Product characterization and preclinical evaluation of the biodistribution and safety of umbilical cord mesenchymal stromal cell-derived extracellular vesicles

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Abstract
Background Extracellular vesicles (EVs) represent a new axis of intercellular communication that can be explored for therapeutic purposes; they have garnered considerable attention for their potential as cell-free therapy. The clinical application of mesenchymal stromal cell (MSC)-derived EVs is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution and toxicity of hUCMSC-EVs in preclinical studies. Methods hUCMSCs were isolated from a single cord tissue donor, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize the hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive male BALB/c mice (6–8 weeks old) after intravenous administration of the product. Results hUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. The comparison of hUCMSC-EVs at 24 and 72 h showed that unique protein-coding gene clusters at 24 h were mainly involved in complement activation, particularly the classic pathway, as well as in innate immune response and positive regulation of B cell activation. The results showed reduced variability of differential proteins and indicated contrasting characteristics (48 versus 72 h). Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Conclusion hUCMSC-EVs were successfully isolated following a cGMP-compliant protocol, with consistent characteristics and safety profiles, supporting their future clinical development as cell-free therapies.

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