High-throughput toxicity testing of chemicals and mixtures in organotypic multi-cellular cultures of primary human hepatic cells

High-throughput screening (HTS) of liver toxicants can bridge the gap in understanding adverse effects of chemicals on humans. Toxicity testing of mixtures is time consuming and expensive, since the number of possible combinations increases exponentially with the number of chemicals. The combination of organotypic culture models (OCMs) and HTS assays can lead to the rapidly evaluation of chemical toxicity in a cost and time-effective manner while prioritizing chemicals that warrant additional investigation. We describe the design, assembly and toxicant response of multi-cellular hepatic organotypic culture models comprised of primary human or rat cells assembled in 96-well plates (denoted as μOCMs). These models were assembled using automated procedures that did not affect hepatocyte function or viability, rendering them ideal for large-scale toxicity evaluations. Rat μOCMs were assembled to obtain insights into deviations from human toxicity. Four test chemicals (acetaminophen, ethanol, isoniazid, and perfluorooctanoic acid) were added to the μOCMs individually or in mixtures. HTS assays were utilized to measure cell death, apoptosis, glutathione depletion, mitochondrial membrane damage, and cytochrome P450 2E1 activity. The μOCMs exhibited increased toxicant sensitivity compared to hepatocyte sandwich cultures. Synergistic and non-synergistic interactions were observed when the toxicants were added as mixtures. Specifically, chemical interactions in the μOCMs were manifested by changes in apoptosis and decreased glutathione. The μOCMs accurately predicted hepatotoxicity for individual and mixtures of toxicants, demonstrating their potential for large-scale toxicity evaluations in the future.