Transmission Electron Microscopy analysis

Far from being conclusive, our data suggest that the use of solvent/detergent addition could be seen as a preferable virus-deactivating method, taking EV’s purity obtained after a single UC step into account and as far as small EVs isolation and analysis are concerned. Non-ionic detergents are indeed relatively mild and usually non-denaturing [ 16 ], but they are known to break lipid–lipid and lipid–protein interactions. This could account for the apparent higher purity of EVs obtained after S/D treatment (see WB analysis), which contrasts with the higher content of lipoparticle contaminants detected following heat treatment. In this sense, their use of upfront UC cycles could be worth further investigating. On the other hand, solvent/detergent treatment led to the depletion of vesicular particles of larger size (>150 nm), and their use should be cautiously pondered if this EVs subpopulation represents the target of analysis. In contrast, heat-based protocols provided higher recovery, and the enrichment in EVs contaminants could be counterbalanced by the introduction of a subsequent step of purification. Overall, the virus-inactivation procedure should be tailored considering the downstream analysis to be undertaken, and further work will be needed in this direction to identify the best possible practices.