Center, Department of Human Genetics, Einthovenweg 20, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail address: V.J.A.van_Harmelen@lumc.nl (V. van Harmelen). | |||||||||||||||||
| 71 | Yoshimura | 2006 | Stromal Vascular Fraction Processed Lipoaspirate Cells (PLA). Liposuction aspirate Fluid Cells (LAF) | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | Collagenase | Wako Pure Chemicals | PBS | 0.08% | 37 | 30 | Shaker | Centrifugation 800g 10 min RBC lysis, 100 micron filter | 100 micron filter | Lysis Buffer | anti-CD4-FITC, CD10-PE, CD13-PE, CD16-PE, CD29-PE, CD31-PE, CD34-PE, CD34-FITC, CD34- PE Cy7, CD36-PE, CD44-PE, CD45-PE, CD45-FITC, CD49d- PE, CD49e-PE, CD54-PE, CD56-PE, CD57-FITC, CD62E-PE, CD62P-PE, CD69-FITC, CD73-PE, CD90-PE, CD106-FITC, CD117-PE, CD135-PE, CD146-PE, CD-151-PE, HLA-A,B,CPE, Tie-2-PE (BD Biosciences, San Diego, CA), CD31-APC (eBioscience, San Diego, CA), CD144-PE (Beckman Coulter, Fullerton, CA), CD59-PE (Ancell, Bayport, MN), CD71-PE, CD105-PE (Serotec, Oxford, UK), CD133-PE, and Flk-1-PE (Techne, Minneapolis, MN). | PLA and LFA – Stopped using SVF | Cell Culture, Differentiation , Characterization | anti-CD4-FITC, CD10-PE, CD13-PE, CD16-PE, CD29-PE, CD31-PE, CD34-PE, CD34-FITC, CD34- PE Cy7, CD36-PE, CD44-PE, CD45-PE, CD45-FITC, CD49d- PE, CD49e-PE, CD54-PE, CD56-PE, CD57-FITC, CD62E-PE, CD62P-PE, CD69-FITC, CD73-PE, CD90-PE, CD106-FITC, CD117-PE, CD135-PE, CD146-PE, CD-151-PE, HLA-A,B,CPE, Tie-2-PE (BD Biosciences, San Diego, CA), CD31-APC (eBioscience, San Diego, CA), CD144-PE (Beckman Coulter, Fullerton, CA), CD59-PE (Ancell, Bayport, MN), CD71-PE, CD105-PE (Serotec, Oxford, UK), CD133-PE, and Flk-1-PE (Techne, Minneapolis, MN). | Yoshimura K, Shigeura T, Matsumoto D, Sato T, Takaki Y, Aiba-Kojima E, Sato K, Inoue K, Nagase T, Koshima I, Gonda K. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol. 2006 Jul;208(1):64-76. doi: 10.1002/jcp.20636. PMID: 16557516. | Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228. | All chemicals were purchased from Wako Pure Chemicals (Osaka, Japan), unless otherwise stated. PLA cells were separated from the fatty portions of liposuction aspirates using a procedure modified from Zuk et al. (2001). Briefly, the suctioned fat was digested with 0.075% collagenase in PBS for 30 min on a shaker at 378C. Mature adipocytes and connective tissues were separated from pellets by centrifugation (800g, 10 min). Pellets were resuspended in erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) and incubated for 5 min at room temperature. The pellets were resuspended and passed through a 100-mm mesh filter (Millipore, Billerica, MA). LAF cells were harvested from the fluid portions of liposuction aspirates. The suctioned fluid was centrifuged (400g, 10 min), and the pellets were resuspended in erythrocyte lysis buffer. After 5 min at room temperature, lysates were passed through a 100-mm mesh filter. The pellets were then processed for density gradient centrifugation with Ficoll (GE Healthcare Bio-Sciences, Piscataway, NJ). After centrifugation (800g, 20 min), cells at the gradient interface were collected, washed with PBS, and passed through a 100-mm mesh filter. For flow cytometry of freshly isolated LAF cells, density gradient centrifugation was not conducted. Nucleated cell counts were performed using a NucleoCounter (Chemometec, Allerod, Denmark). | Correspondence to: Kotaro Yoshimura, Department of Plastic Surgery, University of Tokyo School of Medicine, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: yoshimura-pla@h.u-tokyo.ac.jp | |||||||||||||||
| 72 | Duckers | 2006 | autologous adipose derived regenerative cells (ADRCs) | SVF | Automated | Human | Liposuction | few hundred millilitres | Enzyme | Duckers HJ, Pinkernell K, Milstein AM, Hedrick MH. The Bedside Celution system for isolation of adipose derived regenerative cells. EuroIntervention. 2006 Nov;2(3):395-8. PMID: 19755319. | The process is started with the harvest of a few hundred millilitres of adipose tissue by standard manual liposuction through a stab incision under local anaesthesia using a Toomey syringe and cannula. This procedure can be performed during the primary coronary intervention or at the bedside. During the fully automated isolation using the Celution™ System, the adipose tissue is enzymatically digested into a single cell suspension, and washed. Subsequently, all lipid-laden adipocytes and debris is separated from the therapeutic ADRCs, which eventually contains a combination of adult mesenchymal-like stem cells, endothelial progenitor cells and other adipose tissue stromal cells.5-8 | ||||||||||||||||||||||||||||||
| 73 | Zheng | 2006 | Mouse Adipose derived Stem Cells muADAS | Adipose stromal Cells (ASC) | Manual | Mouse | visceral | surgical | finley minced | collagenase type XI | Sigma | 0.08 | 37 | 45 | Enzyme activity was neutralized by treatment with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). | cell suspension was centrifuged at 1200g for 10 min | filtration of the cell suspension through a 100-mm nylon strainer to remove cellular debris, | ammonium chloride (160mM), incubated at room temperature for 10 min to lyse contaminating red blood cells | 7.0 106 nucleated cells per mouse | Trypan Blue Exclusion | muADAS | In Vitro In Vivo differentiation | FACS | Zheng B, Cao B, Li G, Huard J. Mouse adipose-derived stem cells undergo multilineage differentiation in vitro but primarily osteogenic and chondrogenic differentiation in vivo. Tissue Eng. 2006 Jul;12(7):1891-901. doi: 10.1089/ten.2006.12.1891. PMID: 16889519. | Cell harvest and culture For isolation of murine adipose-derived adult stem (muADAS) cells, visceral adipose tissue was excised separately from six C57BL/10J mice and five BALB/cJ mice (4–8 weeks of age), finely minced, and digested using 0.075% collagenase type XI (Sigma, St. Louis, MO) for 45 min at 378C. Enzyme activity was neutralized by treatment with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). The cell suspension was centrifuged at 1200g for 10 min to separate the floating adipocytes from the SVF. The SVF was resuspended in ammonium chloride (160mM), incubated at room temperature for 10 min to lyse contaminating red blood cells, and then centrifuged as described above. After filtration of the cell suspension through a 100-mm nylon strainer to remove cellular debris, cell number and viability were determined using trypan blue exclusion. The SVF cells were plated in T75 flasks overnight in control medium (DMEM, 10% FBS, 1% penicillin/streptomycin) at 378C in 5%carbon dioxide (CO2). After incubation for 24 h, the flasks were washed extensively uisng phosphate buffered solution (PBS) to remove residual nonadherent cells. The adherent muADAS cells were expanded using serial passaging. | ||||||||||||||||
| 74 | Oedayrajsingh-Varma | 2006 | Stromal Vascular Fraction. SVF | SVF | Manual | Human | SubQ | Liposuction | Collagenase A | Roche Diagnostics GmbH, Mannheim, Germany | PBS | 0.10% | 37 | 45 | Intermittent shaking | Centrifuge 600g 10 minutes, Resuspended pellet passed though 200 micron mesh, Ficoll gradient centrifugation. 1 wash | 1 wash | 200 micron mesh | Ficoll Centrifugation | FACS cultured cells Therefore in the present study we used a CD34+ CD31- CD105+ CD166+ CD45- CD90+ | 0.7 /1069/0.1 /106 cells/g adipose tissue (mean9/SEM, n/16), the hip and thigh region 0.5 /1069/0.07 /106 cells/g (n /11) and the mamma 0.6 /1069/0.3 /106 cells/g (n /4). | 81 +/- 2% (mean +/- SEM) | SVF | Characterization Differentiation | Oedayrajsingh-Varma MJ, Ham SM van, Knippenberg M, Helder MN, Klein-Nulend J, Schouten TE, Ritt MJ, Milligen FJ van (2006) Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy 8:166–177 | The crude lipoaspirate or resected tissue was transported from the operation room to the laboratory in sterile transportation bags within 2 h post-surgery. The resected material was minced using a surgical scalp, and the weight of the tissue was determined. To isolate the stromal vascular fraction, the adipose tissue was washed extensively with PBS, followed by enzymatic digestion with 0.1% collagenase A (Roche Diagnostics GmbH, Mannheim, Germany) in PBS containing 1% BSA, for 45 min at 378C under intermittent shaking. The digested adipose tissue was then washed with DMEM-glucose (BioWhit- taker, Cambrex, Verviers, Belgium) containing 10% FBS and centrifuged for 10 min at 600 g. The cell pellet was resuspended in PBS and passed through a 200-mm mesh (Braun/ Beldico s.a-n.v, Marche-en-Famenne, Belgium) to remove debris. Subsequently, cells were subjected to Ficoll density centrifugation (lymphoprep, r /1.077 g/mL, osmolarity 2809/15 mOsm; Axis-Shield, Oslo, Norway) to remove contaminating erythrocytes. Ficoll density filtration was performed after the first 10 isolations, as it improved the flow cytometric analyses of the samples without affecting the yield of viable cells in the stromal vascular fraction (data not shown). The cell-containing interface was harvested and washed with DMEM contain- ing 10% FBS. The viability of the cells was assessed using trypan blue exclusion assay. Viable, large cells were counted using light-microscopy. After the isolation proce- dure 3 /10 /106 cells were resuspended in a mixture (1:1) of culture medium and cyroprotective medium (Freezing Medium; BioWhittaker), frozen under ‘controlled rate’ conditions in a Kryosave (HCI Cryogenics BV, Hedel, the Netherlands) and stored in the vapor phase of liquid nitrogen according to standard practice in the Department of Pathology of the VU University Medical Center, and following the guidelines of current good manufacturing practice. | Florine J. van Milligen, PhD, Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. | ||||||||||||||
| 75 | Wolbank | 2007 | Human adipose-derived SCs | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | Collagenase I | 20 mg/ mL bovine serum albumin (PAA), and 25mMN-2-hydroxy ethylpiperazine-N0-2-ethanesulfonic acid (PAA) in PBS containing calcium2þ/magnesium2þ) | 1.5 mg/mL collagenase I, | 37 | 60 | Vigourous Shaking | erythrocyte lysis buffer (154mM ammo nium chloride, 10mM potassium bicarbonate, and 0.1mM EDTA (Sigma, Vienna, Austria) in aqua bidest.) for 10 min at 37C | Centrifugation | 100-micron cell strainer (BD, Vienna, Austria) | erythrocyte lysis buffer (154mM ammo nium chloride | FACS Cytochem | ASC | Culture then characterization | FACS Cytochem | Wolbank S, Peterbauer A, Fahrner M, Hennerbichler S, van Griensven M, Stadler G, Redl H, Gabriel C. Dose-dependent immunomodulatory effect of human stem cells from amniotic membrane: a comparison with human mesenchymal stem cells from adipose tissue. Tissue Eng. 2007 Jun;13(6):1173-83. doi: 10.1089/ten.2006.0313. PMID: 17518752. | Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228. | ASC isolation was modified from Zuk et al. Briefly, liposuction material was washed with PBS (PAA) and digested with collagenase (1.5 mg/mL collagenase I, 20 mg/ mL bovine serum albumin (PAA), and 25mMN-2-hydroxy ethylpiperazine-N0-2-ethanesulfonic acid (PAA) in PBS containing calcium2þ/magnesium2þ) for 60 min at 378C under vigorous shaking. After centrifugation, the cell pellet was treated with erythrocyte lysis buffer (154mM ammo- nium chloride, 10mM potassium bicarbonate, and 0.1mM EDTA (Sigma, Vienna, Austria) in aqua bidest.) for 10 min at 378C. After adding PBS, cells were filtered through a 100-micron cell strainer (BD, Vienna, Austria) and cultured at 378C, 5% CO2, and 95% air humidity to a subconfluent state for up to 6 passages. | Susanne Wolbank, Ph.D. Red Cross Blood Transfusion Service of Upper Austria Blumauerstrasse 3-5 A-4020 Linz Austria E-mail: susanne.wolbank@blutz.o.redcross.or.at | |||||||||||||||
| 76 | Wosnitza | 2007 | Stromal Vascular Fraction | Stromal Vascular Fraction | Manual | Human | abdominoplasty, breast reduction, liposuction | Liposuction | Fat lobules from excised adipose tissue were prepared by carefully removing capillaries and connective tissue. After mincing into small pieces of 2–6 mm | CLS type I | collagenase buffer (100mM HEPES, 120mM NaCl, 50mM KCl, 1mM CaCl2, 50mM glucose, pH 7.4) | 0.494 U/ml | 37 | 60 | constant shaking | 250 mm filter and centrifuged at 700 g for 7 min at room temperature | 250 micron filter | Magnetic beads. +/- CD31 | Wosnitza M, Hemmrich K, Groger A, Gräber S, Pallua N. Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation. Differentiation. 2007 Jan;75(1):12-23. | Authors abstract: The SVF also comprises CD31+ cells that, although they have an endothelial phenotype, can be converted into mature adipocytes. | Isolation of SVF cells Preadipocytes were isolated from freshly excised human subcutaneous abdominal or mammary fat tissue at the Department of Plastic Surgery and Hand Surgery—Burn Center from patients who underwent elective operations (e.g., abdominoplasty, breast reduction, liposuction). Harvesting of adipose tissue by liposuction was performed according to Sydney Coleman (manually applied negative pressure using a 10 cc-syringe with a blunt tip cannula, no addition of any solution) from the abdomen and breast (Coleman, 1997). Adipose tissue from aspiration was digested by collagenase treatment after washing with 0.9% NaCl. Fat lobules from excised adipose tissue were prepared by carefully removing capillaries and connective tissue. After mincing into small pieces of 2–6 mm, adipose tissue was washed with 0.9% NaCl and also digested. Digestion of liposuctioned and excised fat tissue was performed with collagenase CLS type I (0.494 U/ml) and 1.5% BSA dissolved in collagenase buffer (100mM HEPES, 120mM NaCl, 50mM KCl, 1mM CaCl2, 50mM glucose, pH 7.4) for 60 min at 371C under constant shaking. The gained suspension was filtered through a 250 mm filter and centrifuged at 700 g for 7 min at room temperature. SVF cells were separated from mature adipocytes by discharging the fat layer on top. | ||||||||||||||||||||
| 77 | Lin | 2008 | Adipose tissue-derived cells | SVF | Automated. Cellution. Cytori | Human | SubQ | Collagenae | Roche Diagnostics, Minatoku, Tokyo, Japan), | Lactated Ringers | 0.08% | Lot number 1 2 3 4 5 6 Average SD system Yield (cells/ Viability Adipose (%) volume (mL) 85.0 50.0 90.0 50.0 88.0 121.0 80.6 55.0 89.7 145.0 86.0 97.0 86.6 adipose 241 800 449 500 278 471 189 000 380 586 231 701 295 176 99 540 mL) 3.5 | Adipose tissue-derived cells | Characterization | Lin K, Matsubara Y, Masuda Y, Togashi K, Ohno T, Tamura T, Toyoshima Y, Sugimachi K, Toyoda M, Marc H, Douglas A. Characterization of adipose tissue-derived cells isolated with the Celution system. Cytotherapy. 2008;10(4):417-26. doi: 10.1080/14653240801982979. PMID: 18574774. | Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228. | The whole system basically mimicked manual isolation steps modified from Zuk et al. | Subcutaneous adipose tissue used in this study was obtained from donors undergoing plastic surgery. The study was approved by the ethical committees of both the medical institution and Olympus Corporation. The cell isolation was performed by the programmed sequence set in the CelutionTM system. The digested enzyme used in this study was collagenase (Roche Diagnostics, Minatoku, Tokyo, Japan), at a concentration of 0.075%. The wash and digestion solution used was a lactated Ringer’s solution called Lactec G injection (Otsuka Pharmaceutical Factory Inc., Naruto, Tokushima). The consumables, including the tissue wash and digestion containers and cell concentration chambers, were sterile and accompanied the CelutionTM system. The final concentrated cell output collected from the CelutionTM system was counted using a NucleoCoun- ter (Chemometec, Allerod, Denmark), which detected only nucleated cells. Thus the red blood cell number was not included in the final cell calculation. | |||||||||||||||||||||||
| 78 | Madonna | 2008 | Adipose Derived Stromal Cells | Adipose Stromal Cells (ASC) | Manual | Mouse and Human | Mouse – Epididymal. Human Omental | Surgical | Mechanical | Type I Collagenase | Worthington | Centrifugation 1200 RPM 5 min | Adipose Derived Stromal Cells | In Vitro Studies | ADSCs featured the expression of early markers of endothelial differentiation [uptake of DiIlabeled acetylated LDL, CD133, CD34, kinase insert domain receptor (KDR)], but not markers for more mature endothelial cells (CD31 and von Willebrand factor). | Madonna R, De Caterina R. In vitro neovasculogenic potential of resident adipose tissue precursors. Am J Physiol Cell Physiol. 2008 Nov;295(5):C1271-80. doi: 10.1152/ajpcell.00186.2008. Epub 2008 Sep 11. PMID: 18787077. | Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228. | ADSCs formed tubes | Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg) and sodium heparin (1,000 U/kg). After anesthesia, a laparotomy was performed, periepididymal and visceral (omental) abdominal adipose tissues were harvested, and ADSCs were isolated with a modification of published methods (47). In brief, the human or murine adipose tissues were mechanically minced and digested with type I collagenase. After adipocyte removal by centrifugation at 1,200 rpm for 5 min, the vascular stromal fraction was plated at a density of 1,000 cells/cm2 in Dulbecco’s modified Eagle’s medium (DMEM)-F-12 medium supplemented with penicillin (100 U/ml), streptomycin sulfate (100 g/ml), and 10% fetal calf serum (FCS). After 24 h, nonadherent cells were removed, and subconfluent adherent, nonpassaged ADSCs were used for the exper- iments. To exclude possible contamination of ADSCs with mesothe- lial cells from the abdominal cavity, which might theoretically also form capillary-like structures in Matrigel and might participate in neoangiogenesis in vivo, the expression profile of the specific markers for mesothelial cells cytokeratin 8 and 18 was analyzed in murine and human ADSCs by flow cytometry (see Supplemental Table S1; Refs. 7, 8).1 | ||||||||||||||||||||||
| 79 | Tang | 2008 | Stromal Vascular Cells | Adipose Stromal Cells (ASC) | Manual | Mouse | inguinal, perigonadal, retroperitoneal, interscapular—the white adipose tissue juxtaposed to the interscapular brown adipose tissue | Surgical | minced into fine pieces (2-5mm2) | Collagenase | adipocyte isolation buffer (100mM HEPES pH7.4, 120mM NaCl, 50mM KCl, 5mM glucose, 1mM CaCl2, 1.5% BSA) | 1 mg/ml | 37 | 120 min | constant slow shaking (~120rpm). During the digestion period, the suspension was triturated several times through a pipet to dissociate the clumps | Digest passed through 80 micron mesh, Effluent centrifuged 500g 10 minutes. Pellet washed 1x | 80 micron mesh | Tang W, Zeve D, Suh JM, Bosnakovski D, Kyba M, Hammer RE, Tallquist MD, Graff JM. White fat progenitor cells reside in the adipose vasculature. Science. 2008 Oct 24;322(5901):583-6. doi: 10.1126/science.1156232. Epub 2008 Sep 18. PMID: 18801968; PMCID: PMC2597101. | Klaus S, Cassard-Doulcier AM, Ricquier D. Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP. J Cell Biol. 1991 Dec;115(6):1783-90. doi: 10.1083/jcb.115.6.1783. PMID: 1684582; PMCID: PMC2289199. | SV fractionation. The fractionation of SV cells and adipocytes was as described (Klaus J Cell Biol 115, 1783 (1991). ) with slight modifications. Briefly, SV cells were isolated from pooled white adipose depots (inguinal, perigonadal, retroperitoneal, interscapular—the white adipose tissue juxtaposed to the interscapular brown adipose tissue) that were explanted and minced into fine pieces (2-5mm2). The adipose pieces were then digested in adipocyte isolation buffer (100mM HEPES pH7.4, 120mM NaCl, 50mM KCl, 5mM glucose, 1mM CaCl2, 1.5% BSA) containing 1mg/ml collagenase at 37°C with constant slow shaking (~120rpm) for 2 hours. During the digestion period, the suspension was triturated several times through a pipet to dissociate the clumps. The suspension was then passed through an 80μm mesh to remove undigested clumps and debris. The effluent was centrifuged at 500xg for 10 minutes and the pellet washed once in 5ml PBS. The resultant isolated cells were subjected to FACS or plated in a 24-well tissue culture dish or on poly D-ornithine-coated coverslips placed in a 24-well culture dish. | |||||||||||||||||||||
| 80 | Tang | 2008 | Stromal Vascular Particulate | Adipose Stromal Cells (ASC) | Manual | Mouse | inguinal, perigonadal, retroperitoneal, interscapular—the white adipose tissue juxtaposed to the interscapular brown adipose tissue | Surgical | minced into fine pieces (2-5mm2) | Collagenase | adipocyte isolation buffer (100mM HEPES pH7.4, 120mM NaCl, 50mM KCl, 5mM glucose, 1mM CaCl2, 1.5% BSA) | 1 mg/ml | 37 | 120 min | constant slow shaking (~120rpm). During the digestion period, the suspension was triturated several times through a pipet to dissociate the clumps | Digest passed through 210 micron mesh and then a 30 micron mesh. The SVP Tubes were washed off the 30 micron mesh, Alternatively the SVP tubes were collected by low speed centrifugation. | 210 micron. 30 micron | Differentiation capability to adipocytes | Tang W, Zeve D, Suh JM, Bosnakovski D, Kyba M, Hammer RE, Tallquist MD, Graff JM. White fat progenitor cells reside in the adipose vasculature. Science. 2008 Oct 24;322(5901):583-6. doi: 10.1126/science.1156232. Epub 2008 Sep 18. PMID: 18801968; PMCID: PMC2597101. | Klaus S, Cassard-Doulcier AM, Ricquier D. Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP. J Cell Biol. 1991 Dec;115(6):1783-90. doi: 10.1083/jcb.115.6.1783. PMID: 1684582; PMCID: PMC2289199. | SVP isolation and culture. Intact retroperitoneal, perigonadal, interscapular and inguinal (cut into 3 pieces) white adipose depot explants were collagenase-treated (1mg/ml, 37°C, 2 hours). The mixture was passed through a 210μm mesh and then a 30μm mesh. The SVP tubes that remained on the 30μm mesh were washed off in DMEM+10%FBS. Alternatively, after passing through 210μm mesh, the SVP tubes were collected with low speed centrifugation (<250rpm, 10 min). SVP tubes were seeded in gelatinized plates or on poly D-ornithine-coated coverslips, or cultured in suspension on non-coated petri dishes. | ||||||||||||||||||||
| 81 | Traktuev | 2008 | Adipose Stromal Cells (ASC) | Stromal Vascular Fraction (SVF) | Manual | Human | SubQ | Liposuction | Collagenase Type I | Worthington | 37 | 120 min | agitation | centrifuged at 300g for 8 minutes to separate the stromal cell fraction (pellet) from adipocytes. The pellet was resuspended in DMEM/F12 containing 10% FBS (Hyclone, Logan, Utah) filtered through 250-micron Nitex filters (Sefar America Inc, Kansas City, Mo) and centrifuged at 300g for 8 minutes. | The cell pellet was treated with red cell lysis buffer (154 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA) for 10 minutes. | Adipose Stromal Cells (ASC) | FACS. Immunofluorescence In Vitro Tube Formation | Traktuev DO, Merfeld-Clauss S, Li J, Kolonin M, Arap W, Pasqualini R, Johnstone BH, March KL. A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circ Res. 2008 Jan 4;102(1):77-85. doi: | The reference provided (#36) is a website: Website: https://www.sigmaaldrich.com/catalog/product/sigma/ c9026?lang=en®ion=US; last accessed 05/14/2019 | Frozen sections of human fat tissue were simultaneously stained with rabbit anti-CD31 and mouse anti-human CD34 or with mouse anti-human CD31 and rabbit anti-CD140b antibodies. See the online data supplement, available at http://circres.ahajournals.org, for detailed procedures. | Isolation and Culture of Human ASCs Human subcutaneous adipose tissue samples (N 10), obtained from lipoaspiration/liposuction procedures were digested in collagenase type I solution (Worthington Biochemical, Lakewood, NJ) under agitation for 2 hours at 37°C and centrifuged at 300g for 8 minutes to separate the stromal cell fraction (pellet) from adipocytes. The pellet was resuspended in DMEM/F12 containing 10% FBS (Hyclone, Logan, Utah) filtered through 250- m Nitex filters (Sefar America Inc, Kansas City, Mo) and centrifuged at 300g for 8 minutes. The cell pellet was treated with red cell lysis buffer (154 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA) for 10 minutes. The final pellet was resuspended in EBM-2/5% FBS or EGM2-MV (Cambrex, Baltimore, Md). | ||||||||||||||||||||
| 82 | Bernacki | 2008 | MSC-rich dense cell fraction | Adipose Stromal Cells (ASC) | Manual | Human | SubQ and Visceral | Liposuction and Surgical | Scissors and scalpel | 50 gm | Collagenase I | Worthington | 0.08% | 15 ml conical | 37 | 30 | rotisserie-style rotator | Serum | Centrifuge 5,000 g 10 min | Centrifuge 1,200 x g 10 min | 100 micron strainer | 160 mM NH4Cl and incubate at room temperature (RT) for 10 min to lyse red blood cells | MSC-rich dense cell fraction | Isolation Method | Bernacki SH, Wall ME, Loboa EG. Isolation of human mesenchymal stem cells from bone and adipose tissue. Methods Cell Biol. 2008;86:257-78. doi: 10.1016/S0091-679X(08)00011-3. PMID: 18442651. | Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228. Lee, R. H., Kim, B., Choi, I., Kim, H., Choi, H. S., Suh, K., Bae, Y. C., and Jung, J. S. (2004). Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell. Physiol. Biochem. 14, 311–324. | 1. If sample is liposuction fluid (lipoaspirate), skip steps (1) and (2) and start at step (3). If sample is resected fat tissue, rinse with an equal volume of sterile PBS with 1 antibiotics to remove excess blood. Repeat until most of the blood has been washed oV. 2. In a sterile Petri dish, mince adipose tissue into small (4–5 mm) pieces with surgical scissors and/or scalpel. Remove any attached skin and as much connective tissue as possible. 3. If using 15-ml tubes for the preparation, dispense 4–5 ml adipose tissue into each tube together with 4 ml of 0.075% type I collagenase solution prepared as above. Minced tissue and lipoaspirate can be drawn up into a 10-ml sterile plastic pipet with the tip broken oV. The procedure can be scaled up to use larger tubes if desired. 4. Incubate adipose tissue-collagenase suspension on a rotisserie-style rotator at 37 C for 30 min. 5. After 30-min incubation, add an equal volume (4 ml or more) of hMSC complete growth medium (with serum) to the digest to neutralize the collagenase. Total volume in a 15-ml conical tube will be 12–14 ml. 6. Centrifuge at 5000 g for 10 min to pellet the MSC-rich dense cell fraction. Adipocytes and fat will be apparent as a yellow oily layer at the top of the tube. 7. Decant supernatant (the oily layer and collagenase solution). A transfer pipet or other sterile tool may be used to dislodge the oily layer. Be careful not to pour oV or disturb the cell pellet. 8. Resuspend each pellet in 2 ml of 160 mM NH4Cl and incubate at room temperature (RT) for 10 min to lyse red blood cells. Samples may be pooled for convenient handling. Add more NH4Cl if necessary to achieve lysis of most of the blood cells. Unlysed red blood cells appear refractive and ‘‘donut’’ shaped by phase microscopy. 9. Transfe r sampl es to new cen trifuge tubes and centrifuge at 1200 g for 10 min to pellet the MS C-rich dense cell fraction. 10. Remo ve su pernata nt. Resus pend pelle ted cell s in hMS C complet e grow th medium. 11. Filter cell suspension through a 100- mm ce ll stra iner. 12. Seed suspensi on into cultur e flasks contai ning the appropri ate amou nt of medium. W e have foun d that cells isol ated from 2.5 g of fat wi ll bec ome 75–80% confluent in 7–10 days if seeded in a 75-cm2 flask (e.g., a 50-g tissue sample would be processed in eight 15-ml tubes, and the resulting c ells seed ed into sixteen 75-cm 2 flasks). Thi s corresp onds to the isolatio n passage (P-ISO ) in Fig. 1. 13. Incubate flasks at 37 C in a humidi fied incubat or with 5% CO 2 for 24 h. Wash fla sks twice wi th PBS to remove nona dherent cells an d ad d fresh grow th me.dia 14. Feed cell s every 3–4 days. When cell s reach 70–80% confl uency (about 7–10 days), harvest using standar d trypsiniza tion method. Harve sted cells may be cryopres erved or seeded at 1500 cells pe r cm 2 for co ntinued expa nsion (P-0, Fig. 1). At this point, cultur es may also be establis hed for surfa ce marker characterization , and to test di Verentia tion capacity (see Sec tions V and VI be low). | ||||||||||||||
| 83 | Bunnel | 2008 | SVF | Adipose Stromal Cells | Manual | Human | SubQ | Liposuction | Mince the adipose tissue sample using two scalpels | 0.075% collagenase Type I prepared in PBS containing 2% P/S for tissue digestion | PBS containing 2% P/S for tissue digestion | 0.00 | 37 | 30 min | neutralize the collagenase Type I activity by adding 5 ml of a-MEM containing 20% heat inactivated fetal bovine serum | Pipette the sample up and down several times to further disintegrate aggregates of the adipose tissue. Upon disintegration, transfer the sample to a 50 ml tube, avoiding the solid aggregates. The SVF, containing the ASCs, is obtained by centrifuging the sample at 2000 rpm for 5 min. Take the samples out of the centrifuge and shake them vigorously to thoroughly disrupt the pellet and to mix the cells | Repeat the centrifugation step | 70 micron strainer Aspirate the supernatant and resuspend the cell pellet in a maximum of 3 ml of stromal medium (alpha- MEM, Mediatech, Herndon, VA) supplemented with 20% FBS, 1% L-glutamine (Mediatech), and 1% P/S and the cell suspension is filtered through 70 mm cell strainer | Resuspend the pellet in 1 ml of lysis buffer, incubate for 10 min on ice and wash with 20 ml of PBS/2% P/S and centrifuge at 2000 rpm for 5 min. | Bunnell BA, Flaat M, Gagliardi C, Patel B, Ripoll C. Adipose-derived stem cells: isolation, expansion and differentiation. Methods. 2008 Jun;45(2):115-20. doi: 10.1016/j.ymeth.2008.03.006. Epub 2008 May 29. PMID: 18593609; PMCID: PMC3668445. | 2. Isolation of mesenchymal stem cells from adipose tissue The initial methods to isolate cells from adipose tissue were pioneered by Rodbell and colleagues in the 1960s [20–22]. They minced rat fat pads, washed extensively to remove contaminating hematopoietic cells, incubated the tissue fragments with collagenase and centrifuged the digest, thereby separating the floating population of mature adipocytes from the pelleted stromal vascular fraction (SVF) (Fig. 1). The SVF consisted of a heterogeneous cell population, including circulating blood cells, fibroblasts, pericytes and endothelial cells as well as ‘‘pre-adipocytes” or adipocyte progenitors [20–22]. The final isolation step selected for the plastic adherent population within the SVF cells, which enriched for the ‘‘pre-adipocytes”. Subsequently, this procedure has been modified for the isolation of cells from human adipose tissue specimens [23,24,14,25,26]. Initially, fragments of human tissue were minced by hand; however, with the development of liposuction surgery, this procedure has been simplified. During tumescent liposuction, plastic surgeons infuse the subcutaneous tissues with a saline solution containing anesthetic and/or epinephrine via a cannula and then remove both the liquid and tissue under suction [27]. The procedure generates finely minced tissue fragments whose size depends on the cannula’s dimensions. Independent studies have determined that liposuction aspiration alone does not significantly alter the viability of isolated SVF cells [28–30]. | Adipose tissue is collected by needle biopsy or liposuction aspiration. The adipose sample can be kept at room temperature for no more than 24 h prior to use (Fig. 1). ASCs can be isolated from adipose tissue by first washing the tissue sample extensively with phosphate-buffered saline (PBS) containing 5% penicillin/streptomycin (P/S). Upon removal of debris, place the sample in a sterile tissue culture plate with 0.075% collagenase Type I prepared in PBS containing 2% P/S for tissue digestion. Mince the adipose tissue sample using two scalpels and pipette the sample up and down with a 25 or 50 ml pipette several times to further facilitate the digestion. Incubate the sample for 30 min at 37 C, 5% CO2, and then neutralize the collagenase Type I activity by adding 5 ml of a-MEM containing 20% heat inactivated fetal bovine serum (FBS, Atlanta Biological, Atlanta, GA), to the tissue sample. Pipette the sample up and down several times to further disintegrate aggregates of the adipose tissue. Upon disintegration, transfer the sample to a 50 ml tube, avoiding the solid aggregates. The SVF, containing the ASCs, is obtained by centrifuging the sample at 2000 rpm for 5 min. Take the samples out of the centrifuge and shake them vigorously to thoroughly disrupt the pellet and to mix the cells. This step completes the separation of the stromal cells from the primary adipocytes. Repeat the centrifugation step. After spinning, aspirate all the collagenase solution above the pellet without disturbing the cells. Resuspend the pellet in 1 ml of lysis buffer, incubate for 10 min on ice and wash with 20 ml of PBS/2% P/S and centrifuge at 2000 rpm for 5 min. Aspirate the supernatant and resuspend the cell pellet in a maximum of 3 ml of stromal medium (alpha- MEM, Mediatech, Herndon, VA) supplemented with 20% FBS, 1% L-glutamine (Mediatech), and 1% P/S and the cell suspension is filtered through 70 mm cell strainer. | |||||||||||||||||||
| 84 | Balwierz | 2008 | stromal-vascular fraction (SVF) | Adipose Stromal Cells ASC | Manual | Human | Adipose Tissue Gynecologic surgery | Surgical | cleared of remaining connective tissue and blood vessels | 5 – 10 gm | Collagenase | 200 U/mL, 3 mL/g tissue | 37 | 60 to 90 min | 700 g for 10 min at room temperature. | cell pellet was re-suspended in inoculation medium (IM, Table 1) before being filtered through a 100- μ m nylon mesh and centrifuged again (700 g , 10 min, room temperature). resulting pellet was again re-suspended in fresh medium and was filtered through a 40- μ m nylon mesh | 100 micron then 40 micron | Balwierz A, Czech U, Polus A, Filipkowski RK, Mioduszewska B, Proszynski T, Kolodziejczyk P, Skrzeczynska-Moncznik J, Dudek W, Kaczmarek L, Kulig J, Pryjma J, Dembinska-Kiec A. Human adipose tissue stromal vascular fraction cells differentiate depending on distinct types of media. Cell Prolif. 2008 Jun;41(3):441-59. doi: 10.1111/j.1365-2184.2008.00531.x. Epub 2008 Apr 14. PMID: 18422701; PMCID: PMC6496779. | Hauner H, Skurk T, Wabitsch M (2001) Cultures of human adipose precursor cells. Methods Mol. Biol. 155, 239–247. | Isolation of stromal-vascular fraction progenitor cells from human adipose tissue The primary progenitor stromal-vascular fraction (SVF) cells were isolated from human adipose tissue according to a modified Hauner’s method (Hauner et al . 2001). Extraneous subcutaneous specimens (5–10 g) were obtained from female patients (age 35–55 years) undergoing gynecological surgery. These were cleared of remaining connective tissue and blood vessels, then collagenase digestion was performed (solution: 200 U/mL, 3 mL/g tissue; approx. 60–90 min, shaking, at 37°C). Samples were centrifuged at 700g for 10 min at room temperature. The supernatant was discarded and the cell pellet was re-suspended in inoculation medium (IM, Table 1) before being filtered through a 100-μm nylon mesh and centrifuged again (700 g, 10 min, room temperature). The resulting pellet was again re-suspended in fresh medium and was filtered through a 40-μm nylon mesh; cells were collected and counted before being seeded in inoculation medium (IM) and incubated for 24 h in a humidified CO2 incubator (Juan) at 37°C. | A. Balwierz, Department Clinical Biochemistry Collegium Medicum, Jagiellonian University, Kopernika 15a, 31-501 Krakow, Poland. Tel.: + 48 12 421 40 06; Fax: + 48 12 421 40 73; E-mail: abalwierz@cm-uj.krakow.pl | ||||||||||||||||||||
| 85 | Rodeheffer | 2008 | Stromal Vascular Cells | Insufficient data to Classify | Mouse | Identification of White Adipocyte Progenitor Cells In Vivo | Rodeheffer MS, Birsoy K, Friedman JM. Identification of white adipocyte progenitor cells in vivo. Cell. 2008 Oct 17;135(2):240-9. doi: 10.1016/j.cell.2008.09.036. Epub 2008 Oct 2. PMID: 18835024. | Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucos-e metabolism and lipolysis. J. Biol. Chem. 239: 375-380. | Subcutaneous and parametrial adipose tissue was excised and the SVF isolated as described (Rodbell, 1964). The isolated SVF was resuspended in ice-cold Dulbecco’s modified Eagle’s medium (DMEM) with 2% fetal bovine serum (FBS) for labeling. | Correspondence: friedj@rockefeller.edu | |||||||||||||||||||||||||||||||
| 86 | Eto/Yoshimura | 2009 | Stromal Vascular Fraction | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | Collagenase | Wako Pure Chemical Industries | PBS | 0.07% | 37 | 30 | Shaker | Centrifugation 800 x g 10 min. 100 micron filter | Whole mount morphology. FACS | SVF | Characterization | Zhu R, Feng Y, Li R, Wei K, Ma Y, Liu Q, Shi D, Huang J. Isolation methods, proliferation, and adipogenic differentiation of adipose-derived stem cells from different fat depots in bovines. Mol Cell Biochem. 2024 Mar;479(3):643-652. doi: 10.1007/s11010-023-04753-9. Epub 2023 May 6. PMID: 37148505. | Yoshimura K, Shigeura T, Matsumoto D, et al. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol. 2006;208:64–76. | Evaluated Fat samples prior to SVF isolation | Stromal vascular fraction was isolated from the fatty portion of liposuction aspirates as described previously.12 Briefly, the aspirated fat tissue was washed with phosphate-buffered saline and digested on a shaker at 37°C in phosphate-buffered saline containing 0.075% collagenase (Wako Pure Chemical Industries, Osaka, Japan) for 30 minutes. Mature adipocytes and connective tissue were separated from pellets by centrifugation at 800 g for 10 minutes. Stromal vascular fraction was obtained through resuspending the pellets and filtering the resuspension with 100- mmesh (Millipore, Billerica, Mass.). The stromal vascular fraction cells were plated (30,000 cells/cm2) on gelatin- coated dishes and cultured at 37°C. | Kotaro Yoshimura, M.D. Department of Plastic Surgery University of Tokyo Graduate School of Medicine 7-3-1, Hongo, Bunkyo-ku Tokyo 113-8655, Japan yoshimura-pla@h.u-tokyo.ac.jp | |||||||||||||||||
