Isolation and Characterization of Extracellular Vesicles

MVs were isolated according to the well-established protocol developed in our laboratory [45 (link)] based on the procedure introduced in the previous study [46 (link)]. HATMSC2 cells were cultured in multi-layer cell culture flasks (Nunc TripleFlasks, Thermo Scientific, Carlsbad, CA, USA) using DMEM + 10% FBS until they reached 75% confluence. Next, the cells were cultured in serum-free media in hypoxic conditions (1% O₂) for 48 h to enhance the release of MVs. The conditioned media collected from the HATMSC2 cultures were mixed to obtain a homogenous starting material before the isolation of MVs. In the next step, the conditioned media were centrifuged at 300× g for 10 min at 4 °C, and at 2000× g for 10 min at 4 °C, in order to remove cellular debris and apoptotic bodies. Subsequently, the supernatants were subjected to double centrifugation at 12,000× g for 30 min at 4 °C using a Sorvall LYNX 6000 ultracentrifuge (Thermo Scientific, Carlsbad, CA, USA) with an intermediate washing step in PBS (IIET, Wroclaw, Poland). The obtained MV pellets were resuspended in 150 μL of PBS and stored at −80 °C.