Isolation and Characterization of BMSC-Derived Exosomes

When passage 3 BMSCs reached a confluency of 80–90%, the culture medium without exosomes was replaced, and the supernatant was collected after 2 days. The exosomes in the supernatant of BMSCs were extracted by differential ultracentrifugation (Rotor: SW 32 Ti, Beckman Coulter, Brea, CA, USA). Briefly, the supernatant was collected by centrifugation at 300, 2000, and 10,000 × g for 10, 10, and 30 min to remove the cells and cell debris. Next, protein contaminants were removed by ultracentrifugation of the supernatant at 110,000 × g for 70 min twice, and the precipitate was resuspended in PBS buffer. Subsequently, exosomes in the precipitate were resuspended in 100 µL PBS and stored at −80 ° C (Théry et al., 2006 ; Chen et al., 2019 (link)).
The approximate concentration and diameter of the exosomes were measured by nanoparticle tracking analysis (NTA) using a ZetaView Particle Metrix (Particle Metrix, Meerbusch, Germany). The shape and size of the exosomes were observed by transmission electron microscopy (TEM) (FEI Tecnai G2 Spirit BioTwin; FEI, Hillsboro, OR, USA). The exosome positive markers (CD81, Hsp70, and TSG101) and purity control (Calnexin) were detected by Western blotting.