改良的原代心肌细胞培养方法

An improved method for primary culture of cardiac myocytes

目的:探索原代心肌细胞培养的条件和方法,建立一套高效快捷的原代心肌细胞培养方案,为组织工程心血管相关研究提供心肌细胞来源。方法:实验于2006-04/07在南京大学生命科学院完成。出生2d内的SD大鼠8只,切取心肌组织,将其反复剪成约1mm3的块状,加入4mL消化液(0.8g/L的胰酶+2×105U/L胶原酶),然后收集细胞放在DMEM(pH7.2)培养基进行培养,以4×108L-1密度接种于培养瓶中,并置于37℃,体积分数为0.05的CO2培养箱中。用4g/L台盼蓝染色检测心肌细胞的存活率,不着色者为存活细胞,并观察心肌细胞的形态学变化。结果:共计数800个细胞,其中42个染色阳性,成活率为95.89%。细胞培养未贴壁时心肌细胞呈圆形;24h后心肌细胞贴壁生长,细胞伸出伪足呈梭形、多角形;3～4d后形成细胞簇,并可出现同簇细胞的同步搏动,最后心肌细胞连接成片生长。结论:该心肌细胞培养方法存活率较高,是一种可靠的的心肌细胞培养方法。

AIM： To explore the condition and approach for the culture of primary cardiac myocyte, and establish an effective method for cardiac myocyte culture, so as to provide cell resource for cardiovascular related study in tissue engineering. METHODS： The experiment was conducted in the College of Life Science, Nanjing University from April to July 2006. Cardiac myocytes were harvested from 8 Sprague-Dawley rats born for 2 days, and cut into pieces of 1 mm^3 and digested with 4 mL digestive juice （0.8 g/L pancreatic enzyme and 2×10^5 U/L collagenase）. Then the cells were cultured in DMEM （pH=7.2） and seeded in culture flask at the density of 4×10^8 L^-1, which was put into the incubator containing 0.05 volume fraction CO2 at 37 ℃. The survival rate of cardiac myocytes was detected by 4 g/L trypan-blue staining. Those without staining were survival cells, and the morphological changes of cardiac myocytes were observed. RESULTS： The cell count was 800 including 42 staining positive ones with the survival rate of 95.89%. The cells were round before adhesion; 24 hours later, the cells of fusiform and polygon began to adhere and stretch out the parapodium; 3-4 days later, cell clusters formed, and the pulsation of cardiac myocytes was observed; finally, the cells grew together flakily. CONCLUSION： This method is an effective one to cultivate cardiac myocyte with high survival rate.