Isolation and primary culture of hepatocytes from Ctenopharyngodon idellus
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KeyWord:Ctenopharyngodon idellus; Hepatocyte; Primary culture
秦洁 上海海洋大学水产与生命学院 上海海洋大学省部共建水产种质资源发掘与利用教育部重点实验室,上海 ;苏州大学基础医学与生物科学学院 江苏省水产动物营养重点实验室,江苏
叶元土 苏州大学基础医学与生物科学学院 江苏省水产动物营养重点实验室,江苏
冷向军 上海海洋大学水产与生命学院 上海海洋大学省部共建水产种质资源发掘与利用教育部重点实验室,上海
蔡春芳 苏州大学基础医学与生物科学学院 江苏省水产动物营养重点实验室,江苏
宋亮 苏州大学基础医学与生物科学学院 江苏省水产动物营养重点实验室,江苏
许凡 苏州大学基础医学与生物科学学院 江苏省水产动物营养重点实验室,江苏
张宝彤 北京市营养源研究所 系统营养工程技术研究中心—水产动物系统营养研究开放实验室,北京
萧培珍 北京市营养源研究所 系统营养工程技术研究中心—水产动物系统营养研究开放实验室,北京
张波 北京市营养源研究所 系统营养工程技术研究中心—水产动物系统营养研究开放实验室,北京
王丽宏 北京市营养源研究所 系统营养工程技术研究中心—水产动物系统营养研究开放实验室,北京
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      ObjectiveTo explore the optimal methods of isolation and culture of hepatocytes from grass carp and serve the studies on nutritional metabolism and injury mechanisms in carp hepatocytes. MethodsProliferation of hepatocytes was tested by MTT assay. Function of THE hepatocytes was examined by measuring the levels of lactic acid dehydrogenase (LDH), albumin (ALB), and urea nitrogen (BUN) in the supernatant at different times, respectively. Results Using 0.25% warm trypsin digestion for 20 minutes each time, and to collect the hepatocytes step by step, the results of trypan blue test and blood cell counting chamber method showed that more than 99% of viable hepatocytes were obtained. ConclusionThe cells were cultivated in M199 medium supplemented with 10% fetal bovine serum and 10μg/mL insulin, at 7×106 cell/mL concentration under the temperature of 28℃, 4.5% CO2 for a long term. Primary culture of carp hepatocytes can be successfully obtained by the following procedure:cells cultured in M199 medium supplemented with 10% fetal bovine serum and 10 μg/mL insulin, at 7×106 cell/mL concentration, under the temperature of 27℃ and 4.5% CO2.
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