Abstract
The accurate prediction of hepatotoxicity demands validated human in vitro models that can close the gap between preclinical animal studies and clinical trials. In this study we investigated the response of primary human liver cells to toxic drug exposure in a perfused microscale 3D liver bioreactor. The cellularized bioreactors were treated with 5, 10, or 30 mM acetaminophen (APAP) used as a reference substance. Lactate production significantly decreased upon treatment with 30 mM APAP (p < 0.05) and ammonia release significantly increased in bioreactors treated with 10 or 30 mM APAP (p < 0.0001), indicating APAP-induced dose-dependent toxicity. The release of prostaglandin E2 showed a significant increase at 30 mM APAP (p < 0.05), suggesting an inflammatory reaction towards enhanced cellular stress. The expression of genes involved in drug metabolism, antioxidant reactions, urea synthesis, and apoptosis was differentially influenced by APAP exposure. Histological examinations revealed that primary human liver cells in untreated control bioreactors were reorganized in tissue-like cell aggregates. These aggregates were partly disintegrated upon APAP treatment, lacking expression of hepatocyte-specific proteins and transporters. In conclusion, our results validate the suitability of the microscale 3D liver bioreactor to detect hepatotoxic effects of drugs in vitro under perfusion conditions.
Highlights
The evaluation of hepatotoxicity of pharmaceutical substances is one major aspect of drug development
Clinical chemistry parameters revealed a dose-dependent effect of APAP on metabolic functions of primary human liver cells maintained in perfused microscale bioreactors (Figure 3)
Our observations emphasize the potential of clinical chemistry parameters, such as lactate production and ammonia release, as sensitive parameters for monitoring dose-dependent hepatotoxic effects throughout the culture period
Summary
The evaluation of hepatotoxicity of pharmaceutical substances is one major aspect of drug development. Conventional 2D models using primary human hepatocytes are impeded by a rapid decrease of hepatic function and by cell dedifferentiation [5] This phenomenon is partly due to loss of the original 3D architecture of the organ, which is characterized by organ-specific cell–cell and cell–extracellular matrix contacts. Perfused culture models enable a constant exposure to test compounds with simultaneous removal of metabolites, in contrast to static 2D cultures with discontinuous medium exchange. In this context, microfluidic culture systems gain increasing importance, since they allow minimization of the amounts of cells and reagents needed while providing characteristics of 3D cultures with physiological cell arrangement [12,13]
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