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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Abstract

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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Materials

Basic Protocol 1: Fat Processing and Cell Harvest

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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 (https://pga.mgh.harvard.edu/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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
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  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.
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