文献类型：期刊文献

英文题名：Three-Dimensional and Serum-Free Culture in Fixed-Bed Bioreactor Enhance Exosome Production by Affecting the Cytoskeleton

作者：Zhou, Yan[1]; Huo, Chuanfu[1]; Zhao, Yuanyuan[1]; Song, Meng[2]; Guo, Yuzhe[1]; Li, Shihao[1]; Tan, Wensong[1]

机构：[1] State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237, China; [2] Shanghai Key Laboratory of Multiphase Materials Chemical Engineering, Department of Product Engineering, School of Chemical Engineering, East China University of Science and Technology, Shanghai, 200237, China

年份：2024

外文期刊名：SSRN

收录：EI(收录号：20240323816)

语种：英文

外文关键词：Bioconversion - Cell culture - Microfiltration - Plastic bottles - Proteins - Stem cells

摘要：Exosomes are nanoscale cellular vesicles containing a variety of bioactive factors, which show great promise for cell-free therapeutic drugs. However, exosomes are administered in large clinical doses, and a scaleable serum-free exosome production system is needed to increase exosome yield. To this end, in this study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs) were cultured in 3D based on polyethylene terephthalate (PET) fiber membranes, and it was found that 3D culture promoted exosome secretion and biogenesis through two pathways, decreasing the expression of Integrin β1, which leads to the decondensation of cortical actin, and increasing the expression of RAC1, which promotes the aggregation of actin on the membranes of multivesicular vesicles (MVBs), respectively, which resulted in a 4.8-fold increase in exosome production per cell compared to 2D culture. Based on the understanding of this mechanism, we simulated the depolymerization/aggregation of the cytoskeleton in 3D culture, and designed and developed a serum-free medium for exosome production, which increased the exosome yield to 8.1-fold under 3D culture conditions compared with 2D culture. Finally, hUCMSCs were cultured in a fixed-bed bioreactor with PET fiber rolls as the fixed-bed layer using the developed serum-free medium for large-scale production of exosomes. The exosome yield per cell was increased to 16.0-fold compared with that of 2D culture, and 2.6×1014 particles of exosomes were harvested. The combination of tangential flow filtration achieved a large-scale separation of exosomes from supernatants, with the exosome recovery rate of 61.5%. The exosome products had better in vitro angiogenic, immunomodulatory and in vivo wound repair capabilities. The results of this study provide strategic guidance for serum-free, large-scale production of exosomes using bioreactors. ? 2024, The Authors. All rights reserved.