ABSTRACT: Background and aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of chronic liver disease worldwide, yet preclinical in vitro models remain suboptimal. MASLD studies predominantly rely on hepatocellular carcinoma–derived cell lines, such as HepG2, which are poorly differentiated and exhibit cancer-associated metabolic reprogramming that suppresses key adult hepatic functions. Primary human hepatocytes, on the other hand, offer greater physiological relevance, but their use is limited by cost, availability, donor variability, and rapid dedifferentiation. Here, we evaluated 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 genomic architecture, lipid-induced steatosis, and pharmacological responsiveness. Results: The two 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, the first U.S. Food and Drug Administration (FDA) approved medication specifically for treating MASH. Resmetirom, a thyroid hormone receptor-β agonist, reduced intracellular triglyceride accumulation in Fa2N-4 cells, but not in HepG2 cells, and this was accompanied by induction of mitochondrial glycolysis and oxidative phosphorylation pathways. Conclusions: Together, 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.
