*Purpose: The aim of our project is to develop an in vitro 3D human liver organoid (3D-HLO) model for studying nonalcoholic fatty liver disease (NAFLD) and its advanced form, nonalcoholic steatohepatitis (NASH), by mimicking the in vivo niche pathophysiological liver microenvironment.

*Methods: 3D-HLOs were created by incorporating primary human hepatic cells isolated from healthy donor livers, including hepatocytes (HCs), hepatic stellate cells, liver sinusoidal endothelial cells, cholangiocytes, and Kupffer cells (Fig. 1A, B). EIA and biochemistry assays were performed to evaluate urea synthesis and secretion of albumin and total bile acids. NAFLD was modeled by subjecting 3D-HLOs to a 7-day treatment of free fatty acid (FFA) and lipopolysaccharide (LPS) to induce steatosis, fibrosis, and inflammation. Lipid accumulation and fibrosis were detected with Nile Red and Sirius Red staining, respectively. 3D-HLOs generated using HCs isolated from a NAFLD patient (Fig. 1C), and treated with obeticholic acid (OCA, 1μM) and lanifibranor (10μM) for 7 days. RT-qPCR and LDH cytotoxicity assay were performed to determine the hepatocyte phenotype, cytotoxicity, and fibrosis in normal and NAFLD 3D-HLOs.

*Results: RT-qPCR and immunofluorescence staining for expression of ALB, CYP3A4, and CYP1A2 demonstrated that 3D-HLOs maintained a mature liver phenotype for up to 30 days. Furthermore, liver functions including urea synthesis, albumin production, and total bile acid secretion were also maintained for up to 30 days (Fig. 1D). Nile Red and Sirius Red staining showed that 3D-HLOs treated with FFA+LPS had increased lipid accumulation and collagen deposition, respectively, compared to FFA alone or control (Fig. 1E). In the NAFLD 3D-HLOs, we found that OCA and lanifibranor upregulated the expression of CYP3A4 and ALB, respectively, and both repressed the expression of Col1A1. LDH cytotoxicity assay further demonstrated that lanifibranor repressed cell death in FFA-treated NAFLD 3D-HLOs.

*Conclusions: Despite the rising incidence of NAFLD/NASH, there is no FDA-approved therapy for treatment, and the disease mechanism is still elusive. Our 3D-HLOs provide an ex vivo human liver model to study the pathogenesis of NAFLD/NASH and screen novel therapeutic strategies.

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