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.

Only little is known about how cells coordinately behave to establish functional tissue structure and restore microarchitecture during regeneration. Research in this field is hampered by a lack of techniques that allow quantification of tissue architecture and its development. To bridge this gap, we have established a procedure based on confocal laser scans, image processing, and three-dimensional tissue reconstruction, as well as quantitative mathematical modeling. As a proof of principle, we reconstructed and modeled liver regeneration in mice after damage by CCl 4 , a prototypical inducer of pericentral liver damage. We have chosen the regenerating liver as an example because of the tight link between liver architecture and function: the complex microarchitecture formed by hepatocytes and microvessels, i.e. sinusoids, ensures optimal exchange of metabolites between blood and hepatocytes. Our model captures all hepatocytes and sinusoids of a liver lobule during a 16 days regeneration process. The model unambiguously predicted a so-far unrecognized mechanism as essential for liver regeneration, whereby daughter hepatocytes align along the orientation of the closest sinusoid, a process which we named "hepatocyte-sinusoid alignment" (HSA). The simulated tissue architecture was only in agreement with the experimentally obtained data when HSA was included into the model and, moreover, no other likely mechanism could replace it. In order to experimentally validate the model of prediction of HSA, we analyzed the three-dimensional orientation of daughter hepatocytes in relation to the sinusoids. The results of this analysis clearly confirmed the model prediction. We believe our procedure is widely applicable in the systems biology of tissues.agent based model | image processing and analysis | mathematical tissue modeling | systems biology | morphogenesis T he liver is the main metabolic organ which removes drugs and toxins from the blood. One of the outstanding features of the liver is its capacity to regenerate hepatocyte loss of up to 70% of its mass within a relatively short period of time (1). Hepatic parenchyma is organized in repetitive functional units called liver lobules, which besides its main constituents, hepatocytes, consists of sinusoidal endothelial cells, Kupffer, stellate, and bile duct cells. Branches of the hepatic artery and portal vein guide blood to the periportal regions of the lobules (Fig. 1A). From there, it flows through microvessels, the sinusoids, along hepatocyte columns that are lined with endothelial cells (generally known as sinusoidal cells), and drains into the central vein. This complex lobule architecture ensures a maximal exchange area between blood and hepatocytes in healthy liver. In liver disease, such as hepatocellular cancer, the contact surface between hepatocytes and sinusoidal cells decreases and contributes to compromised liver function (Fig. 1F). Recent research on liver regeneration has focused on molecular pathways and the mechanisms involved (2). Little is known about...

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CellSys is a modular software tool for efficient off-lattice simulation of growth and organization processes in multi-cellular systems in 2D and 3D. It implements an agent-based model that approximates cells as isotropic, elastic and adhesive objects. Cell migration is modeled by an equation of motion for each cell. The software includes many modules specifically tailored to support the simulation and analysis of virtual tissues including real-time 3D visualization and VRML 2.0 support. All cell and environment parameters can be independently varied which facilitates species specific simulations and allows for detailed analyses of growth dynamics and links between cellular and multi-cellular phenotypes.Availability: CellSys is freely available for non-commercial use at http://msysbio.com/software/cellsys. The current version of CellSys permits the simulation of growing monolayer cultures and avascular tumor spheroids in liquid environment. Further functionality will be made available ongoing with published papers.Contact: hoehme@izbi.uni-leipzig.de; dirk.drasdo@inria.frSupplementary information: Supplementary data are available at Bioinformatics online.

The impairment of hepatic metabolism due to liver injury has high systemic relevance. However, it is difficult to calculate the impairment of metabolic capacity from a specific pattern of liver damage with conventional techniques. We established an integrated metabolic spatial-temporal model (IM) using hepatic ammonia detoxification as a paradigm. First, a metabolic model (MM) based on mass balancing and mouse liver perfusion data was established to describe ammonia detoxification and its zonation. Next, the MM was combined with a spatial-temporal model simulating liver tissue damage and regeneration after CCl 4 intoxication. The resulting IM simulated and visualized whether, where, and to what extent liver damage compromised ammonia detoxification. It allowed us to enter the extent and spatial patterns of liver damage and then calculate the outflow concentrations of ammonia, glutamine, and urea in the hepatic vein. The model was validated through comparisons with (1) published data for isolated, perfused livers with and without CCl 4 intoxication and (2) a set of in vivo experiments. Using the experimentally determined portal concentrations of ammonia, the model adequately predicted metabolite concentrations over time in the hepatic vein during toxin-induced liver damage and regeneration in rodents. Further simulations, especially in combination with a simplified model of blood circulation with three ammonia-detoxifying compartments, indicated a yet unidentified process of ammonia consumption during liver regeneration and revealed unexpected concomitant changes in amino acid metabolism in the liver and at extrahepatic sites. Conclusion: The IM of hepatic ammonia detoxification considerably improves our understanding of the metabolic impact of liver disease and highlights the importance of integrated modeling approaches on the way toward virtual organisms. (HEPATOLOGY 2014;60:2040-2051

