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So far, only few authors addressed the serum-free, defined differentiation of adipocytes. And there are hardly any trials available on the defined maintenance of adipocytes. In this study, the development of a defined culture medium for the adipogenic differentiation of primary human adipose-derived stem cells (ASCs) was aimed. Based on the addition of specific factors for the replacement of serum, ASCs were differentiated to viable and characteristic adipocytes for 14 days, which was proven through the accumulation of lipids, the expression of perilipin A and by the release of leptin and glycerol. Furthermore, a defined maintenance medium was developed, which supported the maturation and stability of cells for a long-term period of additional 42 days until day 56.