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Hep3B Cells: Hepatocellular Carcinoma Research and Discovery

HEP3B is an immortalized human-derived hepatocyte carcinoma cell line. It is widely used in hepatotoxicity and drug metabolism research. HEP3B transfection efficiency is substantial; therefore, these cells are commonly employed to study liver cancer development, progression, and therapeutic interventions. This article covers all the necessary information about the HEP3B cell line to assist you in working with it. Mainly, it will include:

  1. General characteristics and origin of HEP3B cells
  2. Culturing information about HEP3B cell line
  3. HEP3B cell line: Advantages & Limitations
  4. Applications of HEP3B cells in research
  5. Publications on HEP3B cell line
  6. Resources for HEP3B cell line: Protocols, Videos, and More

1.      General characteristics and origin of HEP3B cells

The primary information you require about a cell line is its origin and general characteristics. It can help you decide its use in your research and help you in dealing with it. This article section covers all the essential information about HEP3B's origin and characteristics. Here you will learn: What is Hep 3B cell lines? What is the origin of Hep3B cells? What is the morphology of HEP3B cells?

  • HEP3B, a continuous human hepatoma cell line, originated from the liver tissue of an 8-year-old African boy with hepatocellular carcinoma (HCC). It was established by Aden and colleagues in 1979 at Barbara B. Knowles's lab, Wistar Institute, Philadelphia, USA [1]. 
  • HEP3B cells have an integration of the hepatitis B virus (HBV) genome in its chromosome.
  • These hepatocyte carcinoma cells exhibit an epithelial morphology.
  • The HEP3B cells have a modal chromosome number of 60. Unlike, HepG2, they do not possess a rearranged chromosome 1.

What is the difference between HepG2 and Hep3B?

HepG2 and HEP3B differ in the number of chromosomes per cell. HEP3B contains 60 chromosomes, while HepG2 cells have an average of 55 chromosomes. Moreover, HepG2 is non-tumorigenic and hepatitis B negative, whereas HEP3B is tumorigenic and HBV positive.

Liver carcinoma at high magnification under the microscope.

2.      Culturing information about HEP3B cell line

Knowledge about the culturing information of a cell line can make you go easy with it. This section covers all the key points for culturing the HEP3B HBV cell line, including: What is the doubling time of Hep3B cells? What are HEP3B culture conditions? How do you culture HEP3B HCC cells?

Key Points for Culturing HEP3B Cells

Doubling Time:

The doubling time of HEP3B cells is approximately 36 hours.

Adherent or in Suspension:

This hepatoma cell line is adherent.

Split ratio:

HEP3B cells are sub-cultivated at a ratio of 1:2 to 1:4. Adherent HEP3B cells are rinsed with 1 x PBS and then incubated with the Accutase dissociation solution. After 8 to 10 min, fresh media is added, and cells are centrifuged. The harvested cells are then carefully resuspended and poured into flasks containing culture media at a recommended split ratio.

Growth Medium:

EMEM media containing 10% FBS, 2.2 g/L NaHCO3, 2 mM L-Glutamine, and Earle's Balanced Salt Solution (EBSS) is used to culture HEP3B cells.

Growth Conditions: 

HEP3B cells are cultured in a 37°C humidified incubator with a continuous 5% CO2 supply.


Frozen HEP3B cells are stored at below -150°C temperature in an electric freezer or the vapour phase of liquid nitrogen.

Freezing Process and Medium:

The freezing medium recommended for HEP3B cells is CM-1 or CM-ACF. Cells are frozen using a slow freezing process that permits only a 1°C decrease in temperature per minute to protect the cell viability.

Thawing Process:

Frozen cells are thawed in a water bath pre-set at 37°C for 40 to 60 seconds. Later these cells are added to the fresh growth medium and centrifuged to remove freezing media elements. Collected cells are resuspended and dispensed into new flasks for growth.

Biosafety Level:

A biosafety level 1 laboratory is required for HEP3B cell culturing.


Adherent semi-confluent layer of Hep3B cells at 10× and 20× magnification.

3.      HEP3B cell line: Advantages & Limitations

HEP3B is a widely used hepatocellular carcinoma cell line. This section will focus on some key advantages and limitations of these hepatoma cells.


The main advantages of HEP3B cells are:

Easy to culture

HEP3B cells have no fussy cell culture requirements, making them easy to handle and maintain in a research laboratory. This simplifies experimental procedures.

High transfection efficiency

HEP3B cells exhibit remarkable transfection efficiency, making them widely used in genetic manipulation and gene expression-related studies.


HEP3B is a tumorigenic hepatocellular carcinoma cell line capable of forming tumours when injected into an immunocompromised mouse. This aids in studying cancer progression and development using HEP3B xenograft models.

HEP3B P53 status

HEP3B cells possess a P53 gene mutation similar to hepatocellular carcinoma (HCC) and other cancers, making them relevant for studying the effects of P53 mutation on cancer growth, development, and progression.



The limitation associated with the HEP3B cell line is:

In vitro cell model

HEP3B cell line serves as an in vitro model of hepatocellular carcinoma (HCC) cells. However, it may not fully represent the complexity of HCC as found in living organisms. Consequently, experimental outcomes in vitro may differ from those observed in vivo.