| 87 | Faustini | 2010 | Stromal Vascular Fraction. SVF | Adipose Stromal Cells (ASC) | Manual | Human | SubQ and abdominoplasty | Liposuction and Surgery | Two collagenase from two different NONEXPANDED ADIPOSE MESENCHYMAL CELLS IN REGENERATIVE THERAPY 1517 suppliers were employed (Serva or Sigma-Aldrich), and different collagenase concentration (ranging from 0.05% to 0.20%) were employed for different incubation times (rang- ing from 1 to 12 h). The different digestion protocols were (a) 0.05% collagenase, incubation 12h; (b) 0.075% collagenase, incubation 12 h; (c) 0.10% collagenase, incubation 2 h; (d) 0.15% collagenase, incubation 1 h; and (e) 0.20% collagenase, incubation 1h. | Serva and Sigma | PBS | 0.05 to 0.2 % | 37 | 60 to 720 | Centrifugation | 2 x PBS | 1.3 to 2.6 x 10E6 Cells – Not clear whether per gm fat. | 67 to 82 % | SVF | Yield and Viability | Faustini M, Bucco M, Chlapanidas T, Lucconi G, Marazzi M, Tosca MC, Gaetani P, Klinger M, Villani S, Ferretti VV, Vigo D, Torre ML. Nonexpanded mesenchymal stem cells for regenerative medicine: yield in stromal vascular fraction from adipose tissues. Tissue Eng Part C Methods. 2010 Dec;16(6):1515-21. doi: 10.1089/ten.TEC.2010.0214. Epub 2010 Sep 6. PMID: 20486782. | Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228. | In summary, in males the best collection site in terms of yield is located in the abdomen, whereas in females the bi- opsy region do not influence cell recovery; the collection technique, the age, and the BMI of donor seem not to influ- ence the cell yield. The tissue-processing procedures strongly modify the yield and the vitality of cells: a collagenase con- centration of 0.2% and a digestion time of 1 h could be cho- sen as the best operating conditions. | In the operating room adipose samples were suspended in phosphate-buffered saline (PBS) with penicillin/streptomycin 1%, put into a sterile box, and forwarded to a GMP lab- oratory at the temperature of 48C. Adipose tissue samples were digested using collagenase in PBS without calcium/ magnesium plus penicillin 100 IU/mL and streptomycin 100mg/mL at 378C. Two collagenase from two differentsuppliers were employed (Serva or Sigma-Aldrich), and different collagenase concentration (ranging from 0.05% to 0.20%) were employed for different incubation times (rang- ing from 1 to 12 h). The different digestion protocols were (a) 0.05% collagenase, incubation 12h; (b) 0.075% collagenase, incubation 12 h; (c) 0.10% collagenase, incubation 2 h; (d) 0.15% collagenase, incubation 1 h; and (e) 0.20% collagenase, incubation 1h. Mature adipocytes and connective tissues were separated from SVF by centrifugation as reported by Zuk et al.48 After centrifugation, cells were washed twice with PBS to obtain clean pellets. Cellular yield was calculated as the number of stromal cells obtained per milliliter of bioptical tissue; counts were performed after staining with Trypan Blue in a Bu ̈rker chamber. In each sample the vitality and vital cell yield (live cells/mL treated tissue) were calculated. All samples were counted in triplicate. | Massimo Faustini, Ph.D. Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza Alimentare Universita` di Milano Via Celoria 10 Milan 20133 Italy E-mail: massimo.faustini@unimi.it | ||||||||||||||||
| 88 | Estes/Gimble | 2010 | Adipose Stem Cells ASC | SVF | Manual | Human | Type I collagenase | Worthington Biochemical Products, cat. No. LS004197 | Dulbecco’s phosphate-buffered saline (D-PBS), without calcium chloride and magnesium chloride (Gibco, cat. no. 14190-144) | 0.1% (w/v) collagenase, 1 % (v/v) bovine serum albumin (fraction V), and 2 mM calcium chloride in PBS. The volume of the collagenase solution should equal the volume of lipoaspirate to be processed (usually 100 to 200 ml) | 250 ml Centrifuge Tube | 37 | Place it in the 37° C shaker water bath and gently shake for 1 hour. Shake to mix by hand every 20 minutes if using 250 ml centrifuge bottles; or shake to mix by hand every 5–10 minutes if using 50 ml centrifuge tubes. | 300 g at 21° C for 5 minutes. 4x | 4X | ASC | Estes BT, Diekman BO, Gimble JM, Guilak F. Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc. 2010 Jul;5(7):1294-311. doi: 10.1038/nprot.2010.81. Epub 2010 Jun 17. PMID: 20595958; PMCID: PMC3219531. | ASC Isolation 1 Prepare the water bath shaker at 37 °C, with enough water to cover the bottles containing collagenase mixture. 2 Prepare fresh a solution of 0.1% (w/v) collagenase, 1 % (v/v) bovine serum albumin (fraction V), and 2 mM calcium chloride in PBS. The volume of the collagenase solution should equal the volume of lipoaspirate to be processed (usually 100 to 200 ml). 3 Warm the D-PBS, collagenase solution, and medium to 37 °C. 4 Allow the fat transport container to sit until the fat and blood are well separated. Transfer the fat to a sterile, 250 ml plastic centrifuge tube for separation. Note: cell isolations are routinely performed the day of receipt or performed the next day, after leaving the tissue at room temperature overnight. CAUTION: Procedures must be performed in accordance with Institutional Review Board policies for obtaining human tissue including informed consent by personnel certified and trained to work with blood borne pathogens. All procedures involving the human tissue should be performed at Biosafety Level 2 with appropriate personal protection. 5 Using a 25 ml serological pipette, aspirate off the blood layer from beneath the floating fat. 6 Check the tissue volume; it should be approximately 100 ml in each 250 ml centrifuge tube. The isolated cells will be plated at an equivalent to ~35 ml of liposuction aspirate digest per T225 flask so plan the amount of tissue needed accordingly. If there is less tissue volume, the flask size should be reduced accordingly to achieve approximately 0.16 ml tissue per square centimeter. 7 Wash the fat with D-PBS (1:1 volume) up to 7 times or until the color of the D- PBS layer is about the hue of the fat – a light pink color. Allow the two phases to separate between washes (3–5 min). Gently stir the solution while it sits; this will encourage separation and aid the washing. Continue to remove the blood layer from beneath the yellow fat tissue. After a few washes, a bright yellow layer of extra cellular fat will begin to form on top of the fat cell layer. Carefully aspirate off this thin layer. Note that 4 washes should be sufficient. After the fat layer has been thoroughly washed, mix it 1:1 (v:v) with the warm D-PBS:collagenase solution. Place it in the 37° C shaker water bath and gently shake for 1 hour. Shake to mix by hand every 20 minutes if using 250 ml centrifuge bottles; or shake to mix by hand every 5–10 minutes if using 50 ml centrifuge tubes. Note that if a shaker water bath is not available, 50 ml or 250 ml tubes may be placed on a shaker in a 37° C incubator. TROUBLESHOOTING Centrifuge at 300 g at 21° C for 5 minutes. Shake the tubes vigorously for 10 seconds to ensure that individual cells are released from the strands of fibrous tissue. Centrifuge at 300 g at 21° C for 5 minutes. Aspirate off the floating mature adipocyte layer and the aqueous collagenase/D- PBS supernatants, leaving 5 ml of D-PBS on each pellet of cells. Be careful with the cell pellet as it is gelatinous and does not adhere readily to the centrifuge tubes. TROUBLESHOOTING Resuspend each cell pellet in 10 ml stromal medium and pool the suspensions into sterile, 50 ml centrifuge tubes. Centrifuge at 300 g at 21° C for 5 minutes. Aspirate off the supernatants, leaving 5 ml of stromal medium on each pellet. Resuspend cell pellets in 10 ml stromal medium and pool contents into a sterile, 50 ml centrifuge tubes. Centrifuge at 300 g at 21° C for 5 minutes. Aspirate off the supernatants, leaving 5 ml of stromal medium on each pellet. Note that the pellet can look very different from patient to patient at this stage. Resuspend in a total of 105 ml stromal media per 100 ml of lipoaspirate digest. | |||||||||||||||||||||||
| 89 | Harris | 2010 | Stromal Vascular Pellet (SV) | SVF | Manual | Human | SubQ | Liposuction | 14 ± 0.8 gm | Collagenase I | Worthington | 1 mg/ml | 37 | 60 min | Gentle Agitation | Centrifugation 1,500 xg 10 min | 1 x in 0.1 % BSA | Coulter Z-Series Counter | 2.87±0.34 × 105 total cells per gm of fat | Harris LJ, Zhang P, Abdollahi H, Tarola NA, DiMatteo C, McIlhenny SE, Tulenko TN, DiMuzio PJ. Availability of adipose-derived stem cells in patients undergoing vascular surgical procedures. J Surg Res. 2010 Oct;163(2):e105-12. doi: 10.1016/j.jss.2010.04.025. Epub 2010 May 10. PMID: 20638677; PMCID: PMC2942956. | Isolation of ASC from human subcutaneous fat Human adipose tissue samples were obtained from patients undergoing various elective vascular procedures at Thomas Jefferson University Hospital (Philadelphia, PA). Under sterile conditions, approximately 60 cc of tumescent solution (30mL 1% lidocaine, 30mL 0.5% bupivicaine, 10mL 4.2% sodium bicarbonate, 1mg epinephrine in 1 liter injectable normal saline) was infiltrated into the peri-umbilical region. A Mercedes 3mm × 9cm Aspiration Luer Lock Cannula (Byron Medical, Tucson, AZ) was used to withdraw the liposuction aspirate. The specimen was immediately placed on ice for transportation to the laboratory. The lipoaspirate was washed with PBS to remove all anesthetic and blood contamination followed by filtration through a 250 μm sieve. The adipose tissue was digested with Collagenase I (1mg/ ml; Worthington Biochemical Corp., Lakewood, NJ) for 1h at 37°C with gentle agitation. The slurry was centrifuged for 10 min at 1500 × g and the supernatant containing the mature adipocytes was discarded. The resulting stromal vascular (SV) pellet was washed with 0.1% BSA (Sigma-Aldrich, St. Louis, MO) and suspended in storage media (M-199 (Mediatech, Herndon, VA), fetal bovine serum (12.8%, Gemini BioProducts, Herndon, VA,) HEPES (1M, Fisher Biotech, Fair Lawn, NJ), heparin (Elkins-Sinn, Inc, Cherry Hill, NJ), antibioticantimycotic solution (100x solution; 10,000 U/ml Penicillin G, 25 μg/ml Amphotericin B, 10,000 μg/ml Streptomycin, Mediatech, Herndon, VA)), counted (Coulter Z-Series Counter), and plated onto gelatinized tissue culture flasks and incubated (37°C, 5% CO2). The flasks were washed at 24 hours to remove all non-adherent cells. | Paul J. DiMuzio, MD, 111 South 11th Street, Suite G6270, Philadelphia, PA 19107, 215-955-8304 (phone), 215-923-0835 (fax), paul.dimuzio@jefferson.edu. | |||||||||||||||||||
| 90 | Zimmerlin | 2010 | SVF | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Surgical abdominoplasty | Scissors | Collagenase Type II | Worthington | Hanks’ Balanced Salt Solution (HBSS, Invitrogen, Carlsbad, CA) containing 3.5% Bovine Serum Albumin (BSA, Millipore, Charlottesville, VA) | 1 mg/ml | 37 | 30 min | Shaking Water Bath | Successive 425 lm and 180 lm sieves (W.S. Tyler, Mentor, OH). Centrifugation 400 g. 10 min. RBC lysis. PBS wash, Ficoll- Hypaque | successive 425 lm and 180 lm sieves (W.S. Tyler, Mentor, OH). 100 mesh SS screen before Flow Cytometry | erythrocyte lysis buffer (Beckman Coulter, Miami, FL, Cat No. IM3630d), | endothelial and perivascular markers including CD31, CD34, CD90, CD146, and a-SMA | SVF | endothelial and perivascular markers including CD31, CD34, CD90, CD146, and a-SMA | Zimmerlin L, Donnenberg VS, Pfeifer ME, Meyer EM, Péault B, Rubin JP, Donnenberg AD. Stromal vascular progenitors in adult human adipose tissue. Cytometry A. 2010 Jan;77(1):22-30. doi: 10.1002/cyto.a.20813. Erratum in: Cytometry A. 2010 Apr;77(4):406. PMID: 19852056; PMCID: PMC4148047. | Fat tissue was thoroughly minced with scissors, digested for 30 min in Hanks’ Balanced Salt Solution (HBSS, Invitrogen, Carlsbad, CA) containing 3.5% Bovine Serum Albumin (BSA, Millipore, Charlottesville, VA) and 1 mg/mL collagenase type II (Worthington, Lakewood, NJ) on a shaking water bath at 378C, and finally disaggregated through successive 425 lm and 180 lm sieves (W.S. Tyler, Mentor, OH). Mature adipocytes were eliminated by centrifugation (400g, ambient temperature, 10 min) and cell pellets were resuspended in NH4Clbased erythrocyte lysis buffer (Beckman Coulter, Miami, FL, Cat No. IM3630d), incubated for 10 min at room temperature (RT) and washed in Phosphate Buffer Saline (PBS). Viable cell enrichment and debris depletion was achieved on a Ficoll- Hypaque density gradient (Histopaque1-1077, Sigma- Aldrich, St. Louis, MO). | Hillman Cancer Center Research Pavilion Suite2.42c, 5117 Center Avenue, Pittsburgh, PA 15213-2582, USA. donnenbergad@upmc.edu Phone : 412-623-7780 Fax: 412-623-7778 | ||||||||||||||||
| 91 | Li | 2010 | Stromal Vascular Pellet (SV) | Adipose Stromal Cells (ASC) | Manual | Mouse | Epididymal Fat | Surgical | minced | collagenase . | Sigma | 1 mg/ml | 37 | 30 | Shaking | Heterogenity of Macrophages | Li P, Lu M, Nguyen MTA, Bae EJ, Chapman J, Feng D, Hawkins M, Pessin JE, Sears DD, Nguyen AK, Amidi A, Watkins SM, Nguyen U, Olefsky JM. Functional heterogeneity of CD11c-positive adipose tissue macrophages in diet-induced obese mice. J Biol Chem. 2010 May 14;285(20):15333-15345. doi:10.1074/jbc.M110.100263. Epub 2010 Mar 22. PMID: 20308074; PMCID: PMC2865288. | Stromal Vascular Cell (SVC) Isolation and FACS Analysis— Epididymal fat pads were weighed, rinsed 3 times in phosphatebuffered saline (PBS), and then minced in FACS buffer (PBS 1% low endotoxin bovine serum albumin). Tissue suspensions were centrifuged at 500 g for 5 min and then collagenasetreated (1 mg/ml, Sigma) for 30 min at 37 °C with shaking. Cell suspensions were filtered through a 100- micron filter and centrifuged at 500 g for 5 min. SVC pellets were then incubated with RBC lysis buffer (eBioscience) for 5 min before centrifugation (300 x g for 5 min) and resuspended in FACS buffer. | |||||||||||||||||||||||
| 92 | Almeida | 2010 | Stromal Vascular Fraction SVF – Then become ADSC spontaneously in methods section | Adipose Stromal Cells (ASC) | Manual | Rabbit | Inguinal | Surgical | Collagenase type Ia | Sigma | 0.08% | 37 | 30 | 100 micron strain then centrifuged | Each inguinal fat pad weighted 11–13 g and provided 0.5– 1×103 cells. 1×102/gm fat | Almeida FG, Nobre YT, Leite KR, Bruschini H. Autologous transplantation of adult adipose derived stem cells into rabbit urethral wall. Int Urogynecol J. 2010 Jun;21(6):743-8. doi: 10.1007/s00192-009-1090-8. Epub 2010 Feb 12. PMID: 20151112. | “Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228.” | The ADSCs were isolated as previously described [11, 12]. Briefly, the fat was washed extensively with phosphate buffer saline (PBS), minced, and enzymatically digested at 37°C for approximately 30 min using 0.075% collagenase type IA (Sigma, St. Louis, MO). The digested tissue was filtered using 100 μm strain to obtain a cell suspension containing the stromal vascular fraction. After centrifugation, the pellet was resuspended in culture media (CM) consisting of Dulbecco’s modified Eagle’s media (DMEM; Mediatech, Herndon, VA), 10% fetal bovine serum (FBS; HyClone, Logan, UT) and 1% of a solution of antibiotic/ antimycotic (penicilin G 10,000 U/ml, amphotericin B 25 μg/ml and streptomycin 10,000 μg/ml). Freshly harvested harvested ADSCs were analyzed regarding viability, counted, and plated in 100-mm2 culture dishes at a concentration of 1×105. Cells were allowed to adhere to the flask for 24 h, after which fresh media was added. The cells were incubated at 37°C and 5% CO2 in CM until they reached semi-confluence. Cellular confluence was avoided to prevent potential spontaneous differentiation. Culture media was changed every 2–3 days. Cells were rinsed with PBS and incubated with 1:200 dilution of dialkylcarbocyanine solution, a fluorescent cell membrane marker, (Vybrant DiI; Molecular Probes, Eugene, OR,) for 20 min at 37°C in accordance with the manufacturer’s protocol. The labeled cells were harvested with 0.25% trypsin/1 mM EDTA solution. To perform the autologous transplantation, cells were suspended to a concentration of 1×107 labeled cells in 30 μl of Hank’s balance salt solution (HBSS). | F. G. Almeida (*) R: Pedro de Toledo, 1222 ap: 22, Sao Paulo, Sao Paulo, Brazil 04039-003 e-mail: fernandourologia@hotmail.com | ||||||||||||||||||||||
| 93 | Basu | 2011 | Stromal Vascular Fraction SVF | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction and Surgery | Collagenase I | Worthington | Dulbecco’s modified Eagle’s medium–high glucose (DMEM-HG) | 0.1%% | 37 | 60 | Centrifugation 300g 5 min. Resuspended in PBS Filtered through 100 micron Steriflip vacuum filter. Centrifuged again 300g 5 min | 1x PBS | 100 micron Steriflip | PCR. FACS (CD31, CD73, CD90, CD105, CD117, or CD133 (BD Biosciences) Immunofluorescence Proteomics | SVF | Smooth Muscle Cells for Bladder | Basu J, Genheimer CW, Guthrie KI, Sangha N, Quinlan SF, Bruce AT, Reavis B, Halberstadt C, Ilagan RM, Ludlow JW. Expansion of the human adipose-derived stromal vascular cell fraction yields a population of smooth muscle-like cells with markedly distinct phenotypic and functional properties relative to mesenchymal stem cells. Tissue Eng Part C Methods. 2011 Aug;17(8):843-60. doi: 10.1089/ten.tec.2010.0697. Epub 2011 May 19. PMID: 21595545. | we believe that taken together, the published data as well as our observations are consistent with the perivascular niche of adipose SVF as a source for a broad continuum of SMCs, smooth muscle progenitors, MSC, and partially lineage-committed MSC-like cell types with variable and overlapping degrees of proliferative and differentiation potential. | Human adipose tissue was commercially obtained (Zen- Bio) from subcutaneous harvest or lipoaspiration. Adipose was digested with 0.1% collagenase I (Worthington Biochemical) and 1% bovine serum albumin (Sigma) in Dulbecco’s modified Eagle’s medium–high glucose (DMEM-HG) (Invitrogen) at 37 C for 1 h, and then centrifuged for 5 min at 300g in 50mL conical tubes. The stromal vascular fraction (SVF) pellet was resuspended in phosphate-buffered saline (PBS)/1% bovine serum albumin and filtered through a 100 mm Steriflip vacuum filter. Cells were pelleted again at 300g for 5 min and resuspended in culture media. | Joydeep Basu, Ph.D. Bioprocess Research and Assay Development Tengion Inc. 3929 Westpoint Blvd, Suite G Winston-Salem, NC 27103 E-mail: joydeep.basu@tengion.com | |||||||||||||||||
| 94 | Cheng | 2011 | SVF | SVF | Manual | Human | Biopsy | Biopsy | using a sterile scalpel and forceps, we cut the biopsy into small pieces | collagenase A | 37 | 30 min | The tube was shaken vigorously every 5 minutes until dissociation of the tissue pieces occurred | The collage-nase digestion reaction was stopped by adding 10 mL DMEM þ 10% fetal calf serum medium. | 800 g for 10 minutes at room temperature | SVF | Culture | Kuang-Hung Cheng a,*, Tzu-Lei Kuo a, Kung-Kai Kuo b, Chang-Chun Hsiao Human adipose-derived stem cells: Isolation, characterization and current application in regeneration medicine Genomic Medicine, Biomarkers, and Health Sciences (2011) 3,53e62 | Review | The protocols below describe in detail how we isolated the ADSCs in our laboratory (Fig. 2). In brief, immediately after the biopsy, adipose tissue (0.1e2 mg) was transported from the hospital to the laboratory in 15 mL ice-cold growth medium (K-NAC medium with 10% fetal calf serum). In the laboratory, using a sterile scalpel and forceps, we cut the biopsy into small pieces and maintained these in a digestion buffer in a culture dish under a laminar flow hood. Tissue without fat (white, soft and easy to shred) was discarded (some biopsies contained a majority of nonadipose tissue). Small pieces of tissue were then digested with 5 mL collagenase A solution. Collagenase digestion was per-formed at 37 C in a water bath. The tube was shaken vigorously every 5 minutes until dissociation of the tissue pieces occurred. This took between 20 and 30 minutes depending on the size of the adipose tissue fragments. Collagenase digestion was stopped after 30 minutes even if a few fragments were not totally dissolved. The collage-nase digestion reaction was stopped by adding 10 mL DMEM þ 10% fetal calf serum medium. The crude SVF was separated from the adipocyte frac-tion by centrifugation at 800 g for 10 minutes at room temperature. The fat and oil fractions (the upper fraction) were discarded, and cells from the SVF were seeded onto one (or two if starting with 2 g of tissue) 100-mm culture dish in 10 mL growth medium. | Corresponding author. Institute of Biomedical Sciences, National Sun Yat-Sen University, 70 Lien-hai Road, Kaohsiung 80424, Taiwan. E-mail address: khcheng@faculty.nsysu.edu.tw (K.-H. Cheng). | ||||||||||||||||||||
| 95 | Rhee | 2011 | Stromal Vascular Fraction SVF | SVF | Manual | Rat | Inguinal | Surgical | Collagenase Type I | Sigma | 0.08% | 37 | 60 | Constant Shaking Water Bath | Centrifugation 300 – 500 g 5 min. Supernatant containing adipocytes resuspended and centrifuged 2000 RPM for 10 min. Pellet of SVF resuspended and injected in rats | 1x | SVF | Transplat – Bone Formation | FACS analyses were performed on the SVFs of rat adipose tissues using antibodies against CD4 (FITC), CD25 (PE), CD45 (FITC), CD73 (FITC), CD90 (FITC), and CD11B (FITC). FACS analysis demonstrates that cells expressing the stem cell markers CD4, CD25, CD45, CD73, CD90, and CD11B are present within the SVF of rat adipose tissues. | “STEM CELLS AND DEVELOPMENT Volume 20, Number 2, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089=scd.2009.0525″ “In Vivo Evaluation of Mixtures of Uncultured Freshly Isolated Adipose-Derived Stem Cells and Demineralized Bone Matrix for Bone Regeneration in a Rat Critically Sized Calvarial Defect Model Seung Chul Rhee,1 Yi-hwa Ji,2 Nareg A. Gharibjanian,3 Eun Sang Dhong,4 Seung Ha Park,5 and Eul-Sik Yoon2,4″ | Fat pooled so allogeneic transplant Cells rejected? Methods indicate SVF isolated from floating adipocytes ??? | Each rat was premedicated with an intramuscular injection of Ketalar (Ketamine HCL; Yuhan Corporation; 50mg=mL, 0.2mL=100 g body weight). After an adequate level of sedation had been achieved, the inguinal area was prepped and draped using a sterile technique. Topical anesthesia was performed using 2% lidocaine (lidocaine HCL 20 mL; Huons Co. Ltd.). A 2–3-cm-long incision was placed and fat was harvested from 20 rats. Acquired fat was collected altogether, washed 3 times with sterile phosphatebuffered saline, digested with 0.075% type I collagenase (Sigma), and prewarmed to 378C. The digested fat was placed in a constant shaking water bath for 60 min (165 rpm, 35 min) and centrifuged for 5 min at 300–500 g at room temperature. The supernatant, which contained mature adipocytes, was aspirated and resuspended in an equal volume of high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Sigma Chemical Co.). This solution was then placed in a 50-mL tube and centrifuged for 10 min at 2,000 rpm. The supernatant was removed and SVF was acquired and prepared for injection at 1 105 cells=g of adipose-derived stem cells per syringe. Cell number was determined by Accuri C6 Flow Cytometer (Accur Cytometers; www.accuricytometers.com=) FACS analysis was performed with freshly harvested SVF from rats. | Dr. Eul-Sik Yoon Department of Plastic and Reconstructive Surgery Korea University Ansan Hospital 516, Gojan-Dong, Danwon-Ku, Ansan-Si Gyeonggi-do 425-707 Republic of Korea E-mail: yesanam2@korea.ac.kr | ||||||||||||||||||
| 96 | Eom | 2011 | Adipose Tissue Derived Stem Cells | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | 10g | Collagenase type IA | Sigma | PBS | 0.08% | 37 | 40 | 1,200 g for 5 minutes, and the pellet was resuspended and passed through a 100-μm mesh filter (Cell Strainer, Becton Dickinson, Franklin Lakes, NJ, USA) | 100 micron | 3.06×106 cells/g at three hours post-storage to 0.4×106 cells/g at 36 hours post-storage | Eom YW, Lee JE, Yang MS, Jang IK, Kim HE, Lee DH, Kim YJ, Park WJ, Kong JH, Shim KY, Lee JI, Kim HS. Rapid isolation of adipose tissue-derived stem cells by the storage of lipoaspirates. Yonsei Med J. 2011 Nov;52(6):999-1007. doi: 10.3349/ymj.2011.52.6.999. PMID: 22028166; PMCID: PMC3220256. | Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279-95. | Isolation of adipose tissue-derived stem cells Human adipose tissue was obtained from three healthy female donors with a mean age of 30.7±7.8 and body mass index of 23.7±3.7 at Park’s Cosmetic and Plastic Surgery (Seoul, Korea). The women underwent elective liposuction procedures under anesthesia according to procedures approved by the Institutional Review Board of Ajou University Hospital. Informed consent was obtained from all donors. Mononuclear cells were isolated using a modified protocol described by Zuk, et al.21 In brief, lipoaspirates were extensively washed with PBS to remove contaminating blood cells and local anesthetics. Then, aliquots (10 g) of the washed lipoaspirates were preserved at 4°C without supplying oxygen or nutrients. At the indicated time points, an aliquot was enzymatically digested at 37°C for 40 minutes with 0.075% type IA collagenase (Sigma, St. Louis, MO, USA) in PBS. The red blood cell lysis step was omitted to reduce the isolation time because omission of this step caused no difference in yield of mononuclear cells and isolation efficiency of mesenchymal stem cells. The digested lipoaspirates was centrifuged at 1,200 g for 5 minutes, and the pellet was resuspended and passed through a 100-μm mesh filter (Cell Strainer, Becton Dickinson, Franklin Lakes, NJ, USA) to remove debris. Cells were plated in 100-mm culture dishes at a density of 5×106 mononuclear cells with low glucose Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin G and 100 μg/mL streptomycin). | ||||||||||||||||||||
| 97 | Yang | 2011 | Adipose Derived Stem Cells / ADSCs: adipose-derived mesenchymal stem cells | Adipose Stromal Cells (ASC) | Manual | Human | Abdominal SubQ | Surgical | cut into 1 mm3 pieces | ~ 5 gm | Collagenase Type I | Invitrogen | 0.10% | 37 | 60 | Shaken Vigorously | Low glucose- Dulbecco’s modified Eagle’s medium (L-DMEM, Hyclone, USA) containing 10% fetal bovine serum (FBS, Invitrogen, USA) | Digest Filtered. Centrifugation 1500 rpm 10 min | 2x | Contaminating erythrocytes were lysed with an osmotic buffer | One gram of adipose tissue could give yield up to 1 × 106 hADSCs | Yang XF, He X, He J, Zhang LH, Su XJ, Dong ZY, Xu YJ, Li Y, Li YL. High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells. J Biomed Sci. 2011 Aug 19;18(1):59. doi: 10.1186/1423-0127-18-59. PMID: 21854621; PMCID: PMC3175156. | Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279-95. | Isolation of hADSCs and Cell Culture The procedure followed the description by Zuk et al. [14] with some modifications. The adipose tissue sample was extensively washed with sterile PBS containing 1000 U/ml penicillin and 1000 μg/ml streptomycin to remove contaminating blood cells. The specimen was then cut carefully. Connective tissue and blood vessels were removed and the tissue was cut into 1 mm3 pieces. The extracellular matrix was digested with 0.1% collagenase Type I (Invitrogen, USA) at 37°C, and shaken vigorously for 60 min to separate the stromal cells from primary adipocytes. The collagenase Type I activity was then neutralized by adding an equal volume of Low glucose- Dulbecco’s modified Eagle’s medium (L-DMEM, Hyclone, USA) containing 10% fetal bovine serum (FBS, Invitrogen, USA). Dissociated tissue was filtered to remove debris, and centrifuged at 1500 rpm for 10 min. The suspending portion containing lipid droplets was discarded and the cell pellet was resuspended and washed twice. Contaminating erythrocytes were lysed with an osmotic buffer, and the remaining cells were plated onto 6-well plate at a density of 1 × 106/ml. Plating and expansion medium consisted of L-DMEM with 10% FBS, 100 U/ml penicillin, and 100 mg/L streptomycin. Cultures were maintained at 37°C with 5% CO2. The medium was replaced after 48 hours, and then every 3 days. Once the adherent cells were more than 80% confluent, they were detached with 0.25% trypsin- 0.02% EDTA, and re-plated at a dilution of 1:3. | hexu@jlu.edu.cn; ylli@jlu.edu.cn | ||||||||||||||||
| 98 | Sujii | 2011 | Adipose Stem Cells hADS | Adipose Stromal Cells (ASC) | Manual | Mouse and Human | SuQ and Visceral | Human – Liposuction and Surgical | Scissors | Type I collagenase | (Worthington Biochemical, cat. no. LS004196 | Immediately prior to the digestion, prepare a fresh solution of 2.5 μg/ml collagenase (wt/vol), 1% BSA, 50 μg/ml D-glucose and 200 nM adenosine in HBSS. Filter sterilize. | autoclaved scintillation vials | 37 | 30 to 60 | Water bath shaker | filter the digested solution through a 250 μm nylon filter. Then filter through a 100 μm cell strainer and transfer the filtrate into 15 ml or 50 ml centrifuge tubes, depending on the volume. Centrifuge at 400g for 5 min at room temperature. | 3 washes | Sugii S, Kida Y, Berggren WT, Evans RM. Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells. Nat Protoc. 2011 Mar;6(3):346-58. doi: 10.1038/nprot.2010.199. Epub 2011 Feb 24. PMID: 21372815; PMCID: PMC3089977. | Estes BT, Diekman BO, Gimble JM, Guilak F. Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc. 2010 Jul;5(7):1294-311. doi: 10.1038/nprot.2010.81. Epub 2010 Jun 17. PMID: 20595958; PMCID: PMC3219531. | Primary adherence to remove monocytes macrophages. Culture non-adherent cells. | 1. Isolation of mouse ADS (mADS) cells 5 – 7 hours i. Sacrifice each mouse (C57BL/6J or Oct4-EGFP mice, 8–10 weeks old) by carbon dioxide asphyxiation. Wipe with 70% ethanol and open abdominal area. Using sterilized forceps and scissors, dissect perigonadal (epididymal in male and parametrial in female) and/or subcutaneous inguinal fat pads and place into 6 cm petri dishes containing HBSS. We advise proceeding immediately to digestion of fat tissue on the day of harvest. Chilling or freezing the tissue for storage purposes makes subsequent collagenase digestion more difficult. ii. Add an equal volume of collagenase solution (to weight of fat pads) into autoclaved scintillation vials and warm to 37°C. iii. Rinse fat pads in HBSS, dry with Kimwipes and place into collagenase solution. iv. Finely mince the tissue with a sterilized scissor. v. Shake vials (~100 rpm) in a 37°C water bath shaker for 30 to 60 min. Check digests every 10 min and stop reaction when complete. The digestion efficiency varies with speed and type of shakers and batch of collagenase used. You should see a white fat layer separated and, when the vials settle, it floats on top of the solution. vi. Transfer and filter the digested solution through a 250 μm nylon filter. Then filter through a 100 μm cell strainer and transfer the filtrate into 15 ml or 50 ml centrifuge tubes, depending on the volume. Centrifuge at 400g for 5 min at room temperature. vii. Carefully aspirate the floating layer containing mature adipocytes and aqueous supernatants, leaving the pellet. This pellet is the stromal vascular fraction (SVF). viii. Resuspend the pellet with 10 ml HBSS. Centrifuge again at 400g for 5 min. ix. Repeat the washing step three times. x. If the pellet appears red, rupture the red blood cells by adding 10 ml erythrocyte lysis buffer. Pipet up and down to resuspend the pellet. The solution should become red. Leave for 5 min at room temperature and centrifuge for 5 min. Aspirate the supernatant. xi. Resuspend the pellet in 10 ml Expansion DMEM media and plate onto Petri dishes. xii. Incubate at 37°C in a 5% CO2 incubator for 1 hour. The majority of hematopoietic lineage cells such as monocytes/macrophages will attach to the Petri dish at this stage. xiii. Transfer non-adherent cells (containing ADS cell populations) to 10 cm regular culture dishes and incubate at 37°C in a 5% CO2 incubator. We typically culture cells isolated from 2 – 3 g fat in one 10 cm dish. Sugii et al. Page 7 Nat Protoc. Author manuscript; available in PMC 2011 May 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript xiv. Change the media after 24 h. After that, feed cells every 3 d until cells reach around 80% confluency. mADS cells should exhibit a large and flat fibroblastic morphology (Fig. 1a). xv. Aspirate the media and wash with D-PBS three times. Add a minimum amount of 0.25% trypsin/EDTA just barely covering the bottom surface (e.g. 1 ml for 10 cm dishes). Incubate at 37°C for no longer than 5 min. Gently tap the dish to dislodge the cells and observe by microscope. Ignore firmly adhering cells, if any, as these are often of the hematopoietic lineage. Add an equal amount of Expansion DMEM media to inactivate the trypsin. xvi. Transfer to the 15 ml tube and centrifuge at 400g for 5 min. Aspirate the supernatant and leave cell pellets. 2. Isolation of human ADS (hADS) cells A detailed protocol for isolating human cells was previously published in Nature Protocols17 and other journals9,18–20. Briefly, the protocol for isolating hADS is similar to Step 1 for mADS, except that the working volume is typically much larger. In addition, if lipoaspirate samples are used, no mincing with scissors is necessary as the liposuction procedure itself results in the mincing of the tissue. Alternatively, hADS cell lines isolated from fat tissue can be obtained from commercial sources (See MATERIALS). The typical morphology of hADS culture is shown in Fig. 1b. Commercially available media can also be used for hADS cells in place of Expansion DMEM media. If human tissue samples are used for ADS cell isolation, assure that the procedures is first reviewed and | |||||||||||||||||||
| 99 | Dhanasekaran | 2012 | Stromal Vascular Fraction. SVF | Adipose Stromal Cells (ASC) | Manual | Human | SubQ and Omental | Mouse – Surgical | Minced with Scissor | Collagenase Type I | Himedia Laboratories Pvt. Ltd | PBS | Centrifugation | Two washes | Filtered | Lysis – Osmotic buffer | CD 34 Sialomucin PE 348057 BD Bioscience 2 CD 45 PTPRC APC CY 7 348795 BD Bioscience 3 CD 133 Prominin 1 APC 17-1338-42 eBioscience 4 HLADR MHC-II PER CP 347364 BD Bioscience Mesenchymal stem cell marker 5 CD 90 Thy 1 PE CY 5 15-0909-73 eBioscience 6 CD 105 Endoglin APC 17-1057-73 eBioscience 7 CD 73 NT5E PE 550257 BD Bioscience Cell adhesion molecules 8 CD 29 Integrin Beta 1 PE 555443 BD Bioscience 9 CD 49d Integrin Alpha 4 PE 12-0499-73 eBioscience 10 CD 44 HCELL FITC 555478 BD Bioscience 11 CD 166 ALCAM PE 559263 BD Bioscience 12 CD 13 ANPEP APC 557454 BD Bioscience 13 CD 106 VCAM-1 FITC 551146 BD Bioscience 14 CD 54 ICAM-1 PER CP 555512 BD Bioscience 15 CD 31 PECAM-1 FITC 555445 BD Bioscience Unique MSC marker 16 CD 117 C-Kit/SCF APC 17-1179-73 eBioscience 17 ABCG2 CDw338 PE 12-8888-73 eBioscience 18 ALDH – – 01700 Stemcell Technologies | SVF | In Vitro Studies | CD 34 Sialomucin PE 348057 BD Bioscience 2 CD 45 PTPRC APC CY 7 348795 BD Bioscience 3 CD 133 Prominin 1 APC 17-1338-42 eBioscience 4 HLADR MHC-II PER CP 347364 BD Bioscience Mesenchymal stem cell marker 5 CD 90 Thy 1 PE CY 5 15-0909-73 eBioscience 6 CD 105 Endoglin APC 17-1057-73 eBioscience 7 CD 73 NT5E PE 550257 BD Bioscience Cell adhesion molecules 8 CD 29 Integrin Beta 1 PE 555443 BD Bioscience 9 CD 49d Integrin Alpha 4 PE 12-0499-73 eBioscience 10 CD 44 HCELL FITC 555478 BD Bioscience 11 CD 166 ALCAM PE 559263 BD Bioscience 12 CD 13 ANPEP APC 557454 BD Bioscience 13 CD 106 VCAM-1 FITC 551146 BD Bioscience 14 CD 54 ICAM-1 PER CP 555512 BD Bioscience 15 CD 31 PECAM-1 FITC 555445 BD Bioscience Unique MSC marker 16 CD 117 C-Kit/SCF APC 17-1179-73 eBioscience 17 ABCG2 CDw338 PE 12-8888-73 eBioscience 18 ALDH – – 01700 Stemcell Technologies | Dhanasekaran M, Indumathi S, Kanmani A, Poojitha R, Revathy KM, Rajkumar JS, Sudarsanam D. Surface antigenic profiling of stem cells from human omentum fat in comparison with subcutaneous fat and bone marrow. Cytotechnology. 2012 Feb 1;64(5):497–509. doi: 10.1007/s10616-012-9427-4. Epub ahead of print. PMID: 22294516; PMCID: PMC3432535. | Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228. | “We found from this study that, identification of an ideal source for stem cell therapeutics is strenuous.” | Stromal vascular fraction was obtained from adipose tissue using a previously standardised protocol (Zuk et al. 2002) with modifications. The harvested tissues were washed several times with PBS, minced with scalpels into small pieces with simultaneous removal of visible blood vessels to maximum possible extent. The minced tissues were enzymatically dissociated with collagenase type I (Himedia Laboratories Pvt. Ltd) and centrifuged. The upper aqueous layer containing lipocytes was discarded and the pelleted stromal vascular fraction was washed twice with Phosphate Buffered Saline. The cells were then filtered and the contaminating erythrocytes lysed with an osmotic buffer. The resulting cells were counted and viability assay performed and characterized using flowcytometry. | |||||||||||||||||
| 100 | Laschke | 2012 | Adipose Microvascular Fragments | MVF | Manual | Mouse. GFP | epididymal fat | surgical | finely minced with micro-scissors | collagenase NB4G | Nordmark | 0.5 U/ml% | 37 | 5 – 7 min | vigirous stirring | neutralising the collagenase with PBS containing 20 % foetal calf serum (FCS) | 1g 5min. Repeat 3 x. centrifuge 40 g 10 min | Microvascular Fragments | Laschke MW, Kleer S, Scheuer C, Schuler S, Garcia P, Eglin D, Alini M, Menger MD. Vascularisation of porous scaffolds is improved by incorporation of adipose tissue-derived microvascular fragments. Eur Cell Mater. 2012 Sep 24;24:266-77. doi: 10.22203/ecm.v024a19. PMID: 23007911. | Microvascular fragments were isolated from epididymal fat pads of male C57BL/6-TgN(ACTB-EGFP)1Osb/J donor mice. In these transgenic mice with an enhanced green fluorescent protein (EGFP) cDNA under the control of a chicken β-actin promoter and cytomegalovirus enhancer all of the tissues, with exception of erythrocytes and hair, exhibit a green fluorescence under blue-light excitation (Okabe et al., 1997). Accordingly, microvascular fragments could easily be detected by their GFP-positive signal after implantation into wild-type recipient animals. The donor mice were anaesthetised by i.p. injection of ketamine (75 mg/kg body weight; Pharmacia GmbH, Erlangen, Germany) and xylazine 2 % (25 mg/kg body weight; Rompun; Bayer, Leverkusen, Germany). After midline laparotomy, the epididymal fat pads were harvested and transferred in 10 % Dulbecco’s modified Eagle medium (DMEM; 100 U/mL penicillin, 0.1 mg/mL streptomycin; PAA, Cölbe, Germany). Then, the fat pads were washed three times in phosphate-buffered saline (PBS). They were finely minced with micro-scissors and digested in collagenase NB4G (0.5 U/mL; Serva, Heidelberg, Germany) for 5-7 min with vigorous stirring at 37 °C in a humidified atmosphere with 5 % CO2. This resulted in the destruction of the fat cells. After neutralising the collagenase with PBS containing 20 % foetal calf serum (FCS), the cell suspension was incubated for 5 min at 37 °C and the fat supernatant was removed. The incubation period and removal of fat supernatants was repeated three to five times. The remaining cell suspension contained microvascular fragments, which were enriched in a small volume by centrifugation for 10 min at 40 g. Finally, the fragments were resuspended in PBS containing 20 % FCS to prevent their agglutination. | |||||||||||||||||||||
