Hollow fiber bioreactor allows sustained production of immortalized mesenchymal stromal cell-derived extracellular vesicles

Sergio G Garcia, Marta Sanroque-Muñoz, Marta Clos-Sansalvador, Miriam Font-Morón, M. Monguió-Tortajada, F. Borràs, Marcella Franquesa
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Abstract

Aim: Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have been reported to hold great potential as cell-free therapies due to their low immunogenicity and minimal toxicity. However, the large doses of MSC-EVs that are required for their clinical application highlight the urgency of finding a large-scale system for MSC-EV manufacture. In this study, we aimed to set up a hollow fiber bioreactor system for the continuous homogenous production of functional and high-quality MSC-EVs. Methods: MSC lines from two donors were immortalized (iMSC) and inoculated into hollow fiber bioreactors. Throughout 4 weeks, conditioned medium was daily harvested. iMSC-EVs were purified and characterized for content, immunophenotype, size, and functionality and compared to 2D cultured iMSC. Results: The iMSC inoculated into the bioreactor remained viable during the whole culture period, and they maintained their MSC phenotype at the end of EV production. Our results showed that the bioreactor system allows to obtain 3D-cultured iMSC-derived EVs (3D-EVs) that are comparable to flask (2D)-cultured iMSC-derived EVs (2D-EVs) in terms of protein and lipid content, size, and phenotype. We also confirm that 3D-derived EVs exhibit comparable functionality to 2D-EVs, showing pro-angiogenic potential in a dose-dependent manner. Conclusions: These findings suggest that setting up a hollow fiber bioreactor system inoculating immortalized MSC lines facilitates the large-scale, functional, and high-quality production of iMSC-EVs. Our results emphasize the great potential of this production methodology to standardize EV production in the pursuit of clinical applications.
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中空纤维生物反应器可持续生产永生化间充质基质细胞衍生的细胞外囊泡
目的:据报道,间充质基质细胞衍生的细胞外囊泡(MSC-EVs)因其免疫原性低、毒性小而具有作为无细胞疗法的巨大潜力。然而,其临床应用所需的大剂量间充质干细胞-细胞外小泡突显了寻找大规模间充质干细胞-细胞外小泡制造系统的紧迫性。在本研究中,我们旨在建立一个中空纤维生物反应器系统,用于连续均质生产功能性和高质量间充质干细胞-EV。方法将两名供体的间充质干细胞系永生化(iMSC)并接种到中空纤维生物反应器中。对 iMSC-EV 进行纯化,对其含量、免疫表型、大小和功能进行鉴定,并与二维培养的 iMSC 进行比较。结果接种到生物反应器中的 iMSC 在整个培养期间保持存活,并且在 EV 生产结束时仍保持间充质干细胞的表型。我们的结果表明,生物反应器系统可以获得三维培养的 iMSC 衍生 EV(三维-EV),其蛋白质和脂质含量、大小和表型与烧瓶(2D)培养的 iMSC 衍生 EV(2D-EV)相当。我们还证实,三维衍生的 EVs 具有与二维 EVs 相当的功能,并以剂量依赖的方式显示出促进血管生成的潜力。结论这些研究结果表明,建立中空纤维生物反应器系统接种永生化间叶干细胞系有助于大规模、功能性和高质量地生产 iMSC-EV。我们的研究结果表明,这种生产方法在实现 EV 生产标准化、促进临床应用方面具有巨大潜力。
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