Isolation, Characterization, and Uptake of sEVs

BMSCs were plated in 10 cm culture dishes at a density of 5 × 10⁵ cells/dish, and then the culture medium was replaced with exosome-free serum medium with/without 1 μg/ml PT for 48 h when the cells reached 80% confluence. The culture medium was then collected and centrifuged at 300 ×g for 10 min, 3,000 ×g for 10 min, 10,000 ×g for 30 min, and then 100,000 ×g for 1 h twice, each time discarding the supernatant and resuspending the pellet with PBS. The final pellets were stored at −80°C until further use. The collected samples included two groups: 1) sEVs derived from untreated BMSCs (Un-sEVs); and 2) PT-sEV.

The concentration of sEVs was evaluated by BCA protein assay kits (23,225, Thermo Fisher Scientific, United States), and sEVs were characterized via electron microscopy, a nanoparticle tracking analyzer (NTA), and Western blot, which was performed with anti-CD9 (ab92726, abcam, United States), anti-Alix (NBP1-90201, Novusbio, United States), and anti-Tsg101 (NBP1-80659, Novusbio, United States) antibodies.