In vitro systems with hepatocytes; novel toxicity tests

Improved in vitro toxicity tests are currently required, mainly due to the new European Chemicals Legislation (REACH). As a result, hepatocytes as test models are highly relevant because most xenobiotics are either metabolically activated or detoxified in the liver. However, the cultivation of isolated hepatocytes in vitro is difficult. Depending on culture conditions they may dedifferentiate and massively alter their functions compared to the in vivo situation. Recently, we have identified several factors involved in early signalling which are responsible for the observed in vivo/in vitro discrepancies (Godoy et al., 2009). We have also established techniques to manipulate signalling network constellations in order to guarantee in vivo like functions of cultivated hepatocytes. After introducing these improvements, hepatocyte in vitro systems can also be applied to -omics studies in which toxic substances are classified by gene or protein expression patterns with respect to their molecular mechanisms of action.

Fig. 1: Hepatocytes in vivo and in vitro. A. Reconstruction of mouse liver tissue from confocal laser scans. The reconstruction visualises the interwoven network of bile canaliculi and sinusoids, the microvessels of the liver (immunostaining with antibodies directed against DPPIV and nuclear staining with DAPI). B. Mouse hepatocytes in vitro using an optimized 3D culture system, including adequate extracellular matrix of the liver. Under these conditions hepatocytes form bile canaliculi in vitro as visualized by yellow fluorescence and a dark lumen. C. Reversibility of hepatocyte differentiation and dedifferentiation. When hepatocytes cultivated as described under B. are transferred into a conventional 2D culture system, the polarised structure with bile canaliculi is lost within 24h. This process is reversible. Depending on defined culture conditions hepatocytes can be switched between a polarised (B) and a non-polarised cell state. D. Ras and TGF-beta induce dedifferentiation of hepatocytes. Upper left: differentiated hepatocytes with bile canaliculi at the cell interfaces. Transduction of a constitutively active Ras (upper right) as well as exposure to TGF-beta (lower left) lead to dedifferentiation, loss of the polarised cell structure and formation of stress fibres.

Fig 2: The state of differentiation of hepatocytes in vitro depends on MAP Kinase and PI-3K/Akt signalling. Overactivation of MAP kinase signalling leads to dedifferentiation features of epithelial-to-mesenchymal transition (EMT). Overactivation of PI-3 K/AKT signalling inhibits apoptosis (Godoy et al., 2009). In vivo-like functions of cultivated hepatocytes could be achieved by manipulation of MAP kinase and PI-3 K/Akt early signalling activities.

Video 1: Dynamic process of bile canaliculi formation in cultivated hepatocytes. Morphogenesis of bile canaliculi includes initial formation of pulsating bile canaliculi with highly variable diameters. After establishment of the canaliculi, smaller lumina with constant diameters are observed.

Further videos with hepatocytes, including interactions with endothelial cells and tumour cells, can be seen here.

Novel systems of neurotoxicity testing

Together with the research groups of Christoph van Thriel (IfADo) and Jonathan West (ISAS), we have established screening techniques for neurotoxic compounds inhibiting the formation of neurite connections (funded by the EC project ESNATS). A long term goal of this project is to integrate the above described in vitro systems with hepatocytes into the neurite connection assay in order to achieve neurotoxicity testing with and without hepatic metabolism.

Fig. 3: Cells attach only at defined positions of a cell culture chip. This has been achieved by the novel technique of microprinting. After attachment to pre-defined areas, neuronal cells start forming neurite connections (red), which can be counted using automated procedures. Certain neurotoxic compounds, such as acrylamide, inhibit formation of neurite connections at much lower concentrations than those known to induce cytotoxicity.

Video 2: Formation of neurite connections is a highly dynamic process. This example illustrates that after formation of neurite connections, the latter may serve as guide rails for migration of individual cell bodies between different attachment foci.

Selected publications

Godoy P, Hengstler JG, Ilkavets I, Meyer C, Bachmann A, Müller A, Tuschl G, Mueller SO, Dooley S. Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor beta-induced apoptosis. Hepatology. 2009 Jun;49(6):2031-43.

Brulport M, Schormann W, Bauer A, Hermes M, Elsner C, Hammersen FJ, Beerheide W, Spitkovsky D, Härtig W, Nussler A, Horn LC, Edelmann J, Pelz-Ackermann O, Petersen J, Kamprad M, von Mach M, Lupp A, Zulewski H, Hengstler JG: Fate of extrahepatic human stem and precursor cells after transplantation into mouse livers. Hepatology 2007;46:861-70.

Hewitt NJ, Lechon MJ, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L, Lohmann C, Skonberg C, Guillouzo A, Tuschl G, Li AP, LeCluyse E, Groothuis GM, Hengstler JG. Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 2007;39:159-234.

Hengstler JG, Utesch D, Steinberg P, Ringel M, Swales N, Biefang K, Platt KL, Diener B, Böttger T, Fischer T, Oesch F, Cryopreserved primary hepatocytes as an in vitro model for the evaluation of drug metabolism and enzyme induction. Drug Metab Rev 32, 81-118, 2000.

Aurich H, Sgodda M, Kaltwaßer P, Vetter M, Weise A, Liehr T, Brulport M, Hengstler JG, Dollinger MM, Fleig WE, Christ B. Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo. GUT 2009;58:570-81.

Aurich I, Mueller LP, Aurich H, Luetzkendorf J, Tisljar K, Dollinger M, Schormann W, Walldorf J, Hengstler J, Fleig WE, Christ B. Functional integration of human mesenchymal stem cell-derived hepatocytes into mouse livers. Gut. 2007;56(3):405-415.

Ruhnke M, Ungefroren H, Nussler A, Martin F, Brulport M, Schormann W, Hengstler JG, Klapper W, Ulrichs K, Hutchinson JA, Soria B, Parwaresch RM, Heeckt P, Kremer B, Fändrich F, Reprogramming of Human Peripheral Blood Monocytes into Functional Hepatocyte and Pancreatic Islet-like Cells. Gastroenterology, 128:1774-86, 2005.

Weng HL, Liu Y, Chen JL, Huang T, Xu LJ, Godoy P, Hu JH, Zhou C, Stickel F, Marx A, Bohle RM, Zimmer V, Lammert F, Mueller S, Gigou M, Samuel D, Mertens PR, Singer MV, Seitz HK, Dooley S. The etiology of liver damage imparts cytokines transforming growth factor beta1 or interleukin-13 as driving forces in fibrogenesis. Hepatology. 2009 Jul;50(1):230-43.

Dooley S, Hamzavi J, Ciuclan L, Godoy P, Ilkavets I, Ehnert S, Ueberham E, Gebhardt R, Kanzler S, Geier A, Breitkopf K, Weng H, Mertens PR. Hepatocyte-specific Smad7 expression attenuates TGF-beta-mediated fibrogenesis and protects against liver damage. Gastroenterology. 2008 Aug;135(2):642-59.

• In vitro systems with hepatocytes; novel toxicity tests
• Spatial –temporal modelling of tissue toxicity and regeneration
• Carcinogenesis and tumour development
• Research for applied and regulatory toxicology