| 101 | Guven | 2012 | SVF | SVF | Manual | Human | SubQ | Liposuction | Collagenase NB6 | SERVA | PBS | 0.15% (W/V). | 37 | 60 | Centrifugation. 190xg 10 min | 1 X | 0.15M ammonium chloride, 1mM potassium hydrogen carbonate | Nucleated cells were counted using a Neubauer chamber after staining with Crystal Violet (Sigma), whereas cell viability was determined by counting the blue (dead) and transparent (alive) cells after trypan blue (Sigma) staining. | 1.6 – 0.9 · 105 nucleated cells/mL of liposuction | Güven S, Karagianni M, Schwalbe M, Schreiner S, Farhadi J, Bula S, Bieback K, Martin I, Scherberich A. Validation of an automated procedure to isolate human adipose tissue-derived cells by using the Sepax® technology. Tissue Eng Part C Methods. 2012 Aug;18(8):575-82. doi: 10.1089/ten.TEC.2011.0617. Epub 2012 Apr 2. PMID: 22372873; PMCID: PMC3401386. | Manual Method: Manual isolation of the cells Adipose samples were digested for 60min at 37 C in 0.15% (W/V) collagenase NB 6 GMP Grade from Clostridium histolyticum (0.12U/mg collagenase; SERVA Electrophoresis GmbH) diluted in phosphate-buffered saline (PBS; Gibco). After centrifugation at 190 g for 10min, the lipid-rich layer was discarded and the cellular pellet was washed once with PBS. For analysis, red blood cells were lysed by incubation for 2 min in a solution of 0.15M ammonium chloride, 1mM potassium hydrogen carbonate (both from Merck; www .merck-chemicals.com), and 0.1mM EDTA (Fluka Analytical, Sigma-Aldrich Chemie GmbH). The resulting SVF cells were then resuspended in complete medium (CM), consisting of alpha-minimum essential medium (a-MEM) supplemented with 10% of fetal bovine serum (FBS), 1% HEPES, 1% sodium pyruvate, and 1% of penicillin-streptomycin-glutamin (100 · ) solution (all from Gibco). | ||||||||||||||||||||
| 102 | Guven | 2012 | SVF | Insufficient Data to Classify | Automated. Sepax Device | Human | SubQ | Liposuction | Collagenase NB6 | SERVA | PBS | 0.15% (W/V). | Transfer Bag | 37 | 60 | Centrifugation | Nucleated cells were counted using a Neubauer chamber after staining with Crystal Violet (Sigma), whereas cell viability was determined by counting the blue (dead) and transparent (alive) cells after trypan blue (Sigma) staining. | Sepax-based procedure isolated 2.6 – 1.2 · 105 cells/mL of liposuction (n = 6 | Güven S, Karagianni M, Schwalbe M, Schreiner S, Farhadi J, Bula S, Bieback K, Martin I, Scherberich A. Validation of an automated procedure to isolate human adipose tissue-derived cells by using the Sepax® technology. Tissue Eng Part C Methods. 2012 Aug;18(8):575-82. doi: 10.1089/ten.TEC.2011.0617. Epub 2012 Apr 2. PMID: 22372873; PMCID: PMC3401386. | Automated System: Sepax-based automated isolation of the cells Liposuction samples were introduced in 400mL transfer bags (Terumo) and NB 6 GMP Grade collagenase diluted in PBS was added at a final concentration of 0.15% (W/V). The bag was placed into an incubator at 37 C for 60 min. After digestion (Fig. 1), the bag was connected to a CS-490.1 kit (Biosafe SA) specifically modified for this purpose and the kit was installed on a Sepax device (Biosafe SA; www.biosafe .ch) for automated digestion. After priming and straining of the digested adipose tissue into the separation chamber, a centrifugation step was performed to remove the supernatant. Itwas followed by a washing procedure of the pellets with saline solution.When more than 200mL of adipose was processed, a two-phase digestion by the machine was necessary, which slightly increased the time for procedure. For cell collection, the system diluted the residual pellets (&10mL) and extracted the content into the final bag, and then automatically rinsed the chamber twice for an optimal cell recovery. The minimumfinal volume used was 40mL. | |||||||||||||||||||||
| 103 | Gupta | 2012 | Stromal Vascular cells | Adipose Stromal Cells (ASC) | Manual | Mice | Fat Pads | Surgical | Minced | Dispase II. Collagenase D | Roche | PBS containing 10 mM CaCl | 2.4 units/mL dispase II (Roche), and 1.5 units/mL collagenase D (Roche). | 37 | 45 | Centrifugation. 600 x g 5 min | 1x | 100 micron before first wash then 40 micron | Gupta RK, Mepani RJ, Kleiner S, Lo JC, Khandekar MJ, Cohen P, Frontini A, Bhowmick DC, Ye L, Cinti S, Spiegelman BM. Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells. Cell Metab. 2012 Feb 8;15(2):230-9 | Isolation of adipose SV cells. Dissected fat tissue from two mice (4 fat pads total) were washed, minced, and then digested for 45 minutes at 37 C in PBS containing 10 mM CaCl, 2.4 units/mL dispase II (Roche), and 1.5 units/mL collagenase D (Roche). Digested tissue was then filtered through a 100 m cell strainer to remove undigested tissues. The flow-through was then centrifuged for 5 minutes at 600 x g to pellet the SV cells. The SV cells, resuspended in complete SV culture medium, were then filtered through a 40 m cell strainer to remove clumps and large adipocytes. Following centrifugation as above, SV cells were then resuspended in SV culture medium and plated onto a 6-cm tissue culture dish. | |||||||||||||||||||||
| 104 | Kapur | 2012 | Freshly isolated cells | Insufficient Data to Classify | Manual | Human | Liposuction | Kapur SK, Dos-Anjos Vilaboa S, Llull R, Katz AJ. Adipose tissue and stem/progenitor cells: discovery and development. Clin Plast Surg. 2015 Apr;42(2):155-67. doi: 10.1016/j.cps.2014.12.010. PMID: 25827560. | Katz A J, Tholpady A, Tholpady S S, Shang H and Ogle R C 2005 Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells Stem. Cells 23 412–23. Zuk P A, Zhu M, Mizuno H, Huang J, Futrell J W, Katz A J, Benhaim P, Lorenz H P and Hedrick M H 2001 Multilineage cells from human adipose tissue: implications for cell-based therapies Tissue Eng. 7 211–28 | 2.1. Adipose stem cell isolation and culture Subcutaneous adipose tissue was obtained from patients undergoing elective surgical procedures in the Department of Plastic Surgery, University of Virginia in accordance with the University’s Human Investigation Committee. Discarded excisional abdominoplasty specimens and/or liposuction aspirates from 58 patients (average age 42.4 years, range 24–70 years; average BMI of 30.14, range of 18.4–63.6) were processed as described previously [18, 19]. During the described studies, cells were cultured at 37 ◦C, 5% CO2 in one of the following media (table 1). All the studies described in this paper were performed a minimum of two times unless otherwise specifically mentioned. | |||||||||||||||||||||||||||||||
| 105 | Lee | 2012 | Stromal vascular cells SVC | Adipose Stromal Cells ASC | Manual | Mouse | minced | type II collagenase | KRBB containing 10 mM HEPES (pH 7.4), and 3% BSA | 2 mg/ml | 37 | 60 | Preparations were passed through a 300 mm mesh and centrifuged at 500 g for 5 min. Floating adipocytes were collected by aspiration. Pellets containing the stromal vascular (SV) fraction were incubated in red blood cell lysis buffer for 5 min at room temperature, passed through a 40 mm mesh, and then collected by centrifugation at 500 g for 5 min. | 300 micron. 40 micron | red blood cell lysis buffer for 5 min at room temperature | immunostaining or flow cytometry. Culture | Lee, Y.H., Petkova, A.P., Mottillo, E.P., and Granneman, J.G. (2012). In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding. Cell Metab. 15, 480–491. | Adipose tissues were washed with PBS, minced, and digested with type II collagenase (2 mg/ml) in KRBB containing 10 mM HEPES (pH 7.4), and 3% BSA for 1 hr at 37 C. Preparations were passed through a 300 mm mesh and centrifuged at 500 g for 5 min. Floating adipocytes were collected by aspiration. Pellets containing the stromal vascular (SV) fraction were incubated in red blood cell lysis buffer for 5 min at room temperature, passed through a 40 mm mesh, and then collected by centrifugation at 500 g for 5 min. The resultant cell preparations were subjected to immunostaining or flow cytometry. | |||||||||||||||||||||||
| 106 | Doi | 2013 | Stromal Vascular Fraction. SVF | SVF | Automated Tissue Genesis | Human | SubQ | Liposuction | Adipase™ | Tissue Genesis | Lactated Ringers | Proprietary Bowl | 37 | Oscillation in Bowl | Centrifugation | 250 micron | Yield/ Viability/FACS. The SVF cell yield was counted using a cell counter (NucleoCounter, Chemometec, Allerod, Denmark), | Nucleocounter | 7×105/ml fat | > 80% | SVF | CALS | Doi K, Tanaka S, Iida H, Eto H, Kato H, Aoi N, Kuno S, Hirohi T, Yoshimura K. Stromal vascular fraction isolated from lipo-aspirates using an automated processing system: bench and bed analysis. J Tissue Eng Regen Med. 2013 Nov;7(11):864-70. doi: 10.1002/term.1478. Epub 2012 Mar 22. PMID: 22438241. | The Tissue Genesis Cell Isolation System (Tissue Genesis, Honolulu, HI, USA) was used for automated SVF isolation from aspirated adipose tissue in this study (Figure 1). The automated cell isolation system consists of a machine and a single-use disposable. The sterile disposable unit contains a processing chamber (for tissue digestion, centrifugation and washing), two syringes (for collagenase solution and final product), and other bags and connecting tubes. The whole system basically mimics amanual method and the cell isolation was automatically performed by the programmed sequence set in the machine. In brief, aspirated fat tissue (20–60 ml) was introduced into the chamber and enzymatically digested. The collagenase (Adipase™) used, as well as the whole system and disposables (Adipase™ Loading Kit, Suspension Kit and Front-end Tissue Collection Kit) were provided by the manufacturer. After digestion, the solution was centrifuged and washed in the same chamber. The final cell output was approximately 35ml SVF cell suspension and the whole process time was consistently 65 min. The nucleated cell number was determined using the cell counter as described above. For clinical application, the SVF solution was centrifuged (700 g, 5min) to remove the fluid and isolate the SVF cell pellets. | |||||||||||||||||
| 107 | Hager | 2013 | Stromal Vascular Fraction. SVF | SVF | Manual | Human | SubQ | Liposuction | 300 ml | Collagenase Type I | Worthington | PBS with Ca2þ/ Mg2þ and supplemented with 20 mg/mL bovine serum albumin (BSA) and 0.025 mol/L 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid buffer (PAA Laboratories, Pasching, Austria) | 1.5 mg/ml | 37 | 60 | Shaking | Centrifugation 7 min 1180g | SVF was washed with PBS, filtered through 100-mmicronand 40-micron cell strainers (VWR International, Vienna, Austria) | treated with lysis buffer consisting of 154 mmol/L NH4Cl, 10 mmol/L KHCO3 (both Sigma, St Louis, MO, USA) and 0.1 mmol/L ethylenediamine tetra-acetic acid (PAA Laboratories) for 4 min at 37 C eliminate red blood cells | Hemocytometer | SVF | Isolation of EPCs from SVF | 35% of SVF is EPC CD31+ | Hager G, Holnthoner W, Wolbank S, Husa AM, Godthardt K, Redl H, Gabriel C. Three specific antigens to isolate endothelial progenitor cells from human liposuction material. Cytotherapy. 2013 Nov;15(11):1426-35. doi: 10.1016/j.jcyt.2013.06.018. PMID: 24094492. | Wolbank S, Peterbauer A, Fahrner M, Hennerbichler S, van Griensven M, Stadler G, et al. Dose-dependent immunomodulatory effect of human stem cells from amniotic membrane: a comparison with human mesenchymal stem cells from adipose tissue. Tissue Eng. 2007;13:1173e83. | The SVF was prepared as described previously (30). Briefly, 300 mL of the liposuction material was washed twice with phosphate-buffered saline (PBS) and was subsequently digested with 1.5 mg/mL collagenase type I (Worthington Biochemical, Lakewood, NJ, USA), prepared in PBS with Ca2þ/ Mg2þ and supplemented with 20 mg/mL bovine serum albumin (BSA) and 0.025 mol/L 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid buffer (PAA Laboratories, Pasching, Austria) for 1 hour at 37 C by shaking. The cell suspension was centrifuged for 7 min at 1180g to separate the stromal cell fraction from adipocytes, which were discarded. The cell pellet was treated with lysis buffer consisting of 154 mmol/L NH4Cl, 10 mmol/L KHCO3 (both Sigma, St Louis, MO, USA) and 0.1 mmol/L ethylenediamine tetra-acetic acid (PAA Laboratories) for 4 min at 37 C eliminate red blood cells. Finally, the SVF was washed with PBS, filtered through 100-mm and 40-mm cell strainers (VWR International, Vienna, Austria), and cells were counted in a hemocytometer. | |||||||||||||||
| 108 | Huh | 2013 | Stromal Vascular Cells | Adipose Stromal Cells (ASC) | Manual | Mouse | Epididymal | surgical | chopped | collagenase | 37 | 30 | shaking | adipocytes regulate iNKT cell activity via CD1d and that the interaction between adipocytes and iNKT cells may modulate adipose tissue inflammation in obesity | Huh JY, Kim JI, Park YJ, Hwang IJ, Lee YS, Sohn JH, Lee SK, Alfadda AA, Kim SS, Choi SH, Lee DS, Park SH, Seong RH, Choi CS, Kim JB. A novel function of adipocytes in lipid antigen presentation to iNKT cells. Mol Cell Biol. 2013 Jan;33(2):328-39. doi: 10.1128/MCB.00552-12. Epub 2012 Nov 12. PMID: 23149942; PMCID: PMC3554106. | Li P, Lu M, Nguyen MTA, Bae EJ, Chapman J, Feng D, Hawkins M, Pessin JE, Sears DD, Nguyen AK, Amidi A, Watkins SM, Nguyen U, Olefsky JM. Functional heterogeneity of CD11c-positive adipose tissue macrophages in diet-induced obese mice. J Biol Chem. 2010 May 14;285(20):15333-15345. doi:10.1074/jbc.M110.100263. Epub 2010 Mar 22. PMID: 20308074; PMCID: PMC2865288. | Adipose tissue fractionation. Fractionation of adipose tissue was performed as previously described (Li 2010). Epididymal adipose tissues were weighed and then chopped and incubated in collagenase buffer for 30 min at 37°C with shaking. After centrifugation, adipocytes in the supernatant were collected for RNA extraction or primary cell culture. The pelleted stromal vascular cell (SVC) fraction was used for flow cytometry or RNA extraction. | Address correspondence to Jae Bum Kim, jaebkim@snu.ac.kr. | |||||||||||||||||||||||
| 109 | Moshtagh | 2013 | MSC-rich dense cell fraction | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | Collagenase Type I | Sigma | alpha MEM | 0.08% | 37 | 30 | shaking | 10% FBS | Centrifugation 5,000 x g 10 min | Centrifugation 1,200 x g 10 min | 160 mM NH4Cl at room temperature for 10 min to lyse remaining red blood cells | Moshtagh PR, Emami SH, Sharifi AM. Differentiation of human adipose-derived mesenchymal stem cell into insulin-producing cells: an in vitro study. J Physiol Biochem. 2013 Sep;69(3):451-8. doi: 10.1007/s13105-012-0228-1. Epub 2012 Dec 29. PMID: 23271274. | Isolation and culture of adipose tissue stem cells Human adipose tissue was obtained from liposuction of human adipose tissue as described earlier [3] by informed consent. Briefly, to remove red blood cells and tissue debris, adipose tissue was washed with an equal volume of sterile phosphate-buffered saline (PBS) with 1 % antibiotics. Washed tissue fragments were digested with 0.075 % type I collagenase (Sigma- Aldrich, USA) in α-MEM (Gibco, Invitrogen, UK) without serum plus 1 % antibiotics at 37 °C for 30 min with shaking. Enzyme activity was neutralized by adding an equal volume of α-MEM containing 10 % fetal bovine serum and centrifuged at 5,000×g for 10 min. The pellet was resuspended in 160 mM NH4Cl at room temperature for 10 min to lyse remaining red blood cells and then centrifuged at 1,200×g for 10 min to pellet the MSC-rich dense cell fraction. Afterward, the collected cells were filtered through 100-μm cell strainer (Grainer, USA) and incubated for 48 h at 37 °C in 5 % CO2 in culture medium with serum and antibiotics. Medium was replaced every third day thereafter. | A. M. Sharifi (*) Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran e-mail: sharifal@Yahoo.com A. M. Sharifi e-mail: amsharifi@TUMS.ac.ir | |||||||||||||||||||
| 110 | Van Pham | 2014 | SVF | Insufficient Data to Classify | ADSC Extraction Kit (GeneWorld, Ho Chi Minh City, Vietnam) | Human | Belly | Aspiration | SuperExtract Solution containing collagenase | 37 | 45 min | agitation every 5 min | Centrifugation 3000 g 10 min | Van Pham, P., Vu, N.B., Phan, N.L.-C., Le, D.M., Truong, N.C., Truong, N.H., Bui, K.H.-T., and Phan, N.K. (2014). Good manufacturing practice-compliant isolation and culture of human adipose derived stem cells. Biomed Res Ther 1, 1-9. | Isolation of the SVF from adipose tissue The SVF was isolated from adipose tissues using an ADSC Extraction Kit (GeneWorld, Ho Chi Minh City, Vietnam) according to the manufacturer’s instructions. Briefly, 50–100 mL of lipoaspirate was placed in a sterile disposable 250 mL conical centrifuge tube (Corning, Tewksbury, MA) and washed in Washing Buffer 1 by centrifugation at 1000 g for 5 min at room temperature. Next, the adipose tissue was washed with Washing Buffer 2 as per the procedure for Washing Buffer 1. Then the adipose tissue was digested using SuperExtract Solution containing collagenase at 37°C for 45 min with agitation at 5 min intervals. The cell suspension obtained was centrifuged at 3000 g for 10 min, and the SVF was obtained as the pellet. The pellet was washed with Washing Buffer 3 to remove any residual enzymes, and resuspended in PBS for the determination of cell quantity and viability using an automatic cell counter (NucleoCounter; Chemometec, Denmark). | ||||||||||||||||||||||||||
| 111 | Fraser | 2014 | ADRC Adipose Derived Regenerative Cells | SVF | Automated | Human | SubQ | Liposuction | Celase™ | Cytori | Proprietary | Continuos Agitation | Centrifugation | Nucleocounter | 3.6 – 1.8 · 105 cells/g of tissue processed | 84.70% | Wound Healing | Fraser JK, Hicok KC, Shanahan R, Zhu M, Miller S, Arm DM. The Celution® System: Automated Processing of Adipose-Derived Regenerative Cells in a Functionally Closed System. Adv Wound Care (New Rochelle). 2014 Jan 1;3(1):38-45. doi: 10.1089/wound.2012.0408. PMID: 24761343; PMCID: PMC3900001. | Adipose tissue processing The Celution System (Cytori Therapeutics, San Diego, CA) was used to obtain ADRCs according to the manufacturer’s instructions. Tissue processing within the Celution device uses a single-use sterile disposable set and the Celase processing enzyme reagent (a proprietary proteolytic reagent optimized for isolation of cells from aspirated adipose tissue). The Celase reagent is validated to be free of risk of contamination with bovine spongiform encephalitis and is provided as a sterile USP Class VI AUTOMATED PROCESSING OF ADIPOSE REGENERATIVE CELLS 39 product. Once the Celution disposable is placed within the device, the system performs a wet test to ensure the integrity of the closed system. Tissue is then introduced into the processing canister where it is weighed and then washed with the lactated Ringers solution to remove the residual wetting solution and extravasated blood. The Celution device calculates the amount of Celase reagent to be used (based on tissue weight) and then, at the appropriate stage, prompts the operator to add the required volume of Celase. The tissue is continuously agitated during enzymatic digestion of the connective tissue. Once digestion is complete, the ADRC fraction is pumped into a centrifuge chamber where it is washed and concentrated. The final cell product can then be aspirated from the chamber in a volume of 5 mL. | Correspondence: Cytori Therapeutics, Inc., 3020 Callan Road, San Diego, CA 92121 (e-mail: jfraser@cytori.com). | |||||||||||||||||||||
| 112 | Valadares | 2014 | Pericytes | Adipose Stromal Cells (ASC) | Manual | Human | surgical | ). Tissues were minced with scalpels | 10 mg | collagenase type II in a 1:3 ratio (tissue weight:collagenase volume) | Sigma | DMEM-F12 with 20% fetal bovine serum (FBS), 1 % non-essential amino acids (GIBCO) and 1 % of antibiotics/antimycotics (GIBCO) | 1 mg/ml | 37 | 30 to 40 min | shaker, at 250 RPM | Cells were then passed through a 70μm strainer and incubated in blood lysis solution for 5–10 minutes. PBS was added in a ratio 2:1 and the solution was filtered again through a 40 μm strainer. | 100, 70 and 40 micron strainer | blood lysis solution for 5–10 minutes. | cells were incubated with conjugated antibodies against CD-34 (Percp-Cy5.5), CD-45 (FITC), CD-56 (APC) and CD 146 (PE), all from BD Biosciences. DAPI was added just before the analysis, and all cells positive for DAPI were excluded. The gating strategy was performed All pericyte populations were confirmed to have at least 75% purity after sorting | Pericytes | Valadares MC, Gomes JP, Castello G, Assoni A, Pellati M, Bueno C, Corselli M, Silva H, Bartolini P, Vainzof M, Margarido PF, Baracat E, Péault B, Zatz M. Human adipose tissue derived pericytes increase life span in Utrn (tm1Ked) Dmd (mdx) /J mice. Stem Cell Rev Rep. 2014 Dec;10(6):830-40. doi: 10.1007/s12015-014-9537-9. PMID: 24943487. | Gao, J., Dennis, J., Muzic, R., et al. (2001). The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells, tissues, organs., 169, 12–20. | Tissue and Cell Preparation Four tissue specimens from the same donor, namely: adipose tissue, muscle, fallopian tubes and endometrium were obtained from total hysterectomy procedures. Tissues were stored in PBS or DMEM with 4 % antibiotics at 4 °C and processed within 24 hours after harvest. Tissue digestion was performed as described elsewhere [9], but with smaller quantities of material from each tissue (adipose tissue: ~10 mg; endometrium: ~3 mg; fallopian tubes: ~2 mg; muscle tissue: ~1 mg). Tissues were minced with scalpels and incubated with 1 mg/ mL of collagenase type II (Sigma Aldrich) in a 1:3 ratio (tissue weight:collagenase volume) diluted inDMEM-F12 with 20% fetal bovine serum (FBS), 1 % non-essential amino acids (GIBCO) and 1 % of antibiotics/antimycotics (GIBCO) at 37 °C for 30–40 minutes in a shaker, at 250 RPM. Cells were then passed through a 70μm strainer and incubated in blood lysis solution for 5–10 minutes. PBS was added in a ratio 2:1 and the solution was filtered again through a 40 μm strainer. Later, cells were incubated with conjugated antibodies against CD-34 (Percp-Cy5.5), CD-45 (FITC), CD-56 (APC) and CD 146 (PE), all from BD Biosciences. DAPI was added just before the analysis, and all cells positive for DAPI were excluded. The gating strategy was performed according to Crisan et al.[9]. All pericyte populations were confirmed to have at least 75% purity after sorting. Cells (CD146+/CD34-/ CD45-/CD56-) were sorted into a 24 well plate at a density of 20.000 cells/cm2 and cultivated until the first passage in EBM- 2 complete medium (Lonza). After the first passage, all cells were cultivated in DMEM F-12 medium with 20 % FBS, 1 % NEAA and 1%antibiotics/antimycotic up to passage four and then frozen. After thawing, cells were always cultivated in EBM-2 medium and were injected between passages 7–10. All cells used in the in vivo experiments were derived from a single 46-year-old healthy female donor. | M. C. Valadares : J. P. Gomes : G. Castello : A. Assoni : M. Pellati : C. Bueno : M. Vainzof : M. Zatz (*) Human Genome and stem-cell Center, Institute of Biosciences, University of São Paulo, Rua do Matāo 106, São Paulo CEP 05508-030, Brazil e-mail: mayazatz@usp.br | ||||||||||||||||
| 113 | Schendel | 2015 | Stromal Vascular Fraction | SVF | Manual | Human | Collagenase Class I and II and BP Protease | Vitacyte, Indianapolis IN | Hanks Balanced Salt Solution | 25 Wunsch units for the collagenases and 200,000 neutral protease units for the B. polymyxa neutral protease. | 37 | 30 | Shaker set at 200 RPM | Centrifuge 800 g 5 min | 3 washes 800 g 5 min | The average SVF cell count was 4.97 × 104 per cc | SVF | Cell Assisted Lipotransfer | Stephen A. Schendel, Enriched Autologous Facial Fat Grafts in Aesthetic Surgery: 3D Volumetric Results Aesthetic Surgery Journal 2015, Vol 35(8) 913–919 | Yoshimura K, Shigeura T, Matsumoto D, Sato T, Takaki Y, Aiba-Kojima E, Sato K, Inoue K, Nagase T, Koshima I, Gonda K. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol. 2006 Jul;208(1):64-76. doi: 10.1002/jcp.20636. PMID: 16557516. | The extracted fat was processed via the technique developed by Yoshimura et al12 at the University of Tokyo, Japan, and modified for reduced time of processing. Physiologic buff- ered saline (PBS) was used to neutralize the collagenase. The fat was harvested by using the Lipivage Closed System (Genesis Biosystems, Lewisville, TX) using a 3 mm, two hole under low pressure and then put aside. 50 cc of the fat was then placed into a sterile plastic tube and centrifuged for 5 minutes, to remove the infranatant blood/tumescent fluid and the supernatant oil/disrupted fat. The centrifuged fat was washed with PBS and centrifuged at 700G for 5 minutes. The infranatant solution was pipetted out and discarded. The washed fat was then preheated in an incubator, allowing the fat to reach 37°C prior to the next step. Collagenase GMP grade, non-animal source collagenase from VitaCyte (Indianapolis, IN) was used. The product is a Blend 1 formulation, Cat. No.005-1010. This is a mixture of collagenase I and collagenase II, derived from bacterial production (Clostridium histolyticum) and a neutral prote- ase (derived from B. polymyxa). One vial of Blend 1 formu- lation was used per 100 gm of centrifuged fat processed. The enzyme activity of each vial is 25 Wunsch units for the collagenases and 200,000 neutral protease units for the B. polymyxa neutral protease. Hank’s balanced salt solution (HBSS) was pre-heated in an incubator at 37°C and 5 cc of this was then added to 1 vial of collagenase (100 mg of enzyme) and left in the incubator at 37°C until the fat was ready. This solution was then added to 95 cc of balanced saline, giving a total volume of 100 cc of collagenase solu- tion ready for mixture with fat (1:1 mixing ratio). Equal volumes of the washed fat and collagenase were now mixed and incubated in a shaker set at 200 rpm for 30 minutes at 37°C. The collagenase/fat solution was then cen- trifuged at 800G for 5 minutes at room temperature. The fat was removed from the supranatant layer of the centrifuge tube and discarded. The lower layer was then re-suspended by removing this with a pipette and placing this in a new 50 cc centrifuge tube with PBS. Three cycles of centrifugation at 800G were done for a total of 5 minutes at room temperature. The pellet was removed and resuspended and the cells counted using a nuclecounter and the total cell number recorded. The residual cell mixture with buffered saline was a total volume of 10 cc. The surgeon then mixed the SVF sus- pension with the fat to be injected by transfer to opposing 60 cc syringes until the fat and PBS were well mixed. This mixture was the transferred into 1cc syringes using a female-to-female transfer hub. The SVF-enriched fat grafts where then injected into the face using Coleman cannulae based on the discretion of the surgeon, the underlying deformity, and the patient’s desired correction. | Dr Stephen A. Schendel, 1900 University Avenue, Suite 101, E. Palo Alto, CA 94303, USA. E-mail: sschendel@stanford.edu | |||||||||||||||||||
| 114 | Travers | 2015 | Stromal Vascular Fraction | Adipose Stromal Cells | Manual | Human | SubQ | Aspiration | type I collagenase (Worthington Biochemical | Worthington | PBS 2% BSA | 37 | ~ 45 min | shaking water bath (220 r.p.m.) | The suspension was filtered and subject to brief, gentle centrifugation (10 s at 100 g) before aspiration of adipocytes. After further centrifugation (10 min at 300 g), | Digest Filtered | pellet was resuspended in erythrocyte lysis buffer for 10 min before a final centrifugation step (5 min at 300 g) | Travers RL, Motta AC, Betts JA, Bouloumié A, Thompson D. The impact of adiposity on adipose tissue-resident lymphocyte activation in humans. Int J Obes (Lond). 2015 May;39(5):762-9. doi: 10.1038/ijo.2014.195. Epub 2014 Nov 12. PMID: 25388403; PMCID: PMC4424387. | Curat CA, Miranville A, Sengenes C, Diehl M, Tonus C, Busse R et al. From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes. Diabetes 2004; 53: 1285–1292. | The remaining tissue was digested using type I collagenase (Worthington Biochemical, Lakewood Township, NJ, USA) at 250 Uml − 1 in phosphatebuffered saline and 2% bovine serum albumin (pH 7.4) for ~ 45 min in a shaking water bath (220 r.p.m.) at 37 °C. The suspension was filtered and subject to brief, gentle centrifugation (10 s at 100 g) before aspiration of adipocytes. After further centrifugation (10 min at 300 g), the remaining pellet was resuspended in erythrocyte lysis buffer for 10 min before a final centrifugation step (5 min at 300 g).3 SVF pellets were stored in 1ml phosphate-buffered saline:foetal bovine serum:dimethyl sulphoxide (5:4:1) and frozen at a rate of −1 °Cmin−1 using a freezing container (Thermo Scientific) to −80°C and stored before subsequent analysis by flow cytometry. | 1Department for Health, University of Bath, Bath, UK; 2Unilever R&D Vlaardingen, Vlaardingen, The Netherlands and 3INSERM U1048, Institut des maladies métaboliques et cardiovasculaires and Université Paul Sabatier, Toulouse, France. Correspondence: Dr D Thompson, Department for Health, University of Bath, Claverton Down, Bath BA2 7AY, UK. E-mail: d.thompson@bath.ac.uk | ||||||||||||||||||||
| 115 | Zielins | 2015 | SVF/ASC | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | Type I collagenase | Hank’s Balanced Salt Solution (HBSS). | 0.08% | 37 | 60 min | shaking water bath for 60 min at 37 °C (approximately 180 shakes/min). | Neutralize enzymatic activity by adding an equal volume of FACS buffer and allowing to sit at RT for 5 min | centrifuge at 233 x g for 20 min at 4 °C. Filter the suspension through a 100 μm nylon cell strainer to remove cellular debris. | Centrifuge at 233 x g for 5 min at 4 °C, and discard the supernatant without disrupting the pellet. | Filter the suspension through a 100 μm nylon cell strainer to remove cellular debris. | Resuspend the pellet in 5-10 ml of RT red cell lysis buffer, based on pellet size. Leave solution to sit at RT for 5 min and centrifuge at 233 x g for 5 min at RT | FACS | SVF/ASC | Analysis | FACS | Zielins, E.R., Tevlin, R., Hu, M.S., Chung, M.T., McArdle, A., Paik, K.J., Atashroo, D., Duldulao, C.R., Luan, A., Senarath-Yapa, K., Walmsley, G.G., Wearda, T., Longaker, M.T., Wan, D.C. Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis. J. Vis. Exp. (95), e52181, doi:10.3791/52181 (2015). | 1. Obtain human subcutaneous adipose tissue from healthy female patients undergoing elective lipoaspiration of the abdomen, flank, and/or thigh region under local/general anesthesia. Ensure that Institutional Review Board (IRB) approval has been obtained for the protocol of isolating ASCs from human tissues, and follow institutional safety precautions while working with such materials. 2. To obtain the SVF from the lipoaspirated adipose tissue, first wash the lipoaspirate three times with equal volumes of 1x sterile phosphatebuffered saline (PBS). Carefully aspirate and discard the bottom aqueous layer. 3. Prepare the collagenase digestion buffer: 0.075% Type I collagenase in Hank’s Balanced Salt Solution (HBSS). Prepare FACS buffer: 2% FBS, 1% P188 and 1% Pen-Strep in PBS. Filter both solutions using a commercially available 0.22 μm pore size fast flow polyethersulfone filter. 4. To digest the washed adipose tissue, add an equal volume of collagenase digestion buffer and place digestion vessel securely in a shaking water bath for 60 min at 37 °C (approximately 180 shakes/min). NOTE: It is best to use a larger volume digestion vessel than required, as this allows for maximal digestion during shaking (i.e., 250 ml of collagenase digestion buffer, 250 ml of washed adipose tissue in a 1L sterile flask). 5. Neutralize enzymatic activity by adding an equal volume of FACS buffer and allowing to sit at RT for 5 min. Next, centrifuge at 233 x g for 20 min at 4 °C. 6. Carefully aspirate and discard the supernatant, taking care not to disturb the high-density SVF pellet. 7. Resuspend the pellet in 5-10 ml of RT red cell lysis buffer, based on pellet size. Leave solution to sit at RT for 5 min and centrifuge at 233 x g for 5 min at RT. 8. Aspirate supernatant and resuspend the pellet by adding 5-10 ml of traditional growth media, based on pellet size (Dulbecco’s Modified Eagle Medium [DMEM]/10% FBS/1% penicillin-streptomycin solution [pen-strep]). 9. Filter the suspension through a 100 μm nylon cell strainer to remove cellular debris. 10. Centrifuge at 233 x g for 5 min at 4 °C, and discard the supernatant without disrupting the pellet. 11. Resuspend the pellet in 5-10 ml of traditional growth media, based on pellet size. 12. Place 2 x 106 cells in a 15 cm standard culture dish and establish primary cultures | Correspondence to: Derrick C. Wan at dwan@stanford.edu | |||||||||||||||
| 116 | Aronowitz | 2015 | Stromal Vascular Fraction | SVF | Manual | Human | SubQ | Liposuction | 50 to 800 ml | CIzyme AS and BP Protease Vitacyte, LLC, Indianapolis, IN) | Vitacyte, Indianapolis IN | Lactated Ringers | 35 Wünsch Units (U) of collagenase per 50 mL of washed lipoaspirate. Lipoaspirate was incubated with CIzyme AS (Vitacyte, LLC, Indianapolis, IN), a mixture of clostridial collagenase type I and type II (60% type I, 40% type II) and neutral protease from Bacillus polymyxa resuspended in warm (37°C) lactated Ringer solution | 37 | 20 to 30 | Heated Shaker 200 rpm | Centrifugation 2000 rpm 10 minutes | minimum 3 x with Lactated Ringers | 15,000–790,000 cells/ml lipoaspirate | SVF | Cell Assisted LipoTransfer | Aronowitz JA, Lockhart RA, Hakakian CS, Hicok KC. Clinical Safety of Stromal Vascular Fraction Separation at the Point of Care. Ann Plast Surg. 2015 Dec;75(6):666-71. doi: 10.1097/SAP.0000000000000594. PMID: 26207555. | Aronowitz JA, Ellenhorn JD. Adipose stromal vascular fraction isolation: a headto- head comparison of four commercial cell separation systems. Plast Reconstr Surg. 2013;132:932e–939e | SVF Isolation A nonautomated, collagenase-based isolation was performed by a technician at the point of care under sterile conditions in a previously described19 compact operating room laboratory equipped with a sterile biohood, centrifuge, and heated shaker. Volume of lipoaspirate digested ranged from 50 mL to 800 mL (180 mL average). Lipoaspiratewas first washed 3 times using a lactated Ringer solution (130 mmol/L Na+, 109 mmol/L Cl−, 28 mmol/L lactate, 1.5 mmol/L Ca2+, 4 mmol/L K+) over a period of 10 to 15 minutes to remove contaminating blood cells and tissue debris. Lipoaspirate was then incubated with 35 Wünsch Units (U) of collagenase per 50 mL of washed lipoaspirate. Lipoaspirate was incubated with CIzyme AS (Vitacyte, LLC, Indianapolis, IN), a mixture of clostridial collagenase type I and type II (60% type I, 40% type II) and neutral protease from Bacillus polymyxa resuspended in warm (37°C) lactated Ringer solution in a temperature controlled shaker at 200 rpm for 20 to 30 minutes at 37°C. The SVF cells were separated from the digested adipose tissue via centrifugation at 2000 rpm for 10 minutes. Isolated SVF cells were then washed a minimum of 3 cycles using lactated Ringer solution to remove residual enzymes. The SVF preparation was brought to a final volume of between 5 mL and 25 mL (11.8 mL on average) using lactated Ringer solution and returned to the surgeon for injection. A small sample of SVF cells (0.1-0.2 mL) was analyzed using a cell counting device (Chemometec NC 200; Chemometec A/S, Davis, CA) to determine the cell yield and viability (Table 1). | Joel A. Aronowitz,MD, Cedars-Sinai Medical Center, Los Angeles, University Stem Cell Center, Los Angeles, 8635W. Third St. Suite 1090W, Los Angeles, CA 90048. E-mail: dra@aronowitzmd.com. | ||||||||||||||||
| 117 | Dang | 2015 | Stromal Vascular Fraction | Insufficient Data for Classification | ADSC Extraction Kit (GeneWorld Ltd. Co., Ho Chi Minh City, Vietnam) | Dang, LT., Bui, AT., Pham, V. et al. Production of islet-like insulin-producing cell clusters in vitro from adiposederived stem cells. Biomed Res Ther 2, 3 (2015). https://doi.org/10.7603/s40730-015-0003-3 | Van Pham, P., Vu, N.B., Phan, N.L.-C., Le, D.M., Truong, N.C., Truong, N.H., Bui, K.H.-T., and Phan, N.K. (2014). Good manufacturing practice-compliant isolation and culture of human adipose derived stem cells. Biomed Res Ther 1, 1-9. | ADSC isolation and proliferation Human adipose tissues were collected (with informed consent) from healthy women who underwent aesthetic surgery at the Van Hanh Hospital, Ho Chi Minh City, Vietnam. ADSCs were isolated using the ADSC Extraction Kit (GeneWorld Ltd. Co., Ho Chi Minh City, Vietnam) following the manufacturer’s instructions. Briefly, the obtained adipose tissue was washed three times with buffer solution to remove blood cells. The washed tissue was cut into many pieces and incubated with the enzyme mixture “SuperExtract” at 37°C for 30 min. The digested adipose tissue suspension was centrifuged at 3500 rpm for 10 min to obtain the stromal vascular fraction (SVF). Finally, these SVF cells were washed and cultured as previous published procedure at 37°C, 5% CO2 (Van Pham et al., 2014). Medium was refreshed every 3 days. When the cell confluency reached 70– 80%, cells were sub-cultured. ADSCs in the 3rd passage were used for further experiments. | |||||||||||||||||||||||||||||||||
