Development of a human liver microphysiological co-culture system for higher throughput chemical safety assessment.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites and exhibit altered toxicity compared to their parent compounds. This paper describes a two-chamber liver-organ co-culture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This two-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a two-dimensional (2D) cell mono-layer. Culture medium and compounds freely diffuse between the two chambers. Human differentiated HepaRGTM liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 (CYP3A4) enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this co-culture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ co-culture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals, to better recapitulate the biological effects and potential toxicity of human exposures.

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