Histological alterations often constitute a fingerprint of toxicity and diseases. The extent to which these alterations are cause or consequence of compromised organ function, and the underlying mechanisms involved is a matter of intensive research. In particular, liver disease is often associated with altered tissue microarchitecture, which in turn may compromise perfusion and functionality. Research in this field requires the development and orchestration of new techniques into standardized processing pipelines that can be used to reproducibly quantify tissue architecture. Major bottlenecks include the lack of robust staining, and adequate reconstruction and quantification techniques. To bridge this gap, we established protocols employing specific antibody combinations for immunostaining, confocal imaging, three-dimensional reconstruction of approximately 100-μm-thick tissue blocks and quantification of key architectural features. We describe a standard procedure termed ‘liver architectural staining’ for the simultaneous visualization of bile canaliculi, sinusoidal endothelial cells, glutamine synthetase (GS) for the identification of central veins, and DAPI as a nuclear marker. Additionally, we present a second standard procedure entitled ‘S-phase staining’, where S-phase-positive and S-phase-negative nuclei (stained with BrdU and DAPI, respectively), sinusoidal endothelial cells and GS are stained. The techniques include three-dimensional reconstruction of the sinusoidal and bile canalicular networks from the same tissue block, and robust capture of position, size and shape of individual hepatocytes, as well as entire lobules from the same tissue specimen. In addition to the protocols, we have also established image analysis software that allows relational and hierarchical quantifications of different liver substructures (e.g. cells and vascular branches) and events (e.g. cell proliferation and death). Typical results acquired for routinely quantified parameters in adult mice (C57Bl6/N) include the hepatocyte volume (5,128.3 ± 837.8 μm3) and the fraction of the hepatocyte surface in contact with the neighbouring hepatocytes (67.4 ± 6.7 %), sinusoids (22.1 ± 4.8 %) and bile canaliculi (9.9 ± 3.8 %). Parameters of the sinusoidal network that we also routinely quantify include the radius of the sinusoids (4.8 ± 2.25 μm), the branching angle (32.5 ± 11.2°), the length of intersection branches (23.93 ± 5.9 μm), the number of intersection nodes per mm3 (120.3 × 103 ± 42.1 × 103), the average length of sinusoidal vessel per mm3 (5.4 × 103 ± 1.4 × 103mm) and the percentage of vessel volume in relation to the whole liver volume (15.3 ± 3.9) (mean ± standard deviation). Moreover, the provided parameters of the bile canalicular network are: length of the first-order branches (7.5 ± 0.6 μm), length of the second-order branches (10.9 ± 1.8 μm), length of the dead-end branches (5.9 ± 0.7 μm), the number of intersection nodes per mm3 (819.1 × 103 ± 180.7 × 103), the number of dead-end branches per mm3 (409.9 × 103 ± 95.6 ×...

In this paper, we explore how potential biomechanical influences on cell cycle entrance and cell migration affect the growth dynamics of cell populations. We consider cell populations growing in free, granular and tissuelike environments using a mathematical single-cell-based model. In a free environment we study the effect of pushing movements triggered by proliferation versus active pulling movements of cells stretching cell-cell contacts on the multi-cellular kinetics and the cell population morphotype. By growing cell clones embedded in agarose gel or cells of another type, one can mimic aspects of embedding tissues. We perform simulation studies of cell clones expanding in an environment of granular objects and of chemically inert cells. In certain parameter ranges, we find the formation of invasive fingers reminiscent of viscous fingering. Since the simulation studies are highly computation-time consuming, we mainly study one-cell-thick monolayers and show that for selected parameter settings the results also hold for multi-cellular spheroids. Finally, we compare our model to the experimentally observed growth dynamics of multi-cellular spheroids in agarose gel.

If successfully translated to patients the GDH-based therapy might provide a less aggressive therapeutic alternative for patients with severe hyperammonemia.