Establishment and validation of an in vitro liver model based extracellular matrix for hepatotoxicity prediction.

Abstract

IntroductionThree-dimension (3D) cell culture presents a promising alternative of drug-induced liver injury (DILI). To advance preclinical toxicology research, we developed an in vitro liver model using HepG2 for toxic evaluation.MethodsThe model was constructed based on the extracellular matrix. We assessed its long-term stability by monitoring the morphological change and anabolic capacity for 2 weeks, with functional analyses including albumin/urea production, lipid accumulation with Nile red staining, bile secretion with CLF signal, and transporter/enzyme expression including PGP, MRP2, BSEP, and CYP3A4.ResultsThe model could be maintained for at least 10 days with enhanced hepatic synthetic functions indicated by albumin and urea nitrogen. Compared with the two-dimensional (2D) cultures, 3D culture exhibited enhanced lipid accumulation and biliary excretion. Key hepatic transporters PGP, MRP2, and BSEP and the metabolic enzyme cytochrome P450 3A4 were expressed between day 5 and 7. Single-exposure induced measurable apoptosis, mitochondrial disfunction and viability assays, while repeated treatment replicated impaired bile acid transport by reduced CLF intensity, and cytotoxicity with elevated AST, ALT, and decreased survival.DiscussionOur 3D model surpasses conventional 2D systems in culture duration and functional complexity. It is suitable for DILI prediction after single- and repeated- treatment, which makes it particularly valuable for preclinical drug screening.