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

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)×106vs (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.目的:建立一种稳定、快速的SD新生乳鼠原代心肌细胞分离培养改良方法。 方法:取SD新生乳鼠心室,0.12%Ⅱ型胶原酶消化,Percoll密度梯度离心结合5-溴脱氧尿嘧啶(5-BrdU)化学抑制法纯化心肌细胞,体外培养于含5%马血清的改良DMEM/F12中,次日更换为普通含10%胎牛血清的高糖DMEM继续培养,并比较此改良方法与传统差速贴壁法的差异。 结果:改良法获得的心肌细胞生长良好,接种24 h 后几乎全部贴壁生长,细胞呈三角形、梭形或不规则形,个别细胞出现自主搏动,频率为10～30 beats/min不等。48 h后心肌细胞变长伸出伪足,部分细胞呈现同步搏动, 频率接近50～80 beats/min。72 h后心肌细胞成菊花样交织成网,自发搏动趋于同步,频率加快至80～100 beats/min;96 h后细胞聚集成簇,呈岛屿样,同步搏动频率在100～120 beats/min左右,一周内细胞状态良好。改良法纯化原代心肌细胞的得率((1.17±0.15)×106vs (1.21±0.22)×106,P＞0.05)和存活率与传统差速贴壁法相当(93.3%±1.4% vs 92.2%±0.7%, P＞0.05 ),但是改良方法获得的原代心肌细胞纯度更高 (94.7%±2.1% vs 89.5%±1.3%, P＜0.05),且用时较短((3.1±0.4)h vs (4.3±0.3)h, P＜0.01)。 结论:改良法获得心肌细胞耗时短、纯度高、结构功能保存完整,且实验重复和稳定性好,是一种理想且简单易行的的原代心肌细胞分离培养方法。.