Isolation and Characterization of BM-MSCs Using Serum/Xeno-free GMP-compliant Media

Objectives: The addition of bovine serum in cell culture media may compromise safe and efficient expansion of human Mesenchymal Stem Cells-hMSCs for therapeutic purposes. Development of clinical-grade, xeno-free cell preparations is central to cGMP (Good Manufacturing Practice compliant) conditions. This study aimed to investigate the potential of two serum/xeno-free, cGMP culture media to maintain stemness of hMSCs compared to a conventional serum-containing medium.
Methods: Bone-Marrow MSCs (BM-MSCs) cultures were established using the enzymatic dissociation from osseous biopsies obtained from six healthy individuals during routine implant placement after informed consent. Three different media were used: StemPro MSC SFM XenoFree (Life Technologies, Carlsbad, CA); STEM MACS MSC, XF (Miltenyi Biotek, Bergisch Gladbach, Germany), and a-MEM (Invitrogen, Karlsruhe, Germany) supplemented with 15% FBS, L-ascorbic acid phosphate and antibiotics/antimycotics. Cells were expanded and characterized for mesenchymal- (CD90/Thy-1, CD73, CD29/b1-integrin, CD49f/a6-integrin, CD81-TAPA, CD166/ALCAM, CD146/MUC18, STRO-1, CD34), endothelial- (CD105/endoglin, CD106/VCAM), hematopoietic- (CD45) and embryonic- (SSEA-1, -3, -4, -5) stem cell (SC) markers at passages 2, 6 and 10 by flow cytometry.
Results: BM-MSCs cultured under the two serum-free media showed significantly lower proliferation rates compared to the serum-containing medium. BM-MSCs at passage 2 were positive (>99% of the population) for CD90, CD73, CD29, CD81, CD49f, CD166 and partly for CD105, CD106, CD146, STRO-1 and SSEA-1, -3, 4, -5, with some inter-individual variations. Cells were consistently negative for CD45, whereas low expression of CD34 was occasionally detected. Continuous expansion of cells with both serum-free media was associated with pronounced down-regulation of CD146, Stro-1, CD105, SSEA-4 and occasionally other SSEAs in parallel with upregulation of CD34 and CD106. Similar trends to a lesser extent were observed for the serum-containing medium.
Conclusions: These findings indicate that there is still a vacant role for the optimal cGMP serum-free medium to maintain stemness of BM-MSCs and meet clinical-grade standards.