Rat neonatal cardiomyocyte isolation and culture

Neonatal rat cardiomyocytes were isolated from neonatal male (1-3 days, 5-7 g, n=700) SD rats as previously described (24); rats were purchased from the Experimental Animal Centre of Xuzhou Medical University. The neonatal male SD rats were housed in the Experimental Animal Centre of Xuzhou Medical University and housed in a controlled environment (temperature, 20-25°C; humidity, 50-60%). The neonatal rats and their mother were housed one cage and were maintained at room temperature under a 12 h light/dark cycle. The neonatal rats were provided breast milk and the mother rat was provided free access to sterile food and water. The neonatal rats were anesthetized with sodium pentobarbital and sacrificed by decapitation. Their hearts were rapidly removed into dishes of ice, and the vessels and atria were discarded. The ventricles were then dissected and minced into 1 mm³ pieces, and washed in PBS. The minced tissue was digested in a PBS with 1 mg/ml trypsin, 1 mg/ml collagenase type II, and 0.2 mg/ml glucose for 5 min at 37°C and incubated with 0.1 mmol/l BrdU to selectively enrich for cardiomyocytes by inhibiting the growth of cardiac fibroblasts. The cardiomyocytes were then purified using the differential adhesion method. The cardiomyocytes were evaluated by indirect immunofluorescence staining with α-SCA antibody. The isolated cardiomyocytes were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) with 4.5 g/l glucose and 10% fetal bovine serum at 37°C. OGD was completed by culturing the cardiomyocytes in a glucose-free DMEM (Gibco; Thermo Fisher Scientific, Inc.) without FBS and a tri-gas incubator (Heal Force, Shanghai, China) with 1% O₂/5% CO₂/94% N₂ at 37°C. OGD/R was performed as previously described (25). Following the OGD procedure, the medium was replaced with DMEM with 4.5 g/l glucose, and then transferred into a humidified normoxic atmosphere at 37°C. The cardiomyocytes were treated with or without 10 nM PEDF 1 h prior to ischemia or reperfusion. The following experimental groups were included: Normal group, OGD group (control, OGD 0.5 h), OGD + PEDF group, OGD/R group (control, OGD 0.5 h and recovery 6 h), OGD/R + PEDF group.