Isolation and Characterization of Extracellular Vesicles from Mesenchymal Stem Cells

MSC-EVs were isolated as previously described (29 (link)). In brief, MSCs were cultured to a density of 2×10⁷ cells/T75 flask. Culture media were removed and cells were washed three times with PBS and then cultured in serum-free media for 48 h. The media were collected and cell debris was discarded by gradient centrifugation at 300 g for 10 min and 10,000 g for 30 min at 4°C. Further centrifugation (100,000 g, 4°C, 70 min) was conducted to obtain extracellular vesicles. After resuspending the pellet and washing it with PBS, the pellet was centrifuged at 100,000 g, 4°C, for 70 min. For extracellular vesicle labeling, extracellular vesicles were incubated with 3,3’-dioctadecyloxacarbocyanine perchlorate, DIOC₁₈(3) (Beyotime) or 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide DIR (Beyotime) at 37°C for 30 min. Samples were then centrifuged at 100,000 g, 4°C, for 70 min (30 (link), 31 (link)). EVs were resuspended in PBS and used immediately for experiments. EVs were not frozen and all experiments used freshly prepared EVs. EVs were identified by transmission electron microscopy (H7500; Hitachi, Tokyo, Japan). The same passage numbers of EVs derived from WT and IL-10 knockout MSCs were used for individual experiments.