Characterizations of Molecularly Distinct Subpopu-lations of Evs: In Vitro and In Vivo Studies

Recently, the possibility of replacing cells or liposomes with the naturally secreted micro-and nano scale extracellular vesi-cles (EVs) has sparked a heated debate. With its capacity to be used as therapeutics agent, cell conditioned medium derived EVs (CM-EVs) play an explosive role in the realm of regenera-tive medicine or pharmaceutical science. Increasing and com-pelling evidence additionally render EVs potentially promising options to be used as “commercial off-the-shelf product”. Ac-cepting the heterogeneity in EV subpopulations isolated by dif-ferent methods, in this report, following the isolation of EVs by different methods (ultracentrifuge, sucrose gradient, and ۲۰,۰۰۰ g centrifugation only), we confirmed that all of them met MISEV۲۰۱۸ criteria (size, morphology, and presence of ۳ positive markers and absence of ۱ negative marker). Further assessments has been done to find out the purity (based on albu-min contaminant), vesicular membrane perfectness (phosphati-dylserine exposure), as well as their functions in in-vitro tests (cellular uptake and proliferation), ex vivo test of angiogenesis and in vivo administration to monkey model of diabetes. Large scale proteome analysis of EVs sub-populations has also pro-vided us with some important clues to choose between tests and functions. In conclusion, we demonstrate that molecularly dis-tinct EV subpopulations can be evaluated by quantifiable meth-ods. Further assessments to correlate some functions to each subpopulation prior to designing clinical trials can accelerate the development of EV-based therapeutics.