3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies
Introduction
Toxicity is one of the major reasons for attrition during drug development and drug withdrawal from the market (Schuster et al., 2005). The main sites of drug toxicity are the cardiovascular system and the liver (Guengerich, 2011). Suitable in vitro test systems as well as predictive test methods are urgently needed (Mandenius et al., 2011a, Mandenius et al., 2011b). An accurate in vitro model for toxicity studies should identify affected pathways and mechanisms and must reflect the in vivo situation as closely as possible. The system should also allow long term functional cultivation of the cells. For liver, primary human hepatocytes (PHH) are still considered as the best available in vitro model for toxicity and metabolism studies (Gomez-Lechon et al., 2003). After isolation, PHH show high expression levels of liver-specific genes which are important for drug metabolism (Guillouzo, 1998). However, they dedifferentiate rapidly in standard cultures (Godoy et al., 2009) and cannot be applied to long term studies. Moreover, the availability of fresh PHH is highly limited in contrast to established cell lines.
The HepaRG cell line shows very promising characteristics such as high CYP450 activities (Aninat et al., 2006, Gripon et al., 2002, Guillouzo et al., 2007). When differentiated, this cell line consists of hepatocyte-like cells and biliary cells in a ratio of about 1:1. The hepatocyte-like cells express both phase I and phase II enzymes and hepatic membrane transporters, indicating that this cell line could serve as a surrogate for primary hepatocytes (Aninat et al., 2006, Kanebratt and Andersson, 2008, Le Vee et al., 2006, Lubberstedt et al., 2011).
Drug toxicity studies show that this cell line is more sensitive to metabolism-mediated toxicity than the HepG2 cell line (Guillouzo et al., 2007). It was also shown that acetaminophen-induced toxic mechanisms in HepaRG cells are similar to animal models and humans, indicating that this cell line is a good in vitro model to study hepatotoxicity of acetaminophen and other drugs (Gunness et al., 2013, McGill et al., 2011).
HepaRG cells can be maintained for weeks with high liver-specific activities in conventional 2D cultures (Aninat et al., 2006). However, these cultures do not reflect the complex, three-dimensional architecture of the liver, where cell–cell and cell-matrix interactions as well as cellular and spatial polarity highly influence gene expression profiles and therefore functionality (Chaumontet et al., 1998, Haouzi et al., 2005, Kono and Roberts, 1996, Narayanan et al., 2002). Recently, several 3D culture systems were developed to improve performance of liver cells for clinical and research purposes (Gerlach et al., 2003, Mueller et al., 2011b; Sauer et al., 2003, van de Kerkhove et al., 2003).
The HepaRG cell line has already been applied to different 3D cultivation systems. In a 3D bioreactor, HepaRG cells maintained CYP450 activities which could be induced and inhibited similar to in vivo (Darnell et al., 2011, Darnell et al., 2012). In another 3D bioreactor system, HepaRG cells showed high liver functionality and moreover, this bioreactor (BAL; bioartificial liver) increased survival time of rats with acute liver failure (Hoekstra et al., 2011, Nibourg et al., 2012a, Nibourg et al., 2012b). 3D HepaRG aggregates produced in spinner-bioreactors maintained liver-specific functions for many weeks (Leite et al., 2012). Taken together, these 3D bioreactor systems are promising tools in terms of maintaining liver-specific functionality, not only for HepaRG cells, but also for PHH (Mueller et al., 2012, Mueller et al., 2011a). However, most 3D bioreactor types are low-throughput systems not allowing toxicity screenings. A 96 well plate high-throughput system for the production of multicellular spheroids was developed (Kelm and Fussenegger, 2004, Kelm et al., 2003). This scaffold-free, hanging drop technique which is now commercially available can be applied for production of spheroids of different cell types including liver cells. The initial cell number and therefore the size of the spheroid is adjustable in this system (Drewitz et al., 2011, Kelm and Fussenegger, 2004, Kelm et al., 2003). We have already shown that spheroids of the hepatoma cell line HepG2 show improved liver-specific functionality compared to 2D HepG2 cultures, such as the production of albumin, CYP450 induction and activity of hepatic drug transporters (Mueller et al., 2011a). Moreover, by using the same system, 3D spheroids of the HepaRG cell line were produced and characterized in terms of long-term maintenance, functionality and responses to drugs (Gunness et al., 2013). These 3D cultures show higher long-term functionality than 2D cultures (increased production of albumin, urea and glucose). They also maintain high drug transporter (MRP2) and CYP2E1 activities.
