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.

Several lines of evidence suggest that GATA6 has an integral role in controlling development of the mammalian liver. Unfortunately, this proposal has been impossible to address directly because mouse embryos lacking GATA6 die during gastrulation. Here we show that the early embryonic deficiency associated with GATA6-knockout mice can be overcome by providing GATA6-null embryos with a wild-type extraembryonic endoderm with the use of tetraploid embryo complementation. Analysis of rescued Gata6 ؊/؊ embryos revealed that, although hepatic specification occurs normally, the specified cells fail to differentiate and the liver bud does not expand. Although GATA6 is expressed in multiple tissues that impact development of the liver, including the heart, septum transversum mesenchyme, and vasculature, all are relatively unaffected by loss of GATA6, which is consistent with a cell-autonomous requirement for GATA6 during hepatogenesis. We also demonstrate that a closely related GATA factor, GATA4, is expressed transiently in the prehepatic endoderm during hepatic specification and then lost during expansion of the hepatic primordium. Our data support the proposal that GATA4 and GATA6 are functionally redundant during hepatic specification but that GATA6 alone is available for liver bud growth and commitment of the endoderm to a hepatic cell fate.

The culture of primary hepatocytes as spheroids creates an efficient three-dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell-cell adhesion, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer nonadherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of more than 80% of 242 liver-related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. N ovel systems are needed to facilitate short-term and long-term culture of hepatocytes for diagnostic, discovery, and therapeutic applications. 1 Traditional monolayer culture of primary hepatocytes on tissue culture plastic is problematic and has been associated with a rapid loss of differentiated function. 2 Although single-layer and double-layer surfaces of collagen or other biomatrix materials are associated with improved differentiated functions in vitro, 3 biological surfaces can pose manufacturing hurdles and do not support high cell density culture of primary hepatocytes (exceeding 1 ϫ 10 7 cell/mL). In contrast, spheroids, which are nonadherent multicell aggregates of greater than 40 m diameter, provide a three-dimensional tissue construct which form spontaneously and allow suspension culture of primary hepatocytes at high cell density under oxygenated bioreactor conditions. 4 Spheroid formation allows recapitulation of the cuboidal geometry of primary hepatocytes with relatively stable long-term differentiated function. 5,6 Reports of structural polarity and bile canaliculi formation by primary rat hepatocytes in spheroid aggregates provide further evidence that hepatic spheroids mimic the hepatocellular microanatomy of the liver. 7 Original observations of tissue-like aggregate formation from isolated cells was reported by Moscona in 1961, using fetal liver cells and a rotational technique. 8 The descriptive term "spheroid" was coined years later by Landry in 1985 when multicellular aggregates were formed from isolated rat hepatocytes after 3-5 days of culture on nonadherent plastic surfaces. 9 Later, spheroids

Loss of the nuclear hormone receptor hepatocyte nuclear factor 4␣ (HNF4␣) in hepatocytes results in a complex pleiotropic phenotype that includes a block in hepatocyte differentiation and a severe disruption to liver function. Recent analyses have shown that hepatic gene expression is severely affected by the absence of HNF4␣, with expression of 567 genes reduced by >2.5-fold (P < 0.05) in Hnf4␣ ؊/؊ fetal livers. Although many of these genes are direct targets, HNF4␣ has also been shown to regulate expression of other liver transcription factors, and this raises the possibility that the dependence on HNF4␣ for normal expression of some genes may be indirect. We postulated that the identification of transcription factors whose expression is regulated by HNF4␣ might reveal roles for HNF4␣ in controlling hepatic functions that were not previously appreciated. Here we identify cyclic adenosine monophosphate responsive element binding protein H (CrebH) as a transcription factor whose messenger RNA can be identified in both the embryonic mouse liver and adult mouse liver and whose expression is dependent on HNF4␣. Analyses of genomic DNA revealed an HNF4␣ binding site upstream of the CrebH coding sequence that was occupied by HNF4␣ in fetal livers and facilitated transcriptional activation of a reporter gene in transient transfection analyses. Although CrebH is highly expressed during hepatogenesis, CrebH ؊/؊ mice were viable and healthy and displayed no overt defects in liver formation. However, upon treatment with tunicamycin, which induces an endoplasmic reticulum (ER)-stress response, CrebH ؊/؊ mice displayed reduced expression of acute phase response proteins. Conclusion: These data implicate HNF4␣ in having a role in controlling the acute phase response of the liver induced by ER stress by regulating expression of CrebH. (HEPATOLOGY 2008;48: 1242-1250

Purpose To investigate the correlation between MRE assessed spleen stiffness and direct portal vein pressure gradient (D-HVPG) measurements in a large animal model of portal hypertension. Materials and Methods Cholestatic liver disease was established in adult canines by common bile duct ligation. A spin echo based EPI MRE sequence was used to acquire 3-D/3-axis abdominal MRE data at baseline, four weeks, and eight weeks. Liver biopsies, blood samples, and D-HVPG measurements were obtained simultaneously. Results Animals developed portal hypertension (D-HVPG: 11.0±5.1 mmHg) with only F1 fibrosis after four weeks. F3 fibrosis was confirmed after eight weeks despite no further rise in portal hypertension (D-HVPG: 11.3±3.2 mmHg). Mean stiffnesses of the spleen increased over two-fold from baseline (1.72±0.33 kPa) to four weeks (3.54±0.31 kPa), and stabilized at eight weeks (3.38±0.06 kPa) in a pattern consistent with changes in portal pressure. A positive correlation was observed between spleen stiffness and D-HVPG (r2 = 0.86, p<0.01). Conclusion These findings indicate a temporal relationship between portal hypertension and the development of liver fibrosis in a large animal model of cholestatic liver disease. The observed direct correlation between spleen stiffness and D-HVPG suggest a non-invasive MRE approach to diagnose and screen for portal hypertension.