Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell‐therapy approaches: Further insights in the search for a fetal calf serum substitute

Abstract

There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.

Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts

“…Many studies have been performed to try and substitute the animal serum with human autologous or allogeneic serum or with synthetic media, but have not usually produced satisfactory results. [13][14][15] The addition of the PL has been suggested previously, [16][17][18] but the exact determination of hMSC yield starting from a reduced number of nucleated cells to reach clinically relevant numbers has never been shown previously.…”

Controversial issue: Is it safe to employ mesenchymal stem cells in cell-based therapies?

“…The cells also retained their immune-modulatory potential. More than that, they showed a more efficient suppression of allo-antigen-induced lymphocyte proliferation (Bernardo et al, 2007a). Furthermore, they were void of tumorgenicity when implanted into nude mice .…”

Mesenchymal stromal cells: a novel and effective strategy for facilitating engraftment and accelerating hematopoietic recovery after transplantation?

“…This was further substantiated by the data published by our and other groups, showing that a culture medium added with 5% platelet lysate is superior to 10% FCS in terms of clonogenic efficiency and proliferative capacity of MSCs. 24,25 These results provide the rationale for a more efficient expansion together with a significant time saving, while preserving MSC immunomodulatory functions. 24,25 However, clinical data on the safety and efficacy of MSCs have been obtained, so far, mainly with cells expanded in the presence of FCS, whereas relatively little in vivo experience is available with MSCs cultured in alternative medium supplements.…”

“…24,25 These results provide the rationale for a more efficient expansion together with a significant time saving, while preserving MSC immunomodulatory functions. 24,25 However, clinical data on the safety and efficacy of MSCs have been obtained, so far, mainly with cells expanded in the presence of FCS, whereas relatively little in vivo experience is available with MSCs cultured in alternative medium supplements. Therefore, cells expanded in the presence of alternative expansion media require extensive experimental and clinical testing before being safely and effectively employed to substitute cells generated in the presence of FCS-based media.…”

“…21,22 Platelet lysate has been shown to be a powerful substitute for FCS in MSC expansion owing to its high concentration of natural growth factors. [23][24][25] Doucet et al 23 first showed that the growth factors contained in platelet lysate are capable of promoting MSC expansion in a dose-dependent manner. This was further substantiated by the data published by our and other groups, showing that a culture medium added with 5% platelet lysate is superior to 10% FCS in terms of clonogenic efficiency and proliferative capacity of MSCs.…”