Characterizing the reproducibility in using a liver microphysiological system for assaying drug toxicity, metabolism, and accumulation.

Liver microphysiological systems (MPSs) are promising models for predicting hepatic drug effects. Yet, after a decade since their introduction, MPSs are not routinely used in drug development due to lack of criteria for ensuring reproducibility of results. We characterized the feasibility of a liver MPS to yield reproducible outcomes of experiments assaying drug toxicity, metabolism, and intracellular accumulation. The ability of the liver MPS to reproduce hepatotoxic effects was assessed using trovafloxacin, which increased lactate dehydrogenase (LDH) release and reduced cytochrome P450 3A4 (CYP3A4) activity. These observations were made in two test sites and with different batches of Kupffer cells. Upon culturing equivalent hepatocytes in the MPS, spheroids, and sandwich cultures, differences between culture formats were detected in CYP3A4 activity and albumin production. Cells in all culture formats exhibited different sensitivities to hepatotoxicant exposure. Hepatocytes in the MPS were more functionally stable than those of other culture platforms, as CYP3A4 activity and albumin secretion remained prominent for greater than 18 days in culture, whereas functional decline occurred earlier in spheroids (12 days) and sandwich cultures (7 days). The MPS was also demonstrated to be suitable for metabolism studies, where CYP3A4 activity, troglitazone metabolites, diclofenac clearance, and intracellular accumulation of chloroquine were quantified. To ensure reproducibility between studies with the MPS, the combined use of LDH and CYP3A4 assays were implemented as quality control metrics. Overall results indicated that the liver MPS can be used reproducibly in general drug evaluation applications. Study outcomes led to general considerations and recommendations for using liver MPSs. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Microphysiological systems (MPSs) have been designed to recreate organ- or tissue-specific characteristics of extracellular microenvironments that enhance the physiological relevance of cells in culture. Liver MPSs enable long-lasting and stable culture of hepatic cells by culturing them in three-dimensions and exposing them to fluid flow. WHAT QUESTION DID THIS STUDY ADDRESS? What is the functional performance relative to other cell culture platforms and the reproducibility of a liver MPS for assessing drug development and evaluation questions, such as toxicity, metabolism, and pharmacokinetics? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? The liver MPS systematically detected the toxicity of trovafloxacin. When compared with spheroids and sandwich cultures, this system had a more stable function and different sensitivity to troglitazone, tamoxifen, and digoxin. Quantifying phase II metabolism of troglitazone and intracellular accumulation of chloroquine demonstrated the potential use of the liver MPS for studying drug metabolism and pharmacokinetics. Quality control criteria for assessing chip function were key for reliably using the liver MPS. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Due to its functional robustness and physiological relevance (3D culture, cells expose to fluid flow and co-culture of different cell types), the liver MPS can, in a reproducible manner: (i) detect inflammatory-induced drug toxicity, as demonstrated with trovafloxacin, (ii) detect the toxicity of other drugs, such as troglitazone, tamoxifen, and digoxin, with different effects than those detected in spheroids and sandwich cultures, (iii) enable studies of hepatic function that rely on prolonged cellular activity, and (iv) detect phase II metabolites and drug accumulation to potentially support the interpretation of clinical data. The integration of MPSs in drug development will be facilitated by careful evaluation of performance and reproducibility as performed in this study.

Three-dimensional perfused human in vitro model of non-alcoholic fatty liver disease.

AIM: To develop a human in vitro model of non-alcoholic fatty liver disease (NAFLD), utilising primary hepatocytes cultured in a three-dimensional (3D) perfused platform. METHODS: Fat and lean culture media were developed to directly investigate the effects of fat loading on primary hepatocytes cultured in a 3D perfused culture system. Oil Red O staining was used to measure fat loading in the hepatocytes and the consumption of free fatty acids (FFA) from culture medium was monitored. Hepatic functions, gene expression profiles and adipokine release were compared for cells cultured in fat and lean conditions. To determine if fat loading in the system could be modulated hepatocytes were treated with known anti-steatotic compounds. RESULTS: Hepatocytes cultured in fat medium were found to accumulate three times more fat than lean cells and fat uptake was continuous over a 14-d culture. Fat loading of hepatocytes did not cause any hepatotoxicity and significantly increased albumin production. Numerous adipokines were expressed by fatty cells and genes associated with NAFLD and liver disease were upregulated including: Insulin-like growth factor-binding protein 1, fatty acid-binding protein 3 and CYP7A1. The metabolic activity of hepatocytes cultured in fatty conditions was found to be impaired and the activities of CYP3A4 and CYP2C9 were significantly reduced, similar to observations made in NAFLD patients. The utility of the model for drug screening was demonstrated by measuring the effects of known anti-steatotic compounds. Hepatocytes, cultured under fatty conditions and treated with metformin, had a reduced cellular fat content compared to untreated controls and consumed less FFA from cell culture medium. CONCLUSION: The 3D in vitro NAFLD model recapitulates many features of clinical NAFLD and is an ideal tool for analysing the efficacy of anti-steatotic compounds.