Current 2-dimensional hepatic model systems often fail to predict chemically induced hepatotoxicity due to the loss of a hepatocyte-specific phenotype in culture. For more predictive in vitro models, hepatocytes have to be maintained in a 3-dimensional environment that allows for polarization and cell–cell contacts. Preferably, the model will reflect an in vivo-like multi-cell type environment necessary for liver-like responses. Here, we report the characterization of a multi-cell type microtissue model, generated from primary human hepatocytes and liver-derived non-parenchymal cells. Liver microtissues were stable and functional for 5 weeks in culture enabling, for example, long-term toxicity testing of acetaminophen and diclofenac. In addition, Kupffer cells were responsive to inflammatory stimuli such as LPS demonstrating the possibility to detect inflammation-mediated toxicity as exemplified by the drug trovafloxacin. Herewith, we present a novel 3D liver model for routine testing in 96-well format capable of reducing the risk of unwanted toxic effects in the clinic.
Keywords3-dimensional Spheroids Kupffer cells
Current strategies to test drug-induced liver injury (DILI) are predominantly based on in vivo animal models (Hartung 2009). However, significant species-specific variation between rodents and humans as well as genetic variability in humans impacts the extrapolation to the clinical situation (Hartung 2009). A recent analysis demonstrated that 43 % of toxic effects in humans were correctly predicted by tests in rodents, whereas this increased to 63 % when non-rodent animals were included (Olson et al. 2000). This low correlation highlights the fact that many adverse effects are not detected by traditional in vivo toxicity tests. More organotypic human in vitro models are expected to support toxicity assessment and decrease the risk of DILI in the clinic. Unfortunately, maintaining liver-specific functionality in vitro is a delicate business as hepatocytes have to retain their polarized 3D structure to maintain liver-specific functionality (Lecluyse et al. 2012; Berthiaume et al. 1996). Growing a single layer of hepatocytes between two extracellular matrix layers is the current gold standard method to maintain polarization. However, such hepatocyte cultures are phenotypically and functionally not very stable over time which impedes their use for long-term toxicity testing (Berthiaume et al. 1996). Furthermore, hepatocyte sandwich cultures are difficult to scale down to a 96-well format due to the instability of the overlaying gels and pronounced edge effects. For these reasons, larger well plates are typically used which hampers toxicity testing at early time points in the drug development process.
Primary mammalian cells retain their capacity to reform a tissue without the use of any scaffold material. Gravity-enforced cellular self-assembly in hanging drops is a well-established technology for tissue reformation enabling the formation of size-controlled, multi-cell type microtissues (Kelm and Fussenegger 2004). Assembling primary human hepatocytes into 3D liver microtissues allows cells to maintain extensive cellular contacts. Heterotypic cell–cell contacts in co-cultures further enhance the hepatocellular phenotype, maintaining hepatocytes in their differentiated state (Lecluyse et al. 2012). In addition, the implementation of non-parenchymal cells provides hepatocytes with diffusible growth factors and cytokines. For example, Kupffer macrophages release both pro-proliferative (e.g., TNF-α, IL-6) and anti-proliferative (IL-1, TGF-β) cytokines and signals (Lecluyse et al. 2012). These cytokines were shown to be involved in precipitating idiosyncratic toxicity of certain drugs, such as trovafloxacin (Liguori et al. 2010; Shaw et al. 2007, 2010). Treatment of mice or rats with inflammatory stimuli such as LPS or TNF-α together with trovafloxacin caused toxicity only in the presence of the inflammatory stimulus. However, routine assessment of inflammation-mediated toxicity in vitro has so far been difficult due to lack of commercially available primary human liver model systems incorporating inflammatory cells.
Results and discussion
Most directly hepatotoxic compounds are detected during pre-clinical investigations. However, indirectly hepatotoxic compounds involving the immune system are not detected during pre-clinical phases, such as trovafloxacin (Shaw et al. 2010). Recent animal experiments indicated that trovafloxacin is only hepatotoxic in combination with an inflammatory stimulus, such as lipopolysaccharide (LPS) or TNF-α (Shaw et al. 2007, 2010; Liguori et al. 2010). The mechanism is thought to involve enhanced cytokine secretion and accumulation in the liver, causing caspase activation and subsequent liver injury. Induction of the inflammatory response in liver microtissues by LPS resulted in elevated levels of IL-6 secretion, verifying the responsiveness of incorporated macrophages in the liver microtissues (Fig. 4c). The addition of LPS shifted the hepatotoxic threshold of trovafloxacin about threefold from 220 (without LPS) to 71 μM in the presence of LPS (Fig. 4d).
Developed to overcome the limitations of conventional 2D culture, multi-cell type 3D liver microtissues resemble liver-like cell composition and an extended stability in culture. The long-term viability and functionality of liver microtissues allows for routine compound testing as well as chronic and inflammation-mediated toxicity. The 96-well format allows for microtissue mass production enabling the implementation of an organotypic liver model at an early time point in drug development.
This project was supported within the framework of FP7 (FETOpen), project no. 296257. We further thank Silvia Behnke (Sophistolab AG) for her excellent immunohistological staining and Tony Rutt for proof reading of the manuscript.
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