Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System
Open accessAbstract
HepG2 cells are an inexpensive hepatocyte model that can be used for repeated experiments, but HepG2 cells do not express major cytochrome P450s (CYPs) and UDP glucuronosyltransferase family 1 member A1 (UGT1A1). In this study, we established CYP3A4–POR–UGT1A1–CYP1A2–CYP2C19–CYP2C9–CYP2D6 (CYPs–UGT1A1) knock-in (KI)-HepG2 cells using a PITCh system to evaluate whether they could be a new hepatocyte model for pharmaceutical studies. To evaluate whether CYPs–UGT1A1 KI-HepG2 cells express and function with CYPs and UGT1A1, gene expression levels of CYPs and UGT1A1 were analyzed by using real-time PCR, and metabolites of CYPs or UGT1A1 substrates were quantified by HPLC. The expression levels of CYPs and UGT1A1 in the CYPs–UGT1A1 KI-HepG2 cells were comparable to those in primary human hepatocytes (PHHs) cultured for 48 h. The CYPs and UGT1A1 activity levels in the CYPs–UGT1A1 KI-HepG2 cells were much higher than those in the wild-type (WT)-HepG2 cells. These results suggest that the CYPs–UGT1A1 KI-HepG2 cells expressed functional CYPs and UGT1A1. We also confirmed that the CYPs–UGT1A1 KI-HepG2 cells were more sensitive to drug-induced liver toxicity than the WT-HepG2 cells. CYPs–UGT1A1 KI-HepG2 cells could be used to predict drug metabolism and drug-induced liver toxicity, and they promise to be a helpful new hepatocyte model for drug discovery research.
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More complex knock-in/CRISPR-derived HepG2 lines simultaneously expressing CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, POR, and UGT1A1 have been developed; in such cells, the activity of the corresponding enzymes increased significantly and was maintained in later passages, making them useful for specific tasks in metabolic research.
“More complex knock-in/CRISPR-derived HepG2 lines have also been developed, simultaneously expressing CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, POR, and UGT1A1. In such cells, the activity of the corresponding enzymes increased significantly and was maintained in later passages, making them useful for specific tasks in metabolic research [164].”
From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [164]
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- Authors
- Negoro, R., Tasaka, M., Deguchi, S., Takayama, K., & Fujita, T.
- Affiliations
- Ritsumeikan University (JP)
- Kyoto University (JP)
- Corresponding author
- Negoro, Ryosuke
- Venue
- Cells
- Publisher
- MDPI AG
- Published
- 2022-05-18
- Volume
- 11
- Issue
- 10
- Pages
- 1677
- ISSN
- 2073-4409 (ISSN-L)
- PMC
- PMC9140068
- OpenAlex
- W4280595994
- Cited by
- 16
- References
- 53
- Citations by year
- 2026: 7 · 2025: 2 · 2024: 3 · 2023: 4
- FWCI
- 2.06
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Cells (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Institutional Repositories DataBase (IRDB) (submittedVersion, cc-by-nc-nd)
- Funders
- Japan Agency for Medical Research and Development (AMED) (21mk0101214, 21mk0101214s0101)
- The Nakatomi Foundation (21mk0101214, 21mk0101214s0101)
- Takeda Science Foundation (21mk0101214, 21mk0101214s0101)
- The Mochida Memorial Foundation (21mk0101214, 21mk0101214s0101)
- Topics
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Pharmacogenetics and Drug Metabolism
- Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
- Keywords
- HepG2 cells, pitch system, cytochrome P450, UGT1A1, UDP-glucuronosyltransferase, drug metabolism, drug-induced liver injury, hepatocyte models, drug discovery, CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP1A2, POR
- MeSH terms
- Cytochrome P-450 Enzyme System, Humans, Hepatocytes, Cytochrome P-450 CYP3A, Drug Discovery, Hep G2 Cells
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