Fibroblast isolation and culture

The rats were euthanized by intraperitoneal injection of sodium pentobarbital (150 mg/kg). Fibroblasts were isolated from three tissue types: Skin (ear), muscle (hind limb), and heart (ventricle). Tissues of the same type from the three rats were mixed together. The tissues were cut, washed in phosphate-buffered saline (PBS), and minced in a 250 μL drop of 0.025% collagenase type I (Sigma-Aldrich, MO, USA) in a 100 cm tissue culture plate. The small pieces of tissue were transferred into a 50 mL sterile conical tube (Falcon^®, Corning, NY, USA) containing 2.5 mL of 0.025% collagenase type I and incubated at 37°C for 20 min for the skin tissue, 35 min for the muscle tissue, and 50 min for the heart tissue. Then, 5 mL of 0.25% trypsin-EDTA (ThermoFisher Scientific, MA, USA) were added and incubated at 37°C for 10 min. Thereafter, 5 mL of PBS was added, and the tissues were vortexed briefly before being centrifuged at 1000×g for 5 min. Then, the sediment was resuspended with 5 mL of culture media containing Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (Thermo Fisher Scientific, MA, USA), 10% fetal bovine serum (FBS) (Sigma-Aldrich, MO, USA), and antibiotic-antimycotic solution (Thermo Fisher Scientific, MA, USA). The cells were introduced into a 10-cm cell culture dish and incubated for 2 days at 37°C in a 5% CO₂ incubator (Heal Force, Shanghai, China).