来源于皮下和内脏白脂肪组织腔室的人脂肪源性干细胞的分离和培养

Xiaojia Ge; Shi Chi Leow; Durgalakshmi Sathiakumar; Walter Stünkel; Asim Shabbir; Jimmy Bok Yan  So; Davide Lomanto; Craig McFarlane

doi:10.21769/BioProtoc.2027

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Peer-reviewed

Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments

来源于皮下和内脏白脂肪组织腔室的人脂肪源性干细胞的分离和培养

Xiaojia Ge

Shi Chi Leow

Durgalakshmi Sathiakumar

Craig McFarlane email

发布: 2016年11月20日第6卷第22期 DOI: 10.21769/BioProtoc.2027 浏览次数: 14693

评审: Salma HasanAgnieszka PastulaAnonymous reviewer(s)

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Cover of Development, featuring study using the protocol.

Mar 2016

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Abstract

Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs was performed through surface marker expression profiling using flow cytometry. Adipogenic capacity of the isolated ASCs was confirmed through inducing adipogenic differentiation and Oil Red O staining of lipid. This protocol provides researchers with the tools to culture and assess purity and adipogenic differentiation capacity of human ASCs, which can then be utilized for required downstream in vitro applications.

This protocol has been modified from Baglioni et al. (2009), Baglioni et al. (2012), and van Harmelen et al. (2005) to describe in detail a complete technique to isolate and subsequently characterize human ASCs from human WAT biopsies. This protocol has been utilized to isolate and characterize human ASCs from both subcutaneous and visceral WAT. The isolated human ASCs show high purity and demonstrate adipogenic differentiation capacity in vitro.

Background

Human ASCs are an invaluable in vitro cell model to study molecular pathways important for the etiology of metabolic diseases, including obesity and type 2 diabetes. Human ASC cultures derived from different WAT sources, including subcutaneous and visceral compartments, can also help us to understand functional differences between different WAT compartments. Short protocols have been published previously to describe the isolation of human ASCs from a maximum of two different WAT compartments (Baglioni et al., 2009; 2012; van Harmelen et al., 2005). Here we describe a detailed protocol for both, isolation and characterization of human ASCs from human WAT biopsies collected from several WAT compartments. This protocol has been used to reliably derive human ASCs from four different WAT compartments, including superficial subcutaneous, deep subcutaneous, omental and mesenteric WAT. The isolated primary cultures display homogeneous morphology and are pure, with a high percentage of cells displaying typical MSC marker expression. The isolated human ASCs also have the ability to differentiate into mature adipocytes, with accumulation of intracellular triglyceride droplets. In summary, this protocol reliably results in the isolation of pure human primary ASCs that maintain robust adipogenic differentiation capacity in vitro.

Materials and Reagents

15 ml centrifugation tube (Corning, Falcon^®, catalog number: 352099 )
50 ml centrifugation tube (Corning, Falcon^®, catalog number: 352070 )
10 cm cell culture dish (Greiner Bio One, CellStar^®, catalog number: 664160 )
0.2 μm 25 mm syringe filter (Pall, Acrodisc^®, catalog number: 4612 )
30 ml syringe (BD, Luer-Lok^TM, catalog number: 302832 )
5 ml polystyrene fluorescence activated cell sorting (FACS) tubes (Corning, Falcon^®, catalog number: 352054 )
100 μm nylon mesh cell strainer (Corning, Falcon^®, catalog number: 352360 )
6-well-plates (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 140675 )
Human WAT from patients
OXOID^TM Phosphate buffered saline (PBS) tablets (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: BR0014G )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
Fetal bovine serum (FBS), heat inactivated (Thermo Fisher Scientific, Gibco^TM, catalog number: 16140071 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
Antibodies (see Table 1)
Isopropanol (EMD Millipore, catalog number: 109634 )
Collagenase type IA (Sigma-Aldrich, catalog number: C9891-1G )
Ammonium chloride (NH₄Cl) (Sigma-Aldrich, catalog number: A0171 )
Note: This product has been discontinued.
Potassium bicarbonate (KHCO₃) (Sigma-Aldrich, catalog number: P7682 )
Note: This product has been discontinued.
0.25% trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200056 )
Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F-12) (1:1) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11330032 )
Penicillin-streptomycin (P/S) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
Dexamethasone (Sigma-Aldrich, catalog number: D4902 )
10 mg/ml insulin solution from bovine pancreas (Sigma-Aldrich, catalog number: I0516 )
3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog number: I5879 )
Indomethacin (Sigma-Aldrich, catalog number: 17378 )

