3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies

Mueller, Daniel; Kramer, Lisa A.; Hoffmann, Esther; Klein, Sebastian G.; Fatima, Nosheen

doi:10.1016/j.tiv.2013.06.024

Cited by 139 publications

(113 citation statements)

References 61 publications

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“…This substrate worked well with the rabbit CYP3A6 enzyme, providing further evidence of the functional similarity of the 2 enzymes. The RLU values obtained for all 3 CYP activities in rabbit hepatocytes are very comparable with RLU values found in human hepatocytes HepG2 (Yueh et al, 2005) and HepaRG (Mueller et al, 2014), and in freshly isolated and cryoplateable hepatocytes (Moeller et al, 2011). The Luciferin-PFBE substrate did not produce detectable signal with the rabbit CYP3A6 enzyme.…”

Section: Discussionsupporting

confidence: 74%

Changes in cytochrome P450 gene expression and enzyme activity induced by xenobiotics in rabbits in vivo and in vitro

et al. 2017

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As considerable inter-species differences exist in xenobiotic metabolism, developing new pharmaceutical therapies for use in different species is fraught with difficulties. For this reason, very few medicines have been registered for use in rabbits, despite their importance in inter alia meat and fur production. We have developed a rapid and sensitive screening system for drug safety in rabbits based on cytochrome P450 enzyme assays, specifically CYP1A1, CYP1A2 and CYP3A6, employing an adaptation of the luciferin-based clinical assay currently used in human drug screening. Short-term (4-h) cultured rabbit primary hepatocytes were treated with a cytochrome inducer (phenobarbital) and 2 inhibitors (alphanaphthoflavone and ketoconazole). In parallel, and to provide verification, New Zealand white rabbits were dosed with 80 mg/kg phenobarbital or 40 mg/kg ketoconazole for 3 d. Ketoconazole significantly increased CYP3A6 gene expression and decreased CYP3A6 activity both in vitro and in vivo. CYP1A1 activity was decreased by ketoconazole in vitro and increased in vivo. This is the first report of the inducer effect of ketoconazole on rabbit cytochrome isoenzymes in vivo. Our data support the use of a luciferin-based assay in short-term primary hepatocytes as an appropriate tool for xenobiotic metabolism assays and short-term toxicity testing in rabbits.

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Section: Discussionsupporting

confidence: 74%

Changes in cytochrome P450 gene expression and enzyme activity induced by xenobiotics in rabbits in vivo and in vitro

et al. 2017

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“…Future work may include testing our cell and assay system with more DILI-positive and -negative compounds, although the number of compounds tested is on par with other research in this area, where an average of eight compounds are tested per experiment. 18,19,24,26,[32][33][34][35][36][37][38] Interlaboratory comparison between the few hepatotoxicity studies conducted on 3D HepaRGs and 3D primary hepatocytes revealed similarities and differences in the reported IC 50 values ( Table 3). While the reported IC 50 values for tetracycline were similar in the 3D HepaRG studies, the amiodarone and rosiglitazone results varied, with our 3D HepaRG cells responding more sensitively to the liver toxins by one to two orders of magnitude than those of Mueller et al 36 and Gunness et al 33 The differing IC 50 values for rosiglitazone and amiodarone may be due to the spheroid generation method (e.g., hanging drop method, U-bottom, additive manufacturing, and biomaterial scaffolding), spheroid maturity, cell culture conditions, and viability endpoint measurement (e.g., ATP depletion and albumin production).…”

Section: Discussionmentioning

confidence: 99%

An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3D

Ott¹,

Ramachandran²,

Stehno‐Bittel

2017

SLAS Discovery

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Drug-induced liver injury (DILI) and drug-drug interactions (DDIs) are concerns when developing safe and efficacious compounds. We have developed an automated multiplex assay to detect hepatotoxicity (i.e., ATP depletion) and metabolism (i.e., cytochrome P450 1A [CYP1A] and cytochrome P450 3A4 [CYP3A4] enzyme activity) in two-dimensional (2D) and three-dimensional (3D) cell cultures. HepaRG cells were cultured in our proprietary micromold plates and produced spheroids. HepaRG cells, in 2D or 3D, expressed liver-specific proteins throughout the culture period, although 3D cultures consistently exhibited higher albumin secretion and CYP1A/CYP3A4 enzyme activity than 2D cultures. Once the spheroid hepatic quality was assessed, 2D and 3D HepaRGs were challenged to a panel of DILI-and CYP-inducing compounds for 7 days. The 3D HepaRG model had a 70% sensitivity to liver toxins at 7 days, while the 2D model had a 60% sensitivity. In both the 2D and 3D HepaRG models, 83% of compounds were predicted to be CYP inducers after 7 days of compound exposure. Combined, our results demonstrate that an automated multiplexed liver spheroid system is a promising cell-based method to evaluate DILI and DDI for early-stage drug discovery.

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“…Recent reports have used HepaRG cells in three-dimensional culture configurations and observed similar increases in metabolic capacity/longevity. However, these models can be complex to generate, highly variable, and difficult to normalize (Malinen et al, 2014;Mueller et al, 2014). Overlaying cryoHepaRG may provide a solution for screening and high-content imaging that obviates the need for confocal microscopy by creating a more coplanar environment.…”

Section: Discussionmentioning

confidence: 99%

Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures

Jackson

Chamberlain

et al. 2016

Drug Metabolism and Disposition

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Over the last decade HepaRG cells have emerged as a promising alternative to primary human hepatocytes (PHH) and have been featured in over 300 research publications. Most of these reports employed freshly differentiated HepaRG cells that require timeconsuming culture (∼28 days) for full differentiation. Recently, a cryopreserved, predifferentiated format of HepaRG cells (termed here "cryo-HepaRG") has emerged as a new model that improves global availability and experimental flexibility; however, it is largely unknown whether HepaRG cells in this format fully retain their hepatic characteristics. Therefore, we systematically investigated the hepatocyte functionality of cryo-HepaRG cultures in context with the range of interindividual variation observed with PHH in both sandwich-culture and suspension formats. These evaluations uncovered a novel adaptation period for the cryo-HepaRG format and demonstrated the impact of extracellular matrix on cryo-HepaRG functionality. Pharmacologically important drug-metabolizing alleles were genotyped in HepaRG cells and poor metabolizer alleles for CYP2D6, CYP2C9, and CYP3A5 were identified and consistent with higher frequency alleles found in individuals of Caucasian decent. We observed liver enzyme inducibility with aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor activators comparable to that of sandwich-cultured PHH. Finally, we show for the first time that cryo-HepaRG supports proper CAR cytosolic sequestration and translocation to hepatocyte nuclei in response to phenobarbital treatment. Taken together, these data reveal important considerations for the use of this cell model and demonstrate that cryo-HepaRG are suitable for metabolism and toxicology screening.

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3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies

Cited by 139 publications

References 61 publications

Changes in cytochrome P450 gene expression and enzyme activity induced by xenobiotics in rabbits in vivo and in vitro

Changes in cytochrome P450 gene expression and enzyme activity induced by xenobiotics in rabbits in vivo and in vitro

An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3D

Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures

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