Characterization of the Optimal Culture Conditions for Clinical Scale Production of Human Mesenchymal Stem Cells

TLDR

Mesenchymal stem cells are multipotent, immunosuppressive cells with high in‑vitro expansion potential, yet their low niche abundance necessitates large‑scale production for therapeutic use. The study aimed to identify an optimal protocol for large‑scale MSC production that preserves multilineage differentiation and immunosuppressive functions. This was achieved by systematically comparing various culture conditions, including medium composition, nutrient levels, cell densities, and surface properties. The data revealed that fetal calf serum quality, basal medium, glucose, glutamine, plating and passaging densities, and plastic surface all influence outcomes, and that bFGF addition increases proliferation, upregulates HLA‑I, induces low HLA‑DR without eliciting significant allogeneic T‑cell responses, enhances in‑vivo immunosuppression, and biases differentiation toward osteogenesis while limiting neurogenesis, thereby defining an optimal protocol for high‑yield, therapeutically competent MSCs.

Abstract

Abstract Mesenchymal stem cells (MSCs) are multipotent cells defined by multilineage potential, ease to gene modification, and immunosuppressive ability, thus holding promise for tissue engineering, gene therapy, and immunotherapy. They exhibit a unique in vitro expansion capacity, which, however, does not compensate for the very low percentage in their niches given the vast numbers of cells required for the relative studies. Taking into consideration the lack of a uniform approach for MSC isolation and expansion, we attempted in this study, by comparing various culture conditions, to identify the optimal protocol for the large-scale production of MSCs while maintaining their multilineage and immunosuppressive capacities. Our data indicate that, apart from the quality of fetal calf serum, other culture parameters, including basal medium, glucose concentration, stable glutamine, bone marrow mononuclear cell plating density, MSC passaging density, and plastic surface quality, affect the final outcome. Furthermore, the use of basic fi-broblast growth factor (bFGF), the most common growth supplement in MSC culture media, greatly increases the proliferation rate but also upregulates HLA-class I and induces low HLA-DR expression. However, not only does this upregulation not elicit significant in vitro allogeneic T cell responses, but also bFGF-cultured MSCs exhibit enhanced in vivo immunosuppressive potential. Besides, addition of bFGF affects MSC multilineage differentiation capacity, favoring differentiation toward the osteogenic lineage and limiting neurogenic potential. In conclusion, in this report we define the optimal culture conditions for the successful isolation and expansion of human MSCs in high numbers for subsequent cellular therapeutic approaches.