Human Adipose‐Derived Stromal Cell Isolation Methods and Use in Osteogenic and Adipogenic In Vivo Applications

Elizabeth Brett1, Ruth Tevlin2, Adrian McArdle2, Eun Young Seo2, Charles K.F. Chan2, Derrick C. Wan3, Michael T. Longaker2

1 Technical University Munich, Department of Plastic and Hand Surgery, Munich, 2 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, 3 Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2H.1
DOI:  10.1002/cpsc.41
Online Posting Date:  November, 2017
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Adipose tissue represents an abundant and easily accessible source of multipotent cells, which may serve as excellent building blocks for tissue engineering. This article presents a newly described protocol for isolating adipose‐derived stromal cells (ASCs) from human lipoaspirate, compared to the standard protocol for harvesting ASCs established in 2001.

Human ASC isolation is performed using two methods, and resultant cells are compared through cell yield, cell viability, cell proliferation and regenerative potential. The osteogenic and adipogenic potential of ASCs isolated using both protocols are assessed in vitro and gene expression analysis is performed. The focus of this series of protocols is the regenerative potential of both cell populations in vivo. As such, the two in vivo animal models described are fat graft retention (soft tissue reconstruction) and calvarial defect healing (bone regeneration). The techniques described comprise fat grafting with cell assisted lipotransfer, and calvarial defect creation healed with cell‐seeded scaffolds. © 2017 by John Wiley & Sons, Inc.

Keywords: adipose derived stromal cell; calvarial defect; digest; fat graft; liposuction

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Table of Contents

  • Introduction
  • Basic Protocol 1: Fat Processing and Cell Harvest
  • Basic Protocol 2: FACS Sorting
  • Basic Protocol 3: In Vitro Assays
  • Basic Protocol 4: RNA Harvest and qRT‐PCR
  • Basic Protocol 5: In Vivo Mouse Calvarial Defect Model
  • Basic Protocol 6: In Vivo fat Grafting Model
  • Basic Protocol 7: In Vivo Cell Harvest From fat Grafts
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Fat Processing and Cell Harvest

  • Human lipoaspirate samples (biohazard, obtained using appropriate IRB and associated consent form)
  • Ice
  • Medium 199 (Gibco, cat. no. 11150059)
  • Type I collagenase 2.2 mg/ml (Sigma Aldrich)
  • Collagenase, from Clostridium histolyticum (Sigma Aldrich, cat. no. C6885)
  • DNase I (Roche, cat. no. 10104159001)
  • Calcium Chloride dehydrate (Sigma Aldrich, cat. no. C3306)
  • Bovine Serum Albumin (Sigma Aldrich, cat. no. A2058)
  • P188 (Sigma Aldrich)
  • 50× HEPES (Life Technologies,)
  • 500 ml sterile FACS buffer [1×phosphate‐buffered saline (PBS; pH 7.4, 1× Gibco, 10010023), 2% fetal bovine serum, 1% P188, 1% penicillin‐streptomycin]
  • Histopaque, a commercially available density gradient separation medium (Sigma Aldrich, cat. no. 10771)
  • Hank's balanced salt solution (Cellgro, cat. no. 55022PB)
  • Sterile serological pipettes (5, 10 and 25 ml; Corning, 357543, 357551, 357525)
  • Sterile plastic bottles for centrifuging (250 ml; Corning, 430776)
  • 0.22‐μm filter system
  • 500‐ml sterile PTEG medium bottle
  • Parafilm®
  • 37°C water bath
  • Orbital shaker
  • Centrifuge
  • 100‐μm cell filter
  • Sterile polypropylene centrifuge tubes (50‐ml; Fisher Scientific, cat. no. 1443222)

Basic Protocol 2: FACS Sorting

  • SVF cells (see protocol 1)
  • Human anti‐CD45, anti‐CD31, and anti‐CD34 (BD Biosciences)
  • 500 ml sterile FACS buffer [1×phosphate‐buffered saline (PBS; pH 7.4, 1× Gibco, 10010023), 2% fetal bovine serum, 1% P188, 1% penicillin‐streptomycin]
  • BD FACSDiva™ software
  • FACS Aria II (BD Biosciences)
  • 100‐μm cell strainer (Corning, cat. no. 352360)
  • 15‐ml tubes