| 118 | Tevlin | 2016 | Stromal Vascular Fraction (SVF) | Adipose Stromal Cell | Manual | Human | SubQ | Liposuction | Type 1 Collagenase | Sigma | M199 medium, 2.2 mg/ml type I collagenase (Sigma Aldrich, St Louis, Mo.), 1000 U/ml DNAse, 1000× 1 mM calcium chloride, 10% bovine serum albumin, 100× poloxamer-188 (Sigma Aldrich), and 50× hydroxyethyl piperazineethanesulfonic acid (Life Technologies, Grand Island, N.Y.), | 2.2 mg/ml | 500-ml sterile polyethylene terephthalate copolyester media bottle | 37 | 30 | Water Bath 10 min. Orbital Shaker 20 min | Collagenase activity was then neutralized by addition of an equal volume of fluorescence-activated cell sorting buffer (1× phosphate-buffered saline, 2% fetal bovine serum, 1% poloxamer-188, and 1% penicillin/streptomycin) | 125 ml of the digested fat was subsequently transferred to a 250-ml conical-bottom centrifuge bottle. The solution was then centrifuged at 1500 rpm for 10 minutes at room temperature. Supernatant was aspirated and the stromal vascular fraction pellet resuspended in 15 ml of room temperature fluorescence-activated cell sorting buffer. The solution was strained through a 100-μm cell strainer Fifteen milliliters of Histopaque (Sigma Aldrich), a commercially available density gradient separation medium, was added to a new 50-ml conical-bottom centrifuge bottle, and the strained cell solution was overlaid on top of the Histopaque in a 1:1 ratio. Solution was centrifuged at 1450 rpm for 15 minutes at room temperature with acceleration set to low and deceleration settings inactivated. The cloudy interface was transferred to a new conical-bottom centrifuge bottle and the tube was filled with fluorescence-activated cell sorting buffer. The solution was centrifuged at 1300 rpm at 4°C for 5 minutes. The supernatant was then aspirated and the pellet resuspended in 500 μl of fluorescence-activated cell sorting buffer in preparation for fluorescence-activated cell sorting. | 100 micron | Yes | Characterization | FACS | FACS | Tevlin R, McArdle A, Brett E, Chung MT, Paik K, Seo EY, Walmsley GG, Duldulao CR, Atashroo D, Zielins E, Vistnes S, Chan CKF, Wan DC, Longaker MT. A Novel Method of Human Adipose-Derived Stem Cell Isolation with Resultant Increased Cell Yield. Plast Reconstr Surg. 2016 Dec;138(6):983e-996e. doi: 10.1097/PRS.0000000000002790. PMID: | Lipoaspirate was placed on ice for 1 hour to allow the fat to congeal and to separate out the fat and blood | New Method—Lipoaspirate was placed on ice for 1 hour to allow the fat to congeal and to separate out the fat and blood. Fresh collagenase digestion buffer was prepared using M199 medium, 2.2 mg/ml type I collagenase (Sigma Aldrich, St Louis, Mo.), 1000 U/ml DNAse, 1000× 1 mM calcium chloride, 10% bovine serum albumin, 100× poloxamer-188 (Sigma Aldrich), and 50× hydroxyethyl piperazineethanesulfonic acid (Life Technologies, Grand Island, N.Y.), and filtered using a 0.22-μm filter system. The congealed fat was transferred to a 500-ml sterile polyethylene terephthalate copolyester media bottle and an equal volume of collagenase digestion buffer was added to the fat. The lid was closed and sealed with Parafilm (Bemis NA, Neenah, Wis.). The fat/collagenase mixture was incubated at 37°C in a water bath for 10 minutes to activate the collagenase. The fat/collagenase mixture was then transferred to the orbital shaker for 20 minutes. Then, 125 ml of the digested fat was subsequently transferred to a 250-ml conical-bottom centrifuge bottle. Collagenase activity was then neutralized by addition of an equal volume of fluorescence-activated cell sorting buffer (1× phosphate-buffered saline, 2% fetal bovine serum, 1% poloxamer-188, and 1% penicillin/streptomycin). The solution was then centrifuged at 1500 rpm for 10 minutes at room temperature. Supernatant was aspirated and the stromal vascular fraction pellet resuspended in 15 ml of room temperature fluorescence-activated cell sorting buffer. The solution was strained through a 100-μm cell strainer. Fifteen milliliters of Histopaque (Sigma Aldrich), a commercially available density gradient separation medium, was added to a new 50-ml conical-bottom centrifuge bottle, and the strained cell solution was overlaid on top of the Histopaque in a 1:1 ratio. Solution was centrifuged at 1450 rpm for 15 minutes at room temperature with acceleration set to low and deceleration settings inactivated. The cloudy interface was transferred to a new conical-bottom centrifuge bottle and the tube was filled with fluorescence-activated cell sorting buffer. The solution was centrifuged at 1300 rpm at 4°C for 5 minutes. The supernatant was then aspirated and the pellet resuspended in 500 μl of fluorescence-activated cell sorting buffer in preparation for fluorescence-activated cell sorting. | Michael T. Longaker, M.D., M.B.A., Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University Medical Center, 257 Campus Drive, Stanford, Calif. 94305-5418, longaker@stanford.edu. | ||||||||||||||
| 119 | Tevlin | 2016 | Stromal Vascular Fraction (SVF) | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | collagenase type I | Sigma | Hank’s Balanced Salt Solution (Cellgro, Manassas, Va.) | 0.08% | 37 | 60 | gentle agitation | FACS | Tevlin R, McArdle A, Brett E, Chung MT, Paik K, Seo EY, Walmsley GG, Duldulao CR, Atashroo D, Zielins E, Vistnes S, Chan CKF, Wan DC, Longaker MT. A Novel Method of Human Adipose-Derived Stem Cell Isolation with Resultant Increased Cell Yield. Plast Reconstr Surg. 2016 Dec;138(6):983e-996e. doi: 10.1097/PRS.0000000000002790. PMID: | Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211–228. | The new and conventional methods of adipose-derived stem cell isolation differ in two key areas: the collagenase digestion buffer constituents and the use of an orbital shaker. The collagenase digestion buffer includes calcium chloride, DNAse, bovine serum albumin, poloxamer-188, and hydroxyethyl piperazineethanesulfonic acid. These additional constituents result in a more hospitable environment for the cells released from adipose tissue during mechanochemical dissociation, in comparison with the conventional collagenase digestion buffer, which offers little support to the stromal vascular fraction cells. | Conventional Method—In the conventional method, stromal vascular fraction was isolated as described previously by Zuk et al.6 Briefly, raw lipoaspirates were washed and treated with 0.075% collagenase type I (Sigma Aldrich) in Hank’s Balanced Salt Solution (Cellgro, Manassas, Va.) for 1 hour at 37°C with gentle agitation. The cellular pellet was treated with Histopaque density gradient separation medium as described above and then resuspended in 500 μl of fluorescence-activated cell sorting buffer in preparation for fluorescence-activated cell sorting. | Michael T. Longaker, M.D., M.B.A., Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University Medical Center, 257 Campus Drive, Stanford, Calif. 94305-5418, longaker@stanford.edu. | ||||||||||||||||||||
| 120 | Herrmann | 2016 | SVF | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | surgical | minced into small pieces. | collagenase type II | Sigma | DMEM containing 4.5 g/L glucose (Gibco) and with addition of 3.5 % bovine serum albumin (Sigma-Aldrich) | 1 mg/ml | 37 | kept on a rocker | Digested tissue was centrifuged at 700 ×g for 10 min, the supernatant removed and the cell pellet resuspended in PBS containing 5 mM EDTA. Cells were subsequently filtered through 100 μm and 70 μm cell strainers and centrifuged again at 700 ×g for 10 min | cells were washed in PBS/ EDTA and spun down at 700 ×g for 10 min. The cell pellet, referred to as stromal vascular fraction (SVF), was resuspended in αMEM containing PenStrep and 10 % FBS and cell count determined. | 100 μm and 70 μm cell strainers | The pellet was resuspended in ACK lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA) for erythrocyte lysis. | Herrmann M, Bara JJ, Sprecher CM, Menzel U, Jalowiec JM, Osinga R, Scherberich A, Alini M, Verrier S. Pericyte plasticity – comparative investigation of the angiogenic and multilineage potential of pericytes from different human tissues. Eur Cell Mater. 2016 Apr 10;31:236-49. doi: 10.22203/ecm.v031a16. PMID: 27062725. | Isolation of SVF from human adipose tissue Abdominal adipose tissue (AT) was harvested from female human donors undergoing plastic surgery (n = 4, mean age 53.5 y, range 46-69 y). Written informed consent of the patient and approval by the local ethical authorities was given (Basel, EK 78/07). Harvested tissue was transferred to sterile containers and stored at 4 °C in αMEM containing 10 % FBS, PenStrep (100 U/mL) until dissection within 24 h. Adipose tissue was detached from surrounding fibrotic and skin tissue, washed in PBS and minced into small pieces. Digestion of tissue was performed by incubation with an equal volume of collagenase type II (Sigma-Aldrich, used at 1 mg/mL in DMEM containing 4.5 g/L glucose (Gibco) and with addition of 3.5 % bovine serum albumin (Sigma-Aldrich)). During the 45 min of incubation time, tissue was kept on a rocker at 37 °C. Digested tissue was centrifuged at 700 ×g for 10 min, the supernatant removed and the cell pellet resuspended in PBS containing 5 mM EDTA. Cells were subsequently filtered through 100 μm and 70 μm cell strainers and centrifuged again at 700 ×g for 10 min. The pellet was resuspended in ACK lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA) for erythrocyte lysis. After 10 min incubation, cells were washed in PBS/ EDTA and spun down at 700 ×g for 10 min. The cell pellet, referred to as stromal vascular fraction (SVF), was resuspended in αMEM containing PenStrep and 10 % FBS | *Address for correspondence: Sophie Verrier AO Research Institute Davos Clavadelerstrasse 8 CH-7270 Davos Platz Switzerland Telephone Number: +41 814142448 FAX Number: +41 814142288 E-mail: sophie.verrier@aofoundation.org | |||||||||||||||||||
| 121 | van Dongen, | 2016 | Stromal Vascular Fraction | MFAT | Manual | Human | SubQ. | Liposuction | SVF | van Dongen JA, Stevens HP, Parvizi M, van der Lei B, Harmsen MC. The fractionation of adipose tissue procedure to obtain stromal vascular fractions for regenerative purposes. Wound Repair Regen. 2016 Nov;24(6):994-1003. doi: 10.1111/wrr.12482. Epub 2016 Oct 21. PMID: 27717133. | Liposuction and FAT procedure Adipose tissue harvesting was performed with a Sorenson lipo-harvesting cannula (Tulip, Medical Products, San Diego, CA) during normal liposuction procedures in eleven patients (Figure S1, Supporting Information). Adipose tissue was harvested after infiltration with 500 mL-modified Klein’s solution (per 500 mL of saline, 20 mL of lidocaine, 2% Epinefrine 1 : 200.000 and 2 mL of bicarbonate was added). For the FAT procedure, harvested adipose tissue was centrifuged at 3,000 rpm with a 9.5 cm radius fixed angle rotor for 2.5 minutes (Medilite, Thermo Fisher Scientific, Waltham, MA) at room temperature (RT) after decantation. After one round of centrifugation, the oil and infiltration fluid fractions were discarded, yielding condensed lipoaspirate. One sample of 10 mL of condensed lipoaspirate that was centrifuged only once was used as a control, from heron referred as “control.” One sample of 10 mL of condensed lipoaspirate was used for mechanical dissociation. For mechanical dissociation, two 10 mL syringes with a total volume of 10 mL of condensed lipoaspirate were connected to the Fractionator, a Luer to Luer transfer with three 1.4 mm holes (Tulip) (Figure S2, Supporting Information). Mechanical dissociation was performed by pushing the lipoaspirate through the Fractionator forward and backwards thirty times (Video S1, Supporting Information). After mechanical dissociation, the adipose tissue was centrifuged again at 3,000 rpm with a 9.5 cm radius fixed angle rotor for 2.5 minutes at RT (Medilite). This resulted in four different fractions: an oily fraction, a SVF (from heron referred as “FAT-SVF”) and an aqueous fraction containing a small pellet fraction (Figure 1). | Dr. Hieronymus P. Stevens, MD, PhD, Bergman Clinics, Rijswijk Braillelaan 5 2289 CL, Rijswijk, the Netherlands. Tel: 131624810440; Fax: 010-4471884; Email: stevens.hp@gmail.com | |||||||||||||||||||||||||||||
| 122 | Carstens | 2017 | Stromal Vascular Fraction | Stromal Vascular Fraction | Semi-Automated | Human | SubQ. Abdominal | Liposuction | 250 to 350 cm3 | GMP Grade Collagenase GIDzyme | GID | 125ml of Lactate Ringer’s Solution, | 200 CDU/ml | GID SVF-1 | 37 | 40 min | incubated benchtop orbital shaker (MaxQ 4450, Fisher Scientific) | collagenase was neutralized by addition of 2.5% solution v/v human serum albumin (Baxter Healthcare, Deerfield, IL) | Centrifugation 800g 10 min | The cell suspensionwas then administered using a 26 gauge needle | automatedfluorescencecytometer(LUNA-STEM | >/= 70% | SVF | Carstens MH, Gómez A, Cortés R, Turner E, Pérez C, Ocon M, Correa D. Non-reconstructable peripheral vascular disease of the lower extremity in ten patients treated with adipose-derived stromal vascular fraction cells. Stem Cell Res. 2017 Jan;18:14-21. doi: 10.1016/j.scr.2016.12.001. Epub 2016 Dec 8. PMID: 27984756. | SVF is a heterogeneous population ofMNCs that includes adipose-derived stem cells (ADSCs) of mesenchymal phenotype (analogous to MSCs), endothelial progenitor cells (EPCs), hemopoietic progenitors, monocytes, leukocytes and pericytes (Amos et al., 2008; Nguyen et al., 2016; Guo et al., 2016). Pericytes represent the perivascular phenotype of native MSCs (Crisan et al., 2008; da Silva Meirelles et al., 2008, 2006; Sacchetti et al., 2007) and constitute a key cell component of SVF during angiogenesis, as they stabilize nascent blood vessels (Armulik et al., 2005, 2011; von Tell et al., 2006). | 2.3. Surgical procedure and SVF preparation The SVF cells were obtained after enzymatic digestion of surgically harvested adipose tissue. Liposuction was performed from the flanks and abdomen with the yield of dry fat per case ranging from 250 to 350 cm3. The lipoaspiratewas collected directly into a sterile tissue-processing canister (GID SVF-1, Louisville, CO, USA) for tissue dissociation and processing under closed conditions at all times and following the manufacturer’s instructions. Itwas firstwashed three timeswith sterile Lactated Ringer’s Solution inside the canister to remove red cells and residual oils, and then dissociatedwithGMP-grade collagenase (GIDzyme, GID, Louisville, CO, USA) in 125ml of Lactate Ringer’s Solution, at a concentration of 200 CDU/ml of total volume. The mixture was dissociated for 40 min by placing the canister inside an incubated benchtop orbital shaker (MaxQ 4450, Fisher Scientific) at 37 °C and 150 rpm. After dissociation, the collagenase was neutralized by addition of 2.5% solution v/v human serum albumin (Baxter Healthcare, Deerfield, IL) and then centrifuged (Sorvall ST40, Fisher Scientific) for 10 min at 800g. The resulting SVF cell pellet at the bottom of the device was removed using a 6-inch #14 gauge spinal needle connected to a 20 ml syringe with 15 ml Hartmann solution. Ten microliters of SVF were taken from the final suspension and submitted for differential staining. Two samples were then passed through an image cytometer (ADAM MC, Portsmouth, NH, USA) for counting of MNCs and to assess cell viability. The cell suspensionwas then administered using a 26 gauge needle into the plane between the gastrocnemius and soleusmuscles in a pattern of injections (22 per muscle, 11 in the external and 11 in the internal gastrocnemius, each one 1.5 cm to 2 cm apart) of equal volume each (0.5 ml), on either side of the midline (Fig. 1B). and cell count determined. SVF were either subjected to MACS® enrichment of pericytes or seeded at a density of 3,000 cells/cm2 in αMEM containing 10 % FBS, PenStrep and 5 ng/mL bFGF for adherence selection of MSCs. | ⁎ Correspondence to: M. Carstens, Division of Plastic Surgery, Hospital Metropolitano Vivian Pellas, Managua, Nicaragua. ⁎⁎ Correspondence to: D. Correa, Department of Orthopaedics Surgery, Diabetes Research Institute, University of Miami, Miller School of Medicine, Miami, FL 33136, United States. E-mail addresses: michaelcarstens@mac.com (M.H. Carstens), dxc821@med.miami.- edu (D. Correa). | ||||||||||||||
| 123 | Jones | 2017 | Adipose Derived. SVF. Adipose Stromal Cells. Adipose Stem Cells | SVF | Manual | Human | SubQ | Liposuction | 15 ml per 50 cc centrifuge tube | Type I Collagenase | Worthington Cat # LS004196 | PBS | 1 mg/ml | 50 cc centrifuge tube | 37 | 60 | 1 min vigorous shake evry 5 to 10 min | Centrifuge 300 x g 5min | 2 x 300g 5min | The expected cell yield ranges from 1.6 x 105 to 1.1 x 106 cells per ml of liposuction tissue. | Trypan Blue | Adipose Stromal Cells | Culture – isolation of ASC | Jones RB, Strong AL, Gimble JM, Bunnell BA. Isolation and Primary Culture of Adult Human Adipose-derived Stromal/Stem Cells. Bio Protoc. 2017 Mar 5;7(5):e2161. doi: 10.21769/BioProtoc.2161. PMID: 34458474; PMCID: PMC8376564. | 1. Warm water bath to 37 °C. 2. Collect lipoaspirate from operating room in a sterile specimen collection container. Lipoaspiration samples commonly drain directly into vacuum containers in the operative field. These containers are sterile and can be safely transported to the laboratory where cell isolation can occur. Keep lipoaspirate at room temperature until ready to isolate cells and maintain sterile conditions when transferring to the tissue culture hood. 3. Prepare digestion solution (see Recipe 1). Filter the solution under sterile conditions with a vacuum filter. 4. Prepare resuspension solution (see Recipe 2). Filter the solution under sterile conditions with vacuum filter. Place resuspension solution in a 37 °C water bath. 5. Warm digestion solution and PBS in the water bath to 37 °C. 6. Place lipoaspirate and prepared solutions into a sterile cell culture hood and perform the following steps under sterile conditions. 7. Separate lipoaspirate into sterile 50 ml centrifuge tubes by placing about 15 ml of lipoaspirate in each tube. Allow lipoaspirate to separate into blood and fat layers. The fat layer will float to the top of the tube, and the blood layer will be underneath it (see Figure 1). 8. Pierce the fat layer with the aspiration pipette, and aspirate the blood layer using a 2 ml aspiration pipet. Avoid aspirating the fat layer, as this will clog the aspiration pipette (see Figure 1). 9. Wash the fat layer with PBS until the lipoaspirate is a light reddish-yellow hue. The volume of PBS required will vary depending on how much blood is present in the lipoaspirate sample. The volume of PBS needed will range from 5 to 20 ml, and multiple washes will be required until the wash solution is clear. Centrifuge the solution at 300 x g at room temperature for 5 min to separate the wash from the fat layer. 10. Aspirate the PBS wash below the fat layer, and avoid aspirating the fat layer and the cell pellet at the bottom of the centrifuge tube (see Figure 1). 11. Incubate lipoaspirate in a volume of digestion solution equivalent to the original amount of lipoaspirate (15 ml) for 60 min at 37 °C. For example, 15 ml of lipoaspirate should be incubated in 15 ml of digestion solution. Mix the tube(s) by vigorously shaking the tube for 1 min and mix the tubes intermittently every 5 to10 min throughout the digestion time. 12. Neutralize collagenase by adding an equal amount of resuspension solution to the digestion solution used. For example, 15 ml of lipoaspirate incubated in 15 ml of digestion solution should be neutralized by 15 ml of resuspension solution. 13. Centrifuge the solution at 300 x g at room temperature for 5 min. This step separates floating adipocytes, oil, fat, collagenase solution and the SVF. 14. Discard supernatant, leaving about 5 ml of solution and the SVF cell pellet. Resuspend the SVF cell pellet in 10 ml PBS. Copyright © 2017 The Authors; exclusive licensee Bio-protocol LLC. 5 www.bio-protocol.org/e2161 Vol 7, Iss 05, Mar 05, 2017 DOI:10.21769/BioProtoc.2161 15. Repeat steps A12 and A13 two times or until pellet appears white. These washes remove red blood cells and other non-adherent cells without compromising the yield of ASCs. 16. Resuspend the SVF in 5 ml of resuspension solution. Pipet 10 μl of cells into a 1.5 ml microcentrifuge tube. 17. Add 10 μl of trypan blue. Mix the solution with the cells by pipetting up and down 3-5 times. 18. Add 10 μl of the mixed trypan blue-cell solution to the hemocytometer. Count cells in the four outer quadrants. 19. As an alternative to trypan blue, mix 1 μl of 1:1 solution of 100 μg/ml ethidium bromide and 100 μg/ml acridine orange with 25 μl of cells. Add 10 μl of the mixture to the hemocytometer. Count the cells under fluorescent microscopy. 20. Use the following equations to determine the total number of cells per milliliter and the total number of cells: Where, ‘2’ represents the dilution factor, ‘10,000’ represents the hemocytometer constant. 21. Plate the cells on 145 cm2 culture dishes at a density of 100 cells per cm2 in 20 ml of CCM (approximately 14,500 cells per 145 cm2 culture dish). The expected cell yield ranges from 1.6 x 105 to 1.1 x 106 cells per ml of liposuction tissue. | For correspondence: bbunnell@tulane.edu | |||||||||||||||
| 124 | Williams | 2017 | Stromal Vascular Fraction. SVF | SVF | Automated. Tissue Genesis Icellator | Human | SubQ | Liposuction | 30 to 60 cc | Adipase – Purified Crude Collagenase | Proprietary enzyme made from Worthington Collagenase | Lactated Ringers | Proprietary Bowl | 37 | 35 | Oscillation | Centrifugation | 1x | 250 micron | 1 to 2.6 x10E5 SVF cells per gm fat | > 80 % | SVF | Human Vasacular Graft Sodding | WILLIAMS, S.K. PE Kosnik, LB Kleinert, EM Vossman, K J. Lye, MH Stein. 2013 Adipose Stromal Vascular Fraction Cells Isolated using an Automated Point of Care System Improve the Patency of ePTFE Vascular Grafts, Tissue Eng Part A. 2013 Jun;19(11-12):1295-302. PMID: 23350681 | Briefly, the ‘‘TGI 1000’’ (Fig. 1) is an automated, point- bF1 of-care cell isolation instrument. It has a graphical user interface that requires minimal operator training and has a small footprint designed for the operating room. It comprises an instrument and one-time-use disposable that is functionally closed, which interfaces with the stand-alone desktop instrument to extract SVF from adipose tissue in about an hour. During this process, the tissue is washed and mixed with a proprietary digestive enzyme. The resultant SVF cell population is further washed and separated by centrifugation from the adipose cells and tissue matrix. The cells are suspended in a user-provided medium and provided in a syringe for immediate economical use. A typical yield is about 50 million viable SVF cells suspended in *30mL of medium. Samples of this cell suspensate are taken for analysis. | Stu Williams. cvregen@gmail.com | |||||||||||||||
| 125 | Bajek | 2017 | Stromal Vascular Fraction. SVF | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Surgical Liposuction | Surgical samples minced into small fragments (1–2 mm) | Type I Collagenase | Sigma Aldrich | sterile PBS (phosphate-buffered saline, Sigma–Aldrich, Germany) containing 5 mg/ml amphotericin B, 100 U/ml penicillin, and 100mg/ml streptomycin (Sigma–Aldrich) | 0.08% | 37 | 30 | Centrifugation. 170g 5min. | 2x | 100 micron cell strainer (BD) | ASC Characterization | The average number of ASCs isolated from 1 g of solid adipose tissue or 1ml of lipoaspirate was 2.9 105 2.4 105, 1.1 105 0.8 105, and 1.2 105 0.7 105, respectively, for ASCsR, ASCsPAL, and ASCsLAL. | SVF | In Vitro Characterization | Morphology FACS | Bajek A, Gurtowska N, Olkowska J, Maj M, Kaźmierski Ł, Bodnar M, Marszałek A, Dębski R, Drewa T. Does the Harvesting Technique Affect the Properties of Adipose-Derived Stem Cells?-The Comparative Biological Characterization. J Cell Biochem. 2017 May;118(5):1097-1107. doi: 10.1002/jcb.25724. Epub 2017 Jan 5. PMID: 27608167. | Procedure of ASCs isolation was initiated by washing adipose tissue with sterile PBS (phosphate-buffered saline, Sigma–Aldrich, Germany) containing 5 mg/ml amphotericin B, 100 U/ml penicillin, and 100mg/ml streptomycin (Sigma–Aldrich) to remove blood cells. Subsequently, vessels and fibrous tissue were gently removed and the adipose tissue was minced into small fragments (1–2 mm). Liposuction aspirate was washed with sterile PBS containing antibiotics (as above) to eliminate blood cells, saline, and anesthetics used during tumescent liposuction. Further isolation steps were the same for both resected fat and lipoaspirate. The washed adipose tissue fragments underwent enzymatic digestion, with type I collagenase at a final concentration of 0.075% (Sigma–Aldrich) at 37°C for 30 min. The digestion was interrupted with the addition of an equal volume of complete culture medium DMEM/Ham’s F12 (Dulbecco’s modified essential medium, Sigma–Aldrich) supplemented with 10% FBS, 5mg/ml amphotericin B, 100 mg/ml penicillin, and 100 mg/ml streptomycin (Sigma– Aldrich). Afterwards, samples were centrifuged twice at 170g for 5 min at room temperature, and SVF (Stromal Vascular Fraction) pellet was resuspended in complete DMEM/Ham’s F12 medium. Suspended cells were then passed through 100 mm cell strainer (BD Bioscience, US AP) to separate the undigested tissue fragments and once again centrifuged. SVF pellet was suspended in complete culture medium and isolated cells were plated at an equivalent to 15 gml of fat tissue/liposuction aspirate per T25 flask. The cells were cultured at 37°C, under 5% CO2. The medium was changed every second day until the cells reached 80–90% confluence. | *Correspondence to: Anna Bajek, PhD, Department of Tissue Engineering, Nicolaus Copernicus University, Karlowicza 24, 85-092 Bydgoszcz, Poland. E-mail: a_bajek@wp.pl | |||||||||||||||
| 126 | Brown | 2017 | SVF | SVF | Automated GID System | Human | SubQ | Liposuction | Collagenase Type I | Worthington | Lactated Ringers | 200CDU/ml | Proprietary GID SVF-2 | 38 | 40 | shaker 150 rpm | HSA | Centrifugation 800 x g 10 min | ammonium– chloride–potassium (ACK) lysing buffer (KD Medical RGF- 3015) and dark incubated at ambient room temperature | Nucleocounter NC-100 | 1 x 106 SVF cells/gm fat | 82% | SVF | Brown JC, Shang H, Li Y, Yang N, Patel N, Katz AJ. Isolation of Adipose-Derived Stromal Vascular Fraction Cells Using a Novel Point-of-Care Device: Cell Characterization and Review of the Literature. Tissue Eng Part C Methods. 2017 Mar;23(3):125-135. doi: 10.1089/ten.TEC.2016.0377. PMID: 28177263. | On arrival in the laboratory, tissue processing occurred within a laminar flow hood even though the device enables a closed-system processing method from start to finish. The tissue was washed with warm solution containing 20 mg/mL of ciprofloxacin and 5000 U/L of injectable heparin until the effluent was clear, leaving ‘‘dry’’ and uniformly yellow adipose tissue inside the mesh compartment (Fig. 2). The canister was weighed, and tissue mass and volume determined for further processing. An equal volume of 37 C LR was then added to the canister to create a ‘‘total catalytic volume’’ (TCV). The adipose was disaggregated using type I collagenaseCLS-1(Worthington, Lakewood, NJ) at 200CDU/ mL of the TCV. The collagenase was injected into the canister through a sterile 0.22-mm filter (Millex-MP, Millipore, Cork, Ireland). The device with adipose, buffer, and collagenase was then placed into a 38 Cincubated shaker for 40min at 150 rpm. After disaggregation, human albumin solution was added to achieve a concentration of 2.5%to reduce collagenase activity, and the lipid-laden, mature adipocytes were subsequently separated fromthe remaining cellular and liquid fractions using centrifugation. The device was centrifuged at 800 g for 10min using a standard laboratory centrifuge (Sorvall ST40; Thermo Fisher Scientific, MA) and rotor (BIOLiner swinging-bucket; Thermo Fisher Scientific, MA). The SVF-2 protocol was slightly modified in respect to the centrifugation secondary to difference in lipoaspirate sample size. The SVF-2 device was centrifuged at 600 · 3 g for 6 min, stirred with the internal impeller, and then centrifuged for another 4min for a total of 10min of centrifugation time. After centrifugation of the dissociated cell suspension, the resulting floating layer (i.e., buoyant layer of mature adipocytes and free oil) was easily removed via aspiration through a device port, leaving an aqueous layer and the SVF cell pellet. The cell pellet was accessed through the device’s central port with a 14G spinal needle and suspended in sterile LR (10mL for SVF-1 cells; 4mL for SVF-2 cells) (Fig. 2). | Address correspondence to: Adam J. Katz, MD, FACS Division of Plastic and Reconstructive Surgery Department of Surgery College of Medicine University of Florida 1200 Newell Drive ARB/R4-171 Gainesville, FL 32610 E-mail: adam.katz@surgery.ufl.edu | |||||||||||||||
| 127 | Coatti | 2017 | Mesenchymal Stromal Cells and Pericytes | Adipose Stromal Cells (ASC) | Manual | Human | surgical | minced with scalpels | 1 mg/mL of collagenase type II (Sigma Aldrich) in a 1:3 ratio (tissue weight: collagenase volume) | Sigma | diluted in DMEM-F12 supplemented with 20% fetal bovine serum (FBS), 1% non-essential amino acids (GIBCO) and 1% antibiotics/antimycotics (GIBCO) | 1 mg/mL | 37 | 30 to 40 min | shaker at 250 rpm | passed through a 70 μm strainer and incubated in blood lysis solution for 5–10 min. PBS was added in a 2:1 ratio and the solution was filtered again through a 40 μm strainer. In this step, the sample was divided into two parts: one corresponding to mesenchymal stromal cells, immediately plated and the other corresponding to the pericytes, prepared for cell sorting. For that purpose, cells were incubated with conjugated antibodies for CD-34 (Percp-Cy5.5), CD-45 (FITC), CD-56 (APC) and CD 146 (PE), all from BD Biosciences. DAPI was added just before the analysis, and all cells positive for DAPI were excluded (dead cells). Pericytes (CD146+/CD34 −/ CD45−/CD56-) were sorted into a 24 well plate at a density of 20.000 cells/cm2. | 100, 70 and 40 micron cell strainer | For that purpose, cells were incubated with conjugated antibodies for CD-34 (Percp-Cy5.5), CD-45 (FITC), CD-56 (APC) and CD 146 (PE), all from BD Biosciences. DAPI was added just before the analysis, and all cells positive for DAPI were excluded (dead cells). Pericytes (CD146+/CD34 −/ CD45−/CD56-) were sorted into a 24 well plate at a density of 20.000 cells/cm2. | Coatti GC, Frangini M, Valadares MC, Gomes JP, Lima NO, Cavaçana N, Assoni AF, Pelatti MV, Birbrair A, de Lima ACP, Singer JM, Rocha FMM, Da Silva GL, Mantovani MS, Macedo-Souza LI, Ferrari MFR, Zatz M. Pericytes Extend Survival of ALS SOD1 Mice and Induce the Expression of Antioxidant Enzymes in the Murine Model and in IPSCs Derived Neuronal Cells from an ALS Patient. Stem Cell Rev Rep. 2017 Oct;13(5):686-698. doi: 10.1007/s12015-017-9752-2. PMID: 28710685. | Valadares, M. C., Gomes, J. P., Castello, G., Assoni, A., Pellati,M., Bueno, C., et al. (2014). Human adipose tissue derived Pericytes increase life span in Utrn (tm1Ked) Dmd (mdx) /J mice. Stem cell reviews. doi:10.1007/s12015-014-9537-9. Crisan,M.,Yap, S., Casteilla, L., Chen, C.-W., Corselli, M., Park, T. S., et al. (2008). A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell, 3(3), 301–313. doi:10. 1016/j.stem.2008.07.003. | Cell Preparation The adipose tissue sample was obtained from one healthy female donor that underwent total hysterectomy, after informed consent. The tissue sample was processed according to a protocol described elsewhere [10, 14]. Briefly, the sample was washed extensively with PBS, minced with scalpels and incubated with 1 mg/mL of collagenase type II (Sigma Aldrich) in a 1:3 ratio (tissue weight: collagenase volume) diluted in DMEM-F12 supplemented with 20% fetal bovine serum (FBS), 1% non-essential amino acids (GIBCO) and 1% antibiotics/antimycotics (GIBCO) and incubated for 30– 40 min in a shaker, at 250 RPM and 37 °C. Cells were then passed through a 70 μm strainer and incubated in blood lysis solution for 5–10 min. PBS was added in a 2:1 ratio and the solution was filtered again through a 40 μm strainer. In this step, the sample was divided into two parts: one corresponding to mesenchymal stromal cells, immediately plated and the other corresponding to the pericytes, prepared for cell sorting. For that purpose, cells were incubated with conjugated antibodies for CD-34 (Percp-Cy5.5), CD-45 (FITC), CD-56 (APC) and CD 146 (PE), all from BD Biosciences. DAPI was added just before the analysis, and all cells positive for DAPI were excluded (dead cells). Pericytes (CD146+/CD34 −/ CD45−/CD56-) were sorted into a 24 well plate at a density of 20.000 cells/cm2. Pericytes and MSCs were cultured and trypsinized after they reached 80% confluence. For cell passaging, a density of 5000 cells per cm2 was considered. MSCs were cultured in DMEM-F12 medium containing 1% Glutamax (Gibco), 20% FBS (Gibco), 1% non-essential amino acids (Gibco) and 1% antibiotics/antimycotics (Gibco). Pericytes were cultured in EBM-2 medium (Lonza). For in vitro and in vivo experiments, cells in 8th passage were used. | Mario Sergio Mantovani biomsm@uel.br Lucia Inês Macedo-Souza luciness@usp.br Merari F. R. Ferrari merari@usp.br 1 Human Genome and Stem Cell Research Center, Department of Genetics and Evolutionary Biology, Biosciences Institute, University of Sao Paulo (USP), Rua do Matāo 106, São Paulo, SP CEP 05508-030, Brazil 2 Department of Epidemiology and Biostatistics, CaseWestern Reserve University, Cleveland, OH, USA 3 Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA 4 Department of Pathology, University Federal of Minas Gerais, Belo Horizonte, MG, Brazil 5 Department of Statistics, University of Sao Paulo (USP), Sao Paulo, SP, Brazil 6 Departament of Biology, University of Londrina, Londrina, PR, Brazil | |||||||||||||||||||
| 128 | Cerino | 2017 | SVF | ASC | Manual | Human | Liposuction | Collagenase II | Worthington | PBS | 0.08% | 37 | 60 min | Continuous shaking | Centrifugation 1,500 RPM 10 min | 1 x PBS | 100 and 70 micron | Nucleated cells stained with crystal violet were counted in an improved Neubauer chamber. | SVF | Prevascularization | FACS | Cerino G, Gaudiello E, Muraro MG, Eckstein F, Martin I, Scherberich A, Marsano A. Engineering of an angiogenic niche by perfusion culture of adipose-derived stromal vascular fraction cells. Sci Rep. 2017 Oct 27;7(1):14252. doi: 10.1038/s41598-017-13882-3. PMID: 29079730; PMCID: PMC5660248. | Stromal Vascular Fraction Cell Isolation. Liposuctions were obtained from nine healthy donors undergoing plastic surgery after informed consent and according to a protocol approved by the Ethical Committee of Basel University Hospital. All investigations conform to the declaration of Helsinki. The adipose solution was digested with 0.075% w/v collagenase type II (Worthington Biochemical Corporation) in phosphate buffered saline (PBS, Invitrogen) at 37 °C undergoing continuous shaking for 60 min. After centrifugation at 1,500 rpm for 10 min, the floating lipid-rich layer was discarded and the cellular pellet was washed once with PBS. Cell suspension was strained through a 100 μm followed by a 70 μm nylon-mesh in order to remove fibrous debris. The resulting SVF cells were then re-suspended in growth medium consisting of DMEM high glucose (Sigma–Aldrich), 10% v/v fetal bovine serum (FBS, HyClone), 1% v/v penicillin/ streptomycin, 1% v/v glutamine, and 1% v/v hepes (all from Sigma-Aldrich). Nucleated cells stained with crystal violet (Sigma–Aldrich) were counted in a Neubauer chamber. Freshly isolated SVF cells were frozen in 10% v/v dimethyl sulfoxide (DMSO), 90% v/v FBS medium and stored in liquid nitrogen. | Departments of Biomedicine and Surgery, University of Basel and University Hospital of Basel, 4031, Basel, Switzerland. Correspondence and requests for materials should be addressed to A.M. (email: anna.marsano@usb.ch) | |||||||||||||||||
| 129 | Huh | 2017 | Stromal Vascular Cells | Insufficient Data for Classification | Huh JY, Park J, Kim JI, Park YJ, Lee YK, Kim JB. Deletion of CD1d in Adipocytes Aggravates Adipose Tissue Inflammation and Insulin Resistance in Obesity. Diabetes. 2017 Apr;66(4):835-847. doi: 10.2337/db16-1122. Epub 2017 Jan 12. PMID: 28082459. | Huh JY, Kim JI, Park YJ, et al. A novel function of adipocytes in lipid antigen presentation to iNKT cells. Mol Cell Biol 2013;33:328–339 | The protocol for cell preparation and antibody staining from adipose tissue and spleen was described previously (17) | 1School of Biological Sciences, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea 2Department of Biophysics and Chemical Biology, Seoul National University, Seoul, Korea Corresponding author: Jae Bum Kim, jaebkim@snu.ac.kr. | |||||||||||||||||||||||||||||||||
| 130 | Priglinger | 2017 | Stromal Vascular Cells | ASC | Manual | Human | abdominal wall | liposuction | collagenase NB4 | Serva | PBS containing Ca2+/Mg2+ and 25 mmol/L N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES; Sigma | “0.2 U/mL | blood bag (Macopharma) | 37 | 60 | moderate shaking | “centrifugation at 1200 g for 7 min,” | 100-µm cell strainer (Greiner) | 100 mL erythrocyte lysis buffer for 5 min at 37°C to eliminate red blood cells | Cell number was determined using trypan blue exclusion and quantification in a cell counter (TC-20, Bio-Rad. 1.2 to 2.5 x E105 peer ml fat | trypan blue exclusion in a cell counter (TC-20, Bio-Rad) | SVF | Culture. Lipedema exploration | “Samples were analyzed on a FACSCalibur (BD).” | Priglinger E, Wurzer C, Steffenhagen C, Maier J, Hofer V, Peterbauer A, Nuernberger S, Redl H, Wolbank S, Sandhofer M. The adipose tissue-derived stromal vascular fraction cells from lipedema patients: Are they different? Cytotherapy. 2017 Jul;19(7):849-860. doi: 10.1016/j.jcyt.2017.03.073. Epub 2017 Apr 25. PMID: 28454682. | Wolbank S, Peterbauer A, Fahrner M, et al. Dose-Dependent Immunomodulatory Effect of Human Stem Cells from Amniotic Membrane: A Comparison with Human Mesenchymal Stem Cells from Adipose Tissue. Tissue Engineering. 2007;13(6):1173-1183. doi:10.1089/ten.2006.0313 | “SVF isolation was performed as modified from Wolbank et al. [29]. Briefly, 100 mL of liposuction material was transferred to a blood bag (Macopharma) and washed with an equal volume of phosphate-buffered saline (PBS) to remove blood and tumescence solution. Af-terward, for tissue digestion PBS was replaced with 0.2 U/mL collagenase NB4 (Serva) dissolved in 100 mL PBS containing Ca2+/Mg2+ and 25 mmol/L N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES; Sigma), and the blood bag was incubated at 37°C under moderate shaking (180 rpm) for 1 h. The digested tissue was transferred into 50-mL tubes. After centrifugation at 1200 g for 7 min, the cell pellet was incubated with 100 mL erythrocyte lysis buffer for 5 min at 37°C to eliminate red blood cells. The supernatant was aspirated after centrifugation for 5 min at 500 g. The pellet was washed with PBS and fil-tered through a 100-µm cell strainer (Greiner). After another centrifugation step at 500 g for 5 min, the supernatant was removed, and the isolated SVF cells were cultured in endothelial growth medium (EGM-2) at 37°C, 5% CO2 and 95% air humidity or” | Correspondence: Eleni Priglinger, PhD, Ludwig Boltzmann Institute for Experimental and ClinicalTraumatology, Krankenhausstraße 7, 4010 Linz, Austria. E-mail: Eleni.Priglinger@trauma.lbg.ac.at (Received 17 January 2017; accepted 21 March 2017 | |||||||||||||
