Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease

The liver is a vital organ for synthesis, metabolism and detoxification, but liver diseases and drug-induced liver injury (DILI) can severely impair liver functionality. To study liver biology and function, drug-induced hepatotoxicity and liver diseases, primary human hepatocytes (PHH) are currently considered as the gold standard in vitro model system1. However, when maintained in conventional 2D monolayer cultures, PHH de-differentiate and rapidly lose hepatocyte-specific functions2,3,4. Thus, the utility of conventional 2D PHH cultures for the long-term study of liver biology and assays that require liver-specific functionalities is largely impaired. There is therefore a need for more faithful in vitro models which more accurately reflect in vivo liver biology. To this end, new systems are needed in which stable liver functionality can be maintained for several weeks to enable long-term studies of liver function under normal and diseased conditions. Normal cell physiology and function strongly depend on cell-cell and cell-extracellular matrix (ECM) interactions in the 3D tissue environment5. In an attempt to mimic the hepatic microenvironment, various more complex culture systems have been developed including sandwich cultures, and 3D models such as scaffold-based systems and bioreactors6,7,8,9,10,11,12. However, major drawbacks of these culture systems include lack of scalability, binding of drugs to scaffold, difficulties in handling and batch-to-batch differences of ECM substrates, which affect reproducibility11. To circumvent these problems, hepatocytes can be cultured as 3D microtissues termed spheroids13,14,15,16. In spheroid culture, it has previously been shown that PHH can be maintained for longer periods of time with stable viability and production of essential molecules such as albumin and urea14,15,16. Furthermore, cellular polarity and formation of functional bile ducts has been described14. However, a full phenotypic characterization as well as a comprehensive assessment of the suitability of the PHH spheroid model in the context of studying e.g. liver diseases and chronic DILI is lacking. Here, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in serum-free, chemically-defined conditions, suitable for long-term functional and toxicological studies. Importantly, PHH spheroids closely resembled the in vivo liver tissue from where they originated more than spheroid cultures isolated from other donors, as determined by whole proteome analyses. Thus, inter-individual variability is maintained on a global scale. The PHH spheroids presented here remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. The culture conditions allowed co-culture of PHH spheroids with non-parenchymal cells (NPCs) such as biliary cells, stellate cells and Kupffer cells and supported their long-term viability. Furthermore, liver diseases such as steatosis, cholestasis and viral hepatitis could be induced and the spheroids could predict chronic drug toxicity in particular of fialuridine, a drug which previously caused several deaths in a clinical trial while having previously passed all pre-clinical safety assessments17. Combined, these results indicate that the PHH spheroid system developed here constitutes a promising and versatile in vitro model to study various aspects of liver function, liver disease and DILI.