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Use of 3D Cultured Human iPSC-Derived Hepatocytes for Long-Term Hepatotoxicity Studies

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Brad Larson1, Peter Banks1, Coby Carlson2 and Wini Luty3
1BioTek Instruments, Inc. Winooski, VT USA
2Cellular Dynamics International Madison, WI USA
3Enzo Life Sciences Farmingdale, NY USA

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Drug-induced liver injury (DILI) or injury to the liver caused by prescription or non-prescription medications continues to be a growing public health problem and a challenge for drug development. Effects can be acute or chronic and are compounded not only by the number of new drug entities but also by the growing market for herbal and other non-traditional remedies. Most DILI is the result of unexpected responses to a particular medication or long-term chronic damage that was unseen during standard hepatotoxicity testing.

To test new drug entities for potential DILI, in vivo models remain the gold standard. However, these studies are costly, time-consuming and more importantly, rather poor predictors of human toxicity due to the incorporation of mainly murine hepatocytes. Consequently, in vitro screens using primary hepatocytes are less costly, reduce animal exposure, and are more amenable to higher-throughput platforms. However, limitations such as high inter-individual variability, finite batch sizes and changes in cell morphology, as well as liver specific functions during long-term culture are challenging this model. Human induced pluripotent stem cells (iPSC)-derived hepatocytes, by comparison, are a promising in vitro alternative to in vivo models by demonstrating primary tissue-like phenotype, high levels of consistency and unlimited availability.

When performing toxicity studies, hepatocytes are repeatedly dosed with varying concentrations of a potential drug over multiple days to assess any cumulative effects. This poses particular challenges when incorporating two-dimensional (2D) cell culture of hepatocytes due to the fact that the cells rapidly dedifferentiate and lose metabolic activity when cultured in this manner. Three-dimensional (3D) cell culture models exist that allow cells to aggregate and retain the functionality and communication networks found in vivo. The favorable environment created by the 3D culture model then allows long-term dosing experiments to be performed that accurately analyze a potential drug’s cumulative effects.

Here we demonstrate the suitability of 3D cultured human iPSC-derived hepatocytes for use in hepatotoxicity studies. Hepatocyte spheroids were exposed to multiple concentrations of the DILI category I and III drugs tolcapone, acetaminophen, and mitomycin C. Direct image-based assessment of hepatocyte mitochondrial health, after short-term and long-term exposure to the drugs, was performed. Comparisons were also made to iPSC-derived hepatocytes cultured in 2D.

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