Primary culture of human hepatocytes is an in vitro model widely used to investigate numerous aspects of liver physiology and pathology. The technique used to isolate human hepatocytes is based on two-step collagenase perfusion. Originally performed in situ for obtaining hepatocytes from the adult rat, this technique has been adapted to the ex vivo treatment of human liver from organ donors or from lobectomy resection for medical purposes. This chapter describes experimental protocols for the isolation of hepatocytes from human liver tissue and for the preparation of short- and long-term cultures in which cells retain a differentiated phenotype for at least 1 mo. The various aspects emphasized here include the conditions for obtaining tissue, quality control of tissue for efficient perfusion, collagenase perfusion parameters, solutions for perfusion and culture media, cell substrate, cell plating, specific equipment, and safety conditions.

The wingless-type MMTV integration site family (WNT)/b-catenin/ adenomatous polyposis coli (CTNNB1/APC) pathway has been identified as a regulator of drug-metabolizing enzymes in the rodent liver. Conversely, little is known about the role of this pathway in drug metabolism regulation in human liver. Primary human hepatocytes (PHHs), which are the most physiologically relevant culture system to study drug metabolism in vitro, were used to investigate this issue. This study assessed the link between cytochrome P450 expression and WNT/b-catenin pathway activity in PHHs by modulating its activity with recombinant mouse Wnt3a (the canonical activator), inhibitors of glycogen synthase kinase 3b, and small-interfering RNA to invalidate CTNNB1 or its repressor APC, used separately or in combination. We found that the WNT/ b-catenin pathway can be activated in PHHs, as assessed by universal b-catenin target gene expression, leucine-rich repeat containing G protein-coupled receptor 5. Moreover, WNT/ b-catenin pathway activation induces the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor, but not of CYP3A4, hepatocyte nuclear factor-4a, or pregnane X receptor (PXR) in PHHs. Specifically, we show for the first time that CYP2E1 is transcriptionally regulated by the WNT/b-catenin pathway. Moreover, CYP2E1 induction was accompanied by an increase in its metabolic activity, as indicated by the increased production of 6-OH-chlorzoxazone and by glutathione depletion after incubation with high doses of acetaminophen. In conclusion, the WNT/b-catenin pathway is functional in PHHs, and its induction in PHHs represents a powerful tool to evaluate the hepatotoxicity of drugs that are metabolized by CYP2E1.