Basic Protocol 3: In Vitro Assays

  • SVF cells
  • Growth medium (DMEM, 10% FBS, and 1% penicillin/streptomycin) supplemented with recombinant FGF‐2
  • BrdU kit (BrdU Cell Proliferation Kit, Abcam, ab12556)
  • Trypan blue
  • XTT‐based assay: Cell Proliferation Kit II XTT (Roche Applied Science)
  • Osteogenic differentiation medium [Sigma Aldrich, L‐ascorbic acid (A4403), glycerol phosphate disodium salt hydrate (G6501)]
  • Adipogenic differentiation medium [Sigma Aldrich, Indomethacin (cat. no. I7378), Dexamethasone (cat. no. D4902), IBMX (cat. no. I7018), Insulin (cat. no. 90177C)]
  • Dulbecco's modified Eagle's medium
  • Fetal bovine serum (FBS)
  • Penicillin/streptomycin
  • 96‐well plate/6‐well plate, standard, untreated (Corning, cat. nos. 3898/CLS3516, respectively)
  • Spectrophotometer (Roche Applied Science)
  • GraphPad Prism
  • Light microscope
  • Additional reagents and solutions for alkaline phosphatase quantification (Levi et al., ), Oil Red O staining (Chung et al., ), and quantitative real‐time polymerase chain reaction (qRT‐PCR; see protocol 4)

Basic Protocol 4: RNA Harvest and qRT‐PCR

  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010023)
  • TRIzol RNA Isolation Reagent (Thermo Fisher, cat. no. 10296010)
  • RNA Isolation: RNEasy Mini Kit (Qiagen, cat. no. 74104)
  • Omniscript RT Kit (Qiagen, cat. no. 205111)
  • Specific gene primer sequences were obtained from PrimerBank (
  • HotStarTaq DNA Polymerase (Qiagen)
  • Fast SYBR Green Master Mix (Thermo Fisher, cat. no. 4385610)
  • Pipettes and pipette tips, sterile
  • Cell scraper, sterile
  • 1.5‐ml microcentrifuge tubes (Fisher Brand, catalog no. 05‐408‐129)
  • Applied Biosystems Prism 7900HT Sequence Detection System (Applied Biosystems)
  • LightCycler software
  • Additional reagents and equipment for harvesting RNA (Levi et al., )
NOTE: Steps 1 to 3 should be performed in a sterile cell culture hood.

Basic Protocol 5: In Vivo Mouse Calvarial Defect Model

  • CD‐1 nude mice, 8 to10 weeks old (Charles River Laboratories)
  • Isothesia (Isoflurane, USP, Butler‐Schein)
  • ASCs (see protocol 1)
  • (HA)‐coated poly(lactic‐co‐glycolic acid) (PLGA) scaffold, 4 mm diameter (see Cowan et al., 2014)
  • UV light
  • 96‐well culture plates
  • NSK Z500 drill (Brassler)
  • 4.0 mm circular knife (Xemax, cat. no. CK40)
NOTE: All research must be conducted through APLAC committee–approved protocols. Using your university guidelines, apply for ethical approval by writing a proposed animal protocol.

Basic Protocol 6: In Vivo fat Grafting Model

  • Human lipoaspirate samples (biohazard, obtained using appropriate IRB and associated consent form)
  • Freshly harvested NM‐ or CM‐ASCS (see protocol 1)
  • CD‐1 nude mice (Charles River, Crl:CD1‐Foxn1nu)
  • Isothesia (Isoflurane, USP, Butler‐Schein)
  • Centrifuge
  • Small scissors
  • Absorbent pad
  • 1‐ml Luer‐lock syringe (BD Biosciences, cat. no. 309628)
  • 14‐G blunt‐tipped cannula (Tulip Medical, cat. no. INJ_LL)
  • 6‐0 Vicryl suture (Ethicon)
NOTE: All research must be conducted through APLAC committee–approved protocols.

