HepG2 cells

Hep-G2 is a human liver cancer cell line originating from the liver tissue of a 15-year-old Caucasian male with hepatocellular carcinoma. These cells are frequently utilized in drug metabolism and hepatotoxicity studies. Although HepG2 cells have high proliferation rates and an epithelial-like appearance, they are non-tumorigenic and perform various differentiated hepatic functions.

3d rendered medical animation of a malignant tumor in a mans liver

In 1975, researchers derived HepG2 cells from hepatocellular carcinoma, making it the first hepatic cell line to exhibit the critical characteristics of hepatocytes. In contrast to the previously established SK-Hep1 cell line, which lacks essential liver cell markers, HepG2 cells can secrete various plasma proteins and provide a valuable model for studying the intracellular dynamics of cell surface domains in human hepatocytes. These cells exhibit an epithelial-like morphology, have a modal chromosome number of 55, and can be stimulated with human growth hormone.

1. HepG2 Characteristics
2. Applications of HepG2 Cells
3. Characteristics and Applications of the HepG2 Cell Line in Comparison to Other Tumor Cell Lines
4. Subculturing HepG2 Cells

1.        HepG2 Characteristics

Primary hepatocytes' typical shape is cubic and usually contains two nuclei. In contrast, HepG2 cells have an epithelial-like morphology with a single nucleus and a chromosome count ranging from 48 to 54 per cell. Although HepG2 cells can account for up to 25% of the total cellular protein, their size is larger than that of normal hepatocytes, making up about 10% of the complete protein in the cell. Cellular proteins are critical actors within the cell, executing the functions specified by genes.

Tumor cells, including those with an abnormal number of chromosomes, often exhibit an increase in the number of nuclei, up to seven per cell. Due to their high degree of differentiation in vitro, HepG2 cells provide an ideal model for studying the intracellular trafficking and dynamics of bile canalicular, sinusoidal membrane proteins, and lipids in human hepatocytes.

The average diameter of a HepG2 cell is around 10-20 µm, which is smaller than a hepatocyte with a diameter of 15 µm but similar to tumor cells with Hepatoblastoma (HB), which range from 10-20 µm.

HepG2 genetics

The Hep-G2 cell line exhibits several translocations, including those between the short arms of chromosomes 1 and 21, trisomies of chromosomes 2, 16, and 17, and tetrasomy of chromosome 20. The loss of the chromosome 4q3 region is also observed, associated with translocation t(1;4) often seen in Hepatoblastoma (HB) and other chromosomal abnormalities, such as trisomies 2 and 20. The number of chromosomes in HepG2 cells ranges from 50 to 60, indicating a hyperdiploid karyotype, while some cases exhibit more than 100 chromosomes and are characterized by tetraploid enlargement. HepG2 cells contain approximately 7.5 pg of DNA, 15% more than an average somatic cell. In comparison, primary hepatocytes have a cubic cell shape and typically contain two nuclei [1].

Mutational Profile of HepG2 Cells

The HepG2 cell line carries the TERT promoter region mutation C228T, also present in hepatocellular carcinoma (HCC) and hepatoblastoma (HB). This mutation contributes to immortalization by protecting telomeres in cancer cells. Additionally, HepG2 cells exhibit wild-type TP53, a critical gene for suppressing human cancer, as it plays a role in cell cycle arrest, apoptosis, and aging. Mutations in this gene can promote cell proliferation.

HepG2 cells participate in several pathways, including dysregulation of cell growth, survival pathways such as fetal and embryonal HB, and the Wnt/β-catenin pathway. Furthermore, the cell line has a characteristic deletion of the third exon of the CTNNB1 gene, which is identical to that seen in epithelial type HB [2,3].

HepG2 cells at high and low confluence

2.       Applications of HepG2 Cells

The use of immortalized hepatic tumor cell lines is widespread in cancer research and the study of viral infections such as hepatitis B (HBV) and hepatitis D (HDV). The cell lines HepG2 and Huh7 have been found to exhibit complete cell cycle replication of HDV and expression of HBV [5,6]. Additionally, HepaRG cell lines have been instrumental in discovering mechanisms of HBV viral entry [7].

