Hep2 cells

Hep 2 cells constitute an immortalized human cervical adenocarcinoma cell line widely used in biological research. Notably, it is applied in virology, bacteriology, cell biology, immunology, and cancer research [1].

This article will help you learn the basics of the Hep 2 cell line. Mainly you will go through the following:

  1. Origin and general characteristics of the Hep 2 cells
  2. Hep 2 cell line: Culturing information
  3. Advantages & Limitations of Hep 2 cells
  4. Hep 2 cell line applications in research
  5. Hep 2 cells: Research publications
  6. Resources for Hep2 cell line: Protocols, Videos, and More

1.      Origin and general characteristics of the Hep 2 cells

A cell line's origin and general characteristics define its applicability in research. This section will help you know about the origin and some salient features of Hep 2 cells. For instance, you will find out: What is the HEp-2 cell line? What is the source of Hep 2 cells? And what is the morphology of Hep 2?

  • Hep 2, an immortal human epithelial cell line was first described by H.W. Toolan as laryngeal carcinoma cells in 1954. However, lately, it has been reported that the Hep 2 cell line is composed of cervical adenocarcinoma cells and originated from contamination of the Hela cell line [2].
  • Hep 2 cells contain Hela marker chromosomes and are found positive for Keratin and human papillomavirus DNA sequences as confirmed via immunoperoxidase staining and PCR, respectively.
  • The Hela cell line derivative Hep 2 possesses an epithelial-like morphology.
  • The Hep 2 cell line exhibits both structural and numerical chromosomal abrasions with a near-triploid karyotype [3].

Hela Vs Hep 2

Hep 2 cells are derivative of the Hela cell line but possess distinguishable characteristics. Hela has high proliferation rates, whereas Hep 2 is a slow-growing cell line.

Division of HeLa cervical cancer cells under microscope.

2.      Hep 2 cell line: Culturing information

Before working with a cell line, we must know the following key points for its culturing. This information can be useful for effectively culturing and maintaining the cell line. You should know: What is the doubling time of HEp-2 cells? Are Hep 2 cells adherent? What is the seeding density of Hep2 cells?


Population Doubling Time:

The doubling time reported for Hep 2 cells is approximately 40 hours.

Adherent or in Suspension:

Hep 2 cells are adherent and grow into monolayers.

Seeding Density:

A seeding density of 1 x 104 cells/cm2 is ideal for Hep 2 cell culture. For seeding, adherent Hep 2 cells are rinsed with 1 x PBS solution, followed by incubation with Accutase dissociation solution. After 8–10-minute incubation at ambient temperature, cells were resuspended in a medium and centrifuged. The collected cells were then carefully dispensed in fresh medium and poured into new flasks for culturing.

Growth Medium:

EMEM or Eagle's minimal essential medium is used to culture Hep 2 cells. This media is supplemented with 10% FBS, 1.0 g/L glucose, 2.2 g/L NaHCO3, 2.0 mM L-glutamine, 1% NEAA, and 1 mM sodium pyruvate for ideal cell growth. Media should be renewed 2 to 3 times a week.

Growth Conditions:

Like other mammalian cell lines, Hep 2 is also cultured in a humidified incubator set at 37°C temperature and with a continuous supply of 5% CO2.


Hep 2 cells can be stored in ultra-low temperature electric freezers (below  -150 °C) or in liquid nitrogen vapor phase for longer terms.

Freezing Process and Medium:

The freezing media recommended for Hep 2 cells are CM-1 or CM-ACF. Cells can be frozen in these freezing media using a slow freezing process that allows a gradual 1°C drop in temperature and protects cell viability to the maximum.

Thawing Process:

The frozen cells vial is quickly thawed by agitation in a pre-set 37°C water bath until a small ice clump is left. Cells are added with fresh media and centrifuged to remove freezing media components. Later, the cell pellet was resuspended in media, and cells were dispensed into the flask for culturing. Cells take almost 24 hours to adhere to the surface of the flask.

Biosafety Level

Biosafety level 1 laboratory is recommended for handling and maintenance of Hep 2 cell cultures.

Hep 2 cells before and after reaching confluence.

3.      Advantages & Limitations of Hep 2 cells

Almost all cell lines exhibit a unique combination of advantages and limitations that contribute to their use in the research field. This section will describe a few main pros and cons associated with the Hep 2 cell line.


The principal advantages of the Hep 2 cell line are:

Human origin

Hep 2 is derived from human epithelial cells, making it a valuable in vitro model for studying human diseases and viral infections.

ANA detection

The Hep 2 cell line possesses a native protein array that presents numerous antigens, making it an excellent substrate for detecting antinuclear antibodies (ANA). This feature enables specific and highly sensitive screening of ANA in serum, making it a crucial diagnostic tool for identifying connective tissue diseases.



The limitations of Hep 2 cells are:

Chromosomal abrasions

Hep 2 cells exhibit multiple numerical and structural chromosomal abnormalities. These abnormalities can impact cell behaviour and may restrict their applicability in certain laboratory experiments.


Hep 2, a tumour-derived human epithelial cell line, may possess genetic abnormalities typically absent in epithelial cells. Consequently, the use of Hep 2 cells might be constrained in specific studies focusing on normal cellular physiology.

