Isolation and Characterization of Rat BMSCs

Bone marrow–derived MSCs (BMSCs) were obtained from the bone marrow of 49 eight-week-old male Sprague-Dawley (SD) rats (45 rats in vitro experiment and 4 rats in vivo experiment), which were acquired from the Qinglongshan Experimental Animal Center of Jiangsu Province, China. The MSCs were identified based on their surface phenotypes and multipotency, as described previously.^{14 (link),15 (link)} After the SD rats were euthanized, the bone marrow from the femur was washed out in a Petri dish with low-glucose Dulbecco’s modified Eagle’s medium (DMEM; KeyGEN, Nanjing, Jiangsu, China), containing 10% fetal bovine serum (FBS; KeyGEN), without exosomes. Then, the cells were cultured in culture dishes with 5% CO₂/95% air at 37°C. After 72 hours, the medium was changed, and the cells were resuspended with 0.25% trypsin until the cells reached 80% to 90% confluence. Thereafter, the cells were re-seeded at the density of approximately 2×10⁶ cells per dish. The follow-up experiment was performed using passage 3.