Content release of extracellular vesicles in a cell‐free extract

Extracellular vesicles (EVs) transfer molecules from donor to acceptor cells. The EV‐content delivery process within the acceptor cell is poorly characterized. We developed a new cell‐free assay to assess EV‐content release in vitro. We found that EV‐cytosolic cargoes are released from EVs when isolated vesicles are incubated with purified plasma membrane sheets at acidic pH, a characteristic of the endolysosomal environment. This process is protein dependent. Our results suggest that EV‐content delivery occurs within the endo/lysosomes of acceptor cells and is triggered by acidification. This process resembles virus content delivery and may require membrane fusion. The assay presented here will facilitate investigations into the core machinery and mechanisms underlying EV content delivery.