Hepatic Spheroids for Long-Term Toxicity Studies

Abstract

Abstract

Drug-induced liver injury is a leading cause of drug failure and withdrawal from the market. Current in vitro screens rely on monolayers of primary hepatocytes to predict in vivo liver injury. Hepatocyte monolayers lose their phenotype and metabolic function within days of seeding, thereby creating a defined experimental window that limits the type, nature, and predictive capacity of the assay. Previous reports have shown that spheroids of primary hepatocytes possess structural polarity, functional bile canaliculi, and long-term viability. Common methods of spheroid formation are limited in pharmaceutical screening strategies due to inherent cell loss, cell stress, novel equipment, and assay requirements. The objective of this work is to develop a rat primary hepatocyte spheroid system that can maintain long-term cell viability, phenotype, and metabolic function, allowing for repeat compound exposure. In this article, we show that primary hepatocytes compact to form stable spheroids. At a seeding density of 32 × 10³ cells per well, microscopy and ATP content analyses confirm spheroid viability through the experimental window of 14 days. Formation of bile canaliculi and albumin production indicate functionality of the spheroids. In addition, CYP3A metabolic activity was maintained and cytochrome P450 isozymes were inducible by classical inducers. Using single and repeat dosing schemes, spheroids correctly predicted toxicity of six compounds, including propranolol, rosiglitazone, cyclophosphamide, acetaminophen, diclofenac, and troglitazone. The spheroid system developed in this work shows promise as a predictive system for pharmaceutical screening and for investigating mechanisms of toxicity following a repeat dosing scheme.