Hepatocytes drug-metabolizing system

Maurel P. The use of adult human hepatocytes in primary culture and other in vitro systems to investigate drug metabolism in man. Adv Drug Dev Rev 1996 22 105-132. 

Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as metabolizing system in mutagenicity assays (Hengstler 2000), providing qualitative metabolic information and quantitative pharmacokinetic parameters from key animal species and human at the early stage of drug discovery and drug development. 

Hepatocytes growing in microcarrier systems are used for the study of liver failure and drug metabolism. Transplantation experiments of hepatocytes grown on microcarriers showed detoxification of ammonium and reduction of bilirubin concentrations after induced acute liver failure (Nagaki et aL, 1990). Furthermore, co-cultivation assays using hepatocytes and Balb/c 3T3 fibroblasts as target cells have been applied to analyse the metabolism-mediated toxicity of xenobiotics in vitro (Voss Seibert, 1992). 

The objective of this study is to identify the major metabolites of the drug in question. For this study, the drug in question is incubated with an appropriate in vitro metabolic system to allow the formation of metabolites. Metabolites are then identified using analytical chemical approaches. The in vitro experimental system of choice is human hepatocytes, with high-performance liquid chromatography/mass spectrometry (HPLC/MS) or tandem mass spectrometry (HPLC/MS/MS) as the most convenient analytical tool to identify the metabolites. 

Hepatocytes cultivated between two layers of soft gel collagen represent the most frequently used hepatocyte in vitro system. They establish an apical pole between the cells which contains bile canaliculi (Fig. 3). The hepatocyte membrane facing the collagen gel corresponds to the basolateral side. Therefore, hepatocyte sandwich cultures represent the easiest to handle 3D culture system, although only one sheet of hepatocytes is represented. The hepatocyte phenotype in sandwich culture is characterized by (1) maintenance of susceptibility to apoptosis, (2) a delayed decrease of drug-metabolizing activities compared to monolayer cultures, (3) establishment and maintenance of bile canaliculi, and (4) a resting cell state where stimulation by HGF and EGF induces almost no proliferation events. As previously mentioned, this cultivation system effectively prevents the spontaneous activation of ERK and Akt which occurs in 2D systems [13]. Consistent with the effects of small chemical inhibitors in 2D cultures, expression of a constitutively active form of Ras in sandwich-cultured hepatocytes induces features of EMT and stress fibers. In contrast to Ras, expression of constitutive active Akt in hepatocytes induces an antiapoptotic phenotype and does not cause EMT [13]. 

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