| 131 | Haynes | 2018 | Stromal Vascular Fraction. SVF | SVF | Manual | Human | omental (OM) and subcutaneous (SC) adipose tissue (AT) | Surgical | Finely mince 5 g of the AT in 5 mL of a collagenase solution in a scintillation vial at room temperature, using two pairs of scissors | Collagenase Type 1 | Krebs Ringer Bicarbonate-Buffered Solution (KRBBS): 135 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium sulfate, 0.4 mM potassium phosphate dibasic, 5.5 mM glucose, 1 mM adenosine, 0.01% antibiotic/antimycotic mix (50 μg/mL of penicillin, 50 μg/mL of streptomycin, 30 μg/mL of gentamicin, 15 ng/mL of amphotericin) and 10 mM HEPES (pH = 7.4). | 1 mg/ml | Scintillation Vial | 37 | 60 | continuous shaking | Filter digest through a 250 micron Nylon mesh. 1 g separation of adipocytes from SVF, 5 min RT. Repeat 2 x. | 250 micron nylon mesh | Subsequent isolation of endothelial cells and studies of EC to adipocyte differentiation | Haynes, B.A., Huyck, R.W., James, A.J., Carter, M.E., Gaafar, O.U., Day, M., Pinto, A., Dobrian, A.D. Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue. J. Vis. Exp. (137), e57804, doi:10.3791/57804 (2018). | Adipose Stromal Vascular Fraction Isolation NOTE: The study included a cross-sectional cohort of morbidly obese type 2 diabetic (T2D) and non-diabetic subjects, aged 18–65 years, undergoing bariatric surgery at the Sentara Metabolic and Weight Loss Surgery Center (Sentara Medical Group, Norfolk, VA). Exclusion criteria included an autoimmune disease including type 1 diabetes mellitus, conditions requiring chronic immunosuppressive therapy, anti-inflammatory medications, thiazolinendiones, active tobacco use, chronic or acute infections, or a history of malignancy treated within the last 12 months. T2D was defined as a fasting plasma glucose of 126 mg/dL or greater, a glucose of 200 mg/dL or greater after a 2 h glucose tolerance test, or the use of antidiabetic medications. 1. Collect human omental (OM) and subcutaneous (SC) adipose tissue (AT) from human subjects undergoing bariatric surgery. 2. Keep the OM and SC AT in separate vials containing Hank’s Buffered Salt Solution with 50 μg/mL of penicillin/streptomycin at room temperature immediately after tissue extraction. 3. Carefully clean and remove fibrotic and cauterized sections of the tissue by using 70% ethanol swabbed scissors and tweezers when the sample arrives at the lab after the tissue extraction. 4. Weigh the adipose tissue and partition it into 5 g (or less) aliquots for the collagenase digestion. 5. Finely mince 5 g of the AT in 5 mL of a collagenase solution in a scintillation vial at room temperature, using two pairs of scissors. 6. Add an additional 10 mL of collagenase solution to the minced tissue for 15 mL total. 7. Digest the samples for 1 h, at 37 °C, in a water bath with continuous shaking. 8. Cut the tip off a 20 mL syringe to make a blunt end. 9. Cut a 9 cm x 9 cm square from a 250 μm mesh and push it halfway into a 50 mL conical tube using the blunt end syringe. 10. Pour the samples into the 20 mL blunt end syringe and filter through the 250 μm nylon mesh into the 50 mL conical tube to separate adipocytes and stromal vascular cells from undigested tissue. NOTE: Do not use more than 5 g of AT per 50 mL conical tube, as the mesh will get clogged due to excess fibrotic undigested material. 11. Wash out the scintillation vial with 10 mL of KRBBS and pour it through the filter to collect residual cells. 12. Incubate the filtered samples for 5 min at room temperature. Note: This step is important to allow for the adipocytes to float at the top of the tube and some of the stromal vascular cells to settle on the bottom of the tube. 13. Utilize a 20 mL syringe and a 20 G x 6 in pipetting needle to remove the stromal vascular fraction layer and transfer it to a clean 50 mL conical tube. 14. Add 10 mL of KRBBS and allow the samples to sit on the bench for 5 min, at room temperature. 15. Repeat steps 2.12–2.14 twice. Note: These steps will ensure that there is no contamination of the stromal vascular cells with the floating adipocytes. 16. Keep the stromal vascular fractions from both depots on ice for further processing, as described in the steps below. Note: Do not leave the cells on ice for more than 1 h, as this may have an impact on the cell viability. The adipocytes can be flash-frozen in liquid nitrogen for long-term storage or used fresh for experiments. | Anca D. Dobrian at dobriaad@evms.edu | |||||||||||||||||||
| 132 | Liu | 2018 | SVF | Adipose Stromal Cells | Manual | Human | SubQ | Liposuction | 10 ml | Collagenase type I solution,. | PBS | 0.00 | 50 cc Conical | 37 | 30 to 60 min | water bath | 5 mL of DMEM containing 10% FBS and 1% P/S. Penicillin and streptomycin solution (P/S), 1%. | The digested sample is centrifuged at 1500 rpm (1300 × g) for 5 min to yield the high-density stromal vascular fraction (SVF) layer containing ASCs | Shake the tube vigorously to disrupt the SVF and mix the cells. Repeat the centrifugation step. Aspirate all the supernatant without disturbing the cells, add 5 mL of DMEM containing 10% FBS and 1% P/S to resuspend SVF and filter through a 100-μm cell strainer to remove cellular debris. | Add 1 mL of NH4Cl solution to resuspend the SVF pellet and incubate at room temperature for 10 min to lyse red blood cells. | SVF | Liu G, Chen X. Isolating and Characterizing Adipose-Derived Stem Cells. Methods Mol Biol. 2018;1842:193-201. doi: 10.1007/978-1-4939-8697-2_13. PMID: 30196410. | 1. Human adipose tissue is obtained by liposuction aspiration or surgical procedures (see Note 1). Fat samples can be preserved on ice under aseptic condition for no more than 6 hours prior to processing. 2. The tissue sample is placed into sterile 50-mL centrifuge tubes and washed extensively with equal volumes of prewarmed PBS containing 1% P/S for three times to remove the fat debris, oil, blood cells and the local anesthetics. 3. Transfer 10 mL of the fat sample into a new 50-mL tube with equal volume of 0.075% collagenase type I prepared in PBS and digested in water bath (37 °C) for 30–60 min (see Note 2). 4. Enzyme is neutralized with 5 mL of DMEM containing 10% FBS and 1% P/S. Pipette the sample up and down several times to disintegrate the adipose aggregates. 5. The digested sample is centrifuged at 1500 rpm (1300 × g) for 5 min to yield the high-density stromal vascular fraction (SVF) layer containing ASCs. 6. Shake the tube vigorously to disrupt the SVF and mix the cells. Repeat the centrifugation step. 7. Aspirate the collagenase and DMEM solution above SVF carefully. 8. Add 1 mL of NH4Cl solution to resuspend the SVF pellet and incubate at room temperature for 10 min to lyse red blood cells. 9. Pipette with 10 mL of PBS containing 1% P/S and centrifuge at 1500 rpm for 5 min.10. Aspirate all the supernatant without disturbing the cells, add 5 mL of DMEM containing 10% FBS and 1% P/S to resuspend SVF and filter through a 100-μm cell strainer to remove cellular debris. 11. Plate the filtered cells into a Φ 100 mm cell culture dish and add an additional 5 mL of DMEM containing 10% FBS and 1% P/S. Incubate the cells in a humidified CO2 incubator at 37 °C and 5% CO2. 12. 48 hours after plating, remove the culture medium and wash the cells with prewarmed PBS containing 1% P/S thoroughly to clean tissue fragments and nonadherent cells. Replace 10 mL of fresh DMEM containing 10% FBS and 1% P/S, and maintain the cells at 37 °C and 5% CO2. 13. Change the medium every three days until the remaining cells reach 80–90% confluence. Then aspirate the medium and wash the cells with 2 mL of PBS for two times. 14. Replace the PBS with 1 mL of 0.25% Trypsin-EDTA solution and place the culture dish in the incubator for 5 min. | ||||||||||||||||||
| 133 | Hepler | 2018 | SVF | Adipose Stromal Cells (ASC) | manual | Mouse | scissors | Collagenase D Roche 11088882001 | Roche | (1X HBSS, 1.5% BSA | 1 mg/ml | 50 mL Falcon tube | shaking water bath | The solution of digested tissue was passed through a 100 mm cell strainer, diluted to 30 mL with 2% FBS in PBS, and centrifuged at 500 x g for 5 min. The supernatant was aspirated and red blood cells in the SVF pellet were lysed by brief incubation in 1 mL RBC lysis buffer (Sigma). Next, the mixture was diluted to 10 mL with 2% FBS in PBS, passed through a 40 mm cell strainer, and then centrifuged at 500 x g for 5 min. The supernatant was aspirated, and cells were resuspended in blocking buffer (2% FBS/PBS containing anti-mouse CD16/CD32 Fc Block (1:200)). | 100 micron 40 micron | 1 mL RBC lysis buffer (Sigma). | SVF | Single-cell RNA-sequencing | Hepler C, Shan B, Zhang Q, Henry GH, Shao M, Vishvanath L, Ghaben AL, Mobley AB, Strand D, Hon GC, Gupta RK. Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice. Elife. 2018 Sep 28;7:e39636. doi: 10.7554/eLife.39636. PMID: 30265241; PMCID: PMC6167054. | Isolation of adipose stromal vascular fraction (SVF) and flow cytometry Adipose tissue was minced with scissors in a 1.5 mL tube containing 200 mL of digestion buffer (1X HBSS, 1.5% BSA, and 1 mg/mL collagenase D) and then transferred to a 50 mL Falcon tube contain- ing 10 mL digestion buffer. The mixture was incubated in a 37°C shaking water bath for 1 hr. The solution of digested tissue was passed through a 100 mm cell strainer, diluted to 30 mL with 2% FBS in PBS, and centrifuged at 500 x g for 5 min. The supernatant was aspirated and red blood cells in the SVF pellet were lysed by brief incubation in 1 mL RBC lysis buffer (Sigma). Next, the mixture was diluted to 10 mL with 2% FBS in PBS, passed through a 40 mm cell strainer, and then centrifuged at 500 x g for 5 min. The supernatant was aspirated, and cells were resuspended in blocking buffer (2% FBS/PBS containing anti-mouse CD16/CD32 Fc Block (1:200)). Primary antibodies were added to the cells in blocking buffer for 15 min at 4°C in the dark. After incubation, the cells were washed once with 2% FBS/PBS and then resuspended in 2% FBS/PBS for sorting. Cells were sorted for collection using a BD Biosciences FACSAria cytometer or analyzed using a BD Biosciences LSR II cytometer (UTSW Flow Cytometry Core Facility). Flow cytometry plots were generated with FlowJo (V10). | |||||||||||||||||||||
| 134 | Joseph | 2018 | Adipose Progenitor Cell | Adipose Stromal Cells (ASC) | Manual | Mouse | Epididymal, inguinal, and axillary fat pads | surgical | collagenase II | Gibco | 37 | 60 | filtering through 70 m cell strainer. | metabolite imbalances in APCs as a potential contributor to adipose tissue disequilibrium in DMD | Joseph J, Cho DS, Doles JD. Metabolomic Analyses Reveal Extensive Progenitor Cell Deficiencies in a Mouse Model of Duchenne Muscular Dystrophy. Metabolites. 2018 Oct 3;8(4):61. doi: 10.3390/metabo8040061. PMID: 30282911; PMCID: PMC6315702. | Adipose Progenitor Cell Isolation Epididymal, inguinal, and axillary fat pads were dissected immediately after mice were sacrificed. The fat pads were incubated in Hank’s Balanced Salt Solution (Gibco) containing 3% (w/v) bovine serum albumin (Gold Biotechnology; St. Louis, MO, USA) for 15 min at room temperature, followed by centrifugation at 200 g for 7 min. The fat pads were digested with 0.1% (w/v) collagenase II (Gibco; Waltham, MA, USA) in 37 C for 60 min followed by filtering through 70 m cell strainer. Adipose progenitor cells (APCs) were then enriched using Adipose Tissue Progenitor Isolation Kit (Miltenyi Biotec; Bergisch Gladbach, Germany) according to the manufacturer’s protocol. | |||||||||||||||||||||||||
| 135 | Schwalie | 2018 | SVF | Adipose Stromal Cells (ASC) | Manual | Mouse | Subcutaneous and visceral adipose tissue depots | surgical | finely minced using scissors | collagenase | Sigma-Aldrich #C6885-1G | (25 mM NaHCO3, 12 mM KH2PO4, 1.2 m MgSO4, 4.8 mM KCl, 120 mM NaCl, 1.4 mM CaCl2, 5 mM Glucose, 2.5% BSA, pH = 7.4 | 37 | 60 | agitation | Following a 10-min centrifugation at 400 g at room temperature, floating mature lipid-filled adipocytes were aspirated and the cell suspension was filtered through a 40-μm cell strainer to ensure a single cell preparation. Next, a red blood cell lysis was performed by incubating the pelleted cells in the red blood cell lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 5 min, followed by two washes (a 5-min centrifugation at 400g, room temperature) with FACS buffer (PBS with 3% fetal bovine serum (FBS) (Gibco #10270-106), 1 mM EDTA, 1% penicillin–streptavidin (Gibco #15140122)) | 40 micron | red blood cell lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 5 min | SVF | scRNA-seq | Schwalie PC, Dong H, Zachara M, Russeil J, Alpern D, Akchiche N, Caprara C, Sun W, Schlaudraff KU, Soldati G, Wolfrum C, Deplancke B. A stromal cell population that inhibits adipogenesis in mammalian fat depots. Nature. 2018 Jul;559(7712):103-108. doi: 10.1038/s41586-018-0226-8. Epub 2018 Jun 20. PMID: 29925944. | Isolation of the mouse SVF. Subcutaneous and visceral adipose tissue depots were dissected from male and female 8–11-week-old wild-type, DLK1–RFP or male ob/ob C57BL/6J mice into ice-cold PBS. The tissue was finely minced using scissors, transferred into collagenase (Sigma-Aldrich #C6885-1G, 2 mg/ml of collagenase buffer (25 mM NaHCO3, 12 mM KH2PO4, 1.2 m MgSO4, 4.8 mM KCl, 120 mM NaCl, 1.4 mM CaCl2, 5 mM Glucose, 2.5% BSA, pH = 7.4)) and incubated for 1 h at 37 °C under agitation. Following a 10-min centrifugation at 400 g at room temperature, floating mature lipid-filled adipocytes were aspirated and the cell suspension was filtered through a 40-μm cell strainer to ensure a single cell preparation. Next, a red blood cell lysis was performed by incubating the pelleted cells in the red blood cell lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 5 min, followed by two washes (a 5-min centrifugation at 400g, room temperature) with FACS buffer (PBS with 3% fetal bovine serum (FBS) (Gibco #10270-106), 1 mM EDTA, 1% penicillin–streptavidin (Gibco #15140122)). | |||||||||||||||||||
| 136 | Schwalie | 2018 | SVF | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | Liberase TM Roche #05401119001 | Roche | 50 ml lipoaspirate per 40 ml 1% human albumin in DPBS −/− | 0.28 U/ml | 37 | 45 | agitation | digested tissue was mixed with 1% human albumin (CSL Behring) in DPBS −/− (Gibco #14190094) (50 ml lipoaspirate per 40 ml 1% human albumin in DPBS −/−) and shaken vigorously to liberate the stromal cells (procedure performed twice). The aqueous phase was recovered and centrifuged (all centrifugations performed at 400 g for 5 min at room temperature). The cell pellet was resuspended in 15 ml remaining buffer and filtered through a 100-μm and then a 40-μm cell strainer to ensure a single-cell preparation, centrifuged and resuspended in 5 ml of 5% human albumin (CSL Behring). | 100 micron. 40 micron | VersaLyse solution (Beckman Coulter #A09777) | SVF | scRNA-seq | Schwalie PC, Dong H, Zachara M, Russeil J, Alpern D, Akchiche N, Caprara C, Sun W, Schlaudraff KU, Soldati G, Wolfrum C, Deplancke B. A stromal cell population that inhibits adipogenesis in mammalian fat depots. Nature. 2018 Jul;559(7712):103-108. doi: 10.1038/s41586-018-0226-8. Epub 2018 Jun 20. PMID: 29925944. | Isolation of human SVF. Fresh lipoaspirates were washed twice with DPBS with calcium and magnesium (Gibco #14040091) in 100-ml syringes (VWR International #720-2528) (50 ml lipoaspirate per 40 ml DPBS with calcium and magnesium) and incubated with 0.28 U/ml of liberase TM (Roche #05401119001) for 45 min at 37 °C under agitation. The digested tissue was mixed with 1% human albumin (CSL Behring) in DPBS −/− (Gibco #14190094) (50 ml lipoaspirate per 40 ml 1% human albumin in DPBS −/−) and shaken vigorously to liberate the stromal cells (procedure performed twice). The aqueous phase was recovered and centrifuged (all centrifugations performed at 400 g for 5 min at room temperature). The cell pellet was resuspended in 15 ml remaining buffer and filtered through a 100-μm and then a 40-μm cell strainer to ensure a single-cell preparation, centrifuged and resuspended in 5 ml of 5% human albumin (CSL Behring). The viability and the number of nucleated cells in the cell suspension obtained was determined using a Nucleostainer, after which a red blood cell lysis was performed using VersaLyse solution (Beckman Coulter #A09777) according to the manufacturer’s recommendations. | |||||||||||||||||||
| 137 | Burl | 2018 | Stromal Vascular Cells | Adipose Stromal Cells (ASC) | Manual | Mouse | Epididymal and inguinal white adipose tissues (WAT) | surgical | minced | type 2 collagenase | Worthington | Hanks’ balanced salt’s solution (HBSS; Gibco; Sigma-Aldrich) containing sodium bicarbonate, 10 mM HEPES (pH 7.4, Gibco; Sigma-Aldrich) and 0.5% fatty acid free bovine serum albumin (FF-BSA; Gemini Bio-products, West Sacramento, CA). Ethylenediaminetetraacetic acid (EDTA) was added to a final concentration of 10 mM and the tissues were incubated for an additional 5 minutes to promote full dissociation of SVCs | eWAT: 1 mg/mL; iWAT: 2 mg/mL | 37 | 30 | Dissociated cells were filtered through a cell strainer, washed with a PBS buffer containing 1 mM EDTA, 2.5mM HEPES, and 10% heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals; Flowery Branch, GA) (FACS buffer), then centrifuged at 500 x g for 10 minutes at 4 C. After removing the supernatant, pellets containing the stromal vascular faction were incubated in red blood cell lysis buffer (containing sodium bicarbonate, ammonium chloride, and 0.5 M EDTA; pH 7.4) for 5 minutes at room temperature, then passed through a 100 mm sterile cell strainer and collected by centrifugation at 500xg for at 4 C for 10 minutes. Cellular debris was removed by gradient centrifugation in Iodixanol solution (52.2% and 50% Iodixanol; OptiPrep; Sigma-Aldrich; St. Louis, MO) overlaid with FACS buffer. Cells recovered in the FACS buffer layer were pelleted, washed, and resuspended in PBS containing 5% FF-BSA. | filter then 100 micron filter | Single Cell RNA Sequencing | Burl, R.B., Ramseyer, V.D., Rondini, E.A., Pique-Regi, R., Lee, Y.H., Granneman, J.G., 2018. Deconstructing adipogenesis induced by beta3- adrenergic receptor activation with single-cell expression profiling. Cell Metabolism 28(2):300e309 e304. | Lee, Y.H., Petkova, A.P., Mottillo, E.P., and Granneman, J.G. (2012). In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding. Cell Metab. 15, 480–491. | Ethylenediaminetetraacetic acid (EDTA) was added to a final concentration of 10 mM and the tissues were incubated for an additional 5 minutes to promote full dissociation of SVCs | Isolation of Stromal Vascular Cells from Mouse eWAT and iWAT Epididymal and inguinal white adipose tissues (WAT) from control and CL-treated mice were surgically removed after CL treatment and processed for stromal vascular cell (SVC) isolation. Tissues of 4 mice were pooled for SVC isolation, as previously described (Lee et al., 2012). Briefly, following dissection, eWAT and iWAT were washed with PBS, minced, and digested with type 2 collagenase (eWAT: 1 mg/mL; iWAT: 2 mg/mL; Worthington Biochemical) in Hanks’ balanced salt’s solution (HBSS; Gibco; Sigma-Aldrich) containing sodium bicarbonate, 10 mM HEPES (pH 7.4, Gibco; Sigma-Aldrich) and 0.5% fatty acid free bovine serum albumin (FF-BSA; Gemini Bio-products, West Sacramento, CA) for 30 minutes at 37 C. Ethylenediaminetetraacetic acid (EDTA) was added to a final concentration of 10 mM and the tissues were incubated for an additional 5 minutes to promote full dissociation of SVCs. Dissociated cells were filtered through a cell strainer, washed with a PBS buffer containing 1 mM EDTA, 2.5mM HEPES, and 10% heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals; Flowery Branch, GA) (FACS buffer), then centrifuged at 500 x g for 10 minutes at 4 C. After removing the supernatant, pellets containing the stromal vascular faction were incubated in red blood cell lysis buffer (containing sodium bicarbonate, ammonium chloride, and 0.5 M EDTA; pH 7.4) for 5 minutes at room temperature, then passed through a 100 mm sterile cell strainer and collected by centrifugation at 500xg for at 4 C for 10 minutes. Cellular debris was removed by gradient centrifugation in Iodixanol solution (52.2% and 50% Iodixanol; OptiPrep; Sigma-Aldrich; St. Louis, MO) overlaid with FACS buffer. Cells recovered in the FACS buffer layer were pelleted, washed, and resuspended in PBS containing 5% FF-BSA. | |||||||||||||||||||
| 138 | Winnier | 2019 | Freshly isolated, uncultured, autologous adipose derived regenerative cells (ADRC) | SVF | Lab Kit Transpose RT system | Human | Abdominal | Lipoaspiration | 25 ml | Matrase Reagent | Ingeneron | Lactated Ringers | processing tube | 39 | 30 | repetitive acceleration and deceleration | 200 micron Filter Centrifugation 600 x g 5 minutes | 2 x Centrifugation 600g 5 minutes Saline as wash material | 200 micron | NucleoCounter NC-200 | Winnier GE, Valenzuela N, Peters-Hall J, Kellner J, Alt C, Alt EU. Isolation of adipose tissue derived regenerative cells from human subcutaneous tissue with or without the use of an enzymatic reagent. PLoS One. 2019 Sep 3;14(9):e0221457. doi: 10.1371/journal.pone.0221457. PMID: 31479463; PMCID: PMC6719836. | Processing was performed as described in the tissue processing procedure section found in the 11011E Transpose RT Instructions for Use (11011–01 IFU; InGeneron, Inc.) (Fig 2), com- prising the following steps: (i) The recovered lipoaspirate (25 ml) was loaded together with 2.5 ml reconstituted Matrase (in case of Transpose RT / Matrase isolation) and lactated Ringer solution (preheated to 39˚ C) into a processing tube up to the MAX FILL line (Fig 2A). (ii) The filled processing tubes were subjected in an inverted position inside the Transpose RT system to repetitive acceleration and deceleration for 30 minutes at 39˚ C (Fig 2B). (iii) The processed lipoaspirate solution was filtered through a 200 μm filter (Fig 2C) and transferred into a wash tube. After filling the wash tube with saline (room temperature) up to the MAX FILL line, the cells were separated from the rest of the tissue by centrifugation at 600g for 5 minutes at room temperature (Fig 2D). Then, the SVF/ ADRCs (approximately 2 ml) were extracted through a swabable luer vial adapter at the bottom of the wash tube, and the remaining substances (fat, debris and liquid) were discarded (Fig 2E). (v) The cells were returned into the empty wash tube and (after adding fresh saline up to the MAX FILL line) centrifugated again for 5 minutes (Fig 2F). (vi) The previous washing step was repeated (Fig 2G and 2H). (vii) Finally, the con- centrated SVF/ ADRCs (approximately 3 ml) were extracted (Fig 2I) and slowly pushed through a luer coupler into a new sterile syringe for further application to the patient. | |||||||||||||||||||
| 139 | Haenel | 2019 | fresh, uncultured, unmodified, autologous adipose-derived regenerative cells (UA-ADRCs) | SVF | Lab Kit Transpose RT system | Pig | Surgical | Minced | 6 t0 10 ml | Matrase Reagent | Ingeneron | Lactated Ringers | processing tube | 39 | 60 | repetitive acceleration and deceleration | 200 micron Filter Centrifugation 600 x g 5 minutes | 2 x Centrifugation 600g 5 minutes Saline as wash material | 200 micron | Hemocytometer | On average, 0.98 × 106 ± 0.10 × 106 nucleated cells were isolated from each gram of adipose tissue (cell yield), | 93.3% ± 0.4% cells were viable (live cell yield) | Myocardial infarction | CFU | Haenel A, Ghosn M, Karimi T, Vykoukal J, Shah D, Valderrabano M, Schulz DG, Raizner A, Schmitz C, Alt EU. Unmodified autologous stem cells at point of care for chronic myocardial infarction. World J Stem Cells. 2019 Oct 26;11(10):831-858. doi: 10.4252/wjsc.v11.i10.831. PMID: 31692971; PMCID: PMC6828597. | Winnier G, Valenzuela N, Alt C, Alt EU. Isolation of adipose tissue derived regenerative cells from human subcutaneous tissue with or without the use of enzymatic reagent; 2018. Preprint. Available from: bioRxiv 485318. Cited February 17, 2019. [DOI: 10.1101/485318] | Cells were isolated during catheterization at 4 wk after MI induction (i.e., at time point T1). Following incision with a scalpel, 12-25 g of subcutaneous adipose tissue was harvested from the nuchal region of each pig. The tissue was divided into aliquots of about 6-10 g each. Then, each aliquot was processed using the Transpose RT system (InGeneron, Houston, TX, United States) for isolating UA-ADRCs from adipose tissue. To this end, each aliquot was minced and incubated together with enzymatic Matrase Reagent (InGeneron) for 1 h under agitation in the processing unit at 39°C, according to the manufacturer’s instructions for use. For counting cells, they were stained with fluorescent nucleic acid stain (SYTO13; Life Technologies, Grand Island, NY, United States) following the manufacturer’s instructions, and then counted using a hemocytometer under an Eclipse Ti-E inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan) using a PlanFluor 10 × objective [numerical aperture (NA) = 0.3] (Nikon). The viability of UA-ADRCs was determined by preparing a 3:1 dilution of the cell suspension in 0.4% Trypan Blue solution. Nonviable cells were counted using a hemocytometer under the same microscope, and were correlated to the number of viable nucleated cells. | ||||||||||||||
| 140 | Baer | 2019 | adipose-derived stromal/stem cells (ASCs) | adipose-derived stromal/stem cells (ASCs) | Manual | Human | perirenal | Surgical | minced by using two scalpels and disintegrated as small as possible | collagenase | CellSystems | 1 mg/ml | 37 | 60 min | continuous agitation | centrifugation at 300 g | sedimented cells were washed with phosphate-bu ered saline | 125 micron plastic mesh | density gradient centrifugation with Bicoll | Flow Cytometry | Baer PC, Koch B, Hickmann E, Schubert R, Cinatl J Jr, Hauser IA, Geiger H. Isolation, Characterization, Differentiation and Immunomodulatory Capacity of Mesenchymal Stromal/Stem Cells from Human Perirenal Adipose Tissue. Cells. 2019 Oct 29;8(11):0. doi: 10.3390/cells8111346. PMID: 31671899; PMCID: PMC6928994. | Cell Isolation and Culture Human MSCs were isolated from perirenal adipose tissue from 15 different donors. The tissue was minced by using two scalpels and disintegrated as small as possible for cell isolation. The minced tissue was then digested at 37◦C with collagenase (1 mg/mL; CellSystems, Troisdorf, Germany (from Worthington)) and continuous agitation for 60 min. Cells were then separated from the remaining fibrous material and the floating adipocytes by centrifugation at 300×g. The pelleted cells were collected, and the procedure was repeated twice. The sedimented cells were washed with phosphate-buffered saline (PBS) and filtered through a 125-µm plastic mesh (Millipore, Schwalbach, Germany). Erythrocyte contamination, if required, was reduced by density gradient centrifugation with Bicoll (Biochrom, Berlin, Germany), because high erythrocyte contamination was found to decrease ASCs adherence and proliferation markedly. It was observed in previous experiments, that a preceding density gradient separation provided a better yield of adipose-derived stromal/stem cells (ASCs) than treatment with an erythrocyte lysing buffer [24]. Finally, the cells were plated for initial cell culture and cultured at 37 ◦C in an atmosphere of 5% CO2 in humid air. Dulbecco’s modified Eagle’s medium (DMEM; Sigma, Taufkirchen, Germany) was used with a physiologic glucose concentration (100 mg/dl) supplemented with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany) as the standard culture medium. Primary isolated cells were intensively washed with PBS after 18–24 h of initial plating to remove debris and non-adherent cells. The medium was then replaced every three to four days. Subconfluent cells were passaged by trypsinization. In all experiments we used cultured prASCs in early passages (between 2 and 5). | |||||||||||||||||||
| 141 | Merrick | 2019 | Stromal Vascular Cells | Adipose Stromal Cells (ASC) | Manual | Human | Abdominai fat | surgical abdominoplasty | manually minced | collagenase D (0.75 unit/ml; Roche) and dispase II (1.2 units/ml; Roche) | Roche | Dulbecco’s modified Eagle’s medium (DMEM) with Ham’s F12 medium (DMEM/F12) containing 0.4% fatty acid–free bovine serum albumin (Sigma) | 0.75 U per ml collagenase 1 g of tissue to 1ml of digestion medium | 37 | 45 | agitation | The digestion was quenched with DMEM containing 10% fetal bovine serum (FBS), | the dissociated cells were filtered twice through a single layer of gauze to remove large undigested particles and then subjected to centrifugation at 400 × g for 5min at roomtemperature (RT). The resulting supernatant containing mature adipocytes was aspirated, and the pellet, consisting of SVCs, was resuspended, passed through a 100-mM filter, and subjected to centrifugation at 400 × g for 5 min at RT. Cells were resuspended in red blood cell lysis buffer (Biolegend) for 5 min at RT and then quenched inDMEMcontaining 10% FBS. Cells were then passed through a 40-mM filter and collected by centrifugation at 400 × g for 5 min. Cells were recovered in FACS buffer [Hanks’ balanced salt solution (HBSS) containing 3% FBS; Fisher] and kept on ice for the duration of processing. | SVCs, was resuspended, passed through a 100-mM filter, centrifuged 400 x g 5 min RT | single layer gauze. 100 micron filter. 40 micron filter | red blood cell lysis buffer (Biolegend) for 5 min at RT | Stromal Vascular Cells. SVCs | Single-cell RNA-seq | Merrick D, Sakers A, Irgebay Z, Okada C, Calvert C, Morley MP, Percec I, Seale P. Identification of a mesenchymal progenitor cell hierarchy in adipose tissue. Science. 2019 Apr 26;364(6438):eaav2501. doi: 10.1126/science.aav2501. PMID: 31023895; PMCID: PMC6816238. | A particularly notable finding is that adipose progenitor cells reside in a recently discovered anatomic niche (Fig. 6C). The RI is a large tissue that surrounds many organs but has not been well studied. | Isolation of SVCs from human subcutaneous adipose The skin and cauterized edges of human tissue samples were removed to yield an intact block of subcutaneous fat that was unmanipulated by the surgical procedure. Three hundred to five hundred grams of tissue was manually minced and digested with collagenase D (0.75 unit/ml; Roche) and dispase II (1.2 units/ml; Roche) in Dulbecco’s modified Eagle’s medium (DMEM) with Ham’s F12 medium (DMEM/F12) containing 0.4% fatty acid–free bovine serum albumin (Sigma) at 37°C with agitation for 45 min and shaking for 10 s at 15-min intervals. Digestion was performed at a ratio of 1 g of tissue to 1ml of digestionmedium. The digestion was quenched with DMEM containing 10% fetal bovine serum (FBS), and the dissociated cells were filtered twice through a single layer of gauze to remove large undigested particles and then subjected to centrifugation at 400 × g for 5min at roomtemperature (RT). The resulting supernatant containing mature adipocytes was aspirated, and the pellet, consisting of SVCs, was resuspended, passed through a 100-mM filter, and subjected to centrifugation at 400 × g for 5 min at RT. Cells were resuspended in red blood cell lysis buffer (Biolegend) for 5 min at RT and then quenched inDMEMcontaining 10% FBS. Cells were then passed through a 40-mM filter and collected by centrifugation at 400 × g for 5 min. Cells were recovered in FACS buffer [Hanks’ balanced salt solution (HBSS) containing 3% FBS; Fisher] and kept on ice for the duration of processing. | |||||||||||||||
| 142 | Merrick | 2019 | Stromal Vascular Cells | Adipose Stromal Cells (ASC) | Manual | Mouse | Inguinal and axillary subcutaneous white adipose depots | surgical | manually minced | collagenase D (1.5 units/ml; Roche) and dispase II (2.4 units/ml; Roche) in DMEM/ F12 containing 0.8% fatty acid–free bovine serum albumin (Sigma) | Roche | DMEM/ F12 containing 0.8% fatty acid–free bovine serum albumin (Sigma) | collagenase 1.5 U/ml. Dispase 2.4 U/ml | 37 | 45 | agitation with vortexing 10 s every 15 min | The digestionwas quenched with DMEM/F12 containing 10% FBS, | the dissociated cells were passed through a 100-mM filter and then subjected to centrifugation at 400 × g for 5 min. The resulting supernatant containing mature adipocytes was aspirated, and the pellet, consisting of SVCs, was resuspended in red blood cell lysis buffer (Biolegend) for 5 min at RT and then quenched in DMEM/F12 containing 10% FBS. Cells were then passed through a 40-mMfilter and collected by centrifugation at 400 × g for 5 min. Cells were Merrick et al., Science 364, eaav2501 (2019) 26 April 2019 8 of 11 RESEARCH | RESEARCH ARTICLE Downloaded from https://www.science.org at University of Louisville on July 29, 2025 recovered in FACS buffer (HBSS containing 3% FBS; Fisher). | 100 micron. 40 micron | red blood cell lysis buffer (Biolegend) for 5 min at RT | Stromal Vascular Cells. SVCs | Single-cell RNA-seq | Merrick D, Sakers A, Irgebay Z, Okada C, Calvert C, Morley MP, Percec I, Seale P. Identification of a mesenchymal progenitor cell hierarchy in adipose tissue. Science. 2019 Apr 26;364(6438):eaav2501. doi: 10.1126/science.aav2501. PMID: 31023895; PMCID: PMC6816238. | Isolation of SVCs from mouse adipose Inguinal and axillary subcutaneous white adipose depots were surgically removed from WT mice and processed for SVC enrichment. Briefly, adipose tissues were manually minced and digested with collagenase D (1.5 units/ml; Roche) and dispase II (2.4 units/ml; Roche) in DMEM/ F12 containing 0.8% fatty acid–free bovine serum albumin (Sigma) at 37°Cwith agitation for 45 min and vortexing for 10 s at 15-min intervals. The digestionwas quenched with DMEM/F12 containing 10% FBS, and the dissociated cells were passed through a 100-mM filter and then subjected to centrifugation at 400 × g for 5 min. The resulting supernatant containing mature adipocytes was aspirated, and the pellet, consisting of SVCs, was resuspended in red blood cell lysis buffer (Biolegend) for 5 min at RT and then quenched in DMEM/F12 containing 10% FBS. Cells were then passed through a 40-mMfilter and collected by centrifugation at 400 × g for 5 min. Cells were recovered in FACS buffer (HBSS containing 3% FBS; Fisher). | |||||||||||||||||
| 143 | Rajbhandari | 2019 | SVF | Adipose Stromal Cells (ASC) | Manual | Mouse | Inguinal white adipose tissue (iWAT) | surgical | cut and minced with scissors | Collagenase II | Worthington Biochemical, Lakewood, NJ, USA) | 3 mg/ml | 15 ml conical tubes | 37 | 40 | gentle shaking at 100 rpm. | Following tis- sue digestion 8 ml of resuspension media (DMEM/F12 with glutamax supplemented with 15%FBS and 1% pen/strep; Thermo Scientific, CA) was added to stop enzyme activity. | The digestion mixture was passed through 100 mm cell strainer and centrifuged at 150 x g for 8 min at room temperature. The pellet was resuspended and incubated in RBC lysis buffer (Thermo Scientific, CA) for 3 min at room temperature to remove red blood cells followed by centrifugation at 150 x g for 8 min. The pellet was resuspened in resuspension media and spun down again at 150 x g for 8 min. Finally, the cell pellet was resuspended in 1 ml of 0.01% BSA (in DPBS). This final cell suspension solution was passed through a 40 mm cell strainer (Fisher Scientific, Hampton, NH, USA) to discard debris and cell number was counted for Drop-Seq application. | 100 micron 40 micron | SVF | Single-cell RNA-seq | Rajbhandari P, Arneson D, Hart SK, Ahn IS, Diamante G, Santos LC, Zaghari N, Feng AC, Thomas BJ, Vergnes L, Lee SD, Rajbhandari AK, Reue K, Smale ST, Yang X, Tontonoz P. Single cell analysis reveals immune cell-adipocyte crosstalk regulating the transcription of thermogenic adipocytes. Elife. 2019 Oct 23;8:e49501. doi: 10.7554/eLife.49501. PMID: 31644425; PMCID: PMC6837845. | adaptive immune cell adipocyte communication in the maintenance of adipose subtype identity and function | Single cell isolation from SVF Inguinal white adipose tissue (iWAT) from mice treated with saline or CL were dissected and placed on sterile 6-well tissue culture plate with ice-cold 1X DPBS. Fat pads were blotted on a napkin to removed excess liquid. Tissues were cut and minced with scissors and placed in 15 ml conical tubes containing digestion buffer (2 ml DPBS and Collagenase II at 3 mg/ml; Worthington Biochemical, Lakewood, NJ, USA) and incubated at 37°C for 40 min with gentle shaking at 100 rpm. Following tis- sue digestion 8 ml of resuspension media (DMEM/F12 with glutamax supplemented with 15%FBS and 1% pen/strep; Thermo Scientific, CA) was added to stop enzyme activity. The digestion mixture was passed through 100 mm cell strainer and centrifuged at 150 x g for 8 min at room temperature. The pellet was resuspended and incubated in RBC lysis buffer (Thermo Scientific, CA) for 3 min at room temperature to remove red blood cells followed by centrifugation at 150 x g for 8 min. The pellet was resuspened in resuspension media and spun down again at 150 x g for 8 min. Finally, the cell pellet was resuspended in 1 ml of 0.01% BSA (in DPBS). This final cell suspension solution was passed through a 40 mm cell strainer (Fisher Scientific, Hampton, NH, USA) to discard debris and cell number was counted for Drop-Seq application. | |||||||||||||||||
