# Extracellular Vesicles for Regenerative Medicine Applications
## 4 Clinical Development of EVs
### Future Challenges
To address concerns in EV isolation and purity, the manufacturing trends are shifting from techniques including ultracentrifugation (which is time consuming and can produce contaminants) towards isolation techniques potentially capable of large-scale manufacturing such as tangential flow filtration (TFF) or size-exclusion chromatography (SEC) [159; 157; 156; 158]. However, these techniques still have their limitations and further studies are necessary to determine the optimal technique for the isolation and purification of EVs at the large-scale, commercial level. In addition to concerns regarding the manufacturing of EV therapeutics, the long-term storage and stability of EV therapies pose a significant hurdle to clinical translation. The effects of various preservation techniques on EV stability and bioactivity are not well understood [157; 160]. Initial studies suggested that cryopreservation at \(-80\)\({}^{\circ}\)C was superior to storage at 4 \({}^{\circ}\)C and would preserve EV structure and composition. However, storage at \(-80\)\({}^{\circ}\)C comes with increased costs and technical challenges. The lyophilization of EVs might address this concern for long-term storage as long as the optimal concentration of cryoprotectants is used to protect the biologic activity of the EVs through this process [156; 160]. Altogether, the optimal conditions for the preservation of EVs depend on multiple factors including, but not limited to, the EV source, isolation and purification technique, and the route of administration. The functional and structural characterization of EVs represent an additional and unavoidable hurdle for the translation of EV-based therapies, and fundamental understanding of the heterogeneity and specificity of each kind of EV is critical. Relevant properties, such as size, shape, molecular cargo (i.e., sRNA, miRNA, cytokines, proteins, or DNA), membrane composition, or surface markers, vary not only between different EV kinds or cell sources but also within EVs derived from the same source [159; 161; 70; 155; 162]. Technical limitations for separating EV subpopulations and the lack of specific markers for EV subtypes obstruct the understanding of how these subpopulations affect bioactivity and safety [156; 157; 159]. Further research and technical innovations are necessary to overcome this hurdle. The safety of EV-based therapies remains a major barrier to clinical translation. Many pre-clinical and early clinical studies have shown that EV therapies are generally biocompatible and have a low risk of immunogenicity in humans [156; 163; 164; 165]. However, these clinical studies seldom discuss the safety of repeated administrations as well as the safety of a wide variety of administration routes [156]. Several pre-clinical studies have assessed the biodistribution of EVs in rodents and have shown that EVs are rapidly cleared from the circulatory system [166; 167; 168]. However, the accumulation of EVs in the liver or kidneys, and the EV-mediated effects on these organs, are unknown and therefore must be investigated in future studies. Moreover, the relatively rapid clearance of EVs from the systemic circulation could affect the specificity of EV therapies and increase the frequency of off-target, non-specific effects. To address these non-specific, off-target effects, EV membrane decorating techniques have been developed and have shown early potential for more focused, targeted therapy as seen in specific models of cancer [156; 168]. In addition to the off-target effects, there remain safety concerns including the possibility of viral vector contaminants in EV preparation. These viral contaminants cannot be overlooked as they present similar characteristics to EVs and are often capable of surpassing filtration systems during the harvest of EVs [155; 156; 157].