HuH7 Cells

Guide to HuH7 Cells: Origin, Characteristics, and Culturing

The human hepatoma-derived HuH7 cell line is an immortalized cell line comprised of tumorigenic epithelial-like cells. This cell line and its subclones, HuH7.5 and HuH7.5.1, can be used instead of primary hepatocytes in experiments. HuH7 cells are widely used as a cell culture model to study hepatitis C virus (HCV) infection and hepatoma.

This article will take in all the necessary information you need to start working with this cell line, including:

• HuH7 cells
  • What is the HuH7 cell line?
• Morphology of HuH7 cells
  • Cell size
  • Genome and ploidy (chromosome Number)
  • HuH7 Vs. HuH7.5 cell line comparison
  • Culturing requirements for HuH7 cells
  • Population doubling time
  • Adherent or in suspension
  • Seeding density
  • Growth medium
  • Growth conditions (temperature, CO2)
  • Storage
  • Freezing process and medium
  • Thawing process
  • Biosafety level
• Advantages and Limitations of HuH7 cell line
  • Advantages of HuH7 cells
  • Limitations of HuH7 cell line
• HuH7 applications
• Publications
• HuH7 Resources: Protocols, Videos & More
  • Transfection protocols of HuH7
    
  • Videos related to HuH7 cell line

Before using HuH7 cells, you should learn about the cell line's origin, salient features, and cell culture conditions. 

What is the HuH7 cell line?

HuH7 cells are a permanent cell line established by Sato, J., and Nakabayshi, H., in 1982. The cell line originated from a highly differentiated hepatocyte-derived cancer cell line, initially developed from hepatocellular carcinoma in a 57-year-old Japanese man. This cell line is known for making a number of biologically active substances, such as albumin and alpha-fetoprotein, among others. In addition, HuH7 cells are highly susceptible to HCV infection and thus can be used as an HCV replicon system. Following these characteristics, the HuH7 cell line can be used to screen and develop anti-HCV drugs.

Morphology of HuH7 cells

HuH7 cells have an epithelial morphology. The cells are hexagonal in shape. Moreover, they are adherent cells that typically grow as 2D monolayers.

Cell size

The mean cell size of HuH7 cells is 21.9 𝜇m. They are generally larger and flattened.

Genome and ploidy (chromosome Number)

HuH7 cells show variable chromosome numbers and are thus highly heterogeneous. They exhibit a ploidy (chromosome number) between 55 and 63. The heterogeneity of HuH7 cell line populations develops due to selective pressure during the culturing of cells. This may differ with varying culture conditions between laboratories.

HuH7 Vs. HuH7.5 cell line comparison

The HuH7.5 cell line is a derivative of the HuH7 cell line. Unlike its parental cell line, HuH7, it possesses a missense mutation in the RIG-I or DDX58 gene, which is involved in intracellular antiviral defense. HuH7.5 is more permissive for replicating genomic and subgenomic hepatitis C virus RNA.

For more information regarding the derivatives of the HuH7 cell line, read this recently published research article (https://www.frontiersin.org/articles/10.3389/fgene.2020.546106/full).

Culturing requirements for HuH7 cells

HuH7 cells are widely cultured in laboratories for multiple research investigations. Here, we will talk about the cell culture requirements of the HuH7 cell line.

Population doubling time

The population doubling time of the HuH7 cell line is 24 hours. However, it may extend between 36-48 hours, depending upon the cell culture medium used.

Adherent or in suspension

The HuH7 cells grow in a 2D monolayer and are adhesive to the surface of cell culture plates and flasks. The cell line is rarely cultured in suspension form.

Seeding density

The HuH7 cells' confluency should be kept between 30 and 90%. Avoid more than 90% confluency as it declines cell proliferation rates. Cells can be subcultured using Accutase, a cell detachment solution, and seeded at optimal (2 × 10^4 cells/cm2) and saturation (6 × 10^4 cells/cm2) densities as per experimental requirement.

Growth medium

HuH7 cell line grows best in RPMI 1640 with 10% FBS. The medium should be renewed 2-3 times a week.

Growth conditions (temperature, CO2)

HuH7 cells are grown in a humidified incubator with 5% CO2 at 37 °C.

Storage

HuH7 cells should be stored in the liquid nitrogen vapor phase for the longer term.

