Characterizations of molecularly distinct subpopulations of conditioned medium derived extracellular vesicles prior to be used as medicinal product

Abstract

Background & Aim Somatic cell therapy medicinal products is the largest group of advanced therapy medicinal products used in clinical trials which also resulted in routinely production of huge amount of conditioned medium. With its capacity to be used as therapeutics agents, cell-conditioned medium derived extracellular vesicles (EVs) play an explosive role in the realm of regenerative medicine or pharmaceutical science. However, the heterogeneity of EVs usually resolved by isolation of different sub-populations has sparked a heated debate. The introduction of quantifiable features assessed by reproducible and standardized assays is necessary to render EVs potentially promising options to be used as “commercial off-the-shelf product”. Methods, Results & Conclusion Therefore, in this report, following the isolation of EVs by different methods (ultracentrifuge, sucrose gradient, and 20,000g centrifugation only), we confirmed that all of them met MISEV2018 criteria (size, morphology, and presence of 3 positive markers and absence of 1 negative marker). Further assessments have been done to find out the purity (based on albumin contaminant), vesicular membrane perfectness (phosphatidylserine exposure), lipid content as well as their functions in in-vitro tests (cellular uptake and proliferation) and ex-vivo test of angiogenesis. Large scale proteome analysis of EVs sub-populations has also provided us with some important clues to choose between tests and functions. In conclusion, we demonstrate that molecularly distinct EV subpopulations can be evaluated by quantifiable methods. Further assessments to correlate some functions to each subpopulation prior to designing clinical trials can accelerate the development of EV-based therapeutics.