Basic Protocol 7: In Vivo Cell Harvest From fat Grafts

  • Qtracker 655 Cell Labeling Kit (Thermo fisher Scientific, cat. no. Q25021MP)
  • 500 ml sterile FACS buffer (1×PBS, 2% fetal bovine serum, 1% P188, 1% penicillin‐streptomycin)
  • 4′,6‐Diamidine‐2′‐phenylindole dihydrochloride (DAPI)
  • Medium 199 (Gibco, cat. no. 11150059)
  • Type I collagenase, 2.2 mg/ml (Sigma Aldrich)
  • Full medium (DMEM, 10% FBS, 1% penicillin/streptomycin)
NOTE: All research must be conducted through APLAC committee–approved protocols.
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Literature Cited

Literature Cited
  Carvalho, P. P., Leonor, I. B., Smith, B. J., Dias, I. R., Reis, R. L., & Gimble, J. M. (2013). Undifferentiated human adipose‐derived stromal/stem cells loaded onto wet‐spun starch‐polycaprolactone scaffolds enhance bone regeneration: Nude mice calvarial defect in vivo study. Journal of Biomedical Materials Research Part A, 102(9), 3102–3111. doi: 10.1002/jbm.a.34983.
  Chung, M. T., Liu, C., Hyun, J. S., Lo, D. D., Montoro, D. T., & Hasegawa, M. (2013). CD90 (Thy‐1)‐positive selection enhances osteogenic capacity of human adipose‐derived stromal cells. Tissue Engineering Part A, 19(7‐8), 989–997. doi: 10.1089/ten.tea.2012.0370.
  Cowan, C. M., Shi, Y. Y., Aalami, O. O., Chou, Y. F., Mari, C., & Thomas, R. (2004). Adipose‐derived adult stromal cells heal critical‐size mouse calvarial defects. Nature Biotechnology, 2004;22(5), 560–567. doi: 10.1038/nbt958.
  Fisher, J. N., Peretti, G. M., & Scotti, C. (2016). Stem cells for bone regeneration: From cell‐based therapies to decellularised engineered extracellular matrices. Stem Cells International, 9352598. doi:
  Garza, R. M., Paik, K. J., Chung, M. T., Duscher, D., Gurtner, G. C., & Longaker, M. T. (2014). Studies in fat grafting: Part III. Fat grafting irradiated tissue–improved skin quality and decreased fat graft retention. Plastic and Reconstructive Surgery, 134(2), 249–257. doi: 10.1097/PRS.0000000000000326.
  Gómez‐Barrena, E., Rosset, P., Lozano, D., Stanovici, J., Ermthaller, C., & Gerbhard, F. (2015). Bone fracture healing: Cell therapy in delayed unions and nonunions. Bone, 70, 93–101. doi: 10.1016/j.bone.2014.07.033.
  James, A. W., Xu, Y., Wang, R., & Longaker, M. T. (2008). Proliferation, osteogenic differentiation, and fgf‐2 modulation of posterofrontal/sagittal suture‐derived mesenchymal cells in vitro. Plastic and Reconstructive Surgery, 122(1), 53–63. doi: 10.1097/PRS.0b013e31817747b5.
  Kim, H. P., Ji, Y. H., Rhee, S. C., Dhong, E. S., Park, S. H., & Yoon, E. S. (2012). Enhancement of bone regeneration using osteogenic‐induced adipose‐derived stem cells combined with demineralized bone matrix in a rat critically‐sized calvarial defect model. Current Stem Cell Research & Therapy, 7(3), 165–172. doi: 10.2174/157488812799859847.
  Kim, J. B., Leucht, P., Luppen, C. A., Park, Y. J., Beggs, H. E., & Damsky, C. H. (2007). Reconciling the roles of FAK in osteoblast differentiation, osteoclast remodeling, and bone regeneration. Bone, 41(1), 39–51. doi: 10.1016/j.bone.2007.01.024.
  Koob, S., Torio‐Padron, N., Stark, G. B., Hannig, C., Stankovic, Z., & Finkenzeller, G. (2011). Bone formation and neovascularization mediated by mesenchymal stem cells and endothelial cells in critical‐sized calvarial defects. Tissue Engineering Part A, 17(3‐4), 311–321. doi: 10.1089/ten.tea.2010.0338.
  Korbling, M., & Estrov, Z. (2003). Adult stem cells for tissue repair ‐ a new therapeutic concept? The New England Journal of Medicine, 349(6), 570–582. doi: 10.1056/NEJMra022361.
