Human in vitro hepatic models for steatosis: a harmonized comparison for studying drug metabolism

Metabolic dysfunction-associated steatotic liver disease (MASLD) involves metabolic, inflammatory, and fibrotic processes which are not fully reproducible in vitro. However, MASLD is increasingly investigated using human-relevant in vitro models. This study models the steatosis component using a harmonized free fatty acid (FFA; oleic/palmitic acid) exposure across four human hepatic platforms: 2D primary human hepatocyte (PHH) suspension cultures, 2.5D PHH sandwich cultures, 3D PHH spheroids, and HepaRG/LX-2/HUVEC micro-organoids. Steatosis and function were assessed via lipid imaging, albumin/LDH release, mRNA profiling of drug metabolizing enzymes (DMEs) and transporters using Multiplex technology, and LC-MS/MS-based CYP1A2/CYP2C9 activity. Untreated cultures served as controls; FFA-treated cultures were designated "FFA-induced steatotic", reflecting lipid accumulation rather than full MASLD. PHH suspensions, classified a priori as putatively healthy or MASH based on medical records of the donor, showed rapid functional decline and substantial donor variability. In contrast, 2.5D sandwiches, 3D spheroids, and micro-organoids remained stable for 10-21 days and developed robust FFA-induced steatotic. Spheroids and micro-organoids maintained CYP activity and exhibited distinct metabolic responses; micro-organoids partially recovered after FFA-stimulus withdrawal. Cross-platform differences arise from both biological responses and model-intrinsic characteristics. 3D spheroids and micro-organoids were most suitable for long-term metabolic remodeling studies, while 2.5D sandwich cultures provided a practical system for transporter and enzyme induction research. Despite functional instability, 2D suspensions retained value for rapid short-term screening. These findings support use-case-driven selection of hepatic in vitro models for toxicology and ADME applications under FFA-induced steatosis.