A long-term three dimensional liver co-culture system for improved prediction of clinically relevant drug-induced hepatotoxicity

https://doi.org/10.1016/j.taap.2013.01.012Get rights and content

Abstract

Drug-induced liver injury (DILI) is the major cause for liver failure and post-marketing drug withdrawals. Due to species-specific differences in hepatocellular function, animal experiments to assess potential liabilities of drug candidates can predict hepatotoxicity in humans only to a certain extent. In addition to animal experimentation, primary hepatocytes from rat or human are widely used for pre-clinical safety assessment. However, as many toxic responses in vivo are mediated by a complex interplay among different cell types and often require chronic drug exposures, the predictive performance of hepatocytes is very limited. Here, we established and characterized human and rat in vitro three-dimensional (3D) liver co-culture systems containing primary parenchymal and non-parenchymal hepatic cells. Our data demonstrate that cells cultured on a 3D scaffold have a preserved composition of hepatocytes, stellate, Kupffer and endothelial cells and maintain liver function for up to 3 months, as measured by the production of albumin, fibrinogen, transferrin and urea. Additionally, 3D liver co-cultures maintain cytochrome P450 inducibility, form bile canaliculi-like structures and respond to inflammatory stimuli. Upon incubation with selected hepatotoxicants including drugs which have been shown to induce idiosyncratic toxicity, we demonstrated that this model better detected in vivo drug-induced toxicity, including species-specific drug effects, when compared to monolayer hepatocyte cultures.

In conclusion, our results underline the importance of more complex and long lasting in vitro cell culture models that contain all liver cell types and allow repeated drug-treatments for detection of in vivo-relevant adverse drug effects.

Highlights

► 3D liver co-cultures maintain liver specific functions for up to three months. ► Activities of Cytochrome P450s remain drug- inducible accross three months. ► 3D liver co-cultures recapitulate drug-induced liver toxicity observed in vivo. ► 3D liver co-cultures can detect species-specific drug toxicity observed in vivo. ► This in vitro model may improve assessment of human relevance of preclinical findings.

Introduction

Drug-induced liver injury (DILI) is still the leading cause of acute liver failure and post-market drug withdrawals (Kaplowitz, 2005). Studies have shown that different risk factors can contribute to DILI such as genetic susceptibility factors, non-genetic factors including age, sex, diseases and compound factors including daily dose, metabolism characteristics, and drug-drug interactions (Chalasani and Bjornsson, 2010, David and Hamilton, 2010). Preclinical animal studies cannot fully predict drug-toxicity in humans due to species-specific variations between human and animal hepatocellular functions (Pritchard et al., 2003). Human in vitro liver models currently used for prediction of drug-induced toxicity include microsomes, cell lines, liver slices and primary hepatocytes (Gebhardt et al., 2003, Guillouzo, 1998, Hewitt et al., 2007, LeCluyse, 2001). Microsomes are used in high-throughput systems to assess drug metabolizing enzymes but lack the cellular machinery required for toxicity testing (Donato et al., 2004). Although hepatoma cell lines such as HepG2 cells can be used for high-throughput screening, they have low levels of CYP activities and lack many key liver-specific functions (Wilkening et al., 2003). Specific hepatoma cell clones such as HepaRG have most of the specific liver functions at levels close to those found in primary human hepatocytes but they do not represent the genetic heterogeneity of human populations (Guguen-Guillouzo and Guillouzo, 2010, Lubberstedt et al., 2011, McGill et al., 2011, Pernelle et al., 2011). Liver slices retain in vivo liver architecture but have only short term viability and are not applicable to high-throughput screening (Guillouzo, 1998). Primary hepatocytes growing in monolayer two-dimensional (2D) culture are easy to use but liver specific functions including drug metabolism rapidly decline under standard culture conditions allowing detection of acute drug-induced toxicity only (Guguen-Guillouzo and Guillouzo, 2010, Hewitt et al., 2007, Lecluyse et al., 2012, Sivaraman et al., 2005). Many modifications to standard culture models for primary hepatocytes have been developed to prolong hepatocyte function such as culturing of the cells in collagen type I/IV, fibronectin or other extracellular matrix (ECM)-coated plates (Bissell et al., 1987, Mingoia et al., 2007), or between two layers of collagen type I or matrigel (Dunn et al., 1989, Guguen-Guillouzo and Guillouzo, 2010, Hewitt et al., 2007, Lecluyse et al., 2012, Mingoia et al., 2007). However, these modifications, while increasing CYP activities and prolonging the functional lifespan of primary hepatocytes to a certain extent, do not recapitulate all the important functions of the liver, mainly because of the lack of hepatic non-parenchymal cells (NPC; Hasmall et al., 2001, Roberts et al., 2007).

