Design and validation of a GMP-compliant manufacturing platform of extracellular vesicles from mesenchymal stromal cells

Abstract

There is growing interest in mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) as key components in the development of next-generation regenerative cell-free therapies; however, their clinical translation remains limited by a lack of validated clinical-grade manufacturing platforms. Thus, we have established a good manufacturing practice (GMP)-compliant, semi-automated, mid-scale manufacturing workflow that couples the single-use stirred-tank bioreactor expansion of Wharton's jelly-derived MSCs with tangential flow filtration and automated size-exclusion chromatography, featuring in-line monitoring of critical process parameters. The MSC-EV-based product meets pre-specified critical quality attributes (CQAs), co-expresses parental-cell- and EV-specific markers, and complies with standardized quality and safety regulations. Moreover, the final product CQAs—including CD73-associated immunomodulatory activity—remained within acceptable limits for 12 months of storage in PlasmaLyte 148 at −80°C. Overall, our integrated platform yields MSC-EVs with high batch-to-batch reproducibility, in alignment with current GMP and regulatory standards, thereby providing a robust foundation supporting their potential clinical applicability.