Ex Vivo Expansion of Human Marrow Mesenchymal Stem Cells: A Serum-Free Medium Compared to Classical α-MEM Medium.

BACKGROUND: Bone Marrow mesenchymal stem cells (MSC) are pluripotent cells that have the capacity to differentiate into several tissue lineages and also the ability to support hematopoiesis. Their use in gene and cell therapy requires their in vitro expansion. Maintenance and proliferation of MSC are strongly dependent on the culture conditions such as properties of the bovine serum added in the culture medium. However, duration and culture conditions are critical for the successful clinical use of MSC.

OBJECTIVE: We have evaluated the efficiency of a commercial serum-free medium (UltraCulture, Bio-Whittaker, Walkersville, MD) supplemented with a serum substitute (Ultroser, BioSepra, Cergy-Saint-Christophe, France) in order to work without FBS and to have a more constant composition. This medium (UC) was compared to the classical medium a-MEM containing 10% FBS (Invitrogen, Merelbeke, Belgium).

METHODS: BM-mononuclear cells collected from 11 healthy donors were plated in petri dishes at a concentration of 10⁵/cm². After 3 days, non adherent cells were removed and culture media were added to adherent cells which were maintained at 37°C until they reached confluence. MSC expansion was analysed after the primoculture (PM) and after the first passage (P1). CFU-F (colony forming units-fibroblastic) number, phenotypic analysis and differentiation potential were also evaluated.

RESULTS::The mean culture duration was 13±2 and 8±2 days respectively for PM and P1 but the confluence was reached more rapidly for cells cultured in UC. After PM, 0.35x10⁶ and 1.21x10⁶ cells were obtained for MEM and UC respectively (p<0.005). Moreover, around 20% of cells cultured in MEM were CD45⁺ while the level of CD45⁺ cells was frequently < 5% in UC indicating that UC medium favored the rapid elimination of hematopoietic cells. After P1, the expansion rate was significantly higher in UC than MEM: respectively 5.13 ± 1.2 and 22.6 ± 3 (p<0.0005). The numbers of CFU-F were always higher in UC demonstrating the enhanced proliferation in serum-free medium. The phenotype of MSC was similar in the both media: SH2⁺, SH3⁺, CD44⁺, CD45⁻, CD34⁻, HLA-DR⁻. More importantly, these cells remained able to differentiate into osteocytes, chondrocytes, adipocytes and neuron-like cells in the both media confirming their multipotentiality.

CONCLUSIONS: Our data strongly support UltraCulture medium supplemented with a serum substitute as an optimal medium for MSC culture. Indeed, it allows a better cell expansion, better proliferation and preserves multipotentiality. This medium, reducing culture period and containing low concentration of serum substitute, is of major interest for clinical production of MSC.