Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile
Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile
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DOI：
10.1016/j.bioactmat.2022.07.004
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发表时间：
2023-07-01
影响因子：
18.9
通讯作者：
Li, Yan
中科院分区：
文献类型：
--
作者：
Jeske, Richard;Liu, Chang;Li, Yan
人间充质基质细胞（hMSCs）是机械敏感的经历表型改变时，受到剪切应力，细胞聚集，和基板的变化，在3D动态生物反应器培养。然而，关于生物反应器微环境如何影响hMSC衍生的细胞外囊泡（EV）的分泌和货物概况知之甚少，所述细胞外囊泡（EV）包括子集“外泌体”，其含有来自亲本细胞的治疗性蛋白质、核酸和脂质。在这项研究中，骨髓来源的hMSC在PBS迷你0.1L Vertical-Wheel生物反应器系统中的3D Synthemax II微载体上在25、40和64 RPM（0.1-0.3 dyn/cm 2）的可变剪切应力水平下扩增。与静态2D培养相比，生物反应器系统促进EV从hMSC分泌2.5倍，并上调EV生物发生标志物和糖酵解基因的表达。来自生物反应器培养的EV中的微小RNA货物也发生了改变，包括miR-10、19 a、19 b、21、132和377的上调。通过蛋白质组学分析表征EV蛋白质货物，与2D培养的EV相比，显示代谢、自噬和ROS相关蛋白的上调。此外，在0.5 L生物反应器中证明了垂直轮生物反应器系统的可扩展性，与0.1 L生物反应器相比，显示出相似或更好的hMSC-EV分泌和货物含量。这项研究推进了我们对干细胞衍生EV的生物制造的理解，用于无细胞治疗，以治疗神经系统疾病，如缺血性中风，阿尔茨海默病和多发性硬化症。
Human mesenchymal stromal cells (hMSCs) are mechanically sensitive undergoing phenotypic alterations when subjected to shear stress, cell aggregation, and substrate changes encountered in 3D dynamic bioreactor cultures. However, little is known about how bioreactor microenvironment affects the secretion and cargo profiles of hMSC-derived extracellular vesicles (EVs) including the subset, "exosomes", which contain therapeutic proteins, nucleic acids, and lipids from the parent cells. In this study, bone marrow-derived hMSCs were expanded on 3D Synthemax II microcarriers in the PBS mini 0.1L Vertical-Wheel bioreactor system under variable shear stress levels at 25, 40, and 64 RPM (0.1-0.3 dyn/cm2). The bioreactor system promotes EV secretion from hMSCs by 2.5-fold and upregulates the expression of EV biogenesis markers and glycolysis genes compared to the static 2D culture. The microRNA cargo was also altered in the EVs from bioreactor culture including the upregulation of miR-10, 19a, 19b, 21, 132, and 377. EV protein cargo was characterized by proteomics analysis, showing upregulation of metabolic, autophagy and ROS-related proteins comparing with 2D cultured EVs. In addition, the scalability of the Vertical-Wheel bioreactor system was demonstrated in a 0.5L bioreactor, showing similar or better hMSC-EV secretion and cargo content compared to the 0.1L bioreactor. This study advances our under-standing of bio-manufacturing of stem cell-derived EVs for applications in cell-free therapy towards treating neurological disorders such as ischemic stroke, Alzheimer's disease, and multiple sclerosis.