| 144 | Cho | 2019 | adipose tissue–derived stem cells (ASCs) | Adipose Stromal Cells (ASC) | Manual | Mouse | epididymal | surgical | collagenase II | Thermo Fisher Scientific | 37 | 60 | cells were filtered through a 70-μm cell strainer | adipose tissue–derived stem cells (ASCs) | single-cell RNA sequencing gene expression signatures | Cho DS, Lee B, Doles JD. Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling. Life Sci Alliance. 2019 Nov 25;2(6):e201900561. doi: 10.26508/lsa.201900561. PMID: 31767614; PMCID: PMC6878222. | Joseph J, Cho DS, Doles JD (2018) Metabolomic analyses reveal extensive progenitor cell deficiencies in a mouse model of duchenne | APCs were isolated from epididymal fat pads as previously described (Joseph et al, 2018) with slight modifications as follows. After tissue dissociation with collagenase II (Thermo Fisher Scientific), the cells were filtered through a 70-μm cell strainer. The cells were washed once and resuspended in 45 μl flow cytometry buffer (PBS supplemented with 2-mM EDTA and 0.5% [wt/vol] bovine serum albumin [Gold Biotechnology]). | |||||||||||||||||||||||
| 145 | Francois | 2020 | SVF | SVF | Manual | Human | SubQ | Liposuction | Collagenase NB6 | Nordmark | 0.10 or 0.25 U/mL | Puregraft Device | 37 | 15, 30 and 45 min | Orbital Shaker | Washing | Centrifugation 400 x g 5 min. in Easyflex device | 200 micron filter (BD Cell Strainer) | Number, viability angiogenesis FACS | Nucleocounter | 4.00 105 to 59.2 105 with a median value of 11.2 105 VNCs/mL AT | 70 to 80 %. Then 90% | SVF | François P, Giraudo L, Veran J, Bertrand B, Dumoulin C, Aboudou H, Grimaud F, Vogtensperger M, Velier M, Arnaud L, Lyonnet L, Simoncini S, Guillet B, Dignat-George F, Magalon J, Sabatier F. Development and Validation of a Fully GMP-Compliant Process for Manufacturing Stromal Vascular Fraction: A Cost-Effective Alternative to Automated Methods. Cells. 2020 Sep 24;9(10):2158. doi: 10.3390/cells9102158. PMID: 32987708; PMCID: PMC7598595. | The developed SVF production process, named the LG process after the name of the person who originally developed it, was conducted in a controlled cleanroom in accordance with GMP guidelines e ective for the French hospital exemption for ATMP. Most stages were performed in a sterile, closed fluid circuit in a class B cleanroom, except for open steps requiring grade A laminar airflow. All consumables and materials were marketed for cleanroom use by suppliers and validated by the person in charge of the quality control of the GMP facility. First, under laminar airflow, the harvested AT contained in a MACO bag (Macopharma) was transferred aseptically into a 250-mL Puregraft bag (Bimini Health Technologies, Solana Beach, CA, USA) using 60-mL syringes (Terumo, Tokyo, Japan).AT was washed three times, with a volume of lactate Ringer’s solution (RL; Ringer Lactate Viaflo; Baxter) equivalent to half of the total Puregraft volume, preheated at 37 C, using the Puregraft device, allowing the drainage of residual wetting/tumescent solution, red blood cells, and other debris. After discarding the waste bag of the Puregraft device, the sample was then weighed and diluted with RL 1:1 (v/v) at 37 C (A class). The diluted AT was transiently incubated in a 37 C incubator (Thermo electron corporation,Waltham, MA, USA) for 15 min before the addition of collagenase enzyme (NB6; Nordmark Biochemicals, Uetersen, Germany) directly into the Puregraft device (injection performed under laminar airflow), at concentrations of 0.10 or 0.25 U/mL for 15, 30, or 45 min at 37 C under constant agitation using an orbital shaker. Collagenase was previously sampled and validated for free germs. Then, the cell suspension was transferred aseptically into a new medical device (Easyflex+, Macopharma) to allow for cellular concentration by centrifugation (5 min at 400 g) (Thermo Fisher Scientific, Waltham, MA, USA) and filtration using a cell strainer with porosity of 200 m (Becton Dickinson, Franklin Lakes, NJ, USA) to avoid any remaining debris. The filtration and washes to remove residual enzymes were performed with 2:1 (v/v) RL or saline solution (Baxter) supplemented with 5% of human serum albumin (HAS; LFB Biomedicaments, Courtaboeuf, France) (NaCl 5% HAS). All steps after digestion were aseptically performed in a closed circuit established with subsequent transfers of the cell suspension between transfer bags through sterile connections created using a sterile tubing welder (TSCD; Terumo BCT, Lakewood, CO, USA). Finally, the active substance was concentrated and resuspended with 10 to 15 mL of RL or NaCl 5% HSA. Figure S1 provides images of the process and GMP-compliant criteria. | ||||||||||||||||
| 146 | Teshima | 2020 | SVF | Adipose Stromal Cells (ASC) | Manual | Canine | Falciform Ligament | Surgical | Minced | Collagenase Type I | Sigma | 37 | 45 | Intermittant | Centrifugation. Resuspended pellet filtered through 100 micron nylon mesh | Insulin production by attached cells. | Teshima T, Okamoto K, Dairaku K, Nagashima T, Michishita M, Suzuki R, Matsumoto H, Koyama H. Generation of Insulin-Producing Cells from Canine Adipose Tissue-Derived Mesenchymal Stem Cells. Stem Cells Int. 2020 Oct 18;2020:8841865. doi: 10.1155/2020/8841865. PMID: 33133196; PMCID: PMC7591982. | Differentiation of Canine AT-MSCs into IPCs. Differentiation was performed according to a previously reported protocol [17], with modifications. Canine AT-MSCs at passage 3 were seeded in conventional 6-well attachment plates (1:5 × 106 cells/well) or 96-well ultralow attachment plates (Thermo Fisher Scientific) with or without μ-pieces (cellnest μ-piece, Fujifilm) (1:0 × 105 cells/well with or without μ-pieces of 0.05mg/well) as 3D culture. AT-MSCs were cultured using step 1 (from day 0 to day 2) medium containing FBS-free DMEM with high glucose (25mM) and 0.5mM β-mercaptoethanol (Sigma-Aldrich). At day 3, small clusters that formed in the 96-well ultralow attachment plates were transferred into 6-well ultralow attachment plates (Corning). Next, cells were cultured in step 2 (from day 3 to day 10) medium containing FBS-free DMEM with high glucose (25mM), 1% glutamine supplement (GlutaMAX, Thermo Fisher Scientific), 1% nonessential amino acid (FUJIFILM Wako), 20 ng/ml of recombinant human basic fibroblast growth factor (FUJIFILM Wako), 20 ng/ml of recombinant human epidermal growth factor (FUJIFILM Wako), 1% N2 supplement (Thermo Fisher Scientific), 1% B27 supplement (Thermo Fisher Scientific), and 10nM exendin-4 (Sigma-Aldrich). Finally, cells were cultured in step 3 (from day 11 to day 21) medium containing FBS-free DMEM with high glucose (25mM), 1% N2 supplement, 1% B27 supplement, 10nM exendin-4, 10mM nicotinamide (FUJIFILM Wako), 10 ng/ml betacellulin (Sigma-Aldrich), 50 ng/ml recombinant human activin-A (FUJIFILM Wako), and 50 ng/ml recombinant human hepatocyte growth factor (FUJIFILM Wako) | Isolation and Expansion of AT-MSCs. Adipose tissue was aseptically collected from falciform ligament fat of three anaesthetized dogs (males; mean age, 1.5 years; mean body weight, 10.4 kg). The tissue was washed extensively in phosphate buffer solution (PBS), minced, and digested with collagenase type I (Sigma-Aldrich) at 37°C for 45min with intermittent shaking. After washing with PBS and centrifuging, the pellets containing the stromal vascular fraction were resuspended, filtered through a 100 μm nylon mesh, and incubated overnight in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Nichirei Bioscience) and a 1% antibiotic-antimycotic solution (Thermo Fisher Scientific) in a humidified atmosphere with 5% CO2 at 37°C. Unattached cells were removed by changing the medium, and the attached cells were washed twice with PBS. Thereafter, the medium was replaced every 3-4 days. At 80-90% confluence, the cells were detached with trypsin- EDTA solution (Sigma-Aldrich) and passaged repeatedly. | ||||||||||||||||||||||
| 147 | Vijay | 2020 | Stromal Vascular Fraction. SVF | Adipose Stromal Cells (ASC) | Manual | Human | greater omentum corresponding to VAT and abdominal subcutaneous fat compartment (SAT) | surgical | collagenase type 1 | Not Stated | Krebs-Ringer-Henseleit (KRH) | 37 | 45 | Cell suspensions containing mature adipocytes and SVF were then filtered with a nylon mesh and washed 3 times with KRH buffer. solution containing the SVF was centrifuged 1500 rpm for 5 minutes. The pellet was washed with pre-adipocyte growth medium (PGM) (DMEM-F12 supplemented with 10% calf serum, 1% penicillin-streptomycin, 17μM pantothenic acid, 33μM biotin, 100μM ascorbic acid and 2,5 μm/ml amphoB) followed by a second centrifugation. SVF cells were then cryopreserved using freezing medium (PGM supplemented with 40% FBS and 10% DMSO). The medium was added to the pellet and was frozen with a temperature gradient (-1 °C/ minutes) and stored in liquid nitrogen until analysis. Cells from the SVF were thawed and suspended in PBS-0.1% BSA. They were centrifuged at 1500 rpm for 5 minutes. Erythrocyte lysis buffer was added for 5 minutes to the suspension to get rid of red blood cells. The suspension was centrifuged again at 1500 rpm for 5 minutes. Primary antibody (CD34, PE, eBioscience) was added to the samples and incubated for 60 minutes at 4°C in the dark. PBS-0.1% BSA was added to wash the suspension followed by 80 μm filtration with nylon mesh to remove debris. The suspension was then transfer to a 10ml polypropylene tube, centrifuged at 1500 rpm for 5 minutes and the pellet was suspended with PBS-0.1% BSA. | 1. Nylon Mesh. 2. 80 micron nylon mesh | Erythrocyte lysis buffer was added for 5 minutes to the suspension to get rid of red blood cells | Isolation of CD34+/CD34- SVF cells by fluorescence activated cell sorting | FACS. Single cell RNA sequencing | FACS | SVF | Single cell RNA sequencing | FACS. Single cell RNA sequencing | Vijay, Jinchu & Gauthier, Marie-Frédérique & Biswell, Rebecca & Louiselle, Daniel & Johnston, Jeffrey & Cheung, Warren & Belden, Bradley & Pramatarova, Albena & Biertho, Laurent & Gibson, Margaret & Simon, Marie-Michelle & Djambazian, Haig & Staffa, Alfredo & Bourque, Guillaume & Laitinen, Anita & Nystedt, Johanna & Vohl, Marie-Claude & Fraser, Jason & Pastinen, Tomi & Grundberg, Elin. (2020). Single-cell analysis of human adipose tissue identifies depot- and disease-specific cell types. Nature Metabolism. 2. 1-13. 10.1038/s42255-019-0152-6. | “Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucos-e metabolism and lipolysis. J. Biol. Chem. 239: 375-380.” | Sample Preparation SVF and mature adipocytes were obtained as follows: adipose tissue was digested within 30 min of collection with collagenase according to a modification of the Robdell method51. Briefly, adipose tissue samples were digested with collagenase type 1 in Krebs-Ringer-Henseleit (KRH) buffer for 45 minutes at 37°C. Cell suspensions containing mature adipocytes and SVF were then filtered with a nylon mesh and washed 3 times with KRH buffer. The nature of the buoyancy adipocytes allows them to float to the surface. Mature adipocytes were aliquoted and the remaining solution containing the SVF was centrifuged 1500 rpm for 5 minutes. The pellet was washed with pre-adipocyte growth medium (PGM) (DMEM-F12 supplemented with 10% calf serum, 1% penicillin-streptomycin, 17μM pantothenic acid, 33μM biotin, 100μM ascorbic acid and 2,5 μm/ml amphoB) followed by a second centrifugation. SVF cells were then cryopreserved using freezing medium (PGM supplemented with 40% FBS and 10% DMSO). The medium was added to the pellet and was frozen with a temperature gradient (-1 °C/ minutes) and stored in liquid nitrogen until analysis. Whole adipose tissue samples were following collection quickly frozen in liquid nitrogen and stored until analysis. Cells from the SVF were thawed and suspended in PBS-0.1% BSA. They were centrifuged at 1500 rpm for 5 minutes. Erythrocyte lysis buffer was added for 5 minutes to the suspension to get rid of red blood cells. The suspension was centrifuged again at 1500 rpm for 5 minutes. Primary antibody (CD34, PE, eBioscience) was added to the samples and incubated for 60 minutes at 4°C in the dark. PBS-0.1% BSA was added to wash the suspension followed by 80 μm filtration with nylon mesh to remove debris. The suspension was then transfer to a 10ml polypropylene tube, centrifuged at 1500 rpm for 5 minutes and the pellet was suspended with PBS-0.1% BSA. Viability staining solution has been added 15 minutes before the sorting process. Viability control was performed with live/dead cells to ascertain that the staining solution worked well and for gate adjustment. OneComp Ebeads control was also used to provide positive/negative control for the antibody. The samples were sorted with the BD FACSAria II (BD, San Diego, California, USA) with the following parameters: Cell size, graininess, viability, singlet cell and CD34+/CD34-. Dead and non-singlet cells were automatically discarded. Cells that were positive for CD34 and negative for CD34 were collected in two distinct tubes (Supplementary Figure 7). After sorting, cells were centrifuged at 1500 rpm for 5 minutes and were conserved in freezing medium as described above and stored at -80 °C until further analysis. | ||||||||||||||||
| 148 | Jeon | 2020 | Insufficient Data for Classification | Yong Geun Jeon, Jae Ho Lee, Yul Ji, Jee Hyung Sohn, Dabin Lee, Dong Wook Kim, Seul Gi Yoon, Kyung Cheul Shin, Jeu Park, Je Kyung Seong, Je-Yoel Cho, Sung Sik Choe, Jae Bum Kim; RNF20 Functions as a Transcriptional Coactivator for PPARγ by Promoting NCoR1 Degradation in Adipocytes. Diabetes 1 January 2020; 69 (1): 20–34. https://doi.org/10.2337/db19-0508 | Huh JY, Park J, Kim JI, Park YJ, Lee YK, Kim JB. Deletion of CD1d in adipocytes aggravates adipose tissue inflammation and insulin resistance in obesity. Diabetes 2017;66:835–847 | Adipose tissues were fractionated as described previously (Huh 2017). | |||||||||||||||||||||||||||||||||||
| 149 | von Dongen | 2020 | Stromal Vascular Fraction | Insufficient Data for Classification | Manual | Human | Liposuction | 0.1% collagenase A and 1% bo- vine serum albumin in PBS, | 0.10% | Van Dongen JA, Gostelie OFE, Vonk LA, De Bruijn JJ, Van Der Lei B, Harmsen MC, Stevens HP. Fractionation of Adipose Tissue Procedure With a Disposable One-Hole Fractionator. Aesthet Surg J. 2020 Mar 23;40(4):NP194-NP201. doi: 10.1093/asj/sjz223. PMID: 31402379. | |||||||||||||||||||||||||||||||
| 150 | Shin | 2020 | human adipose tissue-derived mesenchymal stem cells | Insufficient Data To Classify | Shin, K.-O.; Ha, D.H.; Kim, J.O.; Crumrine, D.A.; Meyer, J.M.; Wakefield, J.S.; Lee, Y.; Kim, B.; Kim, S.; Kim, H.-k.; et al. Exosomes from human adipose tissue-derived mesenchymal stem cells promote epidermal barrier repair by inducing de novo synthesis of ceramides in atopic dermatitis. Cells 2020, 9, 680. | After isolating ASCs from the adipose tissue of a healthy donor | |||||||||||||||||||||||||||||||||||
| 151 | Lee | 2020 | adipose tissue-derived mesenchymal stem/stromal cells (ASCs) | Insufficient Data To Classify | Lee JH, Ha DH, Go HK, Youn J, Kim HK, Jin RC, Miller RB, Kim DH, Cho BS, Yi YW. Reproducible Large-Scale Isolation of Exosomes from Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells and Their Application in Acute Kidney Injury. Int J Mol Sci. 2020 Jul 5;21(13):4774. doi: 10.3390/ijms21134774. PMID: 32635660; PMCID: PMC7370182. | Shin, K.-O.; Ha, D.H.; Kim, J.O.; Crumrine, D.A.; Meyer, J.M.; Wakefield, J.S.; Lee, Y.; Kim, B.; Kim, S.; Kim, H.-k.; et al. Exosomes from human adipose tissue-derived mesenchymal stem cells promote epidermal barrier repair by inducing de novo synthesis of ceramides in atopic dermatitis. Cells 2020, 9, 680. | A human ASC cryostock of passage 4 was prepared as described previously [Shine et al 2020) | ||||||||||||||||||||||||||||||||||
| 152 | Sharun | 2021 | Stromal Vascular Fraction. SVF | SVF | Manual | Rabbit | Intracapsular | Surgical | Scissors | 5 gm. Washed 3 x by centrifugation 1200 g 2 min. | Collagenase Type I | MP Biomedicals LLC France | 0.1 %. 1:1 with fat | Centrifuge Tube | 37 | 60 | Continuous Gentle Agitation | equal quantity of control medium containing Dulbecco’s Modified Eagle’s Medium (#D5796; Sigma-Aldrich, MO, USA) supplemented with 10% fetal bovine serum (#16000- 044, Gibco, Life Technologies, USA) and 1% penicillin- streptomycin solution (Sigma-Aldrich) was added to neutralize collagenase activity | Centrifugation 1200g 10 min. Adipocytes and supernatant discarded . Pellet resuspended. | 100 micron Nylon Cell Strainer after RBC lysis. Filtrate centrifuged 1200 g 10 min | red blood cell lysis buffer (G-Biosciences, Geno Technology Inc., USA | 3.15 ×106 cells/g +/- 0.09 | Stromal Vascular Fraction. SVF | Characterization | Hemocytometer | Sharun K, Pawde AM, Kumar R, Kalaiselvan E, Kinjavdekar P, Dhama K, Pal A. Standardization and characterization of adipose-derived stromal vascular fraction from New Zealand white rabbits for bone tissue engineering. Vet World. 2021 Feb;14(2):508-514. doi: 10.14202/vetworld.2021.508-514. Epub 2021 Feb 25. PMID: 33776318; PMCID: PMC7994125. | Extensive Methods | The rabbits were anesthetized by the intramuscular injection of xylazine (Xylaxin, Indian Immunologicals Ltd., Hyderabad, India) at 6 mg/kg body weight followed by ketamine (administered 5 min later) (Aneket, Neon Laboratories Ltd., Thane, Mumbai, India) at 60 mg/kg body weight in the thigh muscles [11]. The dorsal thoracic area was prepared for aseptic surgery (Figure-1a). A 2 cm long incision was made over the interscapular fat depot for the removal of adipose tissue. Using rat-tooth thumb forceps (A2Z SCILAB, A2zscilab Incorporated, VA, USA), the adipose tissue was grasped and exteriorized (Figure-1b). A sufficient quantity (approximately 5-7 g) of adipose tissue was removed by careful dissection and kept in a Petri dish in phosphate-buffered saline (PBS) (Gibco, UK) (Figure-1c). The incision was then sutured in a routine manner [12]. Preparation of adipose-derived SVF Autologous adipose-derived SVF was prepared from the adipose tissue harvested from the interscapular fat depot of the rabbits. The adipose tissue was separated from the tissue debris and connective tissues. After collection, 5 g of adipose tissue was added to a centrifuge tube (Tarsons Products Private Ltd., India), minced using scissors (Vantage, Integra LifeSciences Corporation, USA), and washed thrice with sterile PBS (G-Biosciences, Geno Technology Inc., USA) to reduce contamination. The tissue was then centrifuged (REMI Laboratory Centrifuge R-8M Plus) at 1200× g (relative centrifugal force) for 2 min to remove cellular debris, including erythrocytes. The adipose tissue was then transferred to another tube containing an equal quantity of 0.1% collagenase type I solution (MP Biomedicals, LLC, France), and the contents were incubated at 37°C under conditions of continuous gentle agitation for 1 h in a water bath to prevent clumping of the cells. After incubation, an equal quantity of control medium containing Dulbecco’s Modified Eagle’s Medium (#D5796; Sigma-Aldrich, MO, USA) supplemented with 10% fetal bovine serum (#16000- 044, Gibco, Life Technologies, USA) and 1% penicillin- streptomycin solution (Sigma-Aldrich) was added to neutralize collagenase activity. The contents were then centrifuged at 1200× g for 10 min. The supernatant and the fat layer containing mature adipocytes were discarded, and the SVF pellet was reconstituted in a small quantity of control medium. To this, a red blood cell lysis buffer (G-Biosciences, Geno Technology Inc., USA) was added to remove the red blood cells from the sample. The mixture was filtered using a 100 μm nylon cell strainer (HiMedia Laboratories Pvt. Ltd., India). The filtrate was then centrifuged at 1200× g for 10 min. The supernatant was discarded and the SVF pellet obtained was suspended in 0.5 mL of control medium, ready for use. | |||||||||||||
| 153 | Wysong/Stehno-Bittel | 2021 | Fat cells | Adipose Stromal Cells (ASC) | Manual | Feline | SubQ, Uterine Horn Ovaries | Surgical | Scissors | Liberase | Roche | 0.6 WU/ml | 37 | 30 min | 100 micron Filter, Centrifugation 260 g 5 min, RBC lysis buffer, Centrifuged 260 g 5 min. Supernatant (??) suspended in buffer | 100 mm | 5 mL of red blood cell lysis buffer (Millipore Sigma, Cat #11,814,389,001) | 7 x 106 cells/gm fat | Fat Cells | Wysong A, Ortiz P, Bittel D, Ott L, Karanu F, Filla M, Stehno-Bittel L. Viability, yield and expansion capability of feline MSCs obtained from subcutaneous and reproductive organ adipose depots. BMC Vet Res. 2021 Jul 15;17(1):244. doi: 10.1186/s12917-021-02948-0. PMID: 34266445; PMCID: PMC8281647. | Likarda LLC, 10330 Hickman Mills Drive, Kansas City, MO, USA Lindsey Ott, Francis Karanu & Lisa Stehno-Bittel | Under aseptic conditions, the tissue was rinsed, weighed, and minced using sterile scissors and digested in a 0.6 WU/mL Liberase (Roch, Liberase MNP-S, /cat #06297790001) for 30 min at 37 °C. For the larger tissue amounts of subcutaneous fat, the tissue was passed through a 100 mm filter and centrifuges at 260xg for 5 min. The supernatant was removed and mixed with 5 mL of red blood cell lysis buffer (Millipore Sigma, Cat #11,814,389,001) followed by centrifugation at 260xg for 5 min. The final supernatant was resuspended in DMEM:F12 with 10 % FBS and 1 % Penicillin- Streptomycin and cultured at 37 °C, 5 % CO2 in a humidified chamber. | |||||||||||||||||||
| 154 | Carstens | 2021 | Stromal Vascular Fraction | Stromal Vascular Fraction | Semi-Automated | Human | SubQ. Abdominal | Liposuction | 58 cc | GMP Grade Collagenase | 125 cc of Hartmann’s solution | 70000CDUofcollagenaseenzyme added final conc 300 CDU/ml | GID SVF-2 | 39 | 60 min | Rotary Mixer | Centrifugation 600g 10 min | AdministrationofSVF AvolumeoftheSVFsuspensioncontainingadoseof40£106SVFcellswasisolated.Thecellswerethenfurthersuspendedtoatotalof100ccofHartmann’ssolution.Thecellsuspensionwasadministeredintravenouslyoveraperiodof30minutesthroughabloodfilter. | automatedfluorescencecytometer(LUNA-STEM | >/= 70% | Michael H. Carstens, Francisco J. Quintana, Santos T. Calderwood, Juan P. Sevilla, Arlen B. Ríos, Carlos M. Rivera, Dorian W. Calero, María L. Zelaya, Nelson Garcia, Kenneth A. Bertram, Joseph Rigdon, Severiano Dos-Anjos, Diego Correa, Treatment of Chronic Diabetic Foot Ulcers with Adipose-Derived Stromal Vascular Fraction Cell Injections: Safety and Evidence of Efficacy at 1 Year, Stem Cells Translational Medicine, Volume 10, Issue 8, August 2021, Pages 1138–1147, https://doi.org/10.1002/sctm.20-0497 | Carstens MH, Gómez A, Cortés R, et al. Non-reconstructable peripheral vascular disease of the lower extremity in ten patients treated with adipose-derived stromal vascular fraction cells. Stem Cell Res. 2017;18:14-21. | AdministrationofSVF AvolumeoftheSVFsuspensioncontainingadoseof40£106SVFcellswasisolated.Thecellswerethenfurthersuspendedtoatotalof100ccofHartmann’ssolution.Thecellsuspensionwasadministeredintravenouslyoveraperiodof30minutesthroughabloodfilter. | Isolation of SVF (liposuction and cell processing) The surgical procedure consisted of preparation of the harvest site with local anesthesia and tumescent solution of Ringer’s lactate followed by lipoaspiration into a closed adipose tissue processing system—the GID SVF-2 device (The GID Group). The lipoaspirate collected in the SVF-2 device was then processed by (a) serial washing to restore pH and remove oil, leukocytes, and erythrocytes, thus producing “dry fat” free of fluids; (b) enzymatic digestion with GMP-grade collagenase; (c) neutralization by repeat washing with lactated Ringer’s; (d) aspiration of the SVF pellet from the cell collecting chamber of the device; (e) resuspension of the SVF cells; and (f ) cell count. Details of these procedures are provided in our original report.16 | |||||||||||||||||
| 155 | Hearnden | 2021 | Stromal Vascular Fraction | Adipose Stromal Cells (ASC) | Manual | Human | Collagenase from Clostridium histolyticum (type II). Sigma Cat# C6885 Deoxyribonuclease I from bovine pancreas Sigma Cat# D4527 | Sigma | 200 mL Collagenase II (2844 U) and 2.5 mL DNase (27.75 U) per mL of Wash/Digestion buffer | 37 | 25 – 30 min | shaker-incubator at 37 C and 225 rpm for 25–30 min | strain through 100 micron cell sieve. Centrifuge 800 3 g for 10 min at 4 C. transfer the samples through a 70 mm cell sieve into a 50 mL tube pre-washed with Wash/Digestion buffer. Then fill the tube to the top with Wash/Digestion buffer. 9. Centrifuge samples at 800 3 g for 10 min at 4 C. | 100 micron 70 micron cell sieve | 500 ml of red blood cell lysis buffer (ACK Lysis Buffer) for about 1 min. | Stromal Vascular Fraction | SVF Isolation Method | Hearnden R, Sandhar B, Vyas V, Longhi MP. Isolation of stromal vascular fraction cell suspensions from mouse and human adipose tissues for downstream applications. STAR Protoc. 2021 Mar 30;2(2):100422. doi: 10.1016/j.xpro.2021.100422. PMID: 33870227; PMCID: PMC8044717. | Macdougall, C.E., Wood, E.G., Loschko, J., Scagliotti, V., Cassidy, F.C., and Robinson, M.E. (2018). Visceral adipose tissue immune homeostasis is regulated by the crosstalk between adipocytes and dendritic cell subsets. Cell Metab. 27, 588–601.e4. NOTE: This reference does not utilize isolated SVF – No isolation methods | STEP-BY-STEP METHOD DETAILS Processing and digesting the adipose tissue Timing: [1–2 h] During this step, the adipose tissue is processed and digested. This isolates the stromal vascular fraction cells from the extracellular matrix whilst ensuring their viability. 1. Weigh the adipose tissue and add 2 mL of ice-cold Wash/Digestion buffer per gram of sample. Note: If samples are <1g, add 2 mL of ice-cold Wash/Digestion and continue with all steps. Note: If the adipose tissueweighs up to 2 g thenwe suggest using 7 mL bijou tubes. However, if the samples exceed 2 g then use larger tubes (e.g., 15 mL tubes) or split the sample into two smaller tubes and weigh each separately. 2. Using sterile scissors, mince the adipose tissue and add 200 mL Collagenase II (2844 U) and 2.5 mL DNase (27.75 U) per mL of Wash/Digestion buffer. CRITICAL: Always check LOT number and corresponding enzymatic activity in Units per mg when making up the Collagenase II and DNase stock solutions, as this will vary. As a result, it is not practical to state the enzyme concentrations in mg/mL as we must standardise to U/mL. CRITICAL: Ensure at this point that all the adipose tissue is floating. Any tissue present sinking to the bottom of the tube is likely to be non-adipose tissue and should be removed. Note: Store the Collagenase II and DNase enzyme stock solutions in small aliquots to minimise the effect of repeated freeze/thaw cycles, which will decrease the enzymatic activity. 3. Place samples in a shaker-incubator at 37 C and 225 rpm for 25–30 min. Note: Digested samples should look homogeneous and pinkish white in colour, larger samples can take slightly longer than smaller samples (Figure 1B). 4. Transfer the samples through a 100 mm cell sieve into a 50 mL tube pre-washed with ice cold Wash/Digestion buffer. Then top up the Falcon tube to 30 mL withWash/Digestion buffer. Note: Ensure you rinse the sample tube used for digestion with Wash/Digestion buffer to remove any remaining sample and strain through the same 100 mm cell sieve. 5. Centrifuge samples at 800 3 g for 10 min at 4 C. CRITICAL: After centrifugation, open the tubes and check that the floating portion of the sample is composed of transparent lipid droplets. If you can see a white layer of adipose tissue floating, then this top layer will need removing with a Pasteur pipette and re-digestion (Figure 1C). Re-digest in 2 mL Wash/Digestion buffer, with half the original collagenase volume used i.e., 200 mL Collagenase II in 2 mL Wash/Digestion buffer, with 5 mL DNase for 10 min (as in step 3). This will ensure you obtain all the stromal cells possible, particularly macrophages, from within each sample. 6. Discard supernatant and the remaining pellet contains the stromal vascular fraction cells. Note: Proceed directly to wash step 8 if you will use bead enrichment (recommended), otherwise carry out red blood cell lysis described in step 7. 7. Optional: Resuspend pellets in 500 ml of red blood cell lysis buffer (ACK Lysis Buffer) for about 1 min. 8. Top up with ice cold Wash/Digestion buffer and transfer the samples through a 70 mm cell sieve into a 50 mL tube pre-washed with Wash/Digestion buffer. Then fill the tube to the top with Wash/Digestion buffer. 9. Centrifuge samples at 800 3 g for 10 min at 4 C. 10. Discard supernatant and the remaining pellet contains the stromal vascular fraction cells. | |||||||||||||||||||||
| 156 | Angueira | 2021 | Single Cell Suspension | Adipose Stromal Cells (ASC) | Manual | Mouse | Peri-Aortic Fat | Surgical | Minced | Collagenase D: 6.1mg/ml (Roche), Dispase II: 2.4 mg/ml (Roche) | Roche | DMEM | Collagenase D: 6.1mg/ml (Roche), Dispase II: 2.4 mg/ml (Roche) | 37 | constant agitation at 200 rpm | To enrich for cell populations with differential sensitivity to digestion, we utilized the following procedures. For embryonic/newborn mice, 20% of the digestion was quenched at 15 minutes, 20% at 20 minutes and the remaining 60% at 25 minutes (embryos/perinatal). For adult aortas 50% of the digestion was quenched at 30 minutes, 50% at 60 minutes. For stripped aorta flow cytometry analysis, perivascular adipose tissue was removed from the aorta and digested separately from the cleaned aorta. Tissue digestions were quenched with an equal volume of complete medium (DMEM/10% FBS). | Dissociated cells were suspended using a P1000 pipette and filtered through a 100 μm filter. Cells were then pelleted at 400 g for 4 min and RBCs were lysed in 155mM NH4Cl, 12mM NaHCO3, and 0.1mM EDTA for 4 min. An equal volume of complete medium was added, and the cells were filtered through a 40um filter for downstream analyses | 40 micron | RBCs were lysed in 155mM NH4Cl, 12mM NaHCO3, and 0.1mM EDTA for 4 min. | Flow Cytometry | Adipogenesis. single nucleus RNAseq. | Angueira AR, Sakers AP, Holman CD, Cheng L, Arbocco MN, Shamsi F, Lynes MD, Shrestha R, Okada C, Batmanov K, Susztak K, Tseng YH, Liaw L, Seale P. Defining the lineage of thermogenic perivascular adipose tissue. Nat Metab. 2021 Apr;3(4):469-484. doi: 10.1038/s42255-021-00380-0. Epub 2021 Apr 12. PMID: 33846639; PMCID: PMC8136151. | Generation of single cell suspensions from mouse aorta Aorta were isolated from mice, minced and placed into digestion medium (DMEM, Collagenase D: 6.1mg/ml (Roche), Dispase II: 2.4 mg/ml (Roche) and placed at 37°C with constant agitation at 200 rpm. To enrich for cell populations with differential sensitivity to digestion, we utilized the following procedures. For embryonic/newborn mice, 20% of the digestion was quenched at 15 minutes, 20% at 20 minutes and the remaining 60% at 25 minutes (embryos/perinatal). For adult aortas 50% of the digestion was quenched at 30 minutes, 50% at 60 minutes. For stripped aorta flow cytometry analysis, perivascular adipose tissue was removed from the aorta and digested separately from the cleaned aorta. Tissue digestions were quenched with an equal volume of complete medium (DMEM/10% FBS). Dissociated cells were suspended using a P1000 pipette and filtered through a 100 μm filter. Cells were then pelleted at 400 g for 4 min and RBCs were lysed in 155mM NH4Cl, 12mM NaHCO3, and 0.1mM EDTA for 4 min. An equal volume of complete medium was added, and the cells were filtered through a 40um filter for downstream analyses. | ||||||||||||||||||
| 157 | Hildreth | 2021 | Stromal Vascular Fraction. SVF | Adipose Stromal Cells (ASC) | Manual | Human | deep subcutaneous adipose tissue | surgical | physical homogenization | physical homogenization in adipose harvest medium ((AHM) 1× Hank’s balanced salt solution + CaCl2 + MgCl2, 5% heat-inactivated fetal bovine serum, 1% l-glutamine, 1% penicillin–streptomycin and 50 μg ml−1 of DNAse1) using a blender (900 W, Ninja BL450 Series). | collagenase type II | Worthington | AHM. 1× Hank’s balanced salt solution + CaCl2 + MgCl2, 5% heat-inactivated fetal bovine serum, 1% l-glutamine, 1% penicillin–streptomycin and 50 μg ml−1 of DNAse1) | 2 mg/ml | 37 | 60 | shaking incubation | Post-incubation, digested samples were filtered using 100-μm cell strainers (Corning) and then centrifuged to isolate the SVF pellets. After aspiration of the supernatants, the pellets were lysed using ACK lysis buffer, pooled into one tube, and then filtered again through 100-μm nitex mesh. | 100 micron | AKC buffer | single-cell transcriptomics and flow cytometry | Hildreth AD, Ma F, Wong YY, Sun R, Pellegrini M, O’Sullivan TE. Single-cell sequencing of human white adipose tissue identifies new cell states in health and obesity. Nat Immunol. 2021 May;22(5):639-653. doi: 10.1038/s41590-021-00922-4. Epub 2021 Apr 27. PMID: 33907320; PMCID: PMC8102391. | Adipose tissue harvest and cell dissociation. Patient deep subcutaneous adipose tissue was freshly harvested from abdominoplasty samples within an hour of their respective operations. Adipose tissue samples were kept on ice until processing. Adipose tissues were first washed three times with 1× phosphate-buffered saline before physical homogenization in adipose harvest medium ((AHM) 1× Hank’s balanced salt solution + CaCl2 + MgCl2, 5% heat-inactivated fetal bovine serum, 1% l-glutamine, 1% penicillin–streptomycin and 50 μg ml−1 of DNAse1) using a blender (900 W, Ninja BL450 Series). Homogenized tissue was then distributed into 14-ml round-bottomed tubes and supplemented with collagenase type II digestion medium (AHM + 2 mg ml−1 of collagenase type II (Worthington Biochem)) before shaking incubation at 37 °C for 1 h. Post-incubation, digested samples were filtered using 100-μm cell strainers (Corning) and then centrifuged to isolate the SVF pellets. After aspiration of the supernatants, the pellets were lysed using ACK lysis buffer, pooled into one tube, and then filtered again through 100-μm nitex mesh. | ||||||||||||||||||
| 158 | Rondini | 2021 | Stromal Vascular Cells | Adipose Stromal Cells (ASC) | Manual | Mouse | inguinal white adipose tissue (iWAT) | surgical | minced | Type 2 collagenase | Worthington Biochemical Corp; Lakewood, NJ | Hanks balanced salt solution (pH 7.4, SigmaeAldrich, St. Louis, MO) containing 0.5% fatty acidefree bovine serum albumin (FF-BSA; Gemini Bio, West Sacramento, CA) | 2 mg/mL | 37 | 30 | gentle agitation | The digestate was passed through a 100 mM filter and rinsed with buffer (PBS containing 0.5 mM EDTA, 10 mM HEPES, 10% fetal bovine serum, and 0.5% FF-BSA, pH7.4). Adipocytes were then separated by centrifugation at 200 g for 5 min at room temperature. Floating cells (adipocytes) were collected by removing the infranatant by aspiration. Adipocytes were then washed twice with pre-heated (37 C) Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1% FF-BSA (Gemini Bio) prior to downstream applications. SVC were pelleted from the infranatant by centrifugation (500 g for 10 min at 4 C), and red blood cells (RBC) were lysed in lysis buffer (155 mM NH4Cl, 14 mM NaHCO3, and 0.1 mM EDTA, pH 7.4) for 5 min at room temperature. SVC were then filtered (40 mM), resuspended in PBS containing 0.5% FF-BSA (MACS buffer, pH 7.4), and pelleted again by centrifugation (500 g for 10 min at 4 C). Hematopoietic lineagee positive cells were depleted from the total SVC fraction by cell sorting using the lineage depletion kit (Miltenyi Biotec; Auburn, CA) and a magnetic separator according to manufacturer’s instructions. | 100 micron filter 40 micron filter | lysed in lysis buffer (155 mM NH4Cl, 14 mM NaHCO3, and 0.1 mM EDTA, pH 7.4) for 5 min at room temperature. | Rondini EA, Ramseyer VD, Burl RB, Pique-Regi R, Granneman JG. Single cell functional genomics reveals plasticity of subcutaneous white adipose tissue (WAT) during early postnatal development. Mol Metab. 2021 Nov;53:101307. doi: 10.1016/j.molmet.2021.101307. Epub 2021 Jul 21. PMID: 34298199; PMCID: PMC8385178. | Burl, R.B., Ramseyer, V.D., Rondini, E.A., Pique-Regi, R., Lee, Y.H., Granneman, J.G., 2018. Deconstructing adipogenesis induced by beta3- adrenergic receptor activation with single-cell expression profiling. Cell Metabolism 28(2):300e309 e304. Lee, Y.H., Petkova, A.P., Mottillo, E.P., Granneman, J.G., 2012. In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding. Cell Metabolism 15(4):480e491. | Stromal vascular cells (SVC) and adipocytes were isolated from inguinal white adipose tissue (iWAT) as described previously [19,20]. Briefly, following excision, fat pads (combined from 3 to 6 male offspring per time point) were minced and then dissociated in Hanks balanced salt solution (pH 7.4, SigmaeAldrich, St. Louis, MO) containing 0.5% fatty acidefree bovine serum albumin (FF-BSA; Gemini Bio, West Sacramento, CA) and 2 mg/mL Type 2 collagenase (Worthington Biochemical Corp; Lakewood, NJ) at 37 C for 30 min with gentle agitation. The digestate was passed through a 100 mM filter and rinsed with buffer (PBS containing 0.5 mM EDTA, 10 mM HEPES, 10% fetal bovine serum, and 0.5% FF-BSA, pH7.4). Adipocytes were then separated by centrifugation at 200 g for 5 min at room temperature. Floating cells (adipocytes) were collected by removing the infranatant by aspiration. Adipocytes were then washed twice with pre-heated (37 C) Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1% FF-BSA (Gemini Bio) prior to downstream applications. SVC were pelleted from the infranatant by centrifugation (500 g for 10 min at 4 C), and red blood cells (RBC) were lysed in lysis buffer (155 mM NH4Cl, 14 mM NaHCO3, and 0.1 mM EDTA, pH 7.4) for 5 min at room temperature. SVC were then filtered (40 mM), resuspended in PBS containing 0.5% FF-BSA (MACS buffer, pH 7.4), and pelleted again by centrifugation (500 g for 10 min at 4 C). Hematopoietic lineagee positive cells were depleted from the total SVC fraction by cell sorting using the lineage depletion kit (Miltenyi Biotec; Auburn, CA) and a magnetic separator according to manufacturer’s instructions. | |||||||||||||||||||
| 159 | Dai | 2022 | Stromal Vascular Fraction. SVF | Insufficient Data To Classify | Manual | Human | Liposuction Surgical | 50 ml | Collagenase | 0.10% | 37 | 24 hours | Centrifugation 1500 rpm 5 minutes | sc/nRNA-seq and snATAC-seq | Dai LG, Huang NC, Kang LY, Fu KY, Hsieh PS, Dai NT. An In Vitro Study of the Effects of Mechanical and Enzymatic Isolation of Stromal Vascular Fraction on Wound Healing. Ann Plast Surg. 2022 Mar 1;88(1s Suppl 1):S13-S21. doi: 10.1097/SAP.0000000000003087. PMID: 35225844. | Cherng JH, Chang SC, Chen SG, et al. The effect of hyperbaric oxygen and air on cartilage tissue engineering. Ann Plast Surg. 2012;69:650–655. | The E-SVF method27 can be briefly described as follows: soak 50-mL adipose tissue in 0.1% collagenase/L-15 medium for reaction for 24 hours, and centrifuge at 1500 rpm for 5 minutes. After removing the supernatant, the precipitate consists of E-SVF. | ||||||||||||||||||||||||