In this study, we produced 3D spheroids of HepaRG and HepG2 cells using the hanging drop system and compared them for toxicity of selected drugs. We assessed the acute toxicity (24 h exposure) of four drugs (aflatoxin B1, amiodarone, valproic acid and chlorpromazine) with different mechanisms of toxicity and compared the results to conventional 2D cultures.
We tested Aflatoxin B1, which is a naturally occurring mycotoxin produced by Aspergillus species. This compound is another example of metabolism-mediated toxicity. Aflatoxin B1 is metabolized by different Cytochrome P450 (CYP450) isoforms, mainly by CYP3A4 to reactive intermediates, e.g. a reactive epoxide (McLean and Dutton, 1995). Amiodarone is an anti-arrhythmic drug which causes abnormal liver function in 15–50% of patients. The toxicity of amiodarone does not require metabolic activation (Golli-Bennour et al., 2012, Spaniol et al., 2001).
As fatty acid analogue, valproic acid can also induce steatosis in patients by competitive inhibition of fatty acid metabolism. A wide range of metabolites, generated by CYP450 or other metabolizing enzymes (Eadie et al., 1988, Park et al., 1995) are described. The main urinary metabolite is valproate β-glucuronide which is generated by UDP-glucuronosyltransferase (Argikar and Remmel, 2009). Both the parent compound as well as the metabolites are implicated in valproic-acid induced toxicity (Baillie, 1992).
Chlorpromazine is an anti-psychotic drug which causes cholestasis in vivo (Padda et al., 2012). Again, both the parent compound as well as the metabolites are hepatotoxic, however the exact mechanism of idiosyncratic toxicity is still unknown (Wen and Zhou, 2009). In HepaRG cells, chlorpromazine induces oxidative stress as a major mechanism leading to cholestasis (Antherieu et al., 2012).
We carried out single and repeated dose experiments (3 daily doses, 72 h exposure) with chlorpromazine. The effects of subtoxic chlorpromazine concentrations on the glucose uptake/production and lactate secretion in HepaRG 2D and 3D cultures and on the 3D bile canaliculi network were analyzed as well. Moreover, we show that CYP3A4 activity was significantly higher in the 3D HepaRG spheroids compared to the 2D cultures.
In summary, our results suggest that the 3D HepaRG spheroids have better functionality than HepaRG 2D cultures and therefore are a good model for application in drug toxicity screenings especially for metabolism mediated toxicity. This case study was carried out within the framework of the NOTOX project as one of the six research projects of the SEURAT-1 (Safety Evaluation Ultimately Replacing Animal Testing) initiative jointly funded by EC and Cosmetics Europe. The major focus of SEURAT-1 is to develop in vitro models for long term toxicity assessment. The drugs used in this study were chosen in close collaboration with the SEURAT-1 cluster project namely; TOXBANK.
Section snippets
Cell culture
HepG2 cells, obtained from the German collection of microorganisms and cell cultures (DSMZ, Braunschweig, Germany), were maintained in Williams Medium E supplemented with penicillin/streptomycin (100U/100 μg/ml) and 10% FCS.
Cryopreserved, differentiated HepaRG cells (HPR 116) were obtained from Biopredic International, Saint-Grégoire, France and cultivated according to the company’s instructions. The cells were maintained in an incubator (Memmert GmbH, Schwabach, Germany) at 37 °C, 95% air
Morphology of 2D and 3D HepG2 and HepaRG cultures
The morphology of 2D and 3D HepG2 and HepaRG cultures is shown in Fig. 1. The 2D HepG2 cultures show an epithelial morphology. The 3D HepG2 spheroids increased in size over time and attain a diameter of 500–600 μm at cultivation day 7. 2D HepaRG cultures consist of clusters of hepatocyte like cells and surrounding flat biliary cells. These cells did not proliferate. Bile canaliculi like structure (at cell to cell contacts, white, see Fig. 1c) can be observed. In contrast, it is not possible to
Discussion
In this study, we tested the toxicity of four drugs on 2D and 3D cultures of HepG2 and HepaRG cells in serum free conditions. We performed the drug exposure studies either in conventional 96 well plates (for the 2D cultures) or in the GravityTrap plates, a specifically designed 96 well plate format for harvesting and maintaining the 3D spheroids. The free drug concentration is mainly influenced by evaporation and/or binding to culture plates and proteins especially to albumin. Since we used
Conflict of interest
There are no conflicts of interest whatsoever.
Acknowledgements
The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement No 267038 and Cosmetics Europe within the framework of the “NOTOX” Project of the SEURAT-1 initiative. We thank InSphero AG for technical support and Leon Mujis for excellent technical assistance in confocal microscopy. We would also like to thank the TOXBANK consortium for excellent support on compound selection.
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