Isolation and Characterization of Therapeutic hucMSC-Derived Small Extracellular Vesicles

Small extracellular vesicles derived from hucMSCs were isolated and purified as described previously (Wu et al., 2021 (link)). Cell supernatants were collected and centrifuged at 1,500 × g for 20 mins to remove cell debris and then centrifuged at 10,000 × g for 30 min. Next, the sEVs were concentrated with a 100 kDa molecular weight cutoff ultrafiltration membrane (MWCO) (Millipore, Billerica, MA, United States). Subsequently, the concentrated solution from the upper tube was collected for ultracentrifugation at 100,000 g for 70 min at 4°C to pellet the hucMSC-sEVs. Then pellet containing sEVs was resuspended in phosphate-buffered saline (PBS) and centrifuged again at 100,000 g for 70 min. Finally, the pelleted sEVs were washed and gathered from the bottom of the tube with PBS. sEVs were finally filtered with a 0.22 μm pore filter (Millipore, Billerica, Massachusetts, United States) and stored at −80°C. The diameter of the concentrated sEVs was determined by nanoparticle tracking analysis (NTA) (NanoSight, Amesbury, United Kingdom). sEVs were also identified morphologically by transmission electron microscopy (FEI Tecnai 12, Philips, Netherlands). Western blotting was also used to confirm the presence of characteristic markers of sEVs (CD81, HSP70, and Alix).