Total bone marrow cells were flushed from the femurs and tibias of rats (2- to 4-month-old) with culture medium; the rats were sacrificed via sevoflurane overdose as previously described [31 (link)]. Complete L-DMEM containing 15% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin was used to resuspend the BMSCs, and then, the BMSCs were incubated in a humid chamber. At 48 h, the first medium change was performed to remove the nonadherent cells. When the cells reached 90% confluence, they were harvested with 0.25% trypsin (Sigma) and passaged at a ratio of 1 : 2. FCM was performed to assess the BMSC surface markers. The cells were incubated with the following fluorochrome-conjugated primary antibodies (all from BioLegend, USA): anti-CD90-PE, anti-CD29-APC, and anti-CD45-PE. BMSCs from passage (P) 3 to P5 were used for subsequent experiments.
The cells were stimulated with hypoxia, and cell viability was detected [32 (link)]. Approximately 5 × 105 BMSCs suspended in L-DMEM were plated in 150 mm-diameter culture dishes. The cells were then separately cultured under the conditions below for 12, 24, 48, 72, 96, or 120 h: 10% exosome-free FBS with hypoxia (94% N2, 5% CO2, and 1% O2 gas mixture). BMSC viability was analyzed with CCK-8 assays. Briefly, adherent cells were digested with 0.05% trypsin and collected for CCK-8 assay according to the manufacturer's instructions.
The cells were stimulated with hypoxia, and cell viability was detected [32 (link)]. Approximately 5 × 105 BMSCs suspended in L-DMEM were plated in 150 mm-diameter culture dishes. The cells were then separately cultured under the conditions below for 12, 24, 48, 72, 96, or 120 h: 10% exosome-free FBS with hypoxia (94% N2, 5% CO2, and 1% O2 gas mixture). BMSC viability was analyzed with CCK-8 assays. Briefly, adherent cells were digested with 0.05% trypsin and collected for CCK-8 assay according to the manufacturer's instructions.