BACKGROUNDMultipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements.METHODSAs PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation.RESULTSWe found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP).DISCUSSIONTaken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.

Dendritic cells (DC) have been used in clinical trials for immune therapy of various malignancies. However, activation strategies for DC to date have been insufficient to mount a curative T cell response. We started to investigate the stimulatory DC maturation marker CD83, whose ligand is unknown. We cloned the extracellular domain of CD83 and expressed it in eukaryotic cells to obtain a physiologically glycosylated recombinant protein. As the affinity was too low for flow cytometric analyses, we designed a tetrameric CD83 complex and identified a CD4+ population of PBMC. Currently, we are optimising the expression to obtain larger amounts of CD83 for use in a glycan array in order to biochemically identify the ligand.