Authors: Fan Yanhong He Chenghua Liu Guofang Zhang Haibin
Publish Date: 2008/10/18
Volume: 58, Issue: 2, Pages: 85-92
Abstract
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 twostep collagenase perfusion and pancreatin digestion The hepatocytes or parenchymal cells could be separated from cell debris and from nonparenchymal cells by lowspeed centrifugation Percoll gradient centrifugation The harvested hepatocytes were suspended in DMEM M199 cultured in 5 CO2 or L15 cultured without 5 CO2 medium then cultured at 17 27 or 37 °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 27 × 108 per g liver weight and the viability was 984 and the best medium was M199 cultured in 5 CO2 or L15 cultured without 5 CO2 The optimum culture temperature was 27 °C The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition
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