| 160 | Saito | 2022 | Microvessel Endothelial Progenitor Cells | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | Collagenase Crude Type | FUJIFILM Waco Pure Chemical | HBSS. Hanks Balanced Salt Solution | 0.1 to 0.2 % | 50 cc Tube | 37 | 30 | 120 RPM of Reciprocating Motion | Centrifuge 800 x g 10 min. 100 micron then 40 micron sieve. Then Centrifugation 800 g x 5 min. | 1x HBSS | Red Blood Cell Lysis Buffer | Magnetic Separation CD45 and CD31 magnetic beads | FACS. EC Colony, EC In Vitro Angiogenesis | 3.84 x 10E5 SVF cells / gm fat | Adipose endothelial Progenitor Cells | In Vitro Studies | CD45, CD34, CD31, CD105, CD146, CD157, and CD200 | Sci Rep . 2022 Feb 2;12(1):1775. doi: 10.1038/s41598-022-05760-4. Purification and characterization of human adipose-resident microvascular endothelial progenitor cells Natsumi Saito 1 , Takako Shirado 1 , Hitomi Funabashi-Eto 2 , Yunyan Wu 1 , Masanori Mori 1 , Rintaro Asahi 1 , Kotaro Yoshimura 3 | Bourin, P. et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissuederived stromal/stem cells: A joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy 15, 641–648. https:// doi. org/ 10. 1016/j. jcyt. 2013. 02. 006 (2013). | EPCs Endothelial progenitor cells ECs Endothelial cells IRB Institutional review board SVF Stromal vascular fraction AEPCs Adipose-resident microvascular endothelial progenitor cells DNase1 Deoxyribonuclease 1 Pol188 Poloxamer 188 HBSS Hanks’ balanced salt solution MACS Magnetic-activated cell sorting FBS Fetal bovine serum FVD780 Fixable viability dye eFluor 780 HUVECs Human umbilical vein endothelial cells FACS Fluorescence-activated cell sorting BSA Bovine serum albumin EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid DPBS Dulbecco’s phosphate-buffered saline RT Room temperature vWF Von Willebrand factor DAPI 4′,6-Diamidino-2-phenylindole PFA Paraformaldehyde in phosphate buffer solution. PBS Phosphate-buffered saline CFU-EC EC colony-forming unit ASCs Adipose-derived stem cells SD Standard deviation GMP Good manufacturing practice | After natural gravity sedimentation, lipoaspirates develop three layers: oil, adipose tissue, and tumescent liquid. The floating adipose tissue layer was extracted, measured, and digested with an equivalent volume of collagenase-based enzyme solution (details below) at 37 °C for 30 min at 120 rpm of reciprocating motion (Yamato Scientific), followed by centrifugation at 800×g for 10 min. The resulting cell pellet was designated the SVF (Fig. 1A). After washing with Hanks’ Balanced Salt Solution (HBSS; Thermo Fisher Scientific; #14175-103), the SVF was sequentially passed through 100-μm and 40-μm sieve cell strainers (Corning). The strained cell suspension was centrifuged again at 800×g for 5 min at 4 °C, and the obtained SVF pellet was washed with HBSS. The nucleated cell number, viability, and total cell-sized particle number were measured using a fluorescent cell counter (LUNA-STEM; Logos Biosystems) after double staining with acridine orange and propidium iodide (Logos Biosystems). We used flow cytometry to examine the extraction efficiency of AEPCs using three collagenase-based enzyme formulations: (#1) 0.2% (w/v) collagenase (crude type; FUJIFILM Wako Pure Chemical; #032-22364) and 3 mM CaCl2 (FUJIFILM Wako; #037-24031) in HBSS; (#2) 0.2% (w/v) collagenase, 3 mM CaCl2, and 1000 U/mL of deoxyribonuclease 1 (DNase1; Worthington Biochemical; #LS002139) in HBSS; (#3) 0.2% (w/v) collagenase, 3 mM CaCl2, 1000 U/mL DNase1, and 0.1% (v/v) Poloxamer 188 solution (Pol188; Sigma-Aldrich) in HBSS. Pol188 has previously been reported as an additive to elevate the extraction efficiency of ASCs in the SVF24. | ||||||||||||
| 161 | Karadag | 2022 | Stromal Vascular Fraction | Insufficient Data To Classify | Manual | Human | SubQ | Liposuction | Liposuction a blunt cannula (the “Fat Grater”) with a 2-mm diameter, the tip of which has 10 holes measuring 1.5mm in diameter | SIGMA-ALDRICH, St. Louis, MO, USA | 50 ml Conical Tube | 60 to 90 min | Shaker 100RPM | Centrifugation 500g 10 min. Pellet vortexed. Pellet filtered through Cell Strainer. Washed with Lactated Ringers. Centrifuged 500g 10 minutes. Resuspended in LR. Centrifuged 500g 10 min. | 3 washes | Cell Strainer | No Method provided and unable to determine basis of yield | No Method provided and unable to determine basis of viability | Karadag Sari EC, Ovali E. Factors affecting cell viability and the yield of adipose-derived stromal vascular fraction. J Plast Surg Hand Surg. 2022 Aug;56(4):249-254. doi: 10.1080/2000656X.2022.2097250. Epub 2022 Jul 12. PMID: 35819816. | All operations were performed under sedoanalgesia in a sterile operating room. We used the tumescent technique and harvested lipoaspirates from the abdominal region. The same plastic surgeon performed all operations. Umbilical and suprapubic areas were selected as entry sites. A tumescent solution containing ringer lactate with 1:500.000 units of epinephrine and 20mg bupivacaine hydrochloride was infiltrated through the 3-mm incisions and allowed to rest for 10 min to achieve hemostasis. The volume of the tumescent solution injected was 150 cc. Following the hemostasis, a blunt cannula (the “Fat Grater”) with a 2-mm diameter, the tip of which has 10 holes measuring 1.5mm in diameter, was used to harvest the adipose aspirate (Figure 1). The cannula attached to a 60-ml syringe and gently pulling pack on the syringe’s plunger provided a light negative pressure while the cannula was advanced and retracted through the harvest site. After filling the syringe with harvested tissue, the cannula was removed from the syringe, and the lipoaspirate was transferred into sterile 50-ml Falcon tubes for further enzymatic processing. | |||||||||||||||||||||
| 162 | Dapra | 2022 | Stromal Vascular Fraction | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | Daprà V, Alotto D, Casarin S, Gambarino S, Zavatto C, Dini M, Galliano I, Castagnoli C, Bergallo M. A new protocol for validation of Chondro, Adipo and Osteo differentiation kit of Cultured Adipose-Derived Stem Cells (ADSC) by real-time rt-QPCR. Tissue Cell. 2022 Dec;79:101923. doi: 10.1016/j.tice.2022.101923. Epub 2022 Sep 13. PMID: 36126415. | Isolation of SVF from Adipose Tissue SVFs were isolated from fresh adipose tissue by enzymatic digestion with Collagenase NB6 (Serva Electrophoresis) 0,3 U/mL using a tube rotator for 40 min at 37 ◦C. The enzyme activity was neutralized by the addition of saline solution + 10 % FBS, and then the samples were centrifuged at 1500 rpm for 10 min. The pellet was re-suspended in saline solution, then passed through 100 μm and 70 μm cell strainers, washed by saline solution, collected, centrifuged at 1700 rpm for 5 min and counted. The phenotype of MSCs contained in the SVF was evaluated after isolation. Cell surface markers of ATDMSCs were analysed by flow cytometry on fresh SVF and on SVF derived after thawing of adipose tissue and its collagenase treatment. ATD-MSCs were identified as CD105, CD44, CD73, and CD271 positive cells and negative for CD45 expression. Standard labelling protocol was performed with the following antibodies fluorochrome-conjugated and isotypic controls: human CD105 PE (Invitrogen), CD73 FITC (kindly provided by Professor Malavasi, University of Turin), CD44 FITC, CD45 PerCP, CD3 PerCP, CD271 APC, IgG1 PE, IgG1 APC, and IgG2a PerCP (Miltenyi Biotec), and IgG1 FITC-conjugated (Immunostep). About 105 events/sample were used for capture with CellQuest software. All data were analysed with FlowJo software (Tree Star). The SVF cells were seeded in T25 flasks and cultured in Alpha-MEM (Minimum Essential Medium, Alpha Modification; Sigma-Aldrich) with 10 % HPL (Human Platelet Lysate; Emocomponent Production and Validation Center – CPVE – Citt`a della Salute e della Scienza, Torino), 2 mM glutamine, and 1 % antibiotics (Gibco, Life Technologies) and the medium was replaced to eliminate nonadherent cells after 24 h. The medium was then changed every 48 h. | |||||||||||||||||||||||||||||||
| 163 | Whytock | 2022 | Stromal Vascular Fraction SVF | Adipose Stromal Cells (ASC) | Manual | Human | Abdominal SubQ | Liposuction | Type I collagenase | Worthington M2C13334 | alphaMEM with 1 % BSA | 37 | 45 | centrifugation at 200g for 5 minutes. SVF was isolated by additional centrifugation at 500g for 5 minutes | The SVF solution was subsequently filtered through 100mm and 40mm strainer (BD Falcon) | washed with Red Blood Cell lysis buffer | countess II automated cell counter (Thermofisher Scientific, Waltham, MA | Nuclei was stained with Hoechst 33342 (ReadyProbes Cell Viability Imaging Kit, Thermofisher Scientific) | SVF | full-length sc/single-nuclei (sn) RNA-Seq | full-length sc/single-nuclei (sn) RNA-Seq | Whytock KL, Sun Y, Divoux A, Yu G, Smith SR, Walsh MJ, Sparks LM. Single cell full-length transcriptome of human subcutaneous adipose tissue reveals unique and heterogeneous cell populations. iScience. 2022 Jul 16;25(8):104772. doi: 10.1016/j.isci.2022.104772. PMID: 35992069; PMCID: PMC9385549. | SVF scRNA-Seq provided greater resolution of non-adipogenic cells | SVF isolation and adipocyte collection Tissue was digested with collagenase buffer (Type I collagenase in aMEM supplemented with 1%BSA) at 37 C for 45 min. Adipocytes were collected by centrifugation at 200g for 5 minutes and washed twice with PBS prior to nuclei isolation (see below). After the adipocytes were collected, the SVF was isolated by additional centrifugation at 500g for 5 minutes and washed with Red Blood Cell lysis buffer. The SVF solution was subsequently filtered through 100mm and 40mm strainer (BD Falcon) and counted with a countess II automated cell counter (Thermofisher Scientific, Waltham, MA). Cells were stained with propidium iodine and Hoechst 33342 (ReadyProbes Cell Viability Imaging Kit, Thermofisher Scientific). Following a wash, cells were resuspended in 1% BSA- nuclease free-water. | |||||||||||||||||
| 164 | Nahmgoong | 2022 | Stromal Vascular Fraction SVF | Stromal Vascular Fraction SVF | Manual | Mouse | epididymal adipose tissue (EAT) and subcutaneous inguinal adipose tissue (IAT | surgical | minced | collagenase I | Worthington 49A18993 | 0.1 M HEPES, 0.125 M NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 1.5% (w/v) bovine serum albumin | 0.1% (w/v) | 37 | 30 to 60 | shaking water bath | After centrifugation at 200 3 g, room temperature (RT) for 5 min, the pelleted stromal vascular fraction (SVF) was collected. The SVF was incubated in red blood cell lysis buffer (1.7 M Tris, pH 7.65, and 0.16 M NH4Cl) for 15 min. Then, the SVFs were washed with phosphate-buffered saline (PBS) several times, passed through a 100- mM filter (93100, SPL), and collected by centrifugation at 200 3 g for 5 min. | 100 micron | red blood cell lysis buffer (1.7 M Tris, pH 7.65, and 0.16 M NH4Cl) for 15 min. | single-cell RNA sequencing (scRNA-Seq) | Nahmgoong H, Jeon YG, Park ES, Choi YH, Han SM, Park J, Ji Y, Sohn JH, Han JS, Kim YY, Hwang I, Lee YK, Huh JY, Choe SS, Oh TJ, Choi SH, Kim JK, Kim JB. Distinct properties of adipose stem cell subpopulations determine fat depot-specific characteristics. Cell Metab. 2022 Mar 1;34(3):458-472.e6. doi: 10.1016/j.cmet.2021.11.014. Epub 2022 Jan 11. PMID: 35021043. | Yong Geun Jeon, Jae Ho Lee, Yul Ji, Jee Hyung Sohn, Dabin Lee, Dong Wook Kim, Seul Gi Yoon, Kyung Cheul Shin, Jeu Park, Je Kyung Seong, Je-Yoel Cho, Sung Sik Choe, Jae Bum Kim; RNF20 Functions as a Transcriptional Coactivator for PPARγ by Promoting NCoR1 Degradation in Adipocytes. Diabetes 1 January 2020; 69 (1): 20–34. https://doi.org/10.2337/db19-0508 | EAT and IAT were obtained from age-matched NCD-fed and 2- or 10-week HFD-fed mice. Adipose tissues were fractionated as described previously (Jeon et al., 2020). Briefly, adipose tissues were minced and digested with collagenase buffer [0.1 M HEPES, 0.125 M NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 1.5% (w/v) bovine serum albumin, and 0.1% (w/v) collagenase I (Worthington, 49A18993)] in a shaking water bath at 37 C for 30–60 min. After centrifugation at 200 3 g, room temperature (RT) for 5 min, the pelleted stromal vascular fraction (SVF) was collected. The SVF was incubated in red blood cell lysis buffer (1.7 M Tris, pH 7.65, and 0.16 M NH4Cl) for 15 min. Then, the SVFs were washed with phosphate-buffered saline (PBS) several times, passed through a 100- mM filter (93100, SPL), and collected by centrifugation at 200 3 g for 5 min. | ||||||||||||||||||
| 165 | Nahmgoong | 2022 | Stromal Vascular Fraction SVF | Stromal Vascular Fraction SVF | Manual | Human | laparoscopic bypass surgery | maually minced | collagenase I | Worthington 49A18993 | 0.1 M HEPES, 0.125 M NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 1.5% (w/v) bovine serum albumin | 0.1% (w/v) | 37 | 30 to 60 | shaking water bath | After centrifugation at 200 3 g, room temperature (RT) for 5 min, the pelleted stromal vascular fraction (SVF) was collected. The SVF was incubated in red blood cell lysis buffer (1.7 M Tris, pH 7.65, and 0.16 M NH4Cl) for 15 min. Then, the SVFs were washed with phosphate-buffered saline (PBS) several times, passed through a 100- mM filter (93100, SPL), and collected by centrifugation at 200 3 g for 5 min. | 100 micron | red blood cell lysis buffer (1.7 M Tris, pH 7.65, and 0.16 M NH4Cl) for 15 min. | single-cell RNA sequencing (scRNA-Seq) | Nahmgoong H, Jeon YG, Park ES, Choi YH, Han SM, Park J, Ji Y, Sohn JH, Han JS, Kim YY, Hwang I, Lee YK, Huh JY, Choe SS, Oh TJ, Choi SH, Kim JK, Kim JB. Distinct properties of adipose stem cell subpopulations determine fat depot-specific characteristics. Cell Metab. 2022 Mar 1;34(3):458-472.e6. doi: 10.1016/j.cmet.2021.11.014. Epub 2022 Jan 11. PMID: 35021043. | Yong Geun Jeon, Jae Ho Lee, Yul Ji, Jee Hyung Sohn, Dabin Lee, Dong Wook Kim, Seul Gi Yoon, Kyung Cheul Shin, Jeu Park, Je Kyung Seong, Je-Yoel Cho, Sung Sik Choe, Jae Bum Kim; RNF20 Functions as a Transcriptional Coactivator for PPARγ by Promoting NCoR1 Degradation in Adipocytes. Diabetes 1 January 2020; 69 (1): 20–34. https://doi.org/10.2337/db19-0508 | Human adipose tissues were rinsed in PBS twice, manually minced, and digested with collagenase buffer [0.1 M HEPES, 0.125 M NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 1.5% (w/v) bovine serum albumin, and 0.1% (w/v) collagenase I (Worthington, 49A18993)] in a shaking water bath at 37 C for 30–60 min. The subsequent steps were the same as those for preparing mouse adipose tissue fractionation. | |||||||||||||||||||
| 166 | Strohmeier | 2022 | Culture. FACS analysis | Flow cytometry | Strohmeier K, Hofmann M, Jacak J, Narzt MS, Wahlmueller M, Mairhofer M, Schaedl B, Holnthoner W, Barsch M, Sandhofer M, Wolbank S, Priglinger E. Multi-Level Analysis of Adipose Tissue Reveals the Relevance of Perivascular Subpopulations and an Increased Endothelial Permeability in Early-Stage Lipedema. Biomedicines. 2022 May 18;10(5):1163. doi: 10.3390/biomedicines10051163. PMID: 35625899; PMCID: PMC9138324. | “SVF Isolation A total of 200 mL of liposuction material was washed with an equal volume of PBS. Tissue digestion was carried out with 0.2 U/mL collagenase NB4 (Nordmark, Uetersen, Ger-many) dissolved in 200 mL of PBS, containing Ca2+/Mg2+ and 25 mM of N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES; Sigma-Aldrich, St. Louis, MO, USA) at 37 ◦C under moderate shaking (180 rpm) for 1 h, resulting in an end concentra-tion of collagenase of 0.1 U/mL. The digested tissue was transferred into 50 mL tubes (Greiner, Kremsmünster, Austria). After centrifugation at 1200× g for 7 min, the cell pellets were incubated with 50 mL of erythrocyte lysis buffer (154 mM of ammonium chloride (Sigma-Aldrich), 10 mM of potassium bicarbonate (Sigma-Aldrich), 0.1 mM of ethylenediamine-tetraacetic acid (EDTA; Biochrom, Austria) in Aqua dest for 2 min at room temperature. After centrifugation for 5 min at 500× g, the cell pellet was washed with PBS and filtered through a 100-µm cell strainer (Greiner, Austria). After another centrifugation step at 500× g for 5 min, the supernatant was removed, and cell number and viability were determined using the NucleoCounter® NC-200™ (Chemometec, Allerod, Denmark). To allow accurate quantification of the freshly isolated SVF cells, the protocol “Viability and Cell Count—Aggregated Cells Assay” was performed. ASCs were analyzed using the protocol “Viability and Cell Count Assay” according to the manufacturer’s instructions.” | Correspondence: eleni.priglinger@trauma.lbg.ac.at | ||||||||||||||||||||||||||||||||||
| 167 | Haack-Sørensen | 2022 | Stromal Vascular Fraction SVF | Insufficient Data To Classify | Haack-Sørensen, Mandana, Johansen, Ellen Mønsted, Højgaard, Lisbeth Drozd, Kastrup, Jens, Ekblond, Annette, GMP Compliant Production of a Cryopreserved Adipose-Derived Stromal Cell Product for Feasible and Allogeneic Clinical Use, Stem Cells International, 2022, 4664917, 12 pages, 2022. https://doi.org/10.1155/2022/4664917 | “Lipoaspirate Preparation and SVF Isolation. The lipoas-pirate was stored at CSCC at room temperature (RT) for <24 hr after procurement. SVF was isolated from the lipoas-pirate by manual handling and enzymatic digestion, as pre-viously described [12, 23]. In brief, each lipoaspirate was washed twice with 1x phosphate-buffered saline (PBS, -Ca2+ -Mg2+, and -phenol red) (Gibco, Life Technologies). The tissue was digested by GMP-grade collagenase NB6 (Nordmark, Germany) dissolved in Hank’s Balanced Salt Solution (2 mM Ca2+) and neutralized with a complete medium. Complete medium, as used during the entire manufacturing process, contained minimum essential medium, MEM alpha (αMEM) without ribonucleosides and deoxyribonucleosides, (Gibco, Life Technologies), 1% penicillin/streptomycin (10,000 U/ml and 10,000 μg/ml, respectively) (Gibco, Life Technologies), and 5% heparin-free human platelet lysate (hPL, custom made small batch, ≤16 donors, Sexton Biotechnologies). The SVF suspension was filtered, centrifuged, and resuspended in complete medium. Cell number and viability of cells in the isolated SVF was determined, using a NucleoCounter® NC-100™ (ChemoMetec, Denmark), according to manufacturer’s instructions.” | Mandana Haack-Sørensen; mandana.haack-soerensen@regionh.dk | ||||||||||||||||||||||||||||||||||
| 168 | Jovic | 2023 | Stromal Vascular Fraction | SVF | Automated Unistation 2nd Version | Human | SubQ | Liposuction | 20 ml | Collagenase | 0.10% | Syringe | 37 | 30 | Centrifugation 800 RPM 5 min, mix SVF with PPP and centrifuge 800 RPM 5 min. | 0 to 100%. (Luna) | SVF | Yield and Viability | Darko Jovic, MD, PhD ; Ljubisa Preradovic, PhD; Mico Kremenovic, MD; Filip Jovic; Milica Antonic, MD; Zoran Aleksic, MD, PhD; and Vesna Ljubojevic, MD, PhD Effect of Donor Site Selection for Fat Grafting on the Yield and Viability of the Stromal Vascular Fraction Aesthetic Surgery Journal 2023, Vol 43(9) NP704–NP712 | After injecting the tumescent solution, we obtained the fat by classic vibration-assisted liposuction with 3-mm cannulae, which we placed through small incisions.25 The obtained fat was processed to obtain SVF with the semiautomatic UNISTATION 2nd Version system, as well as manual preparation with common laboratory equipment. The first step was to centrifuge the obtained fat for 5 minutes at 800 revolutions per minute to obtain concentrated adipose tissue and separate the layer of red blood cells (RBCs). We mixed 20 mL of centrifuged adipose tissue with 20 mL of 0.1% collagenase and performed the mixing process at a temperature of 37°C for 30 minutes, after which we centrifuged the resulting mixture for 5 minutes at 800 revolutions per minute. After centrifugation, we took the syringe carefully, because in the lower part of the syringe near the very top there was a concentrate of 5 mL of SVF. We finally mixed that concentrate with 5 mL of platelet-poor plasma (PPP) and 30 mL of NaCl 0.9%, and put it to centrifugation for 5 minutes at 800 revolutions per minute to carry out the collagenase neutralization and washing process. From the centrifuged liquid, we separated 5 mL of SVF concentrate, which was located near the tip of the syringe. | |||||||||||||||||||||
| 169 | Murphy | 2023 | Preadipocytes | Insufficient Data to Classify | Manual | Human | approximately 1 g of SAT | Murphy J, Tam BT, Kirkland JL, Tchkonia T, Giorgadze N, Pirtskhalava T, Tsoukas MA, Morais JA, Santosa S. Senescence markers in subcutaneous preadipocytes differ in childhood- versus adult-onset obesity before and after weight loss. Obesity (Silver Spring). 2023 Jun;31(6):1610-1619. doi: 10.1002/oby.23745. Epub 2023 May 17. PMID: 37194560. | Tchkonia T, Tchoukalova YD, Giorgadze N, et al. Abundance of two human preadipocyte subtypes with distinct capacities for replication, adipogenesis, and apoptosis varies among fat depots. Am J Physiol Endocrinol Metab. 2005;288(1):E267-E277. | Preadipocytes from approximately 1 g of SAT were isolated and cultured as previously described [16]. | |||||||||||||||||||||||||||||||
| 170 | Zou | 2023 | EAT Cells | Adipose Stromal Cells (ASC) | Manual | Human | Epicardium | Surgical | chopped into small pieces (1–2 mm3) | 3 mg/mL type I collagenase, 0.156 mg/mL type XI collagenase, and 60 U/mL DNase I | 3 mg/mL type I collagenase, 0.156 mg/mL type XI collagenase, and 60 U/mL DNase I | 37 | 45 | water bath | Digestion was terminated through the addition of Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. | The cells were filtered out from the cell-enzyme mixture using a 40-μM stainless nylon mesh (Corning Falcon, USA). The filtrate was centrifuged at 300 ×g and 4 °C for 5 min, and cell sediment was resuspended in 1X PBS on ice for single-cell capture. | 40-μM stainless nylon mesh (Corning Falcon, USA) | and activity of cells were measured using a TC20 automatic cell counter (Singleron) | Trypan blue (302643, Sigma) staining | single-cell RNA sequencing (scRNA-Seq) | Zou R, Zhang M, Zou Z, Shi W, Tan S, Wang C, Xu W, Jin J, Milton S, Chen Y, Wang G, Fan X. Single-cell transcriptomics reveals zinc and copper ions homeostasis in epicardial adipose tissue of heart failure. Int J Biol Sci. 2023 Jul 31;19(13):4036-4051. doi: 10.7150/ijbs.82844. PMID: 37705737; PMCID: PMC10496493. | Fresh EAT samples were rinsed over ice with phosphate buffer saline (PBS) and then chopped into small pieces (1–2 mm3). EAT cells were isolated using digestive enzymes (including 3 mg/mL type I collagenase, 0.156 mg/mL type XI collagenase, and 60 U/mL DNase I) in 37°C water bath for 45min. Digestion was terminated through the addition of Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. The cells were filtered out from the cell-enzyme mixture using a 40-μM stainless nylon mesh (Corning Falcon, USA). The filtrate was centrifuged at 300 ×g and 4 °C for 5 min, and cell sediment was resuspended in 1X PBS on ice for single-cell capture. The concentration and activity of cells were measured using a TC20 automatic cell counter (Singleron) following Trypan blue (302643, Sigma) staining. Cells with > 80% viability and concentration > 1×106 cells/mL were used for subsequent library preparation. | |||||||||||||||||||
| 171 | Acosta | 2023 | MVF | MVF | Manual | Rat | SubQ and Visceral | Surgical | Scissors | Collagenase | BSA/PBS | 6 mg/ml | 50 ml conical | 37 | 8 to 20 min | shaking | 37 micron filter | microscopy – visual | morphology | Acosta, F.M., Gonzalez Porras, M.A., Stojkova, K., Pacelli, S., Rathbone, C.R., Brey, E.M. Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments. J. Vis. Exp. (192), e64650, doi:10.3791/64650 (2023). | Hoying Latsche | 4. Microvascular fragment isolation protocol 1. Place 50 mL conical tube(s) containing excised fat from the rat into biohood. 2. Using forceps, place the fat in a standard 100 mm Petri dish (with ~0.5 mL of 1 mg/mL BSA in PBS to keep the tissue hydrated). 3. Clean and remove any visible blood vessels and muscle/ extraneous tissue from fat. 4. Mince the fat with scissors for ~10 min (mince enough so that it can be transferred with a 10 mL pipet). 5. Check for lumps by adding ~0.5 mL of 1 mg/mL BSA in PBS; continue mincing if necessary. 6. Transfer the minced fat to a sterile 250 mL flask with a 10 mL pipet. NOTE: Note the volume using a pipet. 7. Add enough BSA (1 mg/mL) so the final volume is 20 mL. 8. Add 4 mg/mL BSA in PBS to collagenase (collagenase final concentration: 6 mg/mL) (i. e., 12 mL for the 72 mg of collagenase or 24 mL for the 144 mg of collagenase). NOTE: Do not add until the mincing is complete as it is time sensitive.9. Gently shake the conical tube to ensure a homogenous solution and filter sterilize the solution with a 0.22 μm nylon net filter. 10. For the epididymal fat, digest for ~8-10 min; for the inguinal and posterior subcutaneous fat, digest for ~15-20 min in a 37 °C water bath, shaking the flask in a circular motion throughout (stop at the moment the fat gets to where there are only a few clumps remaining). 11. Transfer the digested fat into a 50 mL conical tube (there should be ~30 mL/tube), and label the tube as “digested fat”. 12. Spin the tube at 400 x g for 4 min; post spinning, the pellet must be red (Figure 3A). 13. During the spin, place the sterile 37 μM screen and 500 μM screens in a sterile Petri dish with 5 mL of 1 mg/mL BSA in PBS to presoak before use. 14. After the spin, decant the supernatant into a 50 mL conical tube labeled as “waste”. Perform the decantation gently to remove the superficial fat and to not disturb the pellet made of MVFs. 15. Add 10 mL of 1 mg/mL BSA in PBS to the tube containing the pellet (“digested fat”). Triturate (pipet up and down) the pellet 2x. 16. Avoid being too rough on the pellet in order not to disrupt the fragments. 17. Place the 500 μM screen in a new Petri dish above the plastic screen holder (Figure 3B). 18. Pipet 10 mL from the “digested pellet” tube over a 500 μM screen using concentric circles (Figure 3C). 19. Wash the filter with an additional 5 mL of 1 mg/mL BSA in PBS. The desired cells will filter through into the Petri dish; therefore, discard the 500 μM screen but save the filtered liquid inside the Petri dish. 20. Place the 37 μM screen in a new Petri dish above the plastic screen holder. 21. Change out the pipet to eliminate any clumps before using the 37 μM screen. 22. Pipet the liquid obtained from the first filtration over the 37 μM screen using concentric circles. 23. Wash the filter with an additional 5 mL of 1 mg/mL BSA in PBS. The desired cells will remain in the filter, therefore discard the filtered liquid, but save the 37 μM screen. 24. Slide the 37 μM screen into a new Petri dish containing 5 mL of 1 mg/mL BSA in PBS. 25. Shake the dish by tapping it against a conical holder to dislodge the fragments. Do not shake too vigorously, as the liquid/cells may spill out of the Petri dish. 26. Rinse the filter with an additional 5 mL of 1 mg/mL BSA in PBS. The desired cells will remain in liquid solution in the Petri dish. Save the 37 μM screen and the dislodged fragment containing liquid inside the Petri dish for the following steps. 27. Transfer the BSA + fragment containing liquid to a sterile 50 mL conical tube. 28. Repeat rinsing of the 37 μM screen several more times (each time with ~5 mL of 1 mg/mL BSA in PBS) and add to the conical tube. Repeat until the total volume collected is ~15-20 mL. Ultimately, the desired cells will be collected from the liquid solution in the Petri dish and placed in a conical tube; at this point, discard the 37 μM screen after the final rinse but save the dislodged fragments containing liquid inside the 50 mL conical tube. 29. Clip the end off of a 200 μL pipet tip using scissors. Gently shake the 50 mL tube, remove two samples of 20 μL, and put them in a clean 35 mm Petri dish. 30. Count the number of fragments in each sample in the Petri dish to obtain the total number of MVFs isolated. Total Fragments = 31. Spin the remaining dislodged fragment containing liquid in a 50 mL conical tube at 400 x g for 4 min to collect the MVF. | Eric M. Brey Eric.brey@utsa.edu | ||||||||||||||||||
| 172 | Li | 2024 | Adipose Stromal Vascular Fraction Adipose Stromal/Stem Cells ASC | SVF | Manual | Human | SubQ | Liposuction | > 100 ml | Type I Collagenase | PBS. 2mM CaCl2 | 0.1 % Used 1:1 with fat | 200 cc Nalgene Bottle | 37 | 60 to 80 min | shaking water bath set at 200 rpm | Centrifugation 300xg 5 min RT Shake Bottle, Re-centrifuge | NH4Cl after placing in culture | Stromal Vascular Fraction | Culture | Li in Jeffrey M. Gimble et al. (eds.), Adipose-Derived Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 2783, https://doi.org/10.1007/978-1-0716-3762-3_2, | 1. Warm up the buffer (500 mL or more of PBS or KRB). Line the surface of the biosafety hood with a disposable bench protector. 2. Warm up the freshly prepared collagenase solution in the 37 °C water bath. The volume of the collagenase solution used will be equal to the volume of the lipoaspirates (1:1). 3. Prepare the PBS (or KRB) solution with 1% BSA, filter the solution, and warm it in the 37 °C water bath. 4. To maintain optimal sterile conditions, open the surgical container used for the liposuction procedure under the biosafety hood (see Note 5). Dispense a volume of adipose tissue in sterile plastic bottles for each 175 cm2 flask (0.16 mL tissue/cm2 ). It is recommended that you distribute about 33 mL of tissue; each bottle can accommodate ~100 mL of tissue. We routinely process a total of 200 mL of tissue to be plated in six 175 cm2 flasks. Add an equal volume of warm PBS. Agitate to wash the tissue and then allow phase separation for 3–5 min. Alternatively, you can spin the samples at 300× g in an appropriate centrifuge for 1 min at room temperature. Suction off the infranatant solution (lower liquid phase) while retaining the supernatant solution containing the floating tissue. The wash is repeated several times until a clear infranatant solution is obtained (usually 3–4 times). 5. Add an equal volume (60–70 mL) of warm collagenase solution into the plastic bottles containing the clean adipose tissue sample. Wrap the bottles with parafilm and place them in a 37 ° C shaking water bath at ~200 rpm for 60–80 min until the tissue appears smooth upon visual inspection (see Note 6). 6. After digestion, spin the samples at 300× g in an appropriate centrifuge for 5 min at room temperature. Take the samples out of the centrifuge and shake them vigorously to thoroughly disrupt the pellet and to mix the cells. This is to complete the separation of the stromal cells from the primary adipocytes. Repeat the centrifugation step. 7. After spinning, the stromal vascular fraction will form a pellet at the bottom of the bottle or tube (this will usually include a layer of dark red cells). Carefully remove the top layer of oil and fat, the primary adipocytes (a yellow layer of floating cells), and the underlying layer of collagenase solution. Leave behind a small volume of collagenase solution above the pellet so that the cells are not disturbed. 8. Resuspend the cells in 10 mL of warm PBS (or KRB) solution and transfer the solution containing the cells into a 50 mL conical tube. Centrifuge the cells at 300× g in an appropriate centrifuge for 5 min at room temperature. 9. Aspirate the remaining collagenase solution. When aspirating, the tip of the pipette should aspirate from the top so that the oil is removed as thoroughly as possible. The cell pellet should be at the bottom of the tubes. 10. Resuspend the cells in 15 mL of stromal medium (see Note 7). Divide the cells according to the number of flasks. The cells are plated at a density equivalent to approximately 0.18 mL of liposuction tissue aspirate per cm2 of surface area (volume of ~33 mL of tissue for a 175 cm2 flask). | |||||||||||||||||||
| 173 | Zhu | 2024 | Adipose Derived Stem Cells | SVF | Manual | River Buffaloes (Murrah) | SubQ, Inguinal | Surgical | Finely Minced <1 mm3 | 200 mg | Type I Collagenase | Solarbio, Beijing, China) | Maybe complete medium containing 20% fetal bovine serum (Sijiqing, Hangzhou, China) and 1% penicillin/ streptomycin (Gibco) | 0.02% | Maybe Test Tube | 37 | 120 min | Shaking Water Bath | Digest filtered through 80 micron nylon mesh filter. Sample centrifuged 1000 RPM 10 min. Cell pellet resuspended | 80 micron nylon mesh filter prior to first centrifugation | Adipose Derived Stem cells | Culture – Adipogenesis | RT PCR Oil Red O in culture | Zhu R, Feng Y, Li R, Wei K, Ma Y, Liu Q, Shi D, Huang J. Isolation methods, proliferation, and adipogenic differentiation of adipose-derived stem cells from different fat depots in bovines. Mol Cell Biochem. 2024 Mar;479(3):643-652. doi: 10.1007/s11010-023-04753-9. Epub 2023 May 6. PMID: 37148505. | Compared enzymatic with nonenzymatic explant cultures. | For the enzymatic method, 0.2 g adipose was finely minced (< 1 mm3) and digested with 0.02% type I collagenase (Solarbio, Beijing, China) in a shaking water bath at 37 ℃ for 2 h. The digested sample was filtered with an 80-μm nylon mesh filter to remove undigested tissue. The digested sample was then centrifuged at 1000 rpm for 10 min. The supernatant was discarded; the cell pellet was resuspended with 1 mL complete medium. Cells were seeded in a 35-mm dish and cultured at 37 ℃ with 5% CO2. The culture medium was replaced every 3 days. When reaching 90% confluence, the primary cells were harvested by tryptase. The harvested primary cells were then continuously expanded until passage 3 (P3). The P3 cells were used for the subsequent experiments. | |||||||||||||||
| 174 | Zinger | 2024 | Stromal Vascular Fraction | Insufficient Data to Classify | Semi- Automated | Human | SubQ | Liposuction | fat was passed through a 0.6 mm Adinizer filter (BSL Co., Ltd., | Roche | 11 Wunch Units | 37 | 60 | Centrifugation | 150 ml Lactated Ringers | 100 micron Adinizer Filter | 2.40 ± 1.9 × 106/cc fat | 85 ± 12% | SVF | Characterization | FACS | Zinger G, Gronovich Y, Lotan AM, Sharon-Gabbay R. Pilot Study for Isolation of Stromal Vascular Fraction with Collagenase Using an Automated Processing System. Int J Mol Sci. 2024 Jun 28;25(13):7148. doi: 10.3390/ijms25137148. PMID: 39000252; PMCID: PMC11241134. | Cell Count and Cell Viability The nucleated cell count was measured using an automated cell counter, CellDropTM FL (DeNovix,Wilmington, DE, USA). The SVF sample was mixed with AO/PI (1:1) prior to direct pipette loading; then, an automated counter was used. | Stromal Vascular Fraction (SVF) Sterile Isolation The lipoaspirate was decanted in the two 60 mL syringes for approximately 3 min. The adipose tissue was washed three times with an equal amount of lactated ringer (LR) solution. The washed fat was passed through a 0.6 mm Adinizer filter (BSL Co., Ltd., Republic of Korea, according to the BCL protocol, this is not a required step) prior to the addition of collagenase (Liberas MNP-S GMP grade, Roche, Heidelberg, Germany) containing 11Wunch units. The mixture was incubated for 60 min in the ACS incubator (BSL Co., Ltd., Republic of Korea) at 37 ◦C. The digested fat was passed through a 0.1 mm Adinizer filter (according to the BCL protocol, this step would be with a 0.6 mm Adinizer filter) and injected into the ACPU single-use device. The washing solution chamber in the ACPU was filled with 150 mL of LR solution. The unit was placed inside the ACS centrifuge, and the SVF program was initiated. After 23 min, the final ACPU was removed from the centrifuge and the SVF product was collected from the ACPU using a long-collection blunt needle. The final SVF was analyzed immediately in the operating room for cell count and cell viability, then transferred to the laboratory for additional SVF characterization. | |||||||||||||||||
| 175 | Qin | 2024 | Stromal Vascular Fraction | Adipose Stromal Cells (ASC) | Manual | Human | SubQ | Liposuction | QY Collagenase. NB4 Collagenase | QY Collagenase, NB4 | QY – Normal Saline. NB4 – DMEM | QY – 45U/ml in a ratio 1:1 NB4 0.2 % ratio 1:1 | 37 | 45 | Shaker | Centrifugation 1500 g 10 minutes. 40 micron filter | 2x | 40 micron | Yield, Viability, and Morphology. The SVFs isolated with clinical and laboratory protocols could differentiate into adipogenic, osteogenic, and chondrogenic lineages in vitro, as demonstrated by positive Oil Red O staining, Alizarin red S staining, and Alcian blue staining | Cellometer® auto 2000 | 1.75 106/ml | SVF | Qin J, Cheng C, Huang RL, He J, Zhou S, Tan PC, Zhang T, Fang B, Li Q, Xie Y. Isolation of the Stromal Vascular Fraction Using a New Protocol with All Clinical-Grade Drugs: From Basic Study to Clinical Application. Aesthetic Plast Surg. 2024 Jul 10. doi: 10.1007/s00266-024-04221-9. Epub ahead of print. PMID: 38987318. | The harvested lipoaspirates were washed twice with nor- mal saline (NS) and transferred into a centrifuge tube. For the clinical protocol, the rinsed lipoaspirates were diluted with QY collagenase solutions at a concentration of 45 U/ml in a ratio of 1:1 by volume and incubated at 37 °C for 45 min. QY was prepared with NS. After digestion, the samples were centrifuged at 1500g for 10 min and the upper layer (oil) was removed. The cell pellet was washed twice with NS and the protector [20] that refers to glucose and amino acids were added. Importantly, in the first washing process, the resuspended liquid was passed through a 40-um filter. Finally, the SVF was the cell pellet at the bottom. For the laboratory protocol, the rinsed lipoaspirates were diluted with NB4 collagenase solutions at a concentration of 0.2% in a ratio of 1:1 by volume and incubated at 37 °C for 45 min. NB4 was prepared with Dulbecco’s Modified Eagle Medium (DMEM, HyClone, USA). After digestion, the samples at 1500 g for 10 min were centrifugated and the upper layer (oil) was removed. The cell pellet was washed with phosphate-buffered saline (PBS, Sinopharm Chemical Reagent Co., Ltd., China). Similarly, the resus- pended liquid was passed through a 40-um filter in the first washing process. The SVF was the cell pellet at the bottom. | |||||||||||||||||