In addition to viral infection studies, HepG2 cells have proven to help investigate human liver diseases, including those caused by an incorrect subcellular distribution of cell surface proteins, progressive familial intrahepatic cholestasis (PFIC), hepatocanalicular transport defects such as Dubin-Johnson Syndrome, and familial hypercholesterolemia. Researchers have used HepG2 cells and their derivatives as model systems for studying liver metabolism and toxicity of xenobiotics, detecting environmental and dietary cytotoxic and genotoxic (as well as cytoprotective, genotoxic, and anti-genotoxic) agents, understanding hepatocarcinogenesis, and conducting drug targeting studies [8,9]. Moreover, HepG2 cells are employed in trials with bio-artificial liver devices.

3.       Characteristics and Applications of the HepG2 Cell Line in Comparison to Other Tumor Cell Lines

There are approximately 40 different hepatic tumor cell lines, but the most commonly used ones are HepaRG, Huh7, SK-Hep-1, Hep3B, and HepG2, which are obtained from various tumors. HepG2 has gained popularity among these cell lines due to its widespread applications in scientific research [13.]

Compared to normal hepatocytes, HepG2 cells exhibit weak or absent expression of the cytochrome P450 (CYP) superfamily, including CYP3A4, CYP2C9, CYP2A6, CYP2D6, CYP2C19, and others involved in xenobiotic oxidation in the liver. Reduced expression levels of these genes are also observed in tumor cells in hepatocellular carcinoma (HCC) and hepatoblastoma (HB), which correlate with low survival estimates. Researchers are working on modifying the HepG2 cell line to increase the expression of cytochromes to better use HepG2 cells as a model of hepatocytes in drug metabolism studies.

In comparison to other tumor cell lines, such as MCF7 (angiosarcoma of the breast), PC3 (prostatic adenocarcinoma), 143B (osteosarcoma), and HEK293 (embryonic kidney), HepG2 cells have a lower content of certain amino acids, including glutamine, proline, asparagine, aspartate, arginine, methionine, alanine, lysine, and threonine [14]. On the other hand, HepG2 cells have a higher content of glutamate, phenylalanine, glycine, acetylcarnitine, methionine, and choline derivatives. These changes in amino acid levels affect protein synthesis, particularly the secretion of the total hepatic protein. This shows a positive relationship between the concentration of several amino acids and the amount of complete protein.

Another approach to studying HepG2 cells is to derive three-dimensional spheroid cell cultures, which transform cells into spheroids and create a more physiologically relevant system. Metabolic activity, including cytochromes, is higher in 3D spheroidal HepG2 models than in 2D cells, bringing them closer to normal hepatocytes. According to molecular profiling data, the HepG2 cell line can serve as a model for hepatoblastoma [10-12].

4.       Subculturing HepG2 Cells

Here are five steps for removing adherent cells from cell culture flasks using Accutase:

1. Remove the medium from the cell culture flask and rinse the adherent cells using PBS without calcium and magnesium. Use 3-5 ml of PBS for T25 flasks and 5-10 ml for T75 flasks.
2. Add Accutase to the cell culture flask, using 1-2 ml per T25 and 2.5 ml per T75 flask. Ensure that the Accutase covers the entire cell sheet.
3. Incubate the flask at room temperature for 8-10 minutes.
4. Carefully resuspend the cells with medium, using 10 ml of fresh medium.
5. Centrifuge the resuspended cells for 5 minutes at 300xg, resuspend them in fresh medium and dispense them into new flasks containing fresh medium.

Future Prospects for HepG2 Cells

The quest to unlock the full potential of the HepG2 cell line continues with groundbreaking progress in increasing the expression of cytochromes. Researchers are also exploring the possibility of three-dimensional spheroid cell cultures, which offer a more physiologically relevant system. The metabolic activity, including cytochromes, is remarkably higher in 3D spheroidal HepG2 models than in 2D cells, bringing us closer to creating a model that mirrors normal hepatocytes. Additionally, exploring the dynamic processes underlying the incorrect distribution of cell surface proteins can pave the way for a better understanding of liver diseases.

