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

TLDR

Current in vitro models, such as primary human hepatocyte monolayers, rapidly dedifferentiate, limiting their utility for studying liver biology, drug‑induced liver injury, and liver diseases. The authors developed and extensively characterized an easily scalable 3D primary human hepatocyte spheroid system in chemically defined, serum‑free conditions. They cultured PHH spheroids in chemically defined, serum‑free media, enabling scalable production while preserving hepatocyte phenotype over extended periods. Whole‑proteome analysis showed that the spheroids closely resemble in vivo liver tissue and retain inter‑individual variability; they remain phenotypically stable, morphologically intact, and functionally active for at least five weeks, exhibit heightened sensitivity to chronic hepatotoxin exposure at clinically relevant concentrations (e.g., fialuridine), and can model liver pathologies such as cholestasis, steatosis, and viral hepatitis, demonstrating the system’s versatility for studying liver function, disease, drug targets, and long‑term DILI.

Abstract

Abstract 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. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. 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. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability and hepatocyte-specific functions for culture periods of at least 5 weeks. 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. 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. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. 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.