Extracellular vesicles and lyophilization: getting functionally stable cell-free biomedical products

During the last decade, clinical application of extracellular vesicles (EVs) is of growing interest. Despite the progress in exploring the therapeutic potential of EVs, e.g. as disease markers or the carriers for therapeutic substances, it is important to identify proper storage conditions – this issue is indeed challenging. A subtype of EVs known as exosomes is of great importance in the therapeutic applications because they participate in the regulation of intercellular communication. Currently, exosomes are considered as a promising tool in the regenerative medicine. The therapeutic potential of exosomes and other subtypes of EVs, especially for their using in immunomodulation and drug delivery, dictates great attention to the methods for their storage, in particular for a long periods of time. Lyophilization is one of the best such methods designed to preserve cell-free EV-based products. In our mini-review, we discuss the main methods developed for stabilizing cell-free products and getting stable solid forms of EVs that are capable for long-term storage. We also point out that the methods need to be following both the ease of transportation and the retaining the functionally important properties for in vivo applications. The development of optimal protocols for storing the EVs are therefore crucially important for warranting that structural and functional integrity of EVs, exosomes in particular, are maintained intact or at least modified as less as possible. For comprehensiveness of our review, we refer to original studies which investigated how the storage temperature and freezing methods may affect stability of the final EV product. We summarize advances in the area of freeze-drying EVs (exosomes), the selection of optimal process parameters and lyoprotectants, the interplay between lyophilization parameters and specific functional properties of exosomes, and the preservation of their biological activity after reconstitution before application in vivo.