C57胎鼠、乳鼠及成年小鼠心室肌细胞分离、培养及鉴定

背景：培养的心肌细胞被广泛应用于心肌细胞的生理特性、毒性实验、基因工程、疾病模型和药物筛选等方面的研究。获得纯度较高活性良好的品系小鼠心肌细胞是研究的关键前提。目的：分离和培养C57小鼠胎鼠、乳鼠及成年小鼠心室肌细胞。方法：应用机械切碎心室肌后，胰蛋白酶消化不同发育阶段的C57小鼠心室肌细胞，差速贴壁1 h纯化心室肌细胞，锥虫蓝染色判定心肌细胞活力，体外分别培养48~72 h后分别行倒置显微镜、扫描及透射电镜观察细胞形态，微电极阵列评价细胞电生理指标，免疫组化鉴定。结果与结论：经3~6次消化后，心室组织消化完全，即刻细胞存活率大于85%。倒置显微镜下观察，细胞呈梭形、多角形。12 h有少部分细胞搏动，48 h细胞交织成网，搏动呈同步性，搏动频率30~90次/min。说明用胰蛋白酶组织消化法可以成功地分离、培养，并获得形态、活力良好的胎鼠、乳鼠及成年C57小鼠心室肌细胞。

Isolation,culture and identification of ventricular cardiomyocytes from embryonal,neonate and adult C57 mice

BACKGROUND:Cultured cardiomyocytes are widely utilized in related researches such as physiological and toxicologic experiments, gene engineering, diseases model, drug screening and so on. How to harvest mouse cardiomyocytes with high activity is a key premise for these studies.  OBJECTIVE:To establish a method to isolate and culture ventricular cardiomyocytes from embryonal, neonate and adult C57 mice.METHODS:The ventricular myocardium from fetal, neonatal and adult C57 mice were minced and digested in trypsin, the purified ventricular cardiocytes were obtained by differential adhesion about 1 hour. The survival rate was assessed by trypan staining. Morphology of ventricular cardiomyocytes was observed through the methods of inverted phase contrast microscope, transmission electron microscope, scanning electron microscope, and the electrophysiological properties were identified through immunocytochemistry and microelectrode array.RESULTS AND CONCLUSION:The ventricular tissues were almost completely digested through 3-6 times. Trypan staining showed that the survival rate of the cardiocytes cultured was more than 85%. Under the inverted phase contrast microscope, we found that the cells were fusiform or polyhedron and connected to each other and some cells began beat after 12 hours. The cells monolayer formed a network and beat spontaneously at the 30-90 times per minutes. The ventricular cardiomyocytes from embryonal, neonate and adult C57 mice can be obtained with well morphology and spontaneous beating with trypsin digestion.