Isolation and expansion of adipose-derived stem cells for tissue engineering

Abstract

For treatment of cardiac failure with bone marrow-derived mesenchymal stem cells, several clinical trials are ongoing. However, more attention is gathering on the use of adipose tissue-derived stem cells (ASCs). This paper describes the optimization of isolation and propagation of ASCs for subsequent clinical use. In the isolation step, several enzymes were compared with respect to yield of nucleated cells and precursor cells. Our results showed, that the interdonor variablility was greater than differences between individual enzymes. For propagation of cells, different types of media, sera and serum replacers were evaluated regarding their ability to support cell growth and preserve differentiation potential. Most of serum replacers proved inferior to fetal calf serum. Among the media tested, modified Eagle's media alpha was superior in promoting cell growth while preserving the ability to differentiate. Also, the effect of cell seeding density and hypoxic culture was evaluated. In this study, we show that it is possible to maximize cell yield regardless of donor individual characteristics by simple manipulations of media composition, cell seeding density and gaseous environment.

Defined Xenogeneic-Free and Hypoxic Environment Provides Superior Conditions for Long-Term Expansion of Human Adipose-Derived Stem Cells

“…For example, the self-renewal of human ESCs is best maintained in 5% oxygen, 13,14 while that of limbal stem cells at 2%. 16 With respect to ASCs, our own comparative studies identified 5% oxygen in culture atmosphere as optimal to support growth, [22][23][24] although it is possible that in certain settings, even lower oxygen concentrations may prove useful. 47,48 The somewhat disparate experimental evidence clearly indicates that better standardization of hypoxic experiments together with identification of possible pitfalls is necessary before consensus on a widely accepted value can be reached.…”

“…22,24,25 Three lines of ASCs, designated ASC12, 21, and 23, which have been isolated and characterized previously, 23 were continually grown in standard growth conditions involving an alpha-modified Eagle medium (A-MEM; Invitrogen, Carlsbad, CA) supplemented with 10% FCS (Europa Bioproducts Ltd., Cambridge, United Kingdom), 100 IU/mL penicillin, 100 mg/ mL streptomycin, and 50 mg/mL gentamicin or in defined and xenogeneic-free conditions involving the StemPro Ò MSC SFM XenoFree + CELLstart Ô culture system (StemPro) (all from Invitrogen). The essential feature of StemPro medium is that it is supplemented with a platelet-derived growth factor, transforming growth factor (TGF)-b, and fibroblast growth factor.…”

“…Adipo-, osteo-, and chondrogenic differentiation assays were carried out according to previously published protocols 22,27,28 with minor modifications. Briefly, for adipo-and osteogenic induction, cultures were initiated at a density of 300 cells/cm 2 in 24-well plates and maintained in a standard growth medium for *14 days until they reached confluency.…”

“…Mesenchymal stem cells (MSCs) are also subject to the regulatory effects of a hypoxic atmosphere and have been shown to grow at a faster rate along with a well-preserved differentiation potential under such conditions. [22][23][24] Cell-based therapeutic applications often require very large batches of cells, with doses ranging from 10 7 to 10 8 cells (www.clinicaltrials.gov, trial identifiers NCT00475410, NCT01222039, and NCT01274975). Amplification of such cell numbers from moderate amounts of starting material is both time consuming and costly.…”

Effect of Adipose Tissue-Derived Osteogenic and Endothelial Cells on Bone Allograft Osteogenesis and Vascularization in Critical-Sized Calvarial Defects

“…Adipose tissue-derived stem cells not only differentiate into osteoblastic cells (OBs) or endothelial cells (ECs), but also are obtainable in large quantities and provide an excellent cellular source for engineered constructs. [8][9][10][11] Studies in several animal models have demonstrated that the implantation of osteoprogenitor cells and OBs can improve the osteogenic and osteoinductive potential of allogenic bone, which occurs likely secondary to the production of growth factors and bone morphogenetic proteins (BMPs) that reciprocally stimulate OBs to form mineralized bone matrix. 5,[12][13][14][15] In addition, in order to improve the blood supply of such grafts, angiogenic strategies have been developed and include the direct application of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor as recombinant proteins or by gene therapeutics.…”