College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.
Cytotechnology. 2008 Oct;58(2):85-92. doi: 10.1007/s10616-008-9169-5. Epub 2008 Oct 18.
The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion, and pancreatin digestion. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll gradient centrifugation). The harvested hepatocytes were suspended in DMEM, M199 (cultured in 5% CO(2)), or L-15 (cultured without 5% CO(2)) medium then cultured at 17, 27, or 37 degrees C. Cell yield was counted by use of a hemocytometer, and the viability of the cells was assessed by use of the Trypan blue exclusion test. Results from these studies showed that the best method of isolation was pancreatin digestion (the cell yield was 2.7 x 10(8) per g (liver weight) and the viability was 98.4%) and the best medium was M199 (cultured in 5% CO(2)) or L-15 (cultured without 5% CO(2)). The optimum culture temperature was 27 degrees C. The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition.
水生环境受到众多化学污染物的影响。因此，人们越来越需要识别这些化学物质，并评估它们对水生生物的潜在毒性。在这项研究中，我们优化了鲤鱼原代肝细胞培养技术，将其作为水生环境中外源化学物质潜在危害生态毒理学评价的辅助模型之一。本研究采用机械分离、两步胶原酶灌注和胰蛋白酶消化法从鲤鱼肝脏中分离原代肝细胞。通过低速离心（Percoll 梯度离心），可将肝细胞或实质细胞与细胞碎片和非实质细胞分离。收获的肝细胞悬浮于 DMEM、M199（在 5%CO2 中培养）或 L-15（无 5%CO2 培养）培养基中，然后在 17、27 或 37℃下培养。使用血球计数器计数细胞产量，使用台盼蓝排斥试验评估细胞活力。这些研究结果表明，最佳的分离方法是胰蛋白酶消化（细胞产量为 2.7×108 个/克（肝重），活力为 98.4%），最佳的培养基是 M199（在 5%CO2 中培养）或 L-15（无 5%CO2 培养）。最佳培养温度为 27℃。在这种条件下，鲤鱼原代肝细胞培养生长良好，满足大多数毒理学实验的要求。
