Production of extracellular vesicles from beta cells in stirred tanks enhances maturation and ectosome release with preserved immune properties

Small extracellular vesicles (sEVs) derived from beta cells are promising therapeutics for diabetes through their ability to modulate inflammation, preserve pancreatic function, and limit pathogenic mechanisms. For clinical translation, scalable production in a stirred tank bioreactor (STR) is essential, but shear stress can affect sEV function. Here, we report the influence of culture mode, medium formulation, and process parameters on beta cell performance and sEV quality. Small spheroid culture promotes beta cell maturation without affecting sEV yield, whereas stirring increases cellular stress and decreases viability. An optimized three-step bioprocess maximizes cell viability and sEV yields at high cell density in STR over short production periods. The produced sEVs display high purity, preserved membrane integrity, and immunomodulatory activity by reducing T-lymphocyte proliferation and IFN-γ cytokine secretion in an allogenic mixed lymphocyte reaction. Flow cytometry shows enhanced ectosome production in STR, and low glucose medium promotes insulin sorting into beta cell-derived sEVs.