Substantial improvements in hepatocyte in vitro models were achieved by the development of more complex human liver systems created by co-culturing of parenchymal cells (PC) with NPC or other cell types. For example, human hepatocytes in a 2D micro-patterned co-culture with mouse 3 T3-J2 fibroblasts (Khetani and Bhatia, 2008) maintained hepatocellular function for several weeks. Yet, the model may not be physiologically relevant for detection of species-specific drug toxicity due to the lack of other liver NPC and the fact that a mouse embryonic fibroblast cell line is used for stabilization of human hepatocyte function (Hasmall et al., 2001, Roberts et al., 2007). In this regard, hepatic stellate cells (HSC) and Kupffer cells play a key role in modulating DILI, including idiosyncratic toxicity and hepatocarcinogenesis, probably due to the release of inflammatory mediators, growth factors and reactive oxygen species after their activation by drugs (Hasmall et al., 2001, Lecluyse et al., 2012, Roberts et al., 2007). More sophisticated models containing hepatocytes and NPC are the 3D liver co-culture bioreactors (Dash et al., 2009, Gerlach et al., 2003, Sivaraman et al., 2005, Zeilinger et al., 2011). These models can be kept in culture for several weeks but due to their complexity may not be suited for drug testing in pharmaceutical industry.

At present only few human co-culture models are available which can be used for drug-safety assessment (Dash et al., 2009, Khetani and Bhatia, 2008, Naughton et al., 1994). There is an urgent need to establish and validate human in vitro liver models able to produce clinically-relevant data. We therefore characterized a 3D liver culture model using both human and rat primary cells and evaluated its suitability to assess DILI potential in vitro. The model originally described by Naughton and co-workers is based on an industry-standard multiwell format and is therefore amenable to higher-throughput testing (Naughton et al., 1994, Naughton et al., 1995). We show that hepatocytes inoculated into a pre-established NPC culture grown on 3D nylon scaffolds can be kept in culture for up to 3 months while maintaining some important hepatic functions and metabolic CYP activities. This allows exposure to compounds over longer time and allows repeated drug-treatments which are not possible using short-term 2D hepatocyte cultures or other currently available 3D models. Thus, we challenged the 3D liver co-culture with known drugs having different hepatotoxic characteristics and assessed its ability to reflect drug-induced species differences in rat and human. We conclude that this model better detects drug-induced acute and chronic liver toxicity observed in vivo than monolayer hepatocyte cultures. This underlines the importance of incorporating not only hepatocytes but all liver cell types into such a system and their exposure to compounds over long time to allow in vitro assessment of in vivo-relevant adverse drug effects.

Section snippets

3D liver cell co-cultures

The human and rat 3D liver models were created as a multiwell insert plate system by RegeneMed (San Diego, USA) as follows: All liver NPC, including vascular and bile duct endothelial cells, Kupffer cells and hepatic stellate cells (HSC), were freshly isolated by gradient centrifugation after in situ liver perfusion from human or rat livers and expanded in monolayer culture for 3–4 passages (Naughton et al., 1994, Naughton et al., 1995). The NPC were then seeded above two interconnected nylon

Human and rat 3D liver tissue co-cultures have preserved liver-specific function over an extended period of time compared to 2D hepatocytes

After isolation and expansion of rat and human NPC in monolayer culture cells were inoculated into two nylon scaffolds placed above a porous membrane of inserts of 24-well plates (Fig. 1A). Two days later microscopic examination was performed to check whether the NPC were attached and uniformly distributed over the scaffold. Hepatocytes were seeded later only if the cultures containing NPC uniformly covered the scaffold. One week after NPC were seeded hepatocytes were inoculated into the

Discussion

In this work, we characterized a 3D liver culture model of rat and human for detection of single or repeated drug-treatment induced toxicities. We compared the 3D liver co-culture model with standard monolayer hepatocytes grown on collagen type I, since this model is one of the most frequently used model in pharmacological industry for drug toxicity screening and mechanistic studies. Other culture models expose hepatocytes between two layers of Matrigel and/or collagen-I gels that allow better

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

We gratefully acknowledge Dr. Jean-Christophe Hoflack and Nicholas Flint for the performance of DNA microarray, Michael Erhart for the help with FACS analysis, Sebastian Krasniqi for the measurements of the secretion of inflammatory cytokines, Dr. Agnès Poirier and Renée Portmann for the help on the uptake transport activity assay, Susanne Brenner, Claudine Sarron-Petit and Maria Cristina De Vera Mudry for the measurements of toxicity markers. All the above mentioned people are employees at F.

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