Background: For the accurate determination of gene expression changes during growth and differentiation studies on adipose-derived stem cells (ASCs), quantitative real-time RT-PCR has become a method of choice. The technology is very sensitive, however, without a proper selection of reference genes, to which the genes of interest are normalized, erroneous results may be obtained.

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Our results demonstrate that the highly purified proteases provide a valuable alternative to crude collagenase preparations, especially in scenarios where a high definition and reproducibility of the digestion process is of importance.

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.