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Validation of a 3-Dimensional Human Liver Microtissue Model for Long-term Hepatotoxicity Studies


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September 26, 2014

Authors: Brad Larson, Peter Banks, BioTek Instruments, Inc.,Winooski, Vermont; Stewart Hunt, InSphero, Inc., Cambridge, Massachusetts; Timothy Moeller, BioreclamationIVT, Baltimore, Maryland; Diana Long, Promega Corporation, Madison, Wisconsin

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Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of drug commonly used as analgesics and antipyretics, as well as for management of rheumatological disorders. They are one of the most highly prescribed drug families around the world, and consequently, along with antimicrobial agents, are the most frequent causes of druginduced liver injury (DILI) (Bjornsson et al., 2010). Diclofenac, in particular, frequently used to treat chronic pain and inflammatory disorders, elicited an FDA warning in 2009 concerning potential hepatic effects.

Multiple in vitro animal models have been used to determine the varied mechanisms of action (MOA) of NSAID-related hepatotoxicity. Studies using rat liver mitochondria and freshly isolated rat hepatocytes demonstrated that diphenylamine, a common NSAID structure, uncoupled oxidative phosphorylation, and decreased hepatic ATP content (Masubuchi et al., 2000). Mitochondrial permeability transition (MPT) has also been shown to be important in diclofenac-induced liver injury, as well as the role that oxidative stress plays in MPT induction (Gómez-Lechón et al., 2003). Finally, according to work done by Schmitz et al., 1992, cytochrome P450 (CYP)-related metabolic activation of the drug, and the formation of reactive metabolites is also related to diclofenac hepatotoxicity.

The combined hepatotoxic effects of these mechanisms usually occurs within weeks of therapy commencement. This emphasizes the fact that in vitro safety testing should incorporate dosing experiments that analyze the effects of a potential drug for periods reaching out to 7-14 days or longer. However, primary hepatocytes cultured in a twodimensional (2D) manner on the bottom of a microplate, standard practice for in vitro liver toxicity testing, have been shown to undergo rapid loss of differentiated function and metabolic capacity (Cheng et al., 2008), and have less complex inter-cellular and cell-matrix interactions compared to in vivo. What is required is a cell culture model where cells retain viability and function long-term, by creating a favorable environment for these interactions to reform.

Here we evaluate the suitability of 3D human liver microtissues for use in longterm toxicity studies. Primary human hepatocytes are reaggregated into functional microtissues by hanging drop technology. The microtissues demonstrate in vivo like cellcell and cell-matrix interactions and retained viability over weeks. A panel of assays was run to assess cell health and the different MOA exhibited by diclofenac using the 3D liver microtissues, in addition to primary hepatocytes cultured in 2D. Quantification of the various luminescent and fluorescent emissions via microplate reading and imaging was be carried out by a novel cell imaging multi-mode reader.