4.      Applications of HEP3B cells in research

HEP3B cell line offers several research applications in biomedical research. Some of the key research applications of the HEP3B cells include:

  • Cancer biology: HEP3B is a human hepatocellular carcinoma cell line. It is an invaluable cell model to investigate cell and molecular mechanisms underlying HCC development and progression. Researchers use these cells to study genetic mutations, cellular processes, and cell signalling pathways associated with liver cancer. Such as a study utilized HEP3B cells and found that microRNA-223-3p regulates NLRP3 inflammasome components, inhibits proliferation, and enhances the apoptosis of HEP3B hepatocarcinoma cells [2].
  • Drug screening and development: HEP3B cell line is also used to test, screen, and develop novel therapeutics against liver carcinoma. Moreover, it is used to evaluate the toxicity and efficacy of different anticancer drugs and treatments. Researchers also used these hepatoma cells to study drug metabolism. Researchers used HEP3B cells and assessed the cytotoxic potential of Cotinus coggygria plant extract on HEP3B hepatocyte carcinoma cells [3].
  • Viral infections: HEP3B is a hepatitis B virus-positive cell line; therefore, it is employed to study viral infections that may lead to liver cancer development, i.e., HBV and HCV. This may help better understand viral infections and develop potential antiviral treatments. For instance, a study used HEP3B hepatocyte cancer cells and investigated the importance of ubiquitination for hepatitis c virus propagation. The findings suggested that ubiquitin-specific protease 15 (USP15) is involved in the propagation of HCV via regulating hepatocyte-specific functions, including lipid droplet formation and HCV RNA translation [4].

5.      Publications on HEP3B cell line

This section of the article will cover a few interesting research publications on HEP3B cells.

Regulation of apoptosis and autophagy by luteolin in human hepatocellular cancer Hep3B cells

This publication in Biochemical and Biophysical Research Communications (2019) proposed that luteolin compound-induced endoplasmic reticulum stress may exert anti-tumour effects in P53-null HEP3B cells in a P53-independent manner.

Potential targeting of Hep3B liver cancer cells by lupeol isolated from Avicennia marina

This study in Archiv der Pharmazie (2021) proposed a pentacyclic triterpenoid, lupeol, as a potential anticancer agent against HEP3B cells.

Ethanol extract from Cnidium monnieri (L.) Cusson induces cell cycle arrest and apoptosis via regulation of the p53‑independent pathway in HepG2 and Hep3B hepatocellular carcinoma cells

This article is published in Molecular Medicine Reports (2017). The study findings state that Cnidium monnieri (L.) Cusson ethanol extract induces cell death (apoptosis) and cell cycle arrest in hepatocarcinoma cells HepG2 and HEP3B through regulation of p53 and Akt/GSK-3β signalling pathway.

Auranofin enhances sulforaphane-mediated apoptosis in hepatocellular carcinoma Hep3B cells through inactivation of the PI3K/Akt signaling pathway

This research published in Biomolecules and Therapeutics (2020) proposed that auranofin exhibits synergistic activity and promotes the sulforaphane-mediated apoptosis of HEP3B cells via PI3K/AKT pathway activation.

Circular RNA‐0072309 has antitumor influences in Hep3B cell line by targeting microRNA‐665

This research article in BioFactors (2023) proposed that circular RNA-0072309 exerts antitumor effects in hepatocellular carcinoma cells HEP3B via targeting miRNA-665.

6.      Resources for HEP3B cell line: Protocols, Videos, and More

The following are a few resources available on HEP3B cells:

  • HEP3B transfection: This video will explain the transfection protocol for HEP3B cells.
  • HEP3B transfection efficiency: This link will help you learn HEP3B DMEM medium composition, cell passaging, and transfection protocol for HEP3B cells. In addition, it provides information for optimizing the HEP3B lipofectamine 3000 reagent in your transfection protocol.

The following link contains the HEP3B cell culture protocol:

  • HEP3B culture conditions: This link will help you learn the protocol for handling and maintaining Huh7 and HEP3B cultures.
  • HEP3B cells: This website has plenty of information on HEP3B cells, including HEP3B media, the protocol for cell splitting, thawing, and handling of proliferative and cryopreserved cultures.


  1. Puttahanumantharayappa, L.D., et al., Origin and prop-erties of hepatocellular carcinoma cell lines. Japanese J Gastroenterol Res, 2021. 1(8): p. 1040.
  2. Wan, L., et al., miRNA-223-3p regulates NLRP3 to promote apoptosis and inhibit proliferation of hep3B cells. Experimental and therapeutic medicine, 2018. 15(3): p. 2429-2435.
  3. Danjolli-Hashani, D. and S. Selen-Isbilir, Cytotoxic effect of Cotinus coggygria extract on Hep3B cancer cell line. Natural Product Research, 2022: p. 1-4.
  4. Kusakabe, S., et al., USP15 participates in hepatitis C virus propagation through regulation of viral RNA translation and lipid droplet formation. Journal of Virology, 2019. 93(6): p. 10.1128/jvi. 01708-18.


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