Long‐term maintenance of HepaRG cells in serum‐free conditions and application in a repeated dose study

Abstract

Chronic repeated-dose toxicity studies are still carried out on animals and often do not correlate with the effects in human beings mainly due to species-specific differences in biotransformation. The human hepatoma cell line HepaRG has been used for human relevant toxicity assessment. However, HepaRG cells are commonly maintained in serum containing medium which limits their use in 'omics'-based toxicology. In this study, we compared the maintenance of HepaRG cells in standard serum-supplemented and serum-free conditions. Viability and Cytochrome P450 (CYP) activity during long-term cultivation were assessed. Liver-specific albumin and urea production was measured. The extracellular metabolome (amino acids, glucose, lactate and pyruvate) was measured to compare different cultivation conditions using metabolic flux analysis. Although metabolic flux analysis reveals differences in certain parts of the metabolism, e.g. production of urea, the overall metabolism of serum-free and serum-supplemented cultured HepaRG cells is similar. We conclude that HepaRG cells can be maintained in optimized serum-free conditions for 30 days without viability change and with high CYP activity. We also tested the acute (24 h) and long-term repeated-dose (7 doses, every second day) toxicity of valproic acid. We calculated an EC50 value of 1.4 mM after repeated exposure which is close to the cmax value for valproic acid. Maintenance of HepaRG cells in serum-free conditions opens up the opportunity for the use of these cells in human long-term repeated-dose hepatotoxicity studies and for application in systems toxicology.

Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches

“…The day before desomorphine exposure, medium was changed to Williams E Medium (serum-free) supplemented with additive ADD650, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.5 % DMSO, 10 ng/mL human hepatocyte growth factor, and 2 ng/mL epidermal growth factor [15,16]. HepG2 and HepaRG cells were exposed to desomorphine concentrations of 0.01, 0.1, or 1 mM for 3 or 24 h. All given concentrations are the final concentrations.…”

Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell–Derived Hepatocytes, HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for Drug-Induced Liver Injury

“…Importantly, however, most hepatic cell lines lack relevant hepatic phenotypes, due to limited expression of drug-metabolizing enzymes, which makes extrapolation of the results to humans questionable (Gerets et al, 2012). The HepaRG cell line presents a cell system that has been reported to be phenotypically stable, thus allowing long-term culture and repeated-exposure studies (Klein et al, 2014). Induced pluripotent stem cells (iPSCs) have the advantage that they can be generated from any human cell type, which allows the retrospective acquisition of cellular material from individuals with a particular genotype or phenotype of interest, such as an idiosyncratic adverse drug reaction, providing an interesting model for deciphering mechanisms of genetically determined DILI reactions (Kia et al, 2013).…”

Innovation for hepatotoxicity in vitro research models: A review

“…Serum‐free medium maintains CYP450 at a higher level than serum‐containing medium does. HepaRG cells cultured in serum‐free medium can maintain viability and CYP450 activity for more than 30 days (Catania, McGarrigle, Rittenhouse‐Olson, & Olson, ; Klein, Mueller, Schevchenko, & Noor, ). Nakamura et al found, in a serum‐free culture system, that iPSCs/HLCs and ESCs/HLCs expressed a high level of hepatocyte‐specific proteins, such as albumin, and CYP3A4 (Nakamura et al, ).…”