A Reproducible and Scalable Process for High Yield Mesenchymal Stromal Cell Extracellular Vesicles Production

Mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs) have emerged as a promising cell‐free therapy due to their immunomodulatory and regenerative properties. However, scalable and standardized EV production remains a major bottleneck for clinical translation. A process based on repeated exposition to shear stress for high yield MSC‐EV production is reported. MSCs from two different sources are expanded in a 3D spinner‐flask using microcarriers, and are submitted repeatedly to shear stress in serum‐free media. EVs are harvested from conditioned media and processed by tangential flow filtration (TFF). Quantitative yields, EV size, EV marker expression, secretome composition, and functional properties are assessed. Repeated shear stress‐based EV production does not impact evaluated parameters. Furthermore, variation in secretome composition and functionality is lower in repeated EV productions. Repeated exposition to shear stress provides a robust foundation for a reproducible and scalable process for high yield MSC‐EV production.