Physiologically relevant media are associated with overlapping metabolic responses in primary human hepatocytes and Huh7 cells.

The initiation and progression of metabolic dysfunction-associated steatotic liver disease (MASLD) is challenging to study in vivo in humans, and robust in vitro high-fidelity disease models are limited. Although primary human hepatocytes (PHH) are often considered to be the gold standard, immortalized hepatic cell lines are often utilized because of their scalability and experimental tractability. Therefore, the aim of this study was to compare the metabolic responses of PHHs with our characterized Huh7-based model when exposed to the physiologically relevant fatty acid (FA) mixtures. PHH and Huh7 cells were treated with 2% human serum and a combination of sugar and FAs enriched in either unsaturated (OPLA) or saturated (POLA) FAs for 4 or 7 days, respectively. Stable isotope tracers were utilized to investigate basal changes in hepatocyte metabolism in response to different treatment regimes. Changes in cell viability, media biochemistry, intracellular metabolism, lipid droplet morphology, and gene expression were quantified. Huh7 cells had greater viability than PHH, while NEFA uptake and triglyceride secretion were similar. Both OPLA and POLA increased the proportion of large lipid droplets in Huh7 cells, whereas only OPLA produced comparable effects in PHH. Despite higher baseline TG in PHH, both models showed similar lipid composition, de novo lipogenic responses, and glycogen levels. Compared to Huh7 cells, PHH exhibited higher media 3-hydroxybutyrate, lower media lactate, reduced glucose uptake, and donor-dependent transcriptomic variability. Our data demonstrate that Huh7 cells are metabolically adaptable and, when cultured in physiologically relevant media, metabolic readouts are more similar to those observed in PHH cells, thus making Huh7 a potentially useful workhorse model to investigate relevant pathways that may underpin the development of MASLD. Progress in MASLD research is constrained by limited access to human liver tissue and the scarcity of robust in vitro models. Although PHHs are considered the gold standard, they can be limited by poor viability and donor-to-donor variability. Huh7 cells are metabolically flexible and can recapitulate key metabolic responses of PHH when exposed to physiologically-relevant chronic culture conditions, making them an alternative to PHH that is a scalable and reproducible model for investigating intrahepatic triglyceride (IHCTG) accumulation and MASLD progression.