Mesenchymal stromal cells (MSC) show a mesodermal differentiation and immunosuppressive potential and are suitable for various clinical applications like for treatment of bone and connective tissue disorders or autoimmune diseases. Different substrates and media are used for in vitro expansion and manipulation of MSC, but they may affect MSC-heterogenity and characteristics. Currently, there are no optimal culture conditions available for MSC. In this work, the properties of the culture-media supplement human platelet lysate (HPL) and the effect of transforming growth factor (TGF)-b1 on MSC-culture-expansion was systematically tested. Therefore, their effects on MSC-characteristics like vitality, expansion, differentiation potential and replicative senescence were analyzed. An examination of individual platelet lysates for MSC-culture revealed high variations in their potential to support MSC-proliferation, which showed a positive correlation to the PDGF-AB and IGFBP1/2 cytokine concentrations of the platelet lysates. Nevertheless, each individual HPL supported MSC-characteristics and was suitable for efficient MSC-expansion. An improvement of MSC-proliferation could be achieved by using preferably low concentrations of preservative-free heparin (0.61 IU/mL). This anti-coagulant is necessary to prevent co-agulation of HPL-media, but showed an anti-proliferative and in high concentrations (625 IU/mL) a cytotoxic effect that was enhanced by benzyl alcohol as a preservative. On the basis of its content of fibrinogen and factors of the coagulation cascade coagulated HPL-media, could be further established as a more efficient three-dimensional substrate for the isolation and multi-layered and rapid culture-expansion of MSC. The viscosity allowed for a non-enzymatic passaging method by fragmentation of the gel, thereby facilitating this critical cell-culture step. MSC-proliferation could be further promoted over a short period by addition of recombinant TGF-b1. Yet, TGF-b1 induced reversible changes in MSC growth-pattern and impaired adipogenic and osteogenic differentiation. A comparative transcriptome-wide analysis of the effect of TGF-b1 on MSC of early and late passage additionally indicated that culture-expansion does not change the response towards this cytokine. Taken together, the results of this thesis offer valuable information that can be used for further optimization of MSC-culture-expansion