References

1. R C Vyas, F Darroudi, A T Natarajan, Radiation-induced chromosomal breakage and  rejoining in interphase-metaphase chromosomes of human lymphocytes, Mutat Res, 1991; 249(1):29-35.
2. Woodfield, S.E.; Shi, Y.; Patel, R.H.; Chen, Z.; Shah, A.P.; Srivastava, R.K.; Whitlock, R.S.; Ibarra, A.M.; Larson, S.R.; Sarabia, S.F.; et al. MDM4 Inhibition: A Novel Therapeutic Strategy to Reactivate P53 in Hepatoblastoma. Sci. Rep. 2021, 11, 2967. [CrossRef]
   Hussain, S.P.; Schwank, J.; Staib, F.; Wang, X.W.; Harris, C.C. TP53 Mutations and hepatocellular Carcinoma: Insights into the Etiology and Pathogenesis of Liver Cancer. Oncogene 2004.
3. Gerda Schicht, Lena Seidemann, Rene Haensel, Daniel Seehofer, Georg Damm, Critical Investigation of the Usability of Hepatoma Cell Lines HepG2 and Huh7 as Models for the Metabolic Representation of Resectable Hepatocellular Carcinoma. Cancers2022, 14(17), 4227 7, 26, 2166–2176.
4. Verrier, E.R.; Colpitts, C.C.; Schuster, C.; Zeisel, M.B.; Baumert, T.F. Cell Culture Models for the Investigation of Hepatitis B and D Virus Infection. Viruses 2016, 8, 261.
   Verrier, E.R.; Colpitts, C.C.; Bach, C.; Heydmann, L.; Weiss, A.; Renaud, M.; Durand, S.C.; Habersetzer, F.; Durantel, D.; AbouJaoud é, G.; et al. A Targeted Functional RNA Interference screen Uncovers Glypican 5 as an Entry Factor for Hepatitis B and D
   Viruses. Hepatology 2016, 63, 35–48.
5. Gripon, P.; Rumin, S.; Urban, S.; Le Seyec, J.; Glaise, D.; Cannie, I.; Guyomard, C.; Lucas, J.; Trepo, C.; Guguen-Guillouzo, C. Infection of a Human Hepatoma Cell Line by Hepatitis B Virus. Proc. Natl. Acad. Sci. USA 2002, 99, 15655–15660.
6. Mersch-Sundermann V, Knasmüller S, Wu XJ, Darroudi F, Kassie F (May 2004). "Use of a human-derived liver cell line for the detection of cytoprotective, antigenotoxic and cogenotoxic agents". Toxicology. 198(1–3): 329–340. doi:1016/j.tox.2004.02.009. PMID 15138059.
7. Fanelli A (2016). "HepG2 (liver hepatocellular carcinoma): cell culture". HepG2. Retrieved 3 December 2017.
8. Xuan, J.; Chen, S.; Ning, B.; Tolleson, W.H.; Guo, L. Development of HepG2-Derived Cells Expressing Cytochrome P450s for Assessing Metabolism-Associated Drug-Induced Liver Toxicity. Physiol. Behav. 2017, 176, 139–148.
9. Ooka, M.; Lynch, C.; Xia, M. Application of in Vitro Metabolism Activation in High-Throughput Screening. Int. J. Mol. Sci. 2020, 21, 8182.
10. Huang, L.; Coughtrie, M.W.H.; Hsu, H. Down-Regulation of Dehydroepiandrosterone Sulfotransferase Gene in Human Hepatocellular Carcinoma. Mol. Cell. Endocrinol. 2005, 231, 87–94.
11. Zhu Z, Hao X, Yan M, et al. Cancer stem/progenitor cells are highly
    enriched in CD133 + CD44 + population in hepatocellular carcinoma. Int J Cancer. 2010; 126:2067 ‐2078
12. Christophe Arbus, Amine Benyamina, Pierre-Michel Llorca, Franck Baylé, Norbert Bromet, Frédéric Massiere, Ricardo P Garay, Ahcène Hameg. Characterization of human cytochrome P450 enzymes involved in the metabolism of cyamemazine. Eur J Pharm Sci . 2007 Dec;32(4-5):357-66.