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Comparable Transcriptomic Profiles of HepaRG and HuH7 Cells Cultured in Chemically Defined and Serum-Based Media Using Primary Human Hepatocytes as Reference

ABSTRACT: The composition of culture medium is a major, yet frequently undercontrolled, determinant of hepatic cell state in vitro. For decades, fetal bovine serum (FBS) has been routinely incorporated into liver cell culture. Its undefined and lot-to-lot variable composition can, however, confound cell identity and experimental reproducibility. Serum-free, chemically defined media (CDM) represent an alternative approach that can improve standardization, but the consequences of transitioning from FBS-supplemented media (FBS-SM) to CDM remain insufficiently characterized in hepatic models, particularly with respect to metabolic and detoxification programs that govern xenobiotic handling and hepatotoxicity readouts. Here, we systematically assessed how replacing FBS-SM with CDM remodels transcriptomic profiles in two widely used human hepatic cell lines (HepaRG and HuH7 cells) and compared the results to that obtained in primary human hepatocytes (PHH). Global transcriptomic analyses indicated that cell type was the primary driver of variance, whereas medium induced a model-dependent secondary effect. Functional interpretation showed preferential enhancement of xenobiotic metabolism and transport-associated programs in HepaRG cells, while HuH7 cells responses were dominated by lipid/sterol homeostasis and stress-linked processes. Benchmarking against PHH based on hepatic identity and detoxification gene panels further supported improved PHH alignment for HepaRG cells under CDM compared to cultures with FBS-SM, with limited improvement for HuH7 cells. Collectively, these findings address a key knowledge gap by defining how FBS-SM and CDM impact the transcriptomic profiles of HepaRG and HuH7 cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE324107 | GEO | 2026/07/21

REPOSITORIES: GEO

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Project description:The human hepatic HepaRG cell line is often considered as the closest surrogate to primary culture of normal human hepatocytes (PHH) for toxicity studies. However, differentiated HepaRGcells express very low levels of the cytochrome P450 2D6 (CYP2D6) protein, which is essential for the biotransformation of nearly 25% of drugs on the market. To overcome this limitation, infection of progenitor HepaRG cells were performed using lentiviral particles containing a transgene encoding a single mRNA translated into a polypeptide undergoing proteolytic cleavage via the T2A peptide to produce both human CYP2D6 and GFP proteins. Differentiated HepaRG cells transduced with this lentivirus stably expressed GFP and catalytically active CYP2D6 enzyme at levels close to those found in PHH with high CYP2D6 activities. Using the CYP2D6 transgenic HepaRG cells, we showed that tramadol was metabolized into both, N- and O-desmethyl tramadol as observed in human serum in contrast with the production of N-desmethyl tramadol only in parental HepaRG cells via the CYP3A4 activity. Similarly, after perhexiline (PHX) treatments, higher IC50 were found in CYP2D6 expressing HepaRG cells associated to lower mitochondrial damages compared to those found in parental cells for the same PHX concentrations. Gene profiling between parental and transgenic cells demonstrated that the CYP2D6 expressing HepaRG cells had kept their ability to proliferate and differentiate with low impact on the expression of the hepatocyte specific functions, however, we identified a limited set of genes including NXF3 and TRIM63 that were up-regulated in CYP2D6 expressing HepaRG cells. By knocking-down GFP and CYP2D6 expression using CRISP/Cas9 technology in the transgenic cell line, we demonstrated that NXF3 and TRIM63 induction was triggered by the mRNA encoding GFP and CYP2D6 but not by the lentiviral transgene inserted into the HepaRG cell genome. Together, these data confirmed that the CYP2D6 transgenic HepaRG cells represent a suitable optimized transgenic model of HepaRG cells to evaluate biotransformation and toxicity of specific compounds metabolized by CYP2D6.

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Comparable Transcriptomic Profiles of HepaRG and HuH7 Cells Cultured in Chemically Defined and Serum-Based Media Using Primary Human Hepatocytes as Reference

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