Polyethylene Glycol Mediated Xeno-Free Isolation of Human Umbilical Cord Tissue-derived Mesenchymal Stem cells secreted Small Extracellular Vesicles: An Optimization, Characterization, and its Functional Evaluation Study in Glioblastoma.

The therapeutic promise of small extracellular vesicles (sEV) derived from mesenchymal stem cells lies in their inherent tumor-targeting ability, immunomodulatory functions, and capacity to deliver bioactive molecules. Human umbilical cord tissue-derived MSCs (hUCT-MSCs), due to their immunosuppressive traits, non-invasive sourcing, high yield, and low immunogenicity, are a compelling source for producing hUCT-MSC-derived sEV. This study establishes a xenofree, PEG-based precipitation protocol for isolating sEV from hUCT-MSCs cultured in human platelet lysate (HPL)-supplemented medium, effectively eliminating the risks associated with animal-derived serum. Among the tested concentrations, 8% PEG yielded the highest recovery and purity, as confirmed by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting for CD9 and CD81. Functionally, the isolated sEV significantly reduced the viability and migration of U87 glioblastoma cells, supporting their direct anti-cancer potential. This work presents a robust and clinically relevant strategy for generating functional sEV, potentially advancing their application in targeted therapies for glioblastoma and other cancers.