A collagen hydrogel-based 3D Heparg model for enhanced hepatocyte function and assessment of cholestatic drug-induced liver injury.

Drug-induced liver injury (DILI) is a major clinical and regulatory challenge, with cholestasis representing a frequent mechanism of hepatotoxicity. Conventional two-dimensional (2D) hepatocyte cultures often fail to predict such effects, underscoring the need for advanced models that better replicate liver architecture and function. In this study, a three-dimensional (3D) model was developed by encapsulating HepaRG cells within type I collagen hydrogels to enhance functional maturation and reduce differentiation time compared to standard 2D cultures. The 3D HepaRG model maintained long-term viability and exhibited improved hepatic functions, including urea and albumin production, xenobiotic metabolism, and antioxidant defense, particularly after 14 days of culture with a reduced amount of DMSO (1%) compared to the standard 2D protocol. When incubated with cholestatic antibiotics, such as cloxacillin, flucloxacillin, and nafcillin, the 3D model reproduced cholestatic DILI features, showing increased cytotoxicity in the presence of bile acids and after prolonged exposure. Transcriptomic profiling revealed dose-dependent modulation of genes involved in bile acid homeostasis. A more in-depth analysis of cloxacillin-toxicity using RNA-seq revealed an altered expression of genes involved in bile acid metabolism, oxidative stress, and endoplasmic reticulum stress. Functionally, cloxacillin induced oxidative stress, mitochondrial dysfunction, and activation of apoptotic signaling pathways. Overall, the collagen hydrogel-based 3D HepaRG model provided a robust and physiologically relevant platform for investigating antibiotic-induced cholestatic DILI and its underlying mechanisms, offering improved predictivity over traditional 2D systems and valuable potential for preclinical drug safety assessment.