However, when maintained in conventional 2D monolayer cultures, PHH de-differentiate and rapidly lose hepatocyte-specific functions 2, 3, 4. 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 system 1. 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. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability and hepatocyte-specific functions for culture periods of at least 5 weeks. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions.

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially.

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