Accelerated and safe expansion of human mesenchymal stromal cells in animal serum‐free medium for transplantation and regenerative medicine

Abstract Human bone marrow mesenchymal stromal cells (hMSC) are currently investigated for a variety of therapeutic applications. However, most expansion protocols still use fetal calf serum (FCS) as growth factor supplement which is a potential source of undesired xenogeneic pathogens. We established an expansion protocol for hMSC based on the use of GMP‐produced basic medium LP02 supplemented with 5% of platelet lysate (PL) obtained from human thrombocyte concentrates. Compared to FCS‐supplemented culture conditions, we found a significant increase in both colony forming unit‐fibroblast (CFU‐F) as well as cumulative cell numbers after expansion. This accelerated growth is optimized by pooling of at least 10 thrombocyte concentrates. A minimal requirement is the use of 5% of PL with an optimal platelet concentration of 1.5 × 10 9 /ml, and centrifugation of thawed lysate at high speed. Cells expanded by this protocol meet all criteria for mesenchymal stromal cells (MSCs), e.g. plastic adherence, spindle‐shaped morphology, surface marker expression, lack of hematopoietic markers, and differentiation capability into three mesenchymal lineages. MSC at passage 6 were cytogenetically normal and retained their immune‐privileged potential by suppressing allogeneic reaction of T‐cells. Additionally, gene expression profiles show increased mRNA levels of genes involved in cell cycle and DNA replication and downregulation of developmental and differentiation genes, supporting the observation of increased MSC‐expansion in PL‐supplemented medium. In summary, we have established a GMP‐compatible protocol for safe and accelerated expansion of hMSC to be used in cell and tissue therapy. J. Cell. Physiol. 213: 18–26, 2007. © 2007 Wiley‐Liss, Inc.