1 100 transmission electron microscope tem

After harvesting UC-MSCs via either the manual manufacturing process or the automated culture platform, the cell culture supernatants were collected for extracellular vesicle (EV) collection using the ultracentrifugation method as previously described [53 ]. Briefly, the supernatants were centrifuged at 300×g for 10 min at 4 °C to remove the cell pellet. The collected supernatants were then centrifuged at 2000×g for 20 min at 4 °C to remove dead cells and large EVs and then at 10,000×g for 30 min at 4 °C to remove cell debris. The supernatants were then centrifuged at 100,000×g for 70 min at 4 °C to collect pellets containing EVs and contaminating proteins. The pellets were washed with 10 mL PBS and subjected to an additional centrifugation at 100,000×g for 70 min at 4 °C to remove contaminating protein. The remaining EVs were resuspended in 100 μl PBS and stored at −80 °C for further use. To visualize and measure the EV size, TEM images of EV samples were obtained using a JEOL 1100 Transmission Electron Microscope (TEM, JEOL Ltd., Tokyo, Japan) at 80 kV. For visualization, EV samples were dropped and dried on a formvar- and carbon-coated copper grid (Ted Pella, Inc.).