Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

Summary

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Abstract

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

Introduction

In 2001, a putative population of multipotent stem cells from adipose tissue was described in the journal Tissue Engineering ¹. These cells were given the name Processed Lipoaspirate or PLA cells due to their derivation from processed lipoaspirate tissue obtained through cosmetic surgery. The isolation method described in this article was based on existing enzymatic strategies for the isolation of the stromal vascular fraction (SVF) from adipose tissue ². The SVF has been defined as a minimally processed population of red blood cells, fibroblasts, endothelial cells, smooth muscle cells, pericytes and pre-adipocytes that have yet to adhe....

Protocol

The protocol shown here describes the manual isolation of ASCs from lipoaspirates obtained through cosmetic procedures using enzymatic digestion and differential centrifugation. This protocol was first published in the journal Tissue Engineering in 2001 ¹, where the resulting cells were called Processed Lipoaspirate Cells or PLA cells because of their isolation from lipoaspirates. However, the term PLA cell has now been replaced with the term Adipose-derived Stem Cells or ASCs to give the field some sort of conformity in terms of nomenclature. The cells isolated through this protocol have been shown by many to possess mesodermal potential....

Results

The protocol outline above describes a manual, enzymatic method for the isolation of an SVF from a large volume lipoaspirate sample. Within this SVF are numerous cell populations, including the ASC. Numerous studies propose that culturing this SVF under standard tissue culture conditions will select for an adherent fibroblast population likely to be composed mainly of the ASC type. Consistent with this, we have shown, using flow cytometry and immunofluorescence, that cultured SVF pellets become essentially free of the .......

Discussion

Adipose tissue for the isolation of ASCs can come in many forms: from solid pieces of tissue obtained through resection or lipoplasty to smaller pieces obtained through either syringe extraction or suction-assisted lipoplasty (i.e. liposuction ). Whether more SVF cells (and thereby ASCs) can be obtained from resected or aspirated adipose samples is unclear as conflicting studies have been presented ^{16, 17}. It is possible that either form of adipose tissue is more than suitable for the isolation of SVF.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
DMEM (Dulbecco’s Modification of Eagle’s Medium)	Mediatech Cellgro	10-013-CV	with 4.5 g/ml glucose, L-glutamine, sodium pyruvate
Penicillin/Streptomycin	Mediatech Cellgro	30-002-CI	10,000 IU/ml penicillin/10,000 μg/ml streptomycin
Amphotericin B	Mediatech Cellgro	30-003-CF	250 μg/ml amphotericin B
10X PBS (Phospho-buffered Saline)	Mediatech Cellgro	25-053-CI	without calcium, without magnesium
Trypsin/EDTA	Mediatech Cellgro	20-031-CV	0.25 % trypsin/2.21mM EDTA
Collagenase type IA (from Clostridium histolyticum)	Sigma	C2674	crude preparation; <125 collagen digestion units/mg solid
FBS (Fetal Bovine Serum) heat inactivated	Gemini Bioproducts	100106	USDA source, heat inactivated
10 ml serological pipettes	Genesee Scientific	12-104
25 ml serological pipettes	Genesee Scientific	12-106
50 ml polypropylene centrifuge tubes	Genesee Scientific	21-106
100 mm tissue culture dishes	Genesee Scientific	25-202
150 mm tissue culture dishes	Genesee Scientific	25-203
500 ml Stericup Filter Units	Millipore	SCGPU05RE	PES membrane, 0.22 μm pore
Cell strainers	FisherBrand	22-363-549	100 μm nylon mesh
dexamethasone - water soluble	Sigma	D-2915
L-ascorbic-acid 2 phosphate	Sigma	A-8960
β-glycerophosphate disodium salt	Sigma	G-9422	also known as glycerophosphate
insulin	Sigma	I-6634	made from bovine pancreas
indomethacin	Sigma	I-7378
apo-transferrin	Sigma	T-4382
TGFβ1	R&D Systems	240-B-002	recombinant human
Oil Red O	Sigma	O-0625
Alcian Blue	Sigma	A-5268
Silver nitrate	Sigma	S-0319
Hydrochloric acid	Fisher Scientific	A144
Paraformaldehyde	Fisher Scientific	30525-89-4	supplied as a 16 % stock
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Equipment Needed
Class II A/B Biosafety hood	Thermo Scientific		ensure hood has vacuum lines for aspiration
Benchtop centrifuge	Hermle Labnet	Z383	Swing-out rotor for 50 ml tubes required, capable of 1200xg
Water bath	Fisher Scientific Isotemp	S52602Q	5-10L capacity, capable of 37C
Automated Pipette Aids	Drummond Pipette Aid XL	4-000-105
CO2 Incubator	Thermo Scientific	Forma 310	direct heat or water jacketed