A Hydrodynamic Bioreactor for High‐Yield Production of Extracellular Vesicles from Stem Cell Spheroids with Defined Cargo Profiling

Extracellular vesicles (EVs) have become pivotal in clinical therapeutics, although no consensus exists on optimal production methods. Here, an innovative approach and associated device are introduced that employ hydrodynamic stress to form and stimulate stem cell spheroids. The method involves culturing cellular material in vessels equipped with internal obstacles, subjected to rotational motion at varying speeds and in alternating reversing modes. It achieves EV yields 10 to 20 times greater than those obtained with traditional static culture methods, offering a robust and high‐output method compatible with large‐scale EV manufacturing. This approach not only streamlines spheroid formation and subsequent EV release into a single, integrated process but also ensures the generation of EVs with enhanced biological activity and a distinctive protein cargo signature. Notably, hydrodynamic stimulation enriched EVs with ectosome‐ and mitochondria‐associated proteins, suggesting distinct biogenesis pathways. Additionally, these EVs demonstrated superior functionality in promoting wound healing, angiogenesis, and anti‐inflammatory responses, underscoring their therapeutic potential.