Isolation and Culture of Rat Bone Marrow MSCs

The MSCs derived from bone marrow were obtained from 60 male Sprague-Dawley (SD) rats (aged 8 weeks), and they were identified based on their surface phenotypes and multipotency, as previously described^{13 (link),19 (link)}. The bone marrow in the rat femur was flushed with low-glucose Dulbecco’s modified Eagle’s medium (DMEM; KeyGEN, Nanjing, Jiangsu, China) containing 10% fetal bovine serum (FBS; KeyGEN). The bone marrow fluid was put into 10 ml centrifuge tubes, and 3 ml phosphate-buffered saline (PBS; KeyGEN) was added. Then, it was centrifuged for 10 min at 1000 r/min. The supernatant was abandoned, and the remaining cells were washed twice with PBS and spun at 1000 r/min for 10 min. After washing, 5 ml DMEM was added to the cell suspension, and then the cells were cultured in culture dishes with 5% carbon dioxide (CO₂)/95% air at 37 °C. When the cells reached 80% to 90% confluence, they were resuspended by trypsin. The subculture was plated at about 2 × 106 cells per dish. MSCs from passages 3 to 4 were used for all experiments.