Tissue-like Culture Of Rat Hepatocytes For Research On Drugs

Primary cultures of hepatocytes have been extensively used for evaluation of drug metabolism, drug-drug interactions and drug hepatotoxicities. However, traditional monolayer culture of hepatocytes cannot maintain liver-specific functions and cytochrome P450 activities for a long time, which limited its application in drug testing in vitro. In this paper, we miniaturized the system of the bioartificial liver and further evaluated its feasibility for researches on drug metabolism, drug-drug interactions and drug hepatotoxicities. Because cryopreservation of hepatocytes is urgently required for application of hepatocytes in studies in vitro, we managed to develop an easy-handling methodology for cryopreservation of hepatocytes, and then evaluate the corresponding live-specific functions of cryopreserved hepatocytes. Therefore, this thesis mainly focused on the following four sections:In section 1, drug metabolism was investigated utilizing rat hepatocytes by gel-entrapment culture. The metabolisms of phenacetin, p-nitrophenol and 7-hydroxycoumarin were individually examined in regarding to the activities of CYP1A2, CYP2E1 and phase II enzymes (UDP-glucuronosyl-transferase and sulfotransferase), respectively. It was demonstrated that the activity of phase I and II enzymes decreased quickly in monolayer culture and these parameters in gel-entrapment culture maintained 1.5-fold higher than those in monolayer culture for 7 days. These results indicated that gel entrapped rat hepatocytes should be an appropriate model in vitro for drug metabolism research.In section 2, we focused on drug-drug interactions on rat hepatocytes in both gel-entrapment and monolayer cultures. Firstly, 3 -naphthoflavone (50 μmol/L) was used as an inducer to investigate its inducibiiity on CYP1A2 and phase II enzymes (UDP-glucuronosyl-transferase and sulfotransferase). After exposure to β -naphthoflavone, the activities of CYP1A2 increased by about 2.5-fold in both cultures, while the activities of phase II enzymes didnot increase so significantly. Secondly, diethyldithiocarbamate and cimetidine, two potent inhibitors of CYP2E1, possessed their remarkable inhibitions on CYP2E1 activities in both cultures. The results above presented that gel-entrapment culture of rat hepatocytes might serve as a selective model for investigation on drug-drug interactions.In section 3, the feasibility of applying gel-entrapped rat hepatocytes for hepatotoxicities studies was estimated utilizing sodium salicylate and methotrexate as two hepatotoxin models. The results showed that rat hepatocytes in gel-entrapment culture and monolayer culture were both susceptible to sodium salicylate toxicity. However, rat hepatocytes in gel-entrapment culture were less sensitive to hepatotoxicity of methotrexate than those in monolayer culture, which may result from quick detoxification processes in gel entrapped hepatocytes by their maintenance of metabolizing enzymes activities and liver-specific functions.In section 4, we discussed a simple method for hepatocytes cryopreservation and the liver-specific functions of cryopreserved hepatocytes. Viability of cryopreserved hepatocytes employed our easy-handling methodology decreased by about 15% compared to fresh isolated hepatocytes. Then, the activities of cytochrome P450, albumin secretion and sensitivity to hepatotoxic drug (2.5 mmol/L sodium salicylate) of cryopreserved hepatocytes were measured. The results indicated that there were no significant difference between cryopreserved hepatocytes and freshly harvested hepatocytes on both the activities of cytochrome P450 (CYP1A2 and CYP2E1) and sensitivity to sodium salicylate hepatotoxicity while albumin secretion slightly decreased to a relative level of 87% compared with fresh hepatocytes.