100% ethanol (EMD Millipore, catalog number: 100983 )

Table 1. Antibody information

Antigen		Antibody	Manufacturer	Dilution	Host/Isotype
CD34	hematopoietic cells	Anti-Human CD34 APC	Affymetrix, eBioscience, catalog number: 17-0349	1:100	Mouse IgG1, kappa
CD31	Endothelial cells	Anti-Human CD31 APC	Affymetrix, eBioscience, catalog number: 17-0319	1:100	Mouse IgG1, kappa
CD14	(Macrophages) hematopoietic cells	Anti-Human CD14 APC	Affymetrix, eBioscience, catalog number: 17-0149	1:100	Mouse IgG1, kappa
CD11b	(Leukocytes) Monocytes	Anti-Human CD11b APC	Affymetrix, eBioscience, catalog number: 17-0113	1:100	Mouse IgG1, kappa
CD45	(Nucleated cells of hematopoietic origin) lymphocytes	Anti-Human CD45 APC	Affymetrix, eBioscience, catalog number: 17-9459	1:100	Mouse IgG1, kappa
CD106	(Activated) endothelial cells	Anti-Human CD106 PE	Affymetrix, eBioscience, catalog number: 12-1069	1:100	Mouse IgG1, kappa
CD90	MSCs	Anti-Human CD90 APC	Affymetrix, eBioscience, catalog number: 17-0909	1:100	Mouse IgG1, kappa
CD44	MSCs	Anti-Human CD44 APC	Affymetrix, eBioscience, catalog number: 17-0441	1:100	Rat IgG2b, kappa
CD29	MSCs	Anti-Human CD29 APC	Affymetrix, eBioscience, catalog number: 17-0299	1:100	Mouse IgG1, kappa
CD73	MSCs	Anti-Human CD73 APC	Affymetrix, eBioscience, catalog number: 17-0739	1:100	Mouse IgG1, kappa
CD105	MSCs	Anti-Human CD105 APC	Affymetrix, eBioscience, catalog number: 17-1057	1:100	Mouse IgG1

Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
Oil Red O powder (Sigma-Aldrich, catalog number: O0625 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
Collagenase solution (1 mg/ml) (see Recipes)
Red blood cell (RBC) lysis buffer (10x) (see Recipes)
Proliferation medium (see Recipes)
Differentiation medium (see Recipes)
Induction medium (see Recipes)
Insulin medium (see Recipes)
10 mM dexamethasone stock solution (see Recipes)
1 mM dexamethasone working solution (see Recipes)
0.5 M IBMX stock solution (1,000x) (see Recipes)
200 mM indomethacin stock solution (1,600x) (see Recipes)
4% paraformaldehyde (PFA) (pH = 7.4) (see Recipes)
Oil Red O working solution (see Recipes)

Equipment

Cryogenic vials (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 366656 )
Laminar flow tissue culture hood
Sterilized surgical tools including forceps and scalpel or scissors
Mr. Frosty^TM freezing container (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 5100-0001 )
Locator 6 Plus Rack and Box Systems, liquid nitrogen Dewar (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: CY50985-70 )
BD FACSCanto II flow cytometer (BD, model: BD FACSCanto II ) or similar equipment
Analytical balance (Sartorius, model: CPA124S )
MaxQ^TM 37 °C orbital shaker (Thermo Fisher Scientific, Thermo Scientific^TM, model: 4450 )
Centrifuge with swinging bucket rotor (KUBOTA, model: 2800 )
Automated cell counter (Thermo Fisher Scientific, Countess^®, catalog number: AMQAF1000 )

Software

FACSDiva software (BD)

Procedure

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Ge, X., Leow, S. C., Sathiakumar, D., Stünkel, W., Shabbir, A., So, J. B. Y., Lomanto, D. and McFarlane, C. (2016). Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments. Bio-protocol 6(22): e2027. DOI: 10.21769/BioProtoc.2027.