| 176 | Montano | 2024 | Stromal Vascular Fraction | Adipose Stromal Fraction | Manual | Human | SubQ | Liposuction | 15 ml | Collagenase Type I | PBS | 0.10% | 50 ml Conical | 37 | 60 min | Shaking Incubator 100 rpm | 15 ml Stromal Media 50 mL of medium from a 500 mL bottle of DMEM/Ham’s F-12 medium, add 50 mL of heatinactivated fetal bovine serum (10%) and 5 mL of antibioticantimycotic 100× stock solution (1%) | Centrifugation 300 x g 5 min | 1 x Stromal Media | 100 micron strainer. Then 70 micron | Stromal Vascular Fraction | Preparation of Adipose Stem Cells | Montano E, Al-Ghadban S, Bunnell BA. Isolation of Human Adipose-Derived Stem Cells. Methods Mol Biol. 2024;2783:3-11 | 1. Wipe the biosafety cabinet working area with 70% ethanol and clean the collagenase solution, PBS, and stromal medium bottles before placing them in the biosafety cabinet. 2. Clean the outside of the lipoaspirate container with 70% ethanol and open it under the biosafety cabinet. 3. Pour up to 100 mL of the lipoaspirate into a 250 mL polypropylene beaker. 4. Add an equal volume of PBS to the lipoaspirate, and allow the fat and blood layers to separate for 3–5 min. The fat layer will float to the top and the blood layer will collect at the bottom. Next, aspirate the lower layer with a glass Pasteur pipette, careful not to aspirate any top fat later (see Note 5). Repeat the wash until the lipoaspirate loses its bloody appearance. 5. Divide the clean adipose tissue into 50 mL conical tubes by adding 15 mL of tissue to each conical. 6. Add an equal volume (15 mL) of collagenase solution to the 50 mL conical tubes and secure the caps with parafilm. Invert the conical tubes a few times, then secure them in the prepared shaking incubator for 1 h at 100 rpm. 7. Isolation of stromal vascular fraction (SVF): After 1 h of digestion, transfer the conical tubes back to the biosafety cabinet. Neutralize the digestion solution with 15 mL of stromal medium. Centrifuge the neutralized mixture at 300 × g (1200 rpm) for 5 min at room temperature. 8. Carefully pour the fat layer into a disposable cup. Aspirate the remaining supernatant, leaving approximately 3 mL and being careful not to aspirate the resulting SVF pellet (see Note 6). 9. Resuspend the cells in the remaining supernatant and filter through a 100 μm cell strainer into a 50 mL conical tube. (a) Add PBS to the filtrate and adjust the final volume to ~25 mL. (b) Centrifuge at 300 × g (1200 rpm) for 5 min. 10. Aspirate the supernatant and resuspend the pellet in 5 mL of stromal medium. Filter the suspension through a 70 μm cell strainer into a new 50 mL conical tube. Rinse the filter with an additional 2–3 mL of stromal medium. 11. Plate the cells using 175 cm2 cell culture flasks in the stromal medium. Add approximately 3 mL of cell suspension per flask. | |||||||||||||||||
| 177 | Carstens | 2024 | SVF | Insufficient Data to Classify. Used IV filter | Semi Automated | Human | SubQ | Liposuction | collagenase enzyme Worthington CLS-1 | Worthington | Hartmann’s solution (Lactated Ringers) | 300 CDU/mL of total volume | 39 | 60 | Rotary Shaker 150 RPM | centrifugedfor10minutesat600g.TheSVFcellpellet,locatedatthebottomofthedevice,wasremovedwitha14G6-inchneedle. | IV injected Thecellsuspensionwasadministeredintravenouslyoveraperiodof30minutesthroughabloodfilter. | Carstens M, Trujillo J, Dolmus Y, Rivera C, Calderwood S, Lejarza J, López C, Bertram K. Adipose-derived stromal vascular fraction cells to treat long-term pulmonary sequelae of coronavirus disease 2019: 12-month follow-up. Cytotherapy. 2024 Sep;26(9):1076-1083. doi: 10.1016/j.jcyt.2024.03.491. Epub 2024 Apr 4. PMID: 38639670. | ShiL,WangL,XuR,etal.MesenchymalstemcelltherapyforsevereCOVID-19. SignalTransductTargetTher2021;6:339–44. | No significant short-or long-term Aes were associated with this intervention | PreparationofSVF SVFisolationandintravenousadministrationoftheSVFcellswereconductedineithertheManaguaorLeonhospitals.TheGIDSVF-2device(Figure1)wasusedtoisolateandseparatetheSVFcellsforeachpatient(GIDBio)[25].Fifty-eightycubiccentimetersofrawlip-oaspiratewasharvestedfromtheabdominalsubcutaneousfatandpasseddirectlyintothesterileSVF-2processingdevice.Thelipoaspi-ratewaswashedthreetimestoremoveredbloodcells,leukocytesandfreeoils.Approximately125ccofHartmann’ssolutionwasaddedtotheadiposetissuewith70000CDUofcollagenaseenzyme(WorthingtonCLS-1,Lakewood,NJ,USA)toachieveaconcentrationof300CDU/mLoftotalvolume.Themixturewasdissociatedfor60minutesonarotarymixerinanincubatorat39°Candat150RPM.Afterdissociation,themixturewascentrifugedfor10minutesat600g.TheSVFcellpellet,locatedatthebottomofthedevice,wasremovedwitha14G6-inchneedle.TwoSVFsampleswerepassedthroughanautomatedfluorescencecytometer(LUNA-STEM;LogosBiosystems,Annandale,VA,USA)forcellcountingandviabilityassessment.SVFcellviabilitywas 70%inallinfusions. | ||||||||||||||||||||
| 178 | Al‑Sammarraie | 2024 | SVF | SVF | Manual | Mouse | Visceral | Surgical | Minced | Collagenase Type II | 37 | 10 to 15 min | DPBS (without Mg, Ca) was added to reduce collagenase activity | Centrifugation | Stromal Vascular Fraction | IV infusion for Corona Virus treatment | Al-Sammarraie, S.H.A., Ayaz-Güner, Ş., Acar, M.B. et al. Mesenchymal stem cells from adipose tissue prone to lose their stemness associated markers in obesity related stress conditions. Sci Rep 14, 19702 (2024). https://doi.org/10.1038/s41598-024-70127-w | Visceral adipose tissues of animals were collected, minced, and incubated with type II collagenase for 10–15 min at 37° C to liberalize the cells. DPBS (without Mg, Ca) was added to reduce collagenase activity. After centrifugations, the pellet of adipose originated cells was dissolved in 8 mL 10% FBS containing DMEM Low glucose (1 g/L) medium and transplanted into the flask. During expansion process cells other cell types of adipose niche such as adipocytes, macrophages etc. were eliminated and spindle shape mesenchymal-like cells were enriched. Cells were expanded and 3 × 106 cells were stored by freezing at the end of passage 2 from each animal. | |||||||||||||||||||||||
| 179 | Heyman | 2024 | Not Stated | Adipose Stromal Cells (ASC) | Manual | Bovine | Back Region near shoulder | Surgical | Minced | Colllagenase. IA (Sigma). Liberase (Sigma) IV (Sigma) IA+Trysin | Sigma | PBS | 13 to 1262 U/mg | 4-well tissue culture plate | 38.5 | Hours: 3,6, Over Night, 24 | None | 30% Serum | 5min at 400 × g | 70 micron | Neubauer hemocytometer. Trypan Blue Stain | The average cell yield ranged from 4.38 × 106 to 130 × 106 cells/g AT | Cells | Culture | Heyman E, Devriendt B, De Vlieghere E, Goethals K, Van Poucke M, Peelman L, De Schauwer C. Evaluation of enzymatic protocols to optimize efficiency of bovine adipose tissue-derived mesenchymal stromal cell isolation. NPJ Sci Food. 2024 Oct 1;8(1):70 | Cell isolation methods Bovine MSCs were isolated from subcutaneous AT (at the back region, near shoulder) from eight healthy, adult (2–4 years) female Belgian White Blue cows, collected in the local abattoir. Briefly, AT samples were transported within 1–2 h to the lab in phosphate buffered saline (PBS) without Ca2+/ Mg2+ (Gibco) containing 0.05mg/mL gentamycin (Gibco). Tissue samples were extensively washed to remove blood, cut in small pieces, weighed, transferred into a 4-well plate and minced with a sterile pipet tip for 1–2min in an enzymatic solution at 38.5 °C in a humidified atmosphere containing 5% CO2. The following isolation conditions were evaluated: the enzymes Coll IA (1262 units/mg, Sigma,C9722), LibTM (13 units/mg, Sigma, LIBTMRO) and Coll IV (Sigma, 1267 units/mg, C5138) as well as a combination of Coll IA with trypsin 0.25% (Sigma, 1609 units/mg,T4799)were tested at two concentrations (0.1% and 0.04%) and four incubation times (3 h, 6 h, ON (±15h) and 24h) (Fig. 6). Subsequently, the enzymatic reaction was neutralized with an equal amount of cold culture medium, consisting of low glucose Dulbecco’s Modified Eagle Medium (DMEM-LG; Invitrogen) supplemented with 30% fetal bovine serum (FBS; Sigma), 10–11 M dexamethasone (Sigma), 1% antibiotic-antimycotic solution (Sigma) and 1% L-glutamine (Invitrogen)49. After 1min, the non-buoyant fraction was filtered using a 70 μmcell strainer. Cells were collected after centrifugation for 5min at 400 × g at room temperature (RT) | |||||||||||||||
| 180 | Divoux | 2024 | Adipose Derived Stem Cells | Manual | Human | Abdominal and gluteofemoral subcutaneous adipose tissues | surgical | minced | Collagenase Type I | 1 % BSA | 37 | 45 | the obtained suspension was centrifuged (200 g, room temperature, 5 min). The remaining tissue fragments were eliminated through filtration of resuspended pellets on 100- and 40-μm strainers. Blood contamination was removed by incubating the pellet suspension in red blood cell lysis buffer for 5 min. The resulting cell suspension was depleted of endothelial cells using magnetic beads coupled to CD31 antibody (15) and the cells in the flowthrough fraction were plated in tissue culture flasks with a Dulbecco’s modified Eagle’s Medium (aMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, for adipose progenitor cells isolation by plastic adherence. | 100- and 40-μm strainers | red blood cell lysis buffer for 5 min | Adipose Stem Cells | single-cell RNA sequencing | Divoux A, Whytock KL, Halasz L, Hopf ME, Sparks LM, Osborne TF, Smith SR. Distinct subpopulations of human subcutaneous adipose tissue precursor cells revealed by single-cell RNA sequencing. Am J Physiol Cell Physiol. 2024 Apr 1;326(4):C1248-C1261. doi: 10.1152/ajpcell.00726.2023. Epub 2024 Mar 4. PMID: 38581663; PMCID: PMC11193533. Copy Download .nbib | Isolation of Human Adipose-Derived Stem Cells and In Vitro Experiments Abdominal and gluteofemoral subcutaneous adipose tissues were minced and incubated with 1 mg/mL collagenase type I supplemented with 1% BSA in a shaking water bath at 37 for 45min. To eliminatemature adipocytes, the obtained suspension was centrifuged (200 g, room temperature, 5 min). The remaining tissue fragments were eliminated through filtration of resuspended pellets on 100- and 40-μm strainers. Blood contamination was removed by incubating the pellet suspension in red blood cell lysis buffer for 5 min. The resulting cell suspension was depleted of endothelial cells using magnetic beads coupled to CD31 antibody (15) and the cells in the flowthrough fraction were plated in tissue culture flasks with a Dulbecco’s modified Eagle’s Medium (aMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, for adipose progenitor cells isolation by plastic adherence. After 24 h, the nonadherent cells were washed away by replacing the media with aMEM supplemented with 2.5% FBS, EGF (10 ng/mL), and FGF (1 ng/mL). Adherent cells were maintained at 37 C in a humid atmosphere with 5%CO2 until themonolayer of cells reached confluence, with medium change every 2 days. After confluence was reached at passage 2, adherent cells were harvested with 10% trypsin/EDTA, slow-frozen, and stored at 80 C. These cells, named adipose-derived stem cells (ADSCs) were assessed for mycoplasma contamination using a luciferase enzyme-based assay, the MycoAlert Detection kit (Lonza), according to the manufacturer’s instructions. | ||||||||||||||||||||||
| 181 | Cremona | 2025 | SVF | Adipose Stromal Cells (ASC) | Manual | Human | Collagenase I and VIII | PBS CaCl MgCl | 37 | 40 to 60 min | Constant Gentle | PBS | Centrifugation | Several Times | 100 micron then 40 micron | SVF | Cremona, M.; Gallazzi, M.; Rusconi, G.; Mariotta, L.; Gola, M.; Soldati, G. State of the Art in the Standardization of Stromal Vascular Fraction Processing. Biomolecules 2025, 15, 199. https://doi.org/10.3390/ biom15020199 | Review | SVF Isolation SVF isolation represents a critical step in extracting a viable population of ASCs and stromal cells for regenerative purposes. This process follows the disruption of AT using enzymatic or mechanical dissociation methods to obtain a heterogeneous population of cells, including ASCs, pericytes, fibroblasts, immune cells, and other progenitor cells [43–45]. Enzymatic digestion has long been considered the “gold standard” for SVF isolation due to its ability to effectively dissociate AT and release a high yield of viable cells [46]. This method typically involves the use of collagenase, an enzyme that breaks down collagen of the extracellular matrix, allowing for the separation of stromal cells from the AT. A commonly used collagenases blend consists of type I and VIII collagenases, resuspended in enzyme-activating phosphate-buffered saline (PBS) containing calcium and magnesium ions (Ca2+/Mg2+) [30]. The tissue is incubated at 37 ◦C, followed by serial centrifugation and washing steps using PBS without Ca2+/Mg2+ to block enzyme activity and optimize cell viability [47,48]. Typical enzymatic protocols for SVF isolation involve collagenase I, III, and V, which are incubated for 40–60 min at 37 ◦C under constant and gentle agitation. This is followed by multiple filtration steps through 100 μm and 40 μm cell strainers to remove undigested tissue, cell debris, and extracellular matrix components. These protocols ensure high cell yields, with viability exceeding 70% and high colony-forming unit (CFU) values, which are indicative of functional ASC populations [39,49]. | ||||||||||||||||||||||
| 182 | Reid | 2025 | SVF | Adipose Stromal Cells (ASC) | Manual | Human | Liposuction | Collagenase II | Worthington | PBS | 0.15% | 37 | 60 min | Continuous shaking | Centrifugation 1,500 RPM 10 min | 1 x PBS | 100 and 70 micron | Nucleated cells stained with crystal violet were counted in an improved Neubauer chamber. | FACS | Reid G, Cerino G, Melly L, Fusco D, Zhang C, Reuthebuch O, Milan G, Marsano A. Harnessing the angiogenic potential of adipose-derived stromal vascular fraction cells with perfusion cell seeding. Stem Cell Res Ther. 2025 May 1;16(1):220. doi: 10.1186/s13287-025-04286-6. PMID: 40312732; PMCID: PMC12044990. | Cerino G, Gaudiello E, Muraro MG, Eckstein F, Martin I, Scherberich A, Marsano A. Engineering of an angiogenic niche by perfusion culture of adipose-derived stromal vascular fraction cells. Scientif Rep. 2017. | SVF cell isolation All materials were purchased from Sigma-Aldrich (Merck Millipore, Burlington, USA) unless otherwise stated. The lipoaspirate was digested with equal parts 0.15% w/v collagenase type II (Worthington Biochemical Corporation, Lakewood, USA) and phosphate buffered saline (PBS) (Invitrogen, Waltham, USA) at 37 °C undergoing continuous shaking for 60 min [11, 12]. Following centrifugation at 1,500 rpm for 10 min, the supernatant and the floating lipid-rich layer was discarded and the cellular pellet was washed once with PBS. The resulting cell suspension was strained through a 100 μm and a 70 μm nylon-mesh. The resulting SVF was then re-suspended in cell culture medium consisting of 87% v/v high glucose DMEM, 10% v/v fetal bovine serum, 1% v/v penicillin/streptomycin, 1% v/v glutamine, and 1% v/v N-2-hydroxyethylpiperazine- N-2-ethane sulfonic acid (HEPES) buffer. Nucleated cells stained with crystal violet were counted in an improved Neubauer chamber. Freshly isolated SVF cells were used straight away or frozen in 10% v/v dimethyl sulfoxide and 90% v/v FBS and stored in liquid nitrogen. | |||||||||||||||||||
| 183 | Carstens | 2025 | Stromal Vascular Fraction. AdSVF | Stromal Vascular Fraction | Semi-Automated | Human | SubQ. Abdominal | Liposuction | Approximately 80–120 ml of abdominal fat was aspirated | “Enzyme” | 125 cc of Hartmann’s solution | GID SVF-2 | 39 | 60 min | Rotary Mixer | Centrifugation 600g 10 min | AdministrationofSVF AvolumeoftheSVFsuspensioncontainingadoseof40£106SVFcellswasisolated.Thecellswerethenfurthersuspendedtoatotalof100ccofHartmann’ssolution.Thecellsuspensionwasadministeredintravenouslyoveraperiodof30minutesthroughabloodfilter. | automatedfluorescencecytometer(LUNA-STEM | >/= 80% | AdSVF | Facial Injection for Parkinsons | Carstens M, Martínez-Cerrato J, Garcia L, Rivera B, Bertram K. Safety of adipose-derived stromal vascular fraction cells to treat Parkinson’s disease. Parkinsonism Relat Disord. 2025 Mar;132:107214. doi: 10.1016/j.parkreldis.2024.107214. Epub 2024 Nov 28. PMID: 39658493. | M.H. Carstens, M.H. Carstens, F.J. Quintana, S.T. Calderwood, et al., Treatment of chronic diabetic foot ulcers with adipose-derived stromal vascular fraction injections: safety and evidence of efficacy at 1 year, Stem Cells Transl Med 10 (8) (2021) 1138–1147. | AdministrationofSVF AvolumeoftheSVFsuspensioncontainingadoseof40£106SVFcellswasisolated.Thecellswerethenfurthersuspendedtoatotalof100ccofHartmann’ssolution.Thecellsuspensionwasadministeredintravenouslyoveraperiodof30minutesthroughabloodfilter. | PreparationofSVF SVFisolationandintravenousadministrationoftheSVFcellswereconductedineithertheManaguaorLeonhospitals.TheGIDSVF-2device(Figure1)wasusedtoisolateandseparatetheSVFcellsforeachpatient(GIDBio)[25].Fifty-eightycubiccentimetersofrawlip-oaspiratewasharvestedfromtheabdominalsubcutaneousfatandpasseddirectlyintothesterileSVF-2processingdevice.Thelipoaspi-ratewaswashedthreetimestoremoveredbloodcells,leukocytesandfreeoils.Approximately125ccofHartmann’ssolutionwasaddedtotheadiposetissuewith70000CDUofcollagenaseenzyme(WorthingtonCLS-1,Lakewood,NJ,USA)toachieveaconcentrationof300CDU/mLoftotalvolume.Themixturewasdissociatedfor60minutesonarotarymixerinanincubatorat39°Candat150RPM.Afterdissociation,themixturewascentrifugedfor10minutesat600g.TheSVFcellpellet,locatedatthebottomofthedevice,wasremovedwitha14G6-inchneedle.TwoSVFsampleswerepassedthroughanautomatedfluorescencecytometer(LUNA-STEM;LogosBiosystems,Annandale,VA,USA)forcellcountingandviabilityassessment.SVFcellviabilitywas 70%inallinfusions. | ||||||||||||||||
| 184 | Yang | 2025 | Stromal Vascular Fraction | Adipose Stromal Cells (ASC) | Manual | Human | Varied | Liposuction and surgical | 0.2% collagenase NB4 (Nordmark Biochemicals) | Nordmark | 0.20% | 37 | 60 min | shaking at 150rpm/min | After digestion, the cell suspension was filtered through a 100μm cell strainer and centrifuged at 1500rpm/min for 5 minutes | 100 micron | SVF pellet was then resuspended with red blood cell lysis buffer (ACK Lysis Buffer) for about 1 min to remove red blood cells, then rinsed with 1 ml of PBS+ 1% penicillin/streptomycin solution and centrifuged at 300 × g for 5 min, where the supernatant was discarded. | FACS | FACS | FACS | Yang ZH, Zhang TY, Chen FZ, Xie Y, Tan PC, Li QF, Zhou SB. Effect of age, harvest site and body mass index on the cell composition of the stromal vascular fraction. Plast Reconstr Surg. 2025 Jan 22. doi: 10.1097/PRS.0000000000011970. Epub ahead of print. PMID: 39840818. | Hearnden R, Sandhar B, Vyas V, Longhi MP. Isolation of stromal vascular fraction cell suspensions from mouse and human adipose tissues for downstream applications. STAR Protoc. 2021 Mar 30;2(2):100422. doi: 10.1016/j.xpro.2021.100422. PMID: 33870227; PMCID: PMC8044717. | Fat from different age groups | Stromal vascular fraction (SVF) isolation The SVF was isolated using a modification of published methods[12, 13]. In a first phase, the adipose tissue was washed extensively with phosphate-buffered saline (PBS, pH 7.4, Gibco) to remove free oil and blood, and then thoroughly minced into small pieces using sterile scissors. Afterwards, tissue digestion was induced with 0.2% collagenase NB4 (Nordmark Biochemicals) at 37°C for 1 hour with shaking at 150rpm/min. After digestion, the cell suspension was filtered through a 100μm cell strainer and centrifuged at 1500rpm/min for 5 minutes. Then a separation of fat, collagenase digestion solution, and stromal vascular fraction will be apparent. We carefully aspirate off the fat and collagenase and leave a small amount of the brown collagenase solution so that the pelleted SVF is not disturbed. The obtained SVF pellet was then resuspended with red blood cell lysis buffer (ACK Lysis Buffer) for about 1 min to remove red blood cells, then rinsed with 1 ml of PBS+ 1% penicillin/streptomycin solution and centrifuged at 300 × g for 5 min, where the supernatant was discarded. The SVF cells were suspended at a final concentration of 1 × 106 nucleated cells/mL in PBS determined by automated cell counters (Invitrogen). Freshly isolated cells from the SVF were maintained on ice and stained for analytical flow cytometry and cell sorting experiments. | |||||||||||||||||
| 185 | Westcott | 2025 | Stromal Vascular Fraction (SVF) | Adipose Stromal Cells. (ASC) | Manual | Human | SubQ | Lipoaspirate | collagenase type II | Sigma | Hanks’ balanced salt solution with calcium and magnesium supplemented with 0.5% bovine serum albumin | 1 mg/ml | 37 | 20 plus 5 minutes (EDTA, pH 8.0 at a concentration of 10 mM was then added for an additional 5 minutes | shaker water bath incubator. vigorous shaking by hand every 5 minutes. | Cells were filtered through a 100 μm filter and washed, and if flow cytometry was to be performed, incubated with ACK lysis buffer for 5 minutes. SVF was then washed and filtered through 40 μm filter followed by plating on multiwell plates in growth media (DMEM+glutamax, 15% FBS, 1% penicillin-streptomycin) or suspended in FACS buffer for flow cytometry | wash media (DMEM+glutamax with 10% fetal bovine serum (FBS) and 1% penicillin- streptomycin) or FACS buffer (PBS with 1mM EDTA, 25 mM HEPES and 1% FBS) was added. | 100 micron. 40 micron | SVF | snRNAseq. Lymphodema. Pro lymhphangiogenic ASCs. single- nucleus RNA sequencing | Westcott GP, Emont MP, Gulko A, Zhou Z, Kim C, Varma G, Tsai LL, O’Donnell E, Loureiro ZY, Liang W, Jacobs C, Tsai LT, Padera TP, Singhal D, Rosen ED. Single-nuclear transcriptomics of lymphedema-associated adipose reveals a pro-lymphangiogenic stromal cell population. bioRxiv [Preprint]. 2025 Feb 19:2025.02.18.638907. doi: 10.1101/2025.02.18.638907. PMID: 40027673; PMCID: PMC11870541. | Fresh lipoaspirate was incubated with collagenase type II (1 mg/ml) (Sigma-Aldrich) in digestion buffer (Hanks’ balanced salt solution with calcium and magnesium supplemented with 0.5% bovine serum albumin) on a shaker water bath incubator at 37°C for 20 minutes with vigorous shaking by hand every 5 minutes. EDTA, pH 8.0 at a concentration of 10 mM was then added for an additional 5 minutes, after which wash media (DMEM+glutamax with 10% fetal bovine serum (FBS) and 1% penicillin- streptomycin) or FACS buffer (PBS with 1mM EDTA, 25 mM HEPES and 1% FBS) was added. Cells were filtered through a 100 μm filter and washed, and if flow cytometry was to be performed, incubated with ACK lysis buffer for 5 minutes. SVF was then washed and filtered through 40 μm filter followed by plating on multiwell plates in growth media (DMEM+glutamax, 15% FBS, 1% penicillin-streptomycin) or suspended in FACS buffer for flow cytometry. Human primary dermal lymphatic endothelial cells (HDLEC, Cell Biologics, H-6064) and human umbilical vein endothelial cells (HUVEC, gift from Shingo Kajimura) were maintained on gelatin-coated flasks in complete human endothelial cell media (Cell Biologics, H-1168). | |||||||||||||||||||
| 186 | Liang | 2025 | adipose-derived mesenchymal stem cells ADMSCs | Insufficient Data to Classify | Human | collagenase type I | (Sigma-Aldrich, St. Louis, MO, USA) | 0.10% | Liang Q, Ding Q, Zhao L, Tan J, Niu W. USP15-modified ADMSCs-Exo alleviates chondrocyte damage and effectively relieved osteoarthritis by inducing M2 polarization of macrophages through deubiquitinating FOXC1. J Orthop Surg Res. 2025 Apr 2;20(1):336. doi: 10.1186/s13018-025-05742-y. PMID: 40176111; PMCID: PMC11963356. | Materials and methods Cells culture and transfection This study was approved by the Second Affiliated Hospital of Guangzhou University of Chinese Medicine Ethics Committee and conducted with informed consent from all volunteers. Adipose tissue was obtained from healthy donors and human ADMSCs were isolated from it. Adipose tissues were first washed with PBS buffer and digested with 0.1% collagenase type I (Sigma-Aldrich, St. Louis, MO, USA) and cultured in DMEM (high glucose) medium (Weike Biotechnology, Shanghai, China) containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (P/S) (Invitrogen, Carlsbad, CA, USA), and the medium was replaced with fresh one in a timely manner. | |||||||||||||||||||||||||||||||
| 187 | Kim | 2026 | Stromal Vascular Fraction (SVF) | Insufficient Data to Classify | Manual | Human | SubQ | Lipoaspirate | Cell viability was assessed using the methylene blue dye exclusion test (NanoEntek, Seoul, South Korea). | endotoxin assays (Associates of Cape Cod, MA, USA) | SVF | Osteoarthritis | Kim YS, Suh DS, Kwon YB, Chung JH, Koh YG. Injection of Adipose-Derived Stromal Vascular Fraction Rapidly Relieves Pain in Patients with Knee Osteoarthritis. Medicina (Kaunas). 2026 Feb 20;62(2):409. doi: 10.3390/medicina62020409. PMID: 41752807; PMCID: PMC12943560. | Isolation of SVF from Subcutaneous Adipose Tissue Subcutaneous adipose tissue samples were obtained through tumescent liposuction from the gluteal regions of patients 1 day before SVF injection. We collected 140 mL of adipose tissue, and this was suspended in phosphate-buffered saline (PBS) at the standard working concentration (1×), placed in a sterile box, and transported to the laboratory. A 120 mL of adipose tissue was used for injection and the remaining 20 mL of adipose tissue was processed similarly and used for laboratory analyses to characterize SVF cellular properties, including colony-forming unit fibroblast (CFU-F) formation and multilineage differentiation potential, as described in detail in the Supplementary Materials [20–25]. An overview of the SVF preparation and injection process is shown in Figure 1. | |||||||||||||||||||||||||||
| 188 | Buyukkaya | 2026 | SVF and Nano-Fat | Insufficient Data to Classify | Lipocell SVF kit (Tiss’You, Domagnano, San Marino | SVF and Nan0-Fat | SKIN QUALITY AND PHOTOAGING | Buyukkaya O, Karakol P, Tapki MM, Bozkurt M. Comparison Of Effects Of Stromal Vascular Fraction And Nano-Fat Applications On Skin Quality And Photoaging. Plast Reconstr Surg. 2026 Mar 11. doi: 10.1097/PRS.0000000000013017. Epub ahead of print. PMID: 41811159. | SVF and Nano-fat Preparation: The harvested fat was filtered through a 50-micron filter using the Lipocell SVF kit (Tiss’You, Domagnano, San Marino) with continuous washing with sterile saline. Through filtration, the fat and blood residues were removed from lipoaspirate, then collected into a 10 cc syringe and centrifuged at 2500 rpm for 5 minutes. After centrifugation, the sample was separated into two layers as SVF at the bottom and adipose tissue at the top. The SVF was subsequently transferred to another syringe for further processing. Nano-fat was obtained by emulsifying the harvested fat using the Tulip Nanofat set, filtering it through progressively finer filters. | ||||||||||||||||||||||||||||||||
| 189 | van Dongen | 2026 | Stromal Vascular Fraction (SVF) | MFAT | Manual | Human | SubQ | Liposuction | van Dongen JA, Pouw RE, Bülbül M, Kemming HJL, Coert JH, Hillegersberg RV. Multidisciplinary management of bronchoesophageal fistula using adipose-derived stromal vascular fraction and platelet-rich plasma. VideoGIE. 2025 Oct 9;11(2):37-40. doi: 10.1016/j.vgie.2025.09.007. PMID: 41727389; PMCID: PMC12921678. | One procedure was performed with the patient under general anesthesia, and the other with the patient under propofol sedation. Subcutaneous adipose tissue was harvested (15 mL) and processed according to the fractionation of the adipose tissue procedure (Arthrex, Utrecht, the Netherlands) (Fig. 1).13,14 Briefly, infiltrated adipose tissue was harvested, centrifuged, fractionated (through a 1.4-mm Luer-to-Luer [Arthrex] lock connector), and centrifuged again. Three separate layers were formed: oil, SVF, and infiltration fluid. Oil and infiltration fluid were discarded (Fig. 2). Simultaneously, 15 mL of whole blood was obtained to create PRP. PRP (7 mL) was mixed with adipose SVF (1 mL) (Video 1, available online at www.videogie. org). Preparation of the injectate requires 30 to 45 minutes. | Roos E. Pouw, Department of Gastroenterology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, the Netherlands. E-mail: r.e.pouw-3@umcutrecht.n | ||||||||||||||||||||||||||||||
| 190 | Zhang | 2026 | Stromal Vascular Fraction (SVF) | Stromal Vascular Fraction (SVF) | Manual | Human | SubQ | Liposuction | 50 ml | Centrifuge at 900rpm for 1 minute to extract the upper layer of adipose tissue after centrifugation | Collagenase | Roche | 5 mg/ml | 37 | 30 | shake intermittently | centrifuge at 1500rpm for 10 minutes. | SVF | Femoral Head Necrosis | Zhang J, Zhao T, Wu Y, Li Y, Liu Z, Qian T, Mi Z, Ma B, Bai Y, He Y, Wu J, You J, Zuo L, Luo E, Zhang D, Peng J. Analysis of the efficacy of autologous adipose-derived stromal vascular fraction and calcium phosphate bone cement combined with core decompression in patients with femoral head necrosis: a retrospective study. BMC Musculoskelet Disord. 2026 Mar 4. doi: 10.1186/s12891-026-09619-x. Epub ahead of print. PMID: 41782103. | SVF extraction and preparation: A 0.3 cm incision is made in the patient’s abdomen, and approximately 300 ml of swelling solution (500 ml of saline + 1 mg of norepinephrine + 5 ml of 2% lidocaine hydrochloride) is injected into the subcutaneous fat layer. After 5 minutes, about 50 ml of autologous fat tissue is extracted using a liposuction needle. Centrifuge at 900rpm for 1 minute to extract the upper layer of adipose tissue after centrifugation, and digest with collagenase (Roche, concentration of 5mg/ml, total volume of 1ml) at 37℃ for 30 minutes. During the digestion process, shake intermittently to ensure complete digestion of the fat. After digestion is complete, centrifuge again at 1500rpm for 10 minutes. Remove the upper oily layer and most of the lower blood, leaving a small amount of blood and undigested fat for later use. Then centrifuge again at 1500rpm for 10 minutes to remove the supernatant. To ensure sufficient SVF cells remain in the femoral head, resuspend the bottom layer of SVF cells in 1-2ml of undigested fat and a small amount of blood to prepare a cell suspension of approximately 3-5×10^8/ml for later use | Dawei Zhang Corresponding author. E-mail:zdwasy6161@163.com;pengjiang301@126.com | |||||||||||||||||||
| 191 | Wu | 2026 | adipose-derived mesenchymal stem cells (ADSCs) | ASC | Manual | Human | Breast Fat (Cancer and Normal) | Surgical | Minced | Collagenase Type I | 0.10% | 37 | 120 min | Shaking Incubator | Digestion was terminated by adding an equal volume of complete culture medium (DMEM/F12 supplemented with 10% fetal bovine serum, FBS), | centrifugation at 500 × g for 5 min | 3X Centrifugaton 500 × g for 5 min | 70 micron filter 3 washes with centrifugation 500 × g for 5 min | FACS PE-CD29, PE-CD34, PE-CD45, PE-CD90, and PE-CD105, Alk Phosphatase | adipose-derived mesenchymal stem cells (ADSCs) | RNA Seq. In Vivo Differentiation | ADSCs were harvested and washed twice with PBS. Cells were divided equally into tubes containing 100 μL PBS and stained with the following antibodies: PE-CD29, PE-CD34, PE-CD45, PE-CD90, and PE-CD105, along with corresponding isotype controls. See Table S2 for detailed information about the antibodies. Staining was performed for 30 min at 4 °C in the dark, followed by two PBS washes. Samples were resuspended in 200 μL PBS and analyzed by flow cytometry. | Wu Y, Zhang M, Li P, Chen F, Liu J, Hou Y, Zhao S, Li X. A Stem Cell-Osteogenesis Axis in Malignant Breast Calcification: SQLE-Driven Reprogramming of Adipose-Derived Mesenchymal Stem Cells. Stem Cell Rev Rep. 2026 Apr 21. doi: 10.1007/s12015-026-11124-6. Epub ahead of print. PMID: 42014574. | Fresh specimens were rinsed twice in phosphate-buffered saline (PBS) containing 1%penicillin–streptomycin, with fascia and visible blood vessels removed. The tissue was minced and digested with 0.1% collagenase type I at 37 ℃ in a shaking incubator for 2 h. Digestion was terminated by adding an equal volume of complete culture medium (DMEM/F12 supplemented with 10% fetal bovine serum, FBS), followed by centrifugation at 500 × g for 5 min. The floating mature adipocytes were discarded, and the cell pellet was resuspended and passed through a sterile 70 μm filter. After centrifugation at 500 × g for 5 min and washing 2–3 times, cells were seeded into six-well plates and cultured at 37 ℃ in a humidified incubator with 5% CO2. | Yayi Hou yayihou@nju.edu.cn Shuli Zhao shulizhao79@njmu.edu.cn Xingxing Li lixingxing@stu.njmu.edu.cn | ||||||||||||||||
| 192 | Park | 2026 | adipose-derived mesenchymal stem cells (MSC) | Insufficient Data to Classify | adipose-derived mesenchymal stem cells (MSC) | Park et al. Extracell Vesicles Circ Nucleic Acids. 2026;7:425-40 DOI:10.20517/evcna.2025.173 | Lee JH, Ha DH, Go HK, et al. Reproducible large-scale isolation of exosomes from adipose tissue-derived mesenchymal stem/stromal cells and their application in acute kidney injury. Int J Mol Sci. 2020;21:4774. DOI PubMed PMC | Human MSCs (passage 4) were isolated from adipose tissue under GMP conditions at ExoCoBio Inc., as previously described[13]. | |||||||||||||||||||||||||||||||||
| 193 | Elschner | 2026 | Brown Adipose Tissue – Endothelial Cells (BAT-EC) White Adipose Tissue- Endothelial Cells (WAT-EC) | Adipose Stromal Cells (ASC) then Magnetic Selection for Brown Adipose Tissue – Endothelial Cells (BAT-EC) White Adipose Tissue- Endothelial Cells (WAT-EC) | Manual | Mouse | interscapular BAT and inguinal WAT | Surgical | Tissues were cut into small pieces | 50 μL Enzyme D, 25 μL Enzyme R and 6.25 μL Enzyme A | Miltenyi Biotec, | DMEM (Thermo Fisher Scientific, 11995-065) | gentleMACS� Octo dissociator | 37 | The cell suspension was washed by centrifugation 300×g 5 min at room temperature and resuspension in 10 mL PBS-B. Through two consecutive centrifugation steps, the cell suspension was concentrated down to first 1 mL and then 90 μL cell suspension in PBS-B. Magnetic cell separation using CD-31 Dynabeads. | 2 times | 100 μm and 40 μm cell strainers | Elschner T, Grein S, Sander J, Hildebrand S, Heubach L, Pannwitz N, Mircea M, Raimundez E, Karagiannakou V, Georgiadi A, Heeren J, Hasenauer J, Pfeifer A, Wilhelm-Jüngling K. From brown to white: Brown adipose tissue endothelial cells whiten in culture conditions. Mol Metab. 2026 Mar 12;107:102349. doi: 10.1016/j.molmet.2026.102349. Epub ahead of print. PMID: 41831567; PMCID: PMC13049445. | EC isolation For EC isolation from pups, interscapular BAT and inguinal WAT of early neonates were dissected after decapitation. Adipose tissues of 3—5 neonates were pooled and processed one batch in the EC isolation protocol. Adult mice were killed by cervical dislocation and interscapular BAT, inguinal WAT and gonadal WAT were dissected. Tissues of adult mice were processed individually or pooled for up to three animals. For EC isolation after chronic cold exposure, adipose tissue of single mice was processed. Tissues were cut into small pieces and placed into adipose tissue dissociation mix in gentleMACS � C tubes (Miltenyi Biotec, 130—0930237). For one batch of adipose tissue dissociation mix, 1.25 mL DMEM (Thermo Fisher Scientific, 11995-065) plus 50 μL Enzyme D, 25 μL Enzyme R and 6.25 μL Enzyme A (Miltenyi Biotec, 130-105-808) were used. Tissues were dissociated on gentleMACS� Octo dissociator with heaters on “37C_mr_ATDK_1” setting. The cell suspension was filtered using 100 μm and 40 μm cell strainers, washing both the collection tubes and strainer with sterile 0.5% bovine serum albumin in PBS (PBS—B). The cell suspension was washed by centrifugation 300×g 5 min at room temperature and resuspension in 10 mL PBS-B. Through two consecutive centrifugation steps, the cell suspension was concentrated down to first 1 mL and then 90 μL cell suspension in PBS-B. The day before the intended EC isolation, Dynabeads� (Thermo Fisher Scientific, 11035) were prepared for overnight coupling to CD31 antibody (BD Pharmigen, 553370). For this, 18 μL Dynabeads� were washed with PBS-B on a magnetic separation rack (Cell Signaling Technology, 14654S). | Kerstin Wilhelm-Jüngling Corresponding author. Institute for Cardiovascular Sciences, University Hospital, University of Bonn, Bonn, Germany. E-mail: kerstin.wilhelm-juengling@ukbonn.de (K. Wilhelm-Jüngling). | |||||||||||||||||||||
| 194 | He | 2026 | “adipose-derived mesenchymal stem cells (ADSCs)” | Insufficient Data to Classify | Rat | Epididymal Fat | Type I Collagenase | 37 | 15 to 30 min | Shaker | He J, Liu Y, Zhang L, Nan N, Ba T, Gao Y, Kang J, Hao H. Exosomes secreted by ADMSCs preserve cartilage integrity in knee osteoarthritis via AMPK-mediated mitochondrial dynamics and apoptosis. Stem Cells. 2026 Apr 22:sxag021. doi: 10.1093/stmcls/sxag021. Epub ahead of print. PMID: 42024683. | Liang Q, Ding Q, Zhao L, et al. USP15-modified ADMSCs-Exo alleviates chondrocyte damage and effectively relieved osteoarthritis by inducing M2 polarization of macrophages through deubiquitinating FOXC1. J Orthop Surg Res. 2025 Apr 2;20(1):336. | 2. Material and methods 2.1 Cell culture ADMSCs and chondrocytes were isolated separately. In brief, ADMSCs were obtained from rat epididymal fat and digested with type I collagenase on a shaker at 37℃ for 15-30 minutes [25]. | ||||||||||||||||||||||||||||
| 195 | Savoji | 2026 | Stromal Vascular Fraction (SVF) | Stromal Vascular Fraction (SVF) | Manual | Human | Lower Abdomen | Liposuction | 50 ml | collagenase type I (0.075%) | 0.08% | 37 | 60 | centrifuged at 1,200 x g 10 min | washed twice PBS | 90% | Bozorg Savoji P, Mirbahari SN, Rafiee S, Fakhim T, Nilforoushzadeh MA. Regenerative Scalp Preparation With Stromal Vascular Fraction and Platelet-Rich Fibrin Prior to Hair Transplantation in Lichen Planus: A Case Report. Clin Case Rep. 2026 Apr 17;14:e72084. doi: 10.1002/ccr3.72084. PMID: 42006542; PMCID: PMC13090173. | SVF was obtained from autologous adipose tissue harvested via manual liposuction of the lower abdomen using a 3-mm cannula under local anesthesia (tumescent solution containing 0.1% lidocaine). Fifty milliliters of adipose tissue were enzymatically digested with collagenase type I (0.075%) at 37°C for 60 min, centrifuged at 1200g for 10 min, and the resulting SVF pellet was washed twice with phosphate-buffered saline. A final volume of 5 mL of SVF (~1.5 × 106 cells/mL, 90% viability) was prepared for injection and administered to affected scalp regions, predominantly in the vertex. | |||||||||||||||||||||||
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