Polyelectrolytes\nAre Effective Cryoprotectants for\nExtracellular Vesicles

Extracellular vesicles (EVs) have been widely recognized\nas a heterogeneous\ngroup of membrane-coated submicrometer particles released by different\ntypes of cells, including stem cells (SCs). Due to their ability to\nharbor and transfer bioactive cargo into the recipient cells, EVs\nhave been reported as important paracrine factors involved in the\nregulation of a variety of biological processes. Growing data demonstrate\nthat EVs may serve as potential next-generation cell-free therapeutic\nfactors. However, clinical application of EVs in tissue regeneration\nrequires the development of standardized procedures for their long-term\nstorage, without the loss of structural integrity and biological activity.\nIn the current study, we developed a procedure of EV cryoprotection\nbased on coating them with ultrathin polyelectrolyte bilayer consisting\nof cationic poly(ethylene glycol)-<i>block</i>- poly(3-(methacryloylamino)propyl)trimethylammonium\nchloride) (PEGn-<i>b</i>-PMAPTACm) and anionic of poly(2-acrylamido-2-methylpropanesulfonic\nacid) (PAMPS). Based on the nanoparticle tracking analysis, high-resolution\nflow cytometry, and mass spectrometry, we studied the vesicle integrity\nfollowing single- or multiple freezing-thawing cycles and long-term\nstorage. Additionally, we evaluated the effect of cryopreservation\non the EVs functional activity in vitro. Obtained data indicate that\ncoating with polyelectrolytes improves the structural integrity of\nEVs and preserves their biological activity in vitro. Additionally,\nproteomic analysis confirmed the effect of particle stabilization,\nas well as an enrichment in EV proteins in samples cryopreserved in\nthe presence of tested polymers. Taking together, our study indicates\nthat the application of polyelectrolytes may be a novel, effective\nway of facilitating long-term storage of EV preparations for their\nfurther use in the biomedical applications.