This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and cross-talk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.

In contrast to microsomes, hepatocytes give a disproportionate prediction with increasing clearance suggesting limitations either at the active site, such as cofactor exhaustion, or with intracellular concentration equilibrium, such as rate-limiting cell permeability. A simple log linear empirical relationship can be used to correct hepatocyte predictions.

Although prediction of clearance using hepatocytes and liver microsomes has long played a decisive role in drug discovery, it is widely acknowledged that reliably accurate prediction is not yet achievable despite the predominance of hepatically cleared drugs. Physiologically mechanistic methodology tends to underpredict clearance by several fold, and empirical correction of this bias is confounded by imprecision across drugs. Understanding the causes of prediction uncertainty has been slow, possibly reflecting poor resolution of variables associated with donor source and experimental methods, particularly for the human situation. It has been reported that among published human hepatocyte predictions there was a tendency for underprediction to increase with increasing in vivo intrinsic clearance, suggesting an inherent limitation using this particular system. This implied an artifactual rate limitation in vitro, although preparative effects on cell stability and performance were not yet resolved from assay design limitations. Here, to resolve these issues further, we present an up-to-date and comprehensive examination of predictions from published rat as well as human studies (where = 128 and 101 hepatocytes and = 71 and 83 microsomes, respectively) to assess system performance more independently. We report a clear trend of increasing underprediction with increasing in vivo intrinsic clearance, which is similar both between species and between in vitro systems. Hence, prior concerns arising specifically from human in vitro systems may be unfounded and the focus of investigation in the future should be to minimize the potential in vitro assay limitations common to whole cells and subcellular fractions.