Enhancement of the Function of Mesenchymal Stem Cells by Using a GMP-grade Three-Dimensional Hypoxic Large-Scale Production System

Background: Efficiently increasing the production of clinical-grade mesenchymal stem cells (MSCs) is crucial for clinical applications. Challenges with the current planar culture methods include scalability issues, labour intensity, concerns related to cell senescence, and heterogeneous responses. This study aimed to establish a large-scale production system for MSC generation. In addition, a comparative analysis of the biological differences between MSCs cultured under various conditions was conducted. Methods and materials: We developed a GMP-grade three-dimensional hypoxic large-scale production (TDHLSP) system for MSCs using self-fabricated glass microcarriers and a multifunctional bioreactor. Different parameters, including cell viability, cell diameter, immunophenotype, morphology, karyotype, and tumourigenicity were assessed in MSCs cultured using different methods. Single-cell RNA sequencing (scRNA-seq) revealed pathways and genes associated with the enhanced functionality of MSCs cultured in three dimensions under hypoxic conditions (3D_Hypo MSCs). Moreover, CD142 knockdown in 3D_Hypo MSCs confirmed its in vitro functions. Results: Inoculating 2 × 108 MSCs into a 2.6 L bioreactor in the TDHLSP system resulted in a final scale of 4.6 × 109 3D_Hypo MSCs by day 10. The 3D_Hypo MSCs retained characteristics of the 2D MSCs, demonstrating their genomic stability and non-tumourigenicity. Interestingly, the subpopulations of 3D_Hypo MSCs exhibited a more uniform distribution and a closer relationship than those of 2D MSCs. The heterogeneity of MSCs was strongly correlated with ‘cell cycle’ and ‘stroma/mesenchyme’, with 3D_Hypo MSCs expressing higher levels of activated stroma genes. Compared to 2D MSCs, 3D_Hypo MSCs demonstrated enhanced capabilities in blood vessel formation, TGF-β1 secretion, and inhibition of BV2 proliferation, with maintenance of Senescence-Associated β-Galactosidase (SA-β-gal) negativity. However, the enhanced functions of 3D_Hypo MSCs decreased upon the downregulation of CD142 expression. Conclusion: The TDHLSP system led to a high overall production of MSCs and promoted uniform distribution of MSC clusters. This cultivation method also enhanced key cellular properties, such as angiogenesis, immunosuppression, and anti-aging. These functionally improved and uniform MSC subpopulations provide a solid basis for the clinical application of stem cell therapies.