[Frontiers in Bioscience E3, 256-263, January 1, 2011]

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

Trine Fink1, Jeppe Grondahl Rasmussen1,2, Pia Lund1, Linda Pilgaard1, Kjeld Soballe3, Vladimir Zachar1

1Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Denmark, 2Department of Pharmacology, Aarhus University, Denmark, 3Orthopaedic Research Laboratory, Aarhus University Hospital, Denmark


1. Abstract
2. Introduction
2. Introduction
3. Materials and methods
3.1. Isolation of ASCs with different collagenases
3.2. Colony-forming unit assays
3.3. Cell proliferation in different media, sera, and sera replacers
3.4. Osteogenic differentiation
3.5. Cell proliferation as a function of initial seeding density
3.6. Short term hypoxic cell culture
3.7. Long term hypoxic cell culture
3.8. Statistics
4. Results
4.1. Effect of collagenase on the yield of ASCs
4.2. Selection of sera or sera replacers for the expansion of human ASCs
4.3. Selection of optimal medium for expansion of human ASCs
4.4. Effect of plating density on expansion of human ASCs
4.5. Effect of hypoxic culture on cell growth
5. Discussion
6. Acknowledgements
7. References


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.