Isolation and Expansion of Functional Differentiating Mesenchymal Stem Cells with a Serum Deprived Medium.

The infusion of mesenchymal stem cells (MSC) is an increasingly utilized therapeutic approach for congenital and acquired diseases in regenerative medicine. Recently, MSC have been used for the treatment of graft-versus-host disease after allogeneic stem cell transplantation[1]. These strategies require ex vivo MSC expansion with media containing fetal bovine serum (FBS). Since the use of FBS may be associated with immunological responses against the cultured MSC[2] and may unknowingly transmit prions[4], the development of a serum free medium (^SFM) for MSC isolation and expansion is essential for the widely applicable MSC therapy.

We evaluated a novel serum deprived medium (Quantum 333^R) for the isolation and expansion of MSC as compared to the standard medium (^SM), DMEM + 10% FBS. Fibroblastoid colony forming unit (CFU-F), as MSC progenitors, and MSC expansion potential were considered as measurable parameters. The number of CFU-F found at 14 days of culture in the SFM (average: 1 CFU-F/ 1.78 x 10⁵ bone marrow mononuclear cells, BMMNC) and the SM (1 CFU-F/ 2.3 x 10⁵ BMMNC) were similar (p=0.18 t-test), but the trend suggested that the SFM may be superior. Passage 5 MSC in both culture conditions showed similar percentages of proliferating, i. e. BrdU+ cells: 7,5% (+/− 1,3%) and 10% (+/− 2,1%) in MSF and in SM, respectively. Flow cytometric analysis of MSC for side and forward scatter properties showed that MSC^SFM were smaller in the side scatter than MSC ^SM. Further immunophenotypical characterization showed, in the MSC^SFM, the absence of CD45, CD34, CD31 and the presence of CD90, CD105, CD73 confirming the phenotype of the “classical” MSC^SM. CD73 and CD105 were upregulated in MSC^SFM in comparison to MSC^SM. Still, MSC^SFM retained the capacity to inhibit the cytokine-induced proliferation of PBMC as reported for MSC^SM. In addition, to test the differentiation potential of MSC^SFM, the cells were successfully driven into osteogenic, adipogenic, myogenic and chondrogenic phenotypes. Our data support that Quantum 333^R is able to isolate and expand MSC clonogenic precursors without affecting their proliferation, immunophenotype, differentiation and immunomodulatory potentials, thus suggesting a superior technique to isolate clinical grade cell population for safer clinical applications.