The isolation of sEVs from MSCs was performed by ultracentrifugation as described previously [22 (link)]. Briefly, after 2–3 passages, when the cell reached 70% confluence, the complete culture medium of MSCs was replaced with DMEM/F12 containing 10% sEV-depleted FBS (SBI, USA). Forty-eight hours later, the culture medium was collected, centrifuged at 2000×g for 10 min and 10,000×g for 30 min at 4 °C to remove dead cells, cellular debris and apoptotic bodies. Then the supernatants were passed through a 0.22 μm membrane filters (Millipore, SLGP033RB) and centrifuged at 100,000×g for 75 min. Subsequently, the supernatants were discarded, and the pellet was resuspended in phosphate-buffered saline (PBS). The resuspensions were centrifuged again at 100,000×g for 75 min, and the deposition was resuspended with PBS for following application or stored at −80 °C.
Nanoparticle tracking analyzer (NTA) (Particle Metrix GmbH, Germany) and transmission electron microscope (TEM) (Hitachi, Japan) were used to measure the particle size and concentration of sEVs. Expression of the sEVs markers was determined by Western blot, including CD63, CD9, tumor susceptibility gene 101 (TSG101) and Calnexin.
Nanoparticle tracking analyzer (NTA) (Particle Metrix GmbH, Germany) and transmission electron microscope (TEM) (Hitachi, Japan) were used to measure the particle size and concentration of sEVs. Expression of the sEVs markers was determined by Western blot, including CD63, CD9, tumor susceptibility gene 101 (TSG101) and Calnexin.