Purification of extracellular vesicles with membrane processes

Extracellular vesicles (EVs) are particles that are naturally released from both eukaryotic and prokaryotic cells. They transport a specific cargo, which is surrounded by a lipid bilayer, that can be packed and deliver to neighbouring or distant cells. Nowadays, the golden standard for the isolation of extracellular vesicles is represented by differential ultracentrifugation (UC), which cannot be applied on an industrial scale due to its prolonged duration and the limited volume processable. This experimental work focuses on the identification of a scalable process for the purification of EVs from different sources by membrane microfiltration and ultrafiltration. Two EVs sources were investigated, milk whey and exhausted culture medium of Echinacea purpurea. For each source, different combinations of clarification and purification procedures were tested. For the clarification step, centrifugation and cross-flow microfiltration were tested. For the purification step, several operating modes of cross-flow microfiltration and ultrafiltration were tested, namely concentration and diafiltration. For milk whey only, also dialysis was tested. The product from each stage was subjected to qualitative and quantitative analysis, to evaluate the efficiency of impurities removal and the yield in terms of recovered EVs, specifically SEC – HPLC, DLS, Zeta potential, BCA and RNA assays. The results of this study demonstrate that membrane-based processes represent a highly performant and versatile technique for the isolation of EVs, since they can be adapted to different feed solutions. For exhausted culture medium of Echinacea purpurea, the optimal protocol was found to be the one that combined centrifugation and diafiltration, that allows for the removal of impurities with a 99.73% efficiency, while maintaining an EVs content of 71.46%. The same protocol resulted very effective also with milk whey, allowing for and impurity removal of 98.79% and a full EVs recovery (100%).