Microscale 3D Liver Bioreactor for In Vitro Hepatotoxicity Testing under Perfusion Conditions

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.

Inter-individual differences in the susceptibility of primary human hepatocytes towards drug-induced cholestasis are compound and time dependent

“…In a perfusion bioreactor, PHH spheroids formed functional bile canalicular networks, displayed stable CYP expression during 2 weeks of culture, and were in a long-term setting responsive towards 2 CYP inducers (260). In another study, responsiveness to drug toxicity of PHH spheroids cultured in a microscale bioreactor was shown using APAP as a model hepatotoxin (261). In these perfusion bioreactor set-ups, in vivo hemodynamics can be mimicked, physiological parameters (e.g.…”

Conceptual Design of Micro-Bioreactors and Organ-on-Chips for Studies of Cell Cultures

“…Other more specified user needs for an MBR related to the design objective could, for example, involve longevity of use of the MBR, generation of gradients in pO 2 and nutrients in the device, transformation rates of the cell culture, scalability of units, and flow-through rates. Such needs are highlighted by other authors in this special issue, e.g., by Wrzesinski & Fey [ 24 ] on oxygen supply to liver cells in an MBR, by Freyer et al [ 23 ] on 3D microstructure of liver cell MBRs, and by Fernandez et al [ 37 ] on oxygen measurement in MBRs.…”

“…Successfully applied, this supports the investigation of safety pharmacology and toxicology, and, when possible, the efficacy of drug compounds [ 7 , 8 , 9 , 10 , 21 , 22 ]. If OoCs generates reliable results, it will be important to control the cells’ survival in the artificial milieu of small-scale OoC; their stability relates to the design of liver devices [ 23 , 24 , 25 ]. However, other organs [ 26 , 27 , 28 , 29 ] have attracted almost the same interest, e.g., pancreas, eye, cartilage, heart, and lung cells [ 28 , 29 ], as well as combinations of several organ cell types on the same chip [ 30 , 31 ].…”

<p>Metabolism of remimazolam in primary human hepatocytes during continuous long-term infusion in a 3-D bioreactor system</p>

“…In a further approach, the bioreactor was downscaled to analytical-scale variants to enable its application for pharmacological in vitro studies 10,11. The suitability of the bioreactor technology to assess human-liver drug metabolism and toxicity in vitro has been shown using different model drugs, such as paracetamol or diclofenac 12–14…”