Rapid Volumetric Bioprinting Coupled with Dynamic Perfusion Enhances Human Hepatic Organoid Toxicity Testing

Drug-induced liver injury (DILI) remains a major cause of acute liver failure and drug withdrawal from the market. Recently developed three-dimensional (3D) hepatic in vitro systems exhibit improved functionality and drug sensitivity compared with conventional two-dimensional cultures. These 3D models range from simple physiologic-like culture systems to advanced bioreactors with dynamic flow to provide sufficient nutrients and consistent drug exposure. However, whether dynamic perfusion improves sensitivity and reproducibility of hepatotoxicity testing remains unclear. Here, we developed a tailor-made perfusion platform to support volumetric bioprinted hepatic constructs for hepatotoxicity testing. The constructs consist of intrahepatic cholangiocyte organoids (ICOs) differentiated towards hepatocyte lineage and embedded in a gelatin methacryloyl bioresin. For toxicity evaluation, the hepatocyte-like ICO constructs were exposed to prolonged subtoxic acetaminophen treatment (10 mM, 7 days). The perfusion system effectively maintained and enhanced hepatocyte differentiation, evidenced by upregulated hepatic markers under perfused conditions compared to static controls. Testing of acetaminophen hepatotoxicity revealed that the perfused constructs displayed elevated cellular injury, with markedly higher liver injury markers relative to controls. Collectively, this study demonstrates the successful application of perfusion-based 3D model culture and highlights its potential as a more physiological platform for hepatotoxicity risk assessment in drug discovery and regenerative medicine.