Multi‐Cellular Human Liver Organoids for Modeling Metabolic Dysfunction–Associated Steatotic Liver Disease and Drug‐Induced Liver Injury

In vitro modeling of human liver function is essential for assessing drug-induced toxicity, particularly in the context of metabolic dysfunction-associated steatotic liver disease (MASLD). However, standard liver organoid systems often lack xenobiotic-metabolizing enzyme activity and fail to replicate the pathological features of MASLD. Here, we present a method for generating functional, multi cell-type human liver organoids (HML) organoids from HepaRG cells, primary human macrophages, and LX-2 hepatic stellate cells. These three-dimensional (3D) organoids are cultured under defined conditions that support key hepatic functions. To mimic MASLD, we expose HML organoids to a mixture of stearic and oleic acids for 9 days, inducing steatosis and fibrogenic responses characteristic of the disease. These MASLD-HML organoids retain liver-specific functions and express key fibrosis markers. We further demonstrate the usefulness of this method to produce standardized organoids useful for the evaluation of drug-induced liver injury (DILI) through IC50 and benchmark dose calculations, particularly in the context of MASLD. Together, HML and MASLD-HML organoids provide a robust model for studying drug metabolism, toxicity, and adverse drug reactions in healthy and diseased liver states. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Culture of HepaRG cells Basic Protocol 2: Culture of LX-2 cells Basic Protocol 3: Culture of primary human unstimulated M1 macrophages Basic Protocol 4: Organoid seeding procedure 1 in agarose molds Basic Protocol 5: Organoid seeding procedure 2 in ultra-low attachment 96-well plate Alternate Protocol: Organoid seeding procedure from cryopreserved cells Basic Protocol 6: Preparation of the fatty acid mixture Basic Protocol 7: MASLD induction.