大口黑鲈肝细胞原代培养方法的建立

Study on primary culture of hepatocytes from Micropterus salmoides

取材大口黑鲈肝脏组织,培养其原代细胞,比较研究了组织块法和酶消化法两种培养方法及不同的培养条件,以确定其最佳培养方法和条件,同时观察了长期培养过程中肝细胞的形态变化。结果表明:组织块方法不适于大口黑鲈肝细胞的培养,未见细胞从组织块中迁出。而胰蛋白酶消化法获得良好稳定的培养效果。胰蛋白酶浓度0.25%,消化时间20 min,分步收集肝细胞,经台盼蓝检测和血球计数板计数,平均活力>90%,每克肝重可获得3×106个分离肝细胞。在细胞活力和数量两方面达到最佳平衡。在含20%胎牛血清、10μg/mL胰岛素的M199/L15培养基中,于4%CO2,28℃培养箱中长期培养并传代。

In this study, the liver tissues from Micropterus salmoides were cultivated under different culture conditions with tissue culture and enzymatic dissociation methods respectively, aiming at comparing the influence of culture methods and conditions on the growth of cells and figuring out the optimal. The morphological changes of cells in longterm culture were also observed. It came to the conclusion that trypsin dissociation method was better than tissue culture. Applying the enzymatic dissociation method and controlling the trypsin acting on cells at the concentration of 0.25% for 20 min, the hepatocyte yield of 3 ×10^6 cells per 1 g liver weight had been achieved with initial viabilities routinely exceeding 90%. The cells were cultivated in M199/L15 basal medium supplemented with 20% foetal bovine serum and 10 μg/mL insulin at the temperature of 28℃, the concentration of CO2 being 4% for long term and were sub-cultured.