Enhanced hepatotoxicity assessment through encapsulated HepG2 spheroids in gelatin hydrogel Matrices: Bridging the gap from 2D to 3D culture.

Conventional 2D drug screening often fails to predict clinical outcomes accurately. We present an innovative approach to enhance hepatotoxicity assessment by encapsulating HepG2 spheroids in gelatin hydrogel matrices with varying mechanical properties. Encapsulated spheroids exhibit sustained liver-specific functionality, enhanced drug-metabolizing enzyme expression, and increased drug sensitivity compared to 2D cultures. The platform detects critical variations in drug response, with significant differences in IC50 values between 2D and spheroid cultures ranging from 1.3-fold to > 13-fold, particularly for acetaminophen. Furthermore, drug-metabolizing enzyme expression varies across hydrogel concentrations, suggesting a role for matrix mechanical properties in modulating liver cell function. This novel spheroid-hydrogel platform offers a transformative approach to hepatotoxicity assessment, providing enhanced sensitivity, improved prediction, and a more physiologically relevant environment. Adopting such advanced in vitro models can accelerate drug development, reduce animal testing, and contribute to improved patient safety and clinical outcomes.