4.      Hep 2 cell line applications in research

Hep 2 cells have a wide range of applications in the biomedical research field. They are used in several in vitro experiments, including cell receptor analysis, ANA diagnosis, cancer research, and virus and bacteria studies.

Here we have discussed a few promising applications of Hep 2 cells.

Cancer research: Hep 2 is a tumorigenic cell line. So, it is widely used to study cancer biology involving cancer signalling pathways, mechanistic studies, and drug screening. A recent study used the Hep 2 cell line to explore the role of miRNA-33a in cancer proliferation. The study found that miRNA-33a targets PIM1 (pim-1 oncogene) and exerts antiproliferative effects [4]. Another research used this human cancer cell line for anti-cancer drug screening and evaluation purposes. The study evaluated the antiproliferative and apoptotic potential of biosynthesized Marsdenia tenacissima zinc oxide nanoparticles in Hep 2 cells [5].

Virology: Hep 2 is susceptible to a number of human viral infections. Therefore, it is widely applied to study viral diseases. A recent study used Hep 2 cells for expressing SARS-CoV-2 virus genes and study complicated virus interaction with host cells [6].

Mechanistic studies: The Hep 2 cell line is an ideal in vitro model to study cell and molecular mechanisms involved in many biological processes. Researchers can easily manipulate gene expression patterns in Hep 2 cells and investigate the roles of particular genes in cellular functions. Such as a study, overexpressed RNA binding protein RBM6 through genetic manipulations in the Hep 2 cell line and examined its tumour suppressor role [7].

5.      Hep 2 cells: Research publications

The following are some interesting and most cited research publications on Hep 2 cells.

Synthesis of Zinc oxide nanoparticles from Marsdenia tenacissima inhibits the cell proliferation and induces apoptosis in laryngeal cancer cells (Hep-2)

This article published in the Journal of Photochemistry and Photobiology B: Biology (2019) explored the anti-cancer potential of biosynthesized Marsdenia tenacissima zinc oxide nanoparticles in the Hep 2 cell line.

Bioformulated hesperidin-loaded PLGA nanoparticles counteract the mitochondrial-mediated intrinsic apoptotic pathway in cancer cells

This paper was published in the Journal of Inorganic and Organometallic Polymers and Materials in 2021. This study examined the anti-cancer properties of bio-formulated hesperidin-loaded Poly (lactic-co-glycolic acid) (PLGA) nanoparticles in Hep 2 cells.

Antiviral activity of ethanol extract of Lophatherum gracile against respiratory syncytial virus infection

This publication in the Journal of Ethnopharmacology in 2019 used Hep 2 cells to study respiratory syncytial virus infection and screen antiviral drugs against it. The study reported the promising antiviral potential of ethanol extract of a medicinal plant i.e., Lophatherum gracile against respiratory syncytial virus infection.

Evaluation of aqueous-extracts from four aromatic plants for their activity against Candida albicans adhesion to human HEp-2 epithelial cells

This research is published in the Gene Reports (2020). This study explored the inhibitory potential of aqueous extracts of four aromatic plants against the adhesion of Candida albicans to human Hep 2 epithelial cells.

Wnt1‐inducible signaling protein 1 regulates laryngeal squamous cell carcinoma glycolysis and chemoresistance via the YAP1/TEAD1/GLUT1 pathway

This study was published in the Journal of Cellular Physiology in 2019. The study reports that Wnt1‐inducible signalling protein 1 (WISP1) interacts with YAP1/TEAD1/GLUT1 pathway and regulates glucose metabolism and chemoresistance in the Hep 2 cell line.

6.      Resources for Hep2 cell line: Protocols, Videos, and More

Hep 2 is a well-known cell line. There are several available resources featuring the Hep 2 cell line.

The cell culture protocol for Hep 2 cells is mentioned in the following link.

  • Hep 2 culturing: This link contains basic cell culture information about Hep 2 cells. It includes cell splitting, cell freezing, and thawing.


  1. Fusi, M. and S. Dotti, Adaptation of the HEp-2 cell line to totally animal-free culture systems and real-time analysis of cell growth. Biotechniques, 2021. 70(6): p. 319-326.
  2. Gorphe, P., A comprehensive review of Hep-2 cell line in translational research for laryngeal cancer. Am J Cancer Res, 2019. 9(4): p. 644-649.
  3. Wang, M., et al., Cancer-associated fibroblasts in a human HEp-2 established laryngeal xenografted tumor are not derived from cancer cells through epithelial-mesenchymal transition, phenotypically activated but karyotypically normal. PLoS One, 2015. 10(2): p. e0117405.
  4. Karatas, O.F., Antiproliferative potential of miR-33a in laryngeal cancer Hep-2 cells via targeting PIM1. Head Neck, 2018. 40(11): p. 2455-2461.
  5. Wang, Y., et al., Synthesis of Zinc oxide nanoparticles from Marsdenia tenacissima inhibits the cell proliferation and induces apoptosis in laryngeal cancer cells (Hep-2). Journal of Photochemistry and Photobiology B: Biology, 2019. 201: p. 111624.
  6. Zhang, J., et al., A systemic and molecular study of subcellular localization of SARS-CoV-2 proteins. Signal Transduct Target Ther, 2020. 5(1): p. 269.
  7. Wang, Q., et al., RNA-binding protein RBM6 as a tumor suppressor gene represses the growth and progression in laryngocarcinoma. Gene, 2019. 697: p. 26-34.