Evaluation of the Immortalized Primary Human Hepatocyte Cell Line Fa2N-4 as a Model for Metabolic Dysfunction-Associated Steatotic Liver Disease.

Background and Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of chronic liver disease worldwide. In vitro MASLD studies predominantly rely on hepatocellular carcinoma-derived cell lines such as HepG2 that are poorly differentiated and exhibit cancer-associated metabolic reprogramming that suppresses key adult hepatic functions. Primary human hepatocytes offer greater physiological relevance and capture inter-individual biological variability that reflects human population diversity, but their use is limited by high costs, availability, and rapid dedifferentiation in culture. Here, we characterized the immortalized primary human hepatocyte cell line Fa2N-4 as an alternative in vitro model for MASLD. Methods: We performed a comparative analysis of Fa2N-4 and HepG2 cells, assessing their genomic architecture, lipid-induced steatosis, and pharmacological responsiveness. Results: The Fa2N-4 and HepG2 cell models exhibited pronounced differences, including distinct karyotypes, divergent MASLD-associated genetic risk variant profiles, and markedly different transcriptional responses to lipid loading and drug treatment. We further examined the hepatic response to resmetirom, a first-in-class US Food and Drug Administration-approved therapy for treating metabolic dysfunction-associated steatohepatitis. Resmetirom, a thyroid hormone receptor-β agonist, reduced intracellular triglyceride accumulation in Fa2N-4 cells but not HepG2 cells, and this was accompanied by transcriptional changes in mitochondrial glycolysis and oxidative phosphorylation pathways. Conclusion: These findings demonstrate that hepatocyte model selection critically influences experimental outcomes in MASLD research and highlight Fa2N-4 cells as a physiologically relevant platform for mechanistic and translational studies of MASLD therapeutics.