  Levi, B., James, A. W., Nelson, E. R., Vistnes, D., Wu, B., & Lee, M. (2010). Human adipose derived stromal cells heal critical size mouse calvarial defects. PLoS One, 5(6) doi: 10.1371/journal.pone.0011177.
  Luan, A., Duscher, D., Whittam, A. J., Paik, K. J., Zielins, E. R., Brett, E. A., … Wan, D. C. (2016). Cell‐Assisted lipotransfer improves volume retention in irradiated recipient sites and rescues radiation‐induced skin changes. Stem Cells, 34(3), 668–673. doi: 10.1002/stem.2256.
  McArdle, A., Chung, M. T., Paik, K. J., Duldulao, C., Chan, C., Rennert, R., … Longaker, M. T. (2014). Positive selection for bone morphogenetic protein receptor type‐IB promotes differentiation and specification of human adipose‐derived stromal cells toward an osteogenic lineage. Tissue Engineering Part A, 20(21‐22), 3031–3040. doi: 10.1089/ten.tea.2014.0101.
  Mizuno, H., Tobita, M., & Uysal, A. C. (2012). Concise review: Adipose‐derived stem cells as a novel tool for future regenerative medicine. Stem Cells (Dayton, Ohio), 30(5), 804–810. doi: 10.1002/stem.1076.
  Oedayrajsingh‐Varma, M. J., van Ham, S. M., Knippenberg, M., Helder, M. N., Klein‐Nulend, J., Schouten, T. E., … van Milligen, F. J. (2006). Adipose tissue‐derived mesenchymal stem cell yield and growth characteristics are affected by the tissue‐harvesting procedure. Cytotherapy, 8(2), 166–177. doi: 10.1080/14653240600621125.
  Panetta, N. J., Gupta, D. M., Kwan, M. D., Wan, D. C., Commons, G. W., & Longaker, M. T. (2009). Tissue harvest by means of suction‐assisted or third‐generation ultrasound‐assisted lipoaspiration has no effect on osteogenic potential of human adipose‐derived stromal cells. Plastic and Reconstructive Surgery, 124(1), 65–73. doi: 10.1097/PRS.0b013e3181ab10cd.
  Peer, L. A. (1955). Cell survival theory versus replacement theory. Plastic and Reconstructive Surgery, (1946);16(3), 161–168. doi: 10.1097/00006534‐195509000‐00001.
  Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., … Marshak, D. R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science (New York, NY), 284(5411), 143–147. doi: 10.1126/science.284.5411.143.
  Rieck, B., & Schlaak, S. (2003). Measurement in vivo of the survival rate in autologous adipocyte transplantation. Plastic and Reconstructive Surgery, 111(7), 2315–2323. doi: 10.1097/01.PRS.0000060797.59958.55.
  Schantz, J. T., Hutmacher, D. W., Lam, C. X., Brinkmann, M., Wong, K. M., Lim, T. C., … Teoh, S. H. (2003). Repair of calvarial defects with customised tissue‐engineered bone grafts II. Evaluation of cellular efficiency and efficacy in vivo. Tissue Engineering, 9(Suppl 1), S127–139. doi: 10.1089/10763270360697030.
  Tevlin, R., McArdle, A., Brett, E., Paik, K., Seo, E. Y., Walmsley, G. G., … Longaker, M. T. (2016). A novel method of human adipose derived stem cell isolation with resultant increased cell yield. Plastic and Reconstructive Surgery, 138(6), 983e–996e. doi: 10.1097/PRS.0000000000002790.
  Uzbas, F., May, I. D., Parisi, A. M., Thompson, S. K., Kaya, A., Perkins, A. D., & Memili, E. (2014). Molecular physiognomies and applications of adipose‐derived stem cells. Stem Cell Reviews, 11(2), 298–308. doi: 10.1007/s12015‐014‐9578‐0.
  Zuk, P. A., Zhu, M., Ashjian, P., De Ugarte, D. A., Huang, J. I., Mizuno, H., … Hedrick, M. H. (2002). Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell, 13(12), 4279–4295. doi: 10.1091/mbc.E02‐02‐0105.
  Zuk, P. A., Zhu, M., Mizuno, H., Huang, J., Futrell, J. W., Katz, A. J., … Hedrick, M. H. (2001). Multilineage cells from human adipose tissue: Implications for cell‐based therapies. Tissue Engineering, 7(2), 211–228. doi: 10.1089/107632701300062859.
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