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Adipose tissue-derived stem cells (ASCs) hold significant potential for treating various clinical conditions. To enhance their regenerative properties, ASCs can be chemically stimulated using various in vitro protocols. However, unsatisfactory results persist, partly due to the relatively costly long-term methods. Furthermore, current culturing techniques often rely on the use of xenogenic fetal bovine serum that can be immunogenic, limiting clinical translations. To facilitate clinical translation of ASCs-derived therapeutics, the effect of different stimulation protocols on human ASCs cultured in a xeno-free medium (PRIME-XV MSC Expansion XSFM) is investigated. The xeno-free medium was supplemented with stimulants (forskolin (FSK), basic fibroblast growth factor, platelet-derived growth factor-AA, neuregulin-1) in combinations or individually. Stimulation for 72 h in FSK alone, or together with the growth factors, enhanced the production of urokinase plasminogen activator (uPA), a serine protease involved in tissue remodeling processes. Conditioned medium derived from stimulated ASCs enhanced in vitro angiogenesis and endothelial cells migration. This study shows that pro-angiogenic responses in human ASCs can be enhanced with a defined short stimulation protocol using a xeno-free medium. The protocol, using readily available manufacturing cell therapy grade molecules, may boost the regenerative properties of ASCs secretome which could enhance their efficacy in clinical treatments.

Introduction: Before performing cell therapy clinical trials, it is important to understand how cells are influenced by different growth conditions and to find optimal xeno-free medium formulations. In this study we have investigated the properties of adipose tissue-derived stem cells (ASCs) cultured under xeno-free conditions.

Methods: Human lipoaspirate samples were digested to yield the stromal vascular fraction cells which were then seeded in i) Minimum Essential Medium-α (MEM-α) supplemented with 10 % (v/v) fetal bovine serum (FBS), ii) MEM-α supplemented with 2 % (v/v) human platelet lysate (PLT) or iii) PRIME-XV MSC expansion XSFM xeno-free, serum free medium (XV). Flow cytometry for ASCs markers CD73, CD90 and CD105 together with the putative pericyte marker CD146 was performed. Growth rates were monitored over multiple passages and adipogenic differentiation performed at early and expanded passage culture. Growth factor gene expression was analyzed and an in vitro angiogenesis assay performed.

Results: Cells in FBS and PLT grew at similar rates whereas the cells cultured in XV medium proliferated significantly faster up to 60 days in culture. All cultures were >98 % positive for CD73, CD90 and CD105, whereas CD146 expression was significantly higher in XV cells. Adipogenic differentiation was most pronounced in cells which had been cultured in XV medium whilst cells grown in PLT were inferior compared with cells from the FBS cultures. IGF1 gene expression was highest in cells cultured in PLT whilst cells grown in XV medium showed 10-fold lower expression compared with FBS cells. In contrast, HGF gene expression was 90-fold greater in cells cultured in XV medium compared with those cultured in FBS. Conditioned medium from XV cultured cells showed the most angiogenic activity, inducing the greatest endothelial cell network formation and maturation.

Conclusion: Culture under different conditions alters the ASCs characteristics. Since cells cultured in XV medium showed the best adipogenic and angiogenic profile this might be a preferred medium formulation for preparing cells required for reconstructive surgical applications such as cell-assisted fat grafting.