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Isolation and Primary Culture of Adult Human Adipose-derived Stromal/Stem Cells
Published: Vol 7, Iss 5, Mar 5, 2017 DOI: 10.21769/BioProtoc.2161 Views: 14192
Reviewed by: Federica PisanoNingfei An
Original research article
The authors used this protocol in:
Mar 2016
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Abstract
Adipose-derived stromal/stem cells (ASCs) are multipotent cells that can be isolated from adipose tissue. Studies have shown that cells have the capacity to self-renew and differentiate into adipocyte, chondrocyte, myocyte, and osteoblast lineages. Thus, significant interest regarding their use for regenerative purposes to restore aging or damaged tissue has grown in recent decades. These cells have also been shown to immunomodulate the microenvironment and secrete abundant growth factors, which minimize inflammation and aid repair and regeneration. ASCs can be readily isolated from the stromal vascular fraction (SVF) of lipoaspirates. Given their ease of accessibility, bountiful source, and potential application in regenerative medicine and tissue engineering, there is growing interest in the characterization and utilization of ASCs. This protocol describes the isolation of ASCs from adult human adipose tissue as well as methods for culture maintenance including expansion and cryopreservation.
Keywords: Adipose-derived stem cells
Adipose-derived stromal cells
Cell isolation
Primary cell culture
Cell expansion
Cryopreservation
Background
Adipose-derived stromal/stem cells (ASCs) demonstrate vast potential for the field of stem cells. Following the therapeutic marvel of hematopoietic stem cells transplantation, ASCs represent the future for stem cells due to their more freely accessible source – adipose tissue. The ability of ASCs to self-renew and differentiate into various tissue lineages including adipocyte, chondrocyte, myocyte, and osteoblast lineages, allows restoration of damaged tissue. Additionally, it is speculated that ASCs have the potential to replicate tissue in vitro. In vitro organs will allow more readily available assessment of novel pharmaceuticals and thus reduce drug production costs significantly. However, inconsistencies in the processes of isolation, maintenance, and cryopreservation, prohibit collective analysis of results from different laboratories worldwide. A standard protocol for isolation and culture of ASCs is necessary to ensure consistent data analysis.
Materials and Reagents
- Stericup-GP 0.22 µm polyethersulfone 500 ml radio-sterilized vacuum filtration flask (EMD Millipore, catalog number: SCGPU05RE )
- Centrifuge tubes:
15 ml (Corning, catalog number: 430790 )
50 ml (Corning, catalog number: 430828 ) - Sterile disposable serologic pipets:
2 ml (Corning, Falcon®, catalog number: 357558 )
5 ml (Corning, Falcon®, catalog number: 357543 )
10 ml (Corning, Falcon®, catalog number: 357551 )
25 ml (Corning, Falcon®, catalog number: 357525 )
50 ml (Corning, Falcon®, catalog number: 357550 ) - NuncTM 15 cm diameter, 145 cm2 culture area cell culture/Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 168381 )
- FisherbrandTM Premium microcentrifuge tubes: 1.5 ml (Fisher Scientific, catalog number: 05-408-129 )
- Cryogenic vials, 1.2 ml (Corning, catalog number: 430487 )
- TipOne RPT 10 µl ultra-low retention filter pipet tips, sterile (USA Scientific, catalog number: 1181-3810 )
- ARTTM Barrier pipette 100 µl tips 100E low retention (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2065E-05 )
- Human adipose tissue obtained from liposuction
- 1x phosphate-buffered saline (PBS) (GE Healthcare, HyCloneTM, catalog number: SH30256 )
- Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
- Ethidium bromide
- Acridine orange
- 0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200072 )
- Isopropanol 95% (v/v)
- Liquid nitrogen
- Type I collagenase (Worthington Biochemical, catalog number: LS004196 )
- Bovine serum albumin (BSA), fraction V (Sigma-Aldrich, catalog number: 10735078001 )
- Calcium chloride (CaCl2), ≥ 96.0% anhydrous (Sigma-Aldrich, catalog number: C4901 )
- Dulbecco’s modified Eagle medium (DMEM): Nutrient Mixture F-12 (DMEM/F-12) (Thermo Fisher Scientific, GibcoTM, catalog number: 11330032 )
- α-minimal essential medium (α-MEM), no nucleosides (Thermo Fisher Scientific, GibcoTM, catalog number: 12561056 )
- Fetal bovine serum (FBS), premium select (Atlanta Biologicals, catalog number: S11550 )
- Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
- L-glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
- DMSO
- Digestion solution (see Recipes)
- Resuspension solution (see Recipes)
- Complete culture medium (CCM) (see Recipes)
- Freezing medium (see Recipes)
Equipment
- Precision scale
- IsotempTM digital-control water bath (Fisher Scientific, model: 215 )
- Sterile cell culture hood
- Motorized pipette aid
- Micropipettes: 10 µl, 200 µl, and 1,000 µl (Eppendorf, model: Research® plus )
- Centrifuge (Eppendorf, model: 5810 R )
- Cell culture incubator
- Inverted routine light microscope (Nikon Instruments, model: Eclipse TS100 )
- Hemocytometer
- Nalgene® freezing container (Sigma-Aldrich, catalog number: C1562 )
- Cryogenic storage dewar (Custom BioGenic Systems, model: 6001 Value Added Cryosystem Dewar )
Procedure
Category
Stem Cell > Adult stem cell > Adipose Stem Cell
Cell Biology > Cell isolation and culture > Cell isolation
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