Background/AimNon-alcoholic fatty liver disease (NAFLD) is a global health concern with limited treatment options. The paucity of predictive i n v itro models in preclinical settings seems to be one of the limitations of identifying effective medicines. We therefore aimed to develop an i n v itro model that can display the key hallmarks of NAFLD, such as steatosis, inflammation, and fibrosis.MethodsAn in vitro model of steatohepatitis was developed using organoids prepared from hepatocytes of healthy individuals from a commercial source (HLOs) and the liver tissues collected from needle biopsies of NAFLD patients (HLONAFLD) using defined culture conditions. HLOs were treated with palmitic acid for 72 h to develop an i n v itro model of steatohepatitis, while HLONAFLD served as a natural model of steatohepatitis. Metformin and saroglitazar were used to validate the liver organoid model of steatohepatitis. Saroglitazar was also evaluated in the high-fat, high-fructose (HF-HF) diet-induced model of NAFLD using C57BL/6 mice to validate the findings from the i n v itro model.ResultsHLOs and HLONAFLD exhibited bipotent properties, showing the expression of markers of hepatocytes, ductal cells, and also stem cells. Furthermore, they demonstrated the expression of nonparenchymal cell markers such as stellate cells (CD166) and Kupffer-like cells (CD68 and EMR1). The steatohepatitis models developed using these organoids displayed markers associated with steatosis, inflammation and fibrosis, which were decreased by metformin and saroglitazar.ConclusionThe in vitro models developed in our lab employing HLOs and HLONAFLD display all three key hallmarks of NAFLD: steatosis, inflammation, and fibrosis without the necessity for coculture with other nonparenchymal cells. The implementation of the HLONAFLD-based model is also expected to provide a more realistic assessment of test substances to develop therapeutics for NAFLD.