Polyelectrolytes Are Effective Cryoprotectants for Extracellular Vesicles

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