An improved method for isolation and culture of primary cardiomyocytes from SD neonatal rats

Abstract

Objective: To establish a stable, rapid and improved method for isolation and culture of primary cardiomyocytes from neonatal rats. Methods: Ventricular tissues from neonatal SD rats were digested with 0.12% collagenase Ⅱ, and then subjected to Percoll density gradient centrifugation. The original cardiomyocytes were cultured in modified DMEM/F12 containing 5% horse serum and 5-bromodeoxyuracil(5-BrdU) in vitro for further purification, and medium was changed to normal high glucose DMEM with 10% FBS the next day. The difference between the improved method and traditional differential attachment one used for isolation and culture of primary cardiomyocytes was compared. Results: Cardiacmyocytes obtained through the improved method grew well. 24 hours after plating, most cells adhered to the dishes, with shapes looked triangular, fusiform or irregular, and some of them showed spontaneously contract at a frequency varying from 10～30 times/min. After 48 h culture, the cardiomyocytes became longer and stretched out pseudopodia. Some cells showed synchronous beats with the frequency close to 50～80 times/min. 72 hours later, cardiomyocytes were interwoven into a network in chrysanthemum patterns, and spontaneous beats tended to be more synchronous, with a frequency of 80-100 times/min. After 96 h, cells gathered into clusters as islands, with synchronous beat at a frequency of around 100～120 times/min. All cardiomyocytes were in good condition within one week. Yields((1.17±0.15)×106 vs (1.21±0.22)×106,P＞0.05)and survival rate of primary cardiomyocytes obtained by the improved method was comparable to that gained using traditional differential attachment way (93.3%±1.4% vs 92.2%±0.7%, P＞0.05), but the purity of primary cardiomyocytes obtained through the improved method was much higher (94.7%±2.1% vs 89.5%±1.3%, P＜0.05), while with less time consuming ((3.1±0.4)h vs (4.3±0.3)h, P＜0.01). Conclusion: This improved method is an ideal and simple method for the isolation and culture of primary cardiomyocytes with shorter time-consuming, high purity, intact structure and function, and with great repeatability and stability.