Optimized Ex Vivo Human Liver Slice Culture Maintains Extended Viability and Function for Hepatotoxicity Testing

ABSTRACT Human precision‐cut liver slices (hPCLS) are widely used to study liver physiology and disease, but their short lifespan limits long‐term functional studies and drug testing. Here, we describe an optimized ex vivo culture system, hPCLS‐EV (Extended Viability), that maintains tissue viability, cellular composition, and liver‐specific functions for up to five days. Optimization combined reduced slice thickness, high oxygen partial pressure, and air‐liquid interface culture, preserving hepatocyte integrity, stromal stability, and immune cell populations. Functional assays confirmed sustained albumin secretion, glutathione content, and cytochrome P450 activity. Transcriptomic profiling revealed rapid downregulation of metabolic pathways by day 3, followed by stabilization up to day 5, with limited advanced injury or fibrosis‐related remodeling. We applied hPCLS‐EV to model drug‐induced liver injury using acetaminophen and troglitazone. The model captured compound‐specific injury patterns, including early glutathione depletion with acetaminophen and delayed oxidative stress with troglitazone. Together, these findings support optimized hPCLS‐EV as a useful ex vivo human liver model that preserves key tissue architecture and selected liver functions over an extended culture period, enabling longitudinal assessment of hepatotoxic injury. This platform may provide a useful experimental tool for preclinical hepatotoxicity testing, mechanistic studies of liver injury, and patient‐specific applications in precision medicine.