Transitioning from static to suspension culture system for large-scale production of xeno-free extracellular vesicles derived from mesenchymal stromal cells
Natália Cristine Dias dos Santos, Paula Bruzadelle-Vieira, Nádia de Cássia Noronha, Amanda Mizukami-Martins, Maristela Delgado Orellana, Maria Vitória L. B. Bentley, Dimas Tadeu Covas, Kamilla Swiech, Kelen Cristina Ribeiro Malmegrim
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引用次数: 0
Abstract
Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC-EV in suspension cultures using spinner flasks and human collagen-coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T-flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold-increase was 5.7-fold for the 3D culture system and 4.6-fold for the 2D culture system, signifying a fold-change of 1.2 (calculated as the ratio of fold-increase 3D to fold-increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T-flask cultures. The total cell production in the spinner flask cultures was 5.2-fold higher than that in T-flask cultures. While the EV specific production (particles/cell) in T-flask cultures (4.40 ± 1.21 × 108 particles/mL, p < 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 108 particles/mL, p < 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC-EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC-EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno-free conditions.
近年来,从间质基质细胞(MSCs)中提取的细胞外囊泡(EVs)显示出越来越大的治疗潜力。然而,治疗目的需要大量生产 EV。因此,扩大间充质干细胞在生物反应器中的培养规模对于监测培养参数至关重要。在这项研究中,我们报道了一种可扩展的生物工艺的建立,该工艺利用旋转瓶和人胶原包被微载体(三维培养系统)在悬浮培养物中生产间充质干细胞-EV。我们比较了这种三维培养系统与使用 T 型烧瓶的标准静态培养(二维培养系统)中的 EV 生产情况。我们通过 Western 印迹和纳米粒子跟踪分析对两种培养体系中产生的 EV 进行了表征和量化。两种培养体系中产生的 EV 均通过 Western 印迹分析证实了典型蛋白标记物 CD9、CD63 和 CD81 的存在。三维培养系统的细胞倍增率为 5.7 倍,二维培养系统为 4.6 倍,倍增率为 1.2(以三维倍增率与二维倍增率之比计算)。此外,值得注意的是,纺锤形培养瓶的细胞总产量是 T 型培养瓶的 4.8 倍。旋转瓶培养物的细胞总产量是 T 型烧瓶培养物的 5.2 倍。而 T 型烧瓶培养物中的 EV 特定产量(颗粒/细胞)(4.40 ± 1.21 × 108 个颗粒/毫升,p 8 个颗粒/毫升,p 10 个颗粒/毫升)是 T 型烧瓶培养物的 4.8 倍。
期刊介绍:
Biotechnology Progress , an official, bimonthly publication of the American Institute of Chemical Engineers and its technological community, the Society for Biological Engineering, features peer-reviewed research articles, reviews, and descriptions of emerging techniques for the development and design of new processes, products, and devices for the biotechnology, biopharmaceutical and bioprocess industries.
Widespread interest includes application of biological and engineering principles in fields such as applied cellular physiology and metabolic engineering, biocatalysis and bioreactor design, bioseparations and downstream processing, cell culture and tissue engineering, biosensors and process control, bioinformatics and systems biology, biomaterials and artificial organs, stem cell biology and genetics, and plant biology and food science. Manuscripts concerning the design of related processes, products, or devices are also encouraged. Four types of manuscripts are printed in the Journal: Research Papers, Topical or Review Papers, Letters to the Editor, and R & D Notes.