Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures

Abstract

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.

Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor

“…Induction of CYP2B6 and CYP3A4 by PK11195 in PXR-KO HepaRG Cells. HepaRG cells have been validated as a promising surrogate for HPHs, and importantly, fully differentiated HepaRG cells exhibit proper CAR cellular localization and maintain physiologically relevant metabolic capacity, which are not present in most immortalized cell models (Jackson et al, 2016). The PXR-KO HepaRG cell line obtained from Sigma-Aldrich is a newly generated cell line that does not express functional PXR (Williamson et al, 2016).…”

Human hepatocyte systems for in vitro toxicology analysis

“…Interestingly, using 38 drugs with classified DILI risk according to FDA standard and a drug concentration for cell culture experiments set at 100-fold the therapeutic maximum plasma concentration, HepaRG were shown to predict the DILI risk with a sensitivity of 87% sensitivity and 87% specificity [53]. The following disadvantages are known for HepaRG: First, the cell line was derived from an individual with poor metabolizer alleles for CYP2D6, CYP3A5 and, to a lesser extent, also CYP2C9 [50,54,55]. Next to CYP3A4, CYP2D6 is the second most important phase I enzyme metabolizing about 30 % of all approved drugs [13].…”

Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells

“…Differentiated HepaRG cells seem to have normal CAR signaling since they mimic ligand-stimulated CAR trafficking from the cytoplasm to the nucleus as observed in PHHs (Jackson et al, 2016); however, CAR expression in fully differentiated HepaRG cells is 5-to 10-fold lower compared with PHH and requires the addition of dimethylsulfoxide (DMSO) during differentiation (Aninat et al, 2006). In HepaRG cells, the high expression of drug-metabolizing enzymes and xenobiotic receptors other than CAR is also dependent on the addition of DMSO (Aninat et al, 2006;Kanebratt and Andersson, 2008).…”