Multiparametric and Pathomimetic In Vitro Platform Mimicking Human Hepatic Steatosis, Lipotoxicity, and Associated Metabolic Stress.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by hepatocellular lipid accumulation and lipotoxicity. However, there are currently limited well-characterized in vitro models that mimic these early pathogenic mechanisms in terms of molecular and metabolomic features. Here, we developed a customized fatty acid conjugate that facilitates efficient lipid accumulation in the cultured hepatocytes without loss of viability. This conjugate reflects the prominent fatty acids found in the serum of patients with chronic metabolic syndrome, MASLD. This conjugate consistently induces the formation of steatotic bodies in HepG2 cells within a short exposure. The model was validated using a comprehensive analytical framework that measured inflammatory markers (e.g., iNOS, TNF-α, IL-6, IL-1β, and MCP-1), alongside intracellular triglyceride and cholesterol accumulation, intracellular oxidative stress, and lipid peroxidation. The lipid conjugate significantly alters hepatocellular metabolism, as observed by intracellular untargeted metabolomic profiling. The mRNA expression of hepatocellular lipogenic genes (e.g., CD26, SERBP, FASN, SCD5, DAGT1/2) was elevated, indicating impaired lipid homeostasis under lipotoxic conditions. Pathway analysis revealed enrichment of metabolic pathways related to steroid and bile acid biosynthesis, as well as glycerolipid metabolism, consistent with compensatory lipid-metabolism responses. These coordinated metabolic changes confirm that the early MASLD-like metabolic phenotype is recapitulated in this in vitro model. Collectively, these results confirm this fatty acid conjugate as a physiologically relevant in vitro model for MASLD, suitable for therapeutic screening and mechanistic research.