Freezing process and medium

The freezing process should be slow to maintain HuH7 cell viability. Cells can be frozen in the freeze medium CM-1, available at the CLS website.

Thawing process

HuH7 cells should be thawed in a pre-warmed water bath at 37°C temperature. Later, the cells are added to a pre-warmed growth medium and centrifuged to remove freezing media components. The resulting cell pellet is again resuspended in fresh medium and cultured. 

Biosafety level

Biosafety level 1 is recommended for handling and propagating the HuH7 cell line.

Advantages and Limitations of HuH7 cell line

The HuH7 cell line offers both advantages and limitations at the same time. Let's go through this cell line's potential pros and cons, as they may help you decide if it is appropriate for your experiments.

Advantages of HuH7 cells

HuH7 cells possess many characteristic features that make them attractive for research use.

• Easy propagation: Unlike other human hepatoma cell lines, the HuH7 cell line can be grown without adding serum. However, CLS recommends using RPMI 1640 with 10% FBS and 2 mM L-glutamine. HuH-7 cells secrete a growth factor that helps cells grow without serum.
• Transfection flexibility: HuH7 cell line is widely used for transfection purposes due to high receptiveness for the HCV genome. Therefore, these cells are pivotal for anti-HCV drug screening and development.
• Alternative to human hepatocytes: HuH7 cell line is an excellent substitute for human hepatocytes. It can help researchers study hepatic drug transporters and understand the interaction of certain drugs with MRPs (multi-drug resistance-associated proteins).

• Developing Xenograft mouse model: HuH7 cells have been used to produce a cell line-originated xenograft mouse model. This animal model may provide valuable insights to researchers about protein kinase inhibitors and anti-cancer therapies.
• Cancer research: HuH7 cell line is a human liver cancer cell line. Thus, it helps researchers to conduct liver cancer studies.

Limitations of HuH7 cell line

There are some limitations associated with the culturing of the HuH7 cell line.

Microbial contamination: Bacterial contamination is the most common problem in cell culture. When an infection happens, it can be distinguished by the pH change and turbidity of the culture media. Among bacterial contamination, mycoplasma contamination is usually not apparent. However, it slowly affects the cell line's morphology, gene expression, and other characteristics.

Culture period: Although the HuH7 cell line is immortal, like many other cell lines, exceeding the culture period can affect the health of cells gradually. It may affect the proliferation rate and gene expression, reducing the reproducibility of the results. For the HuH7 cell line, the passage number should be kept below 20–25.

HuH7 Cells applications

This human hepatoma-derived cell line offers many research applications. Here we are describing a few research areas using the HuH7 cell line.

• Drug resistance: HuH7 is a hepatocellular carcinoma (HCC) cell line that can be used to study drug resistance against potential liver cancer treatments. A study conducted in 2021 used HuH7 cells, which were experimentally made resistant to sorafenib, to develop new and effective therapeutics, i.e., mitochondria-accumulating self-assembly peptides.
• Gene expression/Receptor signaling: HuH7 cells can be used in gene expression studies. The expression of different oncogenic markers can be investigated to understand their behavior in response to any treatment, such as a study that used HuH7 cells to examine the effect of the Kaempferol compound on the expression pattern of MMP9 and AKT pathway genes. These pathways regulate the metastasis and invasiveness of hepatocellular carcinoma. Another recent study also investigated the role of the Wnt/β‐catenin signaling cascade gene in the survival and growth of HCC cells.
• Drug development: HuH7 cells are widely used in drug screening and development studies against liver cancer. Multiple studies have investigated the inhibitory potential of many plant extracts, plant-based compounds, nanoparticles, and other therapeutic agents against HCC. A study conducted in 2022 studied the pro-apoptotic and anti-cancer properties of a radionuclide against hepatocellular carcinoma using a HuH7 cell line.
• HCV replicon system: HuH7 cell line is highly susceptible to hepatitis C virus infection. Thus, the cell line can be used as an HCV replicon system and generate infectious HCV particles in vitro. This may help better understand the HCV infection and effective drug development to combat the infection.

Publications

There are a plethora of publications using the HuH7 cell line for different purposes. Here we have mentioned a few recent and significant publications.

