3D multi-cell-type liver organoids: A new model of non-alcoholic fatty liver disease for drug safety assessments.

The development of in vitro models that recapitulate critical liver functions is essential for accurate assessments of drug toxicity. Although liver organoids can be used for drug discovery and toxicology, they are limited by (i) the lack of expression and activity of xenobiotic-metabolizing enzymes, and (ii) the difficulty of mimicking non-alcoholic fatty liver disease (NAFLD, which influences the expression of these enzymes) in vitro. Here, we generated three-dimensional multi-cell-type liver organoids (hereafter "HML organoids") from HepaRG cells, primary human macrophages, and hepatic-stellate-cell-derived LX-2 cells. We also developed an NAFLD model by culturing HML organoids for 9 days with a mixture of stearic and oleic acids. The exposed organoids showed typical features of steatosis and expressed fibrosis markers. We subsequently used HML and NAFLD-HML organoids to model drug-induced liver injury. By estimating the IC50 and benchmark doses, we were able to improve the in vitro detection of drugs likely to be toxic in fatty livers. Thus, HML and NAFLD-HML organoids exhibited most of the liver's functions and are relevant in vitro models of drug metabolism, drug toxicity, and adverse drug event in NAFLD.