Isolation of Cell-Derived Extracellular Vesicles

Vesicles were isolated from MSCs (MSC CIMVs) and SNB-19 cells (SNB-19 CIMVs) using cytochalasin B (cytochalasin B from Drechslera dematioidea, #C6762-5MG, Sigma-Aldrich, St. Louis, MO, USA) as previously described [17 (link)]. When the cell culture reached a monolayer density of 90%, the medium was removed, the culture was washed twice with PBS and the cells were detached with a 0.25% trypsin-EDTA solution (PanEco, Moscow, Russia). Then the cells were washed with PBS and incubated in modified serum-free DMEM containing 10 µg/mL cytochalasin B (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C in a humidified atmosphere with 5% CO₂. After incubation, the cells were vortexed for 60 s. Next, a series of subsequent centrifugations were carried out. Firstly, the cells were centrifuged at 700 rpm for 10 min, then the supernatant was collected and centrifuged at 1400 rpm for 10 min, after which the supernatant was passed through a filter (1 µm) and centrifuged at 12,000 rpm for 15 min. The precipitate containing CIMVs was washed with PBS. After centrifugation, the supernatant was removed, and the resulting CIMV pellet was dissolved in the culture medium, PBS, or another buffer, depending on the purpose of further experiments.