Three-dimensional and serum-free culture in fixed-bed bioreactor enhance exosome production by affecting the cytoskeleton through integrin β1 and RAC1

Exosomes are nanoscale vesicles containing a variety of bioactive factors, showing significant potential as cell-free therapeutic agents. However, the large clinical doses required for exosome administration highlight the need for a scalable serum-free production system to increase exosome yield. In this study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs) were cultured in three-dimensional (3D) environments using polyethylene terephthalate (PET) fiber membranes. Two key pathways promoting exosome secretion and biogenesis through 3D culture were identified. Specifically, 3D culture reduced the expression of Integrin β1, leading to the decondensation of cortical actin, and increased the expression of RAC1, which promoted actin aggregation on the membranes of multivesicular bodies (MVBs). Building on this mechanistic understanding, we simulated the depolymerization/aggregation of the cytoskeleton in 3D culture and designed a serum-free medium optimized for exosome production. For large-scale exosome production, hUCMSCs were cultured in a fixed-bed bioreactor using PET fiber rolls as the fixed-bed layer with the developed serum-free medium. This setup increased exosome production per cell by 16-fold compared to 2D culture, yielding 2.6 × 10^14 exosome particles. The exosome products demonstrated enhanced in vitro angiogenesis, immunomodulation, and in vivo wound healing capabilities. This study provides valuable insights and strategic guidance for the serum-free, large-scale production of exosomes using bioreactors, with significant implications for therapeutic applications.