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bioRxiv · 10.64898/2026.03.16.712092

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

Cross, E.·Westcott, F.·Smith, K.·Nagarajan, S. R.·Sanna, F.·Dennis, K. M.·Hodson, L.
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Abstract

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is challenging to study in vivo in humans and in vitro models are limited. Although primary human hepatocytes (PHHs) are considered the gold-standard, immortalized hepatic cell lines are utilised due to scalability. This study compared the metabolic responses of PHHs with our Huh7-based model cultured in physiologically-relevant fatty acid (FA) mixtures. MethodsPHH and Huh7 cells were treated with 2% human serum, sugars and FAs enriched in either unsaturated (OPLA) or saturated (POLA) FAs for 4 or 7 days, respectively. Stable isotope tracers investigated basal metabolic changes in response to treatment. Cell viability, media biochemistry, intracellular metabolism, lipid droplet morphology and gene expression were quantified. ResultsHuh7 cells had greater viability than PHHs, while NEFA uptake and triglyceride secretion were similar. OPLA and POLA increased large lipid droplets in Huh7 cells, whereas only OPLA produced comparable effects in PHHs. Despite higher baseline TG in PHHs, both models showed similar lipid composition, de novo lipogenic responses, and glycogen levels. Compared to Huh7 cells, PHHs exhibited higher 3-hydroxybutyrate, lower lactate, reduced glucose uptake, and donor-dependent transcriptomic variability. ConclusionsHuh7 cells are metabolically adaptable and when cultured in physiologically-relevant media, produce metabolic readouts similar PHH cells.

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Cross, E., Westcott, F., Smith, K., Nagarajan, S. R., Sanna, F., Dennis, K. M., Hodson, L.. 2026-03-18. Physiologically relevant media are associated with overlapping metabolic responses in primary human hepatocytes and Huh7 cells. https://doi.org/10.64898/2026.03.16.712092

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