Qi Yiyao, Wang Xicheng, Bai Zhihui, Xu Ying, Lu Tingting, Zhu Hanyu, Zhang Shoumei, Wu Zhihong, Liu Zhongmin, He Zhiying, Jia Wenwen
Institute for Regenerative Medicine, School of Life Sciences and Technology, School of Medicine, Tongji University, Shanghai, 200123, China.
National Stem Cell Translational Resource Center, Shanghai East Hospital, Tongji University, Shanghai, 200123, China.
Heliyon. 2024 May 18;10(10):e30968. doi: 10.1016/j.heliyon.2024.e30968. eCollection 2024 May 30.
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.
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 functions.
Inoculating 2 × 10 MSCs into a 2.6 L bioreactor in the TDHLSP system resulted in a final scale of 4.6 × 10 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.
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.
有效提高临床级间充质干细胞（MSC）的产量对临床应用至关重要。当前的平面培养方法面临诸多挑战，包括可扩展性问题、劳动强度大、与细胞衰老相关的问题以及异质性反应。本研究旨在建立一个用于MSC生成的大规模生产系统。此外，还对在不同条件下培养的MSC之间的生物学差异进行了比较分析。
我们使用自制的玻璃微载体和多功能生物反应器开发了一种用于MSC的GMP级三维低氧大规模生产（TDHLSP）系统。在使用不同方法培养的MSC中评估了不同参数，包括细胞活力、细胞直径、免疫表型、形态、核型和致瘤性。单细胞RNA测序（scRNA-seq）揭示了与在低氧条件下三维培养的MSC（3D_Hypo MSC）功能增强相关的途径和基因。此外，3D_Hypo MSC中CD142的敲低证实了其功能。
在TDHLSP系统中，将2×10个MSC接种到2.6 L生物反应器中，到第10天最终可获得4.6×10个3D_Hypo MSC。3D_Hypo MSC保留了二维MSC的特征，证明了它们的基因组稳定性和非致瘤性。有趣的是，3D_Hypo MSC的亚群比二维MSC表现出更均匀的分布和更紧密的关系。MSC的异质性与“细胞周期”和“基质/间充质”密切相关，3D_Hypo MSC表达更高水平的活化基质基因。与二维MSC相比，3D_Hypo MSC在血管形成、TGF-β1分泌和抑制BV2增殖方面表现出增强的能力，同时维持衰老相关β-半乳糖苷酶（SA-β-gal）阴性。然而，CD142表达下调后，3D_Hypo MSC的增强功能下降。
TDHLSP系统导致MSC的总体产量较高，并促进了MSC簇的均匀分布。这种培养方法还增强了关键的细胞特性，如血管生成、免疫抑制和抗衰老。这些功能改善且均匀的MSC亚群为干细胞疗法的临床应用提供了坚实的基础。
