Establishing Immortalized Hepatocytes By Hybridoma Technique For Drug Metabolism Study

Background: Primary hepatocytes contain almost all of drug-metabolizing enzyme and cofactors in the liver, whose metabolic ability resemble with the hepatocytes in vivo and become a classic modal for in vitro drug metabolism study constantly. But, the isolation and culture of primary hepatocytes in vitro is elaborate and the metabolic activity keeps too short, which causes the high cost and the failure in flexibility of hepatocyte application. So, it is the urgent request of drug metabolism study area to establish ideal substitute models of primary hepatocytes.Objective: To select suitable tumor cells derived from proper species and tissue, and fuse them with primary hepatocytes to establish hepat-hybridoma cells of contain both drug metabolism activities and the infinite reproductive activity, which could be utilized in vitro for long-term.Contents: To isolate, culture and cryopreserve of rat primary hepatocytes with good metabolic activities; to select and mutate tumor cells of different species and tissues to investigate which would be the proper parent tumor cell of fusion; using hybridoma Technique to make the primary hepatocyte and the mutated tumor cells fused to generate immortalized hepat-hybridoma cells which might contain good metabolic activities and consistent with the requirement of pharmacokinetic study; to identify the bio-function and evaluate the application value of the hepat-hybridoma cells in drug metabolism study.Methods: Rat hepatocytes were isolated by a improved two-step collagenase perfusion; Using chemical mutagen method, cell clone culture, and HAT filter to obtain suitable tumor cells as parent cells in cell fusion; Cell fusion were conducted by PEG hybridoma technique; Morphology, HAT screening, chromosome karyotype analysis, double-fluorescent labelled confocal laser scanning analysis and typical biochemical indicator determination such as glycogen, albumin, urea were used to evaluate cell functions. The metabolic activity evaluation of hepat-hybridoma cells were conducted by HPLC and LC-MS technique. Results: An improved method of isolating and culturing of hepatocytes was established. The primary hepatocytes obtained kept the abilities to excrete glycogen, albumin, and urea and contained good cytochrome P450 activities; The mutant HGPRT~-type of SMMC-7721 hepatoma carcinoma cell line were established, and the 3 filted tumor cell lines including the HGPRT~- type of SMMC-7721, H4TG and SP2/0 cells, were fused with primary hepatocytes respectively, and the obtained hybridoma cells were different in the culture character.The obtained hepat-7721 and hepat-H4TG hybridoma cells contained drug metabolic activities both. And hepat-H4TG hybridoma cells represented fine stability in morphology and biosynthesis ability; it had been cultured for more than 5 months and passaged for 25 times, keeping the activities of typical cytochrome P450 isozymes. After the purification period, the 5th passage of hybridoma cells contained about 40-50% metabolic activities compared with primary hepatocytes, activities of CYP2C, CYP3A of the 10th passage cells became decreased while CYP1A,CYP2D remained the similar levels through the culture period.Conclusions: Fusion results of the three different tumor cells indicated that hepatoma carcinoma cells was better than tumor cells of other tissues, tumor cells of the same species was better than that derived from other species, when fused with primary hepatocytes. It was to say that hepatoma carcinoma cells derived from the same species of primary hepatocytes would be the suitable parent cells for the fusion.According to the above results, to some degree, the hepat-H4TG hybridoma cells may become a useful substitute model of primary hapatocytes for a given time period, at least. But study further should be done in the future to investigate the stability maintenance of metabolic activities which some influential factor unknown may be involved.