Isolation of Primary Neonatal Rat Ventricular Cardiomyocytes

Primary neonatal rat ventricular cardiomyocytes (NRVMs) were isolated from 1-day to 2-day old Sprague-Dawley rat pups (Envigo, #002 timed mated), cultured on gelatin-coated dishes^{19 (link),64 (link)}, and maintained in M199 medium (ThermoFisher Scientific, #SH30253FS) supplemented with 2% bovine growth serum (ThermoFisher Scientific, #SH3054103) and 1× penicillin-streptomycin (Cellgro, #30-0002-CI) at 37 °C in 5% CO₂. For the isolation procedure, neonatal hearts were collected, the atria were removed, and the ventricles were cut up in HBSS prior to enzymatic digestion. The ventricular tissue was subjected to 5 rounds of enzymatic digestion using 0.05% pancreatin (Sigma) and 84 units/ml of collagenase (Worthington, Lakewood, NJ). Cells were collected by centrifugation at 500×g for 5 min at 4 °C and resuspended in M199 medium. The cells were then differentially plated for 1 h on culture dishes to reduce fibroblasts. The cardiomyocytes were then plated on gelatinized cell culture dishes.