A 3D bioengineered human liver for the study of acute and chronic drug-induced hepatotoxicity and fibrosis.

The liver is frequently exposed to acute and chronic insults that disrupt its function, leading to inflammation, fibrosis, and serious conditions like cirrhosis and hepatocellular carcinoma. Fibrosis, driven by the activation of hepatic stellate cells is a key feature of chronic liver damage. Traditional 2D liver models face limitations in maintaining liver functions and accurately replicating chronic conditions like fibrosis. In response to these needs, 3D models have emerged as more physiologically relevant platforms for studying liver disease and drug testing. However, most existing 3D models still lack chronic injury features or immune elements. In this study, we developed a 3D human-liver model to investigate both acute and chronic drug-induced liver injury. The model consists of encapsulated human hepatocytes (HepaRG) and hepatic stellate cells (LX-2) within a gelatin methacryloyl (GelMA) and carboxymethyl cellulose methacrylate matrix. Over a 30-day culture period, the 3D liver constructs maintained physiologically relevant functions, including albumin secretion and cytochrome P450 activity, which are lost rapidly in conventional 2D models. The model enabled reliable simulation of low-level, long-term, and high-dose, short-term damage by administering lipopolysaccharide and Paracetamol. Chronic liver injury was characterized by progressive fibrosis, elevated expression of COL1A1, and persistent hyperammonemia. In contrast, acute models showed significant but transient injury. Moreover, dexamethasone treatment successfully reversed fibrotic markers and restored hepatocyte functionality, indicating the model's predictive power for evaluating potential therapies. Incorporating human monocytes (THP-1) allowed investigation of the immune response and macrophage activation, which are critical contributors to liver disease pathogenesis. Overall, this 3D liver model offers a physiologically relevant and versatile platform for studying complex multi-cellular interactions under acute and chronic injury. It has broad implications for drug safety screening, disease modeling, and personalized therapy. The tri-culture design extends the model's capacity to elucidate immune-driven hepatic pathology.