Lung single-cell suspensions were prepared from the pooled lungs of at least 5 C57BL/6 mice (4–6 weeks old). In brief, mice were sacrificed by cervical dislocation. The lung parenchyma from the mice was digested by fine mincing with a razor blade, followed by incubation in an enzyme mixture containing 0.2% collagenase I (Sigma, USA), 2.4 U/mL dispase (Sigma, USA) and 0.001% DNase (Sigma, USA) for 1 h at 37 °C with shaking. This suspension was filtered through 100-μm and 40-μm filters, centrifuged, and depleted of red blood cells by lysis. Cells were resuspended in PBS at 1 × 107 cells/mL and stained for stem cell antigen (Sca)-1, CD45 and CD31 followed by sorting using the AutoMACS cell separator system (Miltenyi Biotec, Bergisch Gladbach, Germany).
Freshly isolated LR-MSCs were cultured at a concentration higher than 105 cells/mL with DMEM containing 15% fetal bovine serum, 4%l -glutamine, 1% nonessential amino acids, and 1% penicillin and streptomycin, and maintained in a humidified atmosphere of 95% air, 5%CO2 at 37 °C. The cells were passaged 1:2 using 0.25% trypsin when they reached 70–90% confluence.
Freshly isolated LR-MSCs were cultured at a concentration higher than 105 cells/mL with DMEM containing 15% fetal bovine serum, 4%