1. “Hong, T. H., Jeena, M. T., Kim, O. H., Kim, K. H., Choi, H. J., Lee, K. H., ... & Kim, S. J. (2021). Application of self-assembly peptides targeting the mitochondria as a novel treatment for sorafenib-resistant hepatocellular carcinoma cells. Scientific Reports, 11(1), 1-15.” This study was published in Nature Scientific Reports in 2021. The study used experimentally induced sorafenib-resistant hepatocellular carcinoma cells (HuH7 cell line) to investigate the pro-apoptotic and anti-tumor effects of recently developed Mito-FF (mitochondria-accumulating self-assembly peptides).
2. „Ju, P. C., Ho, Y. C., Chen, P. N., Lee, H. L., Lai, S. Y., Yang, S. F., & Yeh, C. B. (2021). Kaempferol inhibits the cell migration of human hepatocellular carcinoma cells by suppressing MMP‐9 and Akt signaling. Environmental Toxicology, 36(10), 1981-1989.” This article was published in the Environmental Toxicology journal by Po-Chung Ju and colleagues in 2021. In this study, the researchers explored the anti-metastatic potential of a plant-based compound, kaempferol, against hepatocellular carcinoma cells (HuH7). In addition, they also studied the signaling pathways regulating the metastasis and invasion of liver cancer cells.
3. „Liu, C., Pan, J., Liu, H., Lin, R., Chen, Y., & Zhang, C. (2022). Daphnetin inhibits the survival of hepatocellular carcinoma cells through regulating Wnt/β‐catenin signaling pathway. Drug Development Research, 83(4), 952-960.” This paper was published in the Drug Development Research journal in 2022. This study explains the anti-cancer role of the daphnetin compound against HCC. This plant-derived compound inhibits Wnt/β‐catenin signaling to restrict the cancer cell's growth.
4. “Yu, X., Yang, F., Jiang, H., & Fan, L. (2020). RGFP966 suppresses tumor growth and migration through inhibition of EGFR expression in hepatocellular carcinoma cells in vitro. Drug Design, Development and Therapy, 121-128.” This research paper, published in the Drug Design, Development, and Therapy journal, describes the role of EGFR signaling in hepatocellular carcinoma growth and metastasis. The study utilized HuH7 cells to investigate the inhibitory effect of RGFP966, a histone deacetylase 3 (HDAC3) inhibitor, against HCC via suppressing EGFR pathway genes.
5. “Asadian, S., Piryaei, A., Gheibi, N., Aziz Kalantari, B., Reza Davarpanah, M., Azad, M., ... & Vosough, M. (2022). Rhenium Perrhenate (188ReO4) induced apoptosis and reduced cancerous phenotype in liver cancer cells. Cells, 11(2), 305.” This publication in the Cells journal indicates the use of HuH7 cells in investigating the anti-HCC potential of a radionuclide, i.e., rhenium perrhenate.
6. “Loutfy, S. A., Elberry, M. H., Farroh, K. Y., Mohamed, H. T., Mohamed, A. A., Mohamed, E. B., ... & Mousa, S. A. (2022). Antiviral Activity of Chitosan Nanoparticles Encapsulating Curcumin Against Hepatitis C Virus Genotype 4a in Human Hepatoma Cell Lines [Retraction]. International Journal of Nanomedicine, 17, 2891-2892.” This study was published in the International Journal of Nanomedicine. The study used a human hepatoma cell line (HuH7 cells) to explore the antiviral activity of curcumin-containing chitosan nanoparticles against HCV genotype 4a.

HuH7 Resources: Protocols, Videos & More

There are many resources available for HuH7 cell line culturing and maintenance. Herein, you will learn about the resources available for explaining cell culturing and transfection protocols for HuH7 and educational videos.

HuH7 Resources: Protocols, Videos & More

There are many resources available for HuH7 cell line culturing and maintenance. Herein, you will learn about transfection protocols for HuH7 cells, and you can find educational videos.

Transfection protocols of HuH7

HuH7 cells can be transfected in many ways. The following resources provide a bulk of information regarding transfection protocols, required reagents and chemicals, valuable tips, and precautions.

HuH 7 Transfection Tutorial:  This video explains the transfection protocol for the HuH7 cell line.

The information provided in this article will help you learn the basics and advanced knowledge of the HuH7 cell line before working on it. If you want to use these cells in your research, you can order them from us.