HuH7 Cells: A Gateway to Hepatoma and HCV Research
The HuH7 cell line, derived from human hepatoma, represents an immortalized collection of tumorigenic epithelial-like cells. This line, along with its derivatives HuH7.5 and HuH7.5.1, serves as a practical surrogate for primary hepatocytes in experimental contexts. It is predominantly utilized in cell culture models to investigate hepatitis C virus (HCV) infection and hepatoma pathophysiology.
Origin and Characteristics of HuH7 Cells
Understanding the origins, key features, and optimal culture conditions of the HuH7 cell line is essential prior to its application in research.
Definition and Establishment
The HuH7 cell line was established by Sato, J, and Nakabayshi, H in 1982, derived from a highly differentiated hepatocyte cancer lineage. It was originally sourced from hepatocellular carcinoma in a 57-year-old Japanese male. Characterized by its capacity to produce various physiologically relevant substances, such as albumin and alpha-fetoprotein, HuH7 cells exhibit a high susceptibility to HCV infection, making them suitable for use in HCV replicon systems. This feature renders the cell line valuable for the screening and development of anti-HCV therapeutics.
Morphological Features
HuH7 cells display an epithelial-like morphology, with a hexagonal shape, and adhere to surfaces to form 2D monolayers during growth.
Cell Dimension
On average, HuH7 cells have a diameter of 21.9µm, tending towards a larger, flattened appearance.
Genomic Composition and Ploidy
The HuH7 cell line is characterized by a heterogeneous chromosome number, typically ranging between 55 and 63, which reflects the genetic variability within the cell population. This heterogeneity is a consequence of selective pressures encountered during cell culture, and may vary across different laboratory conditions.
Comparative Analysis: HuH7 vs. HuH7.5 Cell Lines
The HuH7.5 cell line, a derivative of HuH7, differs primarily due to a missense mutation in the RIG-I (DDX58) gene, crucial for the intracellular antiviral response. This mutation enhances the permissiveness of HuH7.5 cells for the replication of genomic and subgenomic HCV RNA, distinguishing it from the parent HuH7 cell line in terms of its utility in virological research.
Cultivating HuH7 Cells: Essential Protocols for Optimal Growth
HuH7 cells, integral to a myriad of research studies, necessitate precise culture conditions to thrive. This section delves into the fundamental aspects of HuH7 cell culture, ensuring the maintenance of their physiological relevance in scientific investigations.
Doubling Time Dynamics
The doubling time for HuH7 cells is typically 24 hours, although variations in cell culture media can extend this period to 36-48 hours, highlighting the influence of environmental conditions on cellular proliferation.
Adhesion Characteristics
HuH7 cells predominantly form a two-dimensional monolayer, adhering to the surfaces of culture plates and flasks, underscoring their preference for a stable physical substrate over suspension cultures.
Optimal Seeding Density
Maintaining HuH7 cell confluency within the 30-90% range is critical to prevent a decline in proliferation rates beyond 90% confluency. Subculturing with accutase facilitates detachment, enabling seeding at densities tailored to the experimental needs, ranging from optimal (2×10⁴ cells/cm²) to saturation (6×10⁴ cells/cm²) levels.
Nutritional Requirements
Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose content and supplemented with 10% fetal bovine serum (FBS) provides an ideal nutritional environment for HuH7 cells. Regular medium renewal, ideally 2-3 times weekly, is crucial for sustained cell health.
Environmental Conditions
A humidified incubator set to 5% CO₂ and 37°C optimally supports HuH7 cell culture, simulating physiological conditions conducive to cellular activities.
Long-Term Storage
For extended preservation, HuH7 cells should be stored in the vapor phase of liquid nitrogen, safeguarding their viability for future research.
Cryopreservation and Recovery
Employing a gradual freezing technique is paramount for maintaining cell viability during cryopreservation. Cells should be suspended in a specifically designed freeze medium, such as CM-1, ensuring a smooth transition to cryogenic storage. Upon thawing, a swift yet gentle revival in a 37°C water bath, followed by centrifugation to discard the freeze medium, and resuspension in fresh growth medium, is essential for re-establishing cell cultures.
Biosafety Considerations
Adhering to a Biosafety Level 1 protocol is advised for the handling and propagation of the HuH7 cell line, aligning with safety and ethical standards in laboratory settings.
By adhering to these meticulously outlined culture conditions, researchers can ensure the robust growth and physiological relevance of HuH7 cells in their scientific explorations, paving the way for groundbreaking discoveries in hepatoma and hepatitis C virus research.
Explore HuH7 cells for your scientific breakthroughs
HuH7 cells: Advantages & Disadvantages
The HuH7 cell line offers both advantages and limitations at the same time. Let’s go through the potential pros and cons of this cell line, as it may help you decide if it is appropriate for your experiments.
Advantages of HuH7 cell line
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 the addition of serum easily; 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: The HuH7 cell line is widely used for transfection purposes due to its high receptiveness to the HCV genome. Therefore, these cells are pivotal for anti-HCV drug screening and development.
- Alternative to human hepatocytes: The 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 a xenograft mouse model: HuH7 cells have been used to produce a cell line-originated xenograft mouse model. This animal model may provide valuable insights for researchers about protein kinase inhibitors and anti-cancer therapies.
- Cancer research: The HuH7 cell line is a human liver cancer cell line, which helps researchers conduct liver cancer studies.
Limitations of the HuH-7 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 through the pH change and turbidity of the culture medium. Among bacterial contamination, mycoplasma contamination is usually not apparent. However, it slowly affects the morphology, gene expression, and other characteristics of the cell line.
Culture period: Although the HuH7 cell line is immortal, exceeding the culture period can affect the health of cells gradually. It may affect the proliferation rate and gene expression, which may reduce the reproducibility of results. For the HuH7 cell line, the passage number should be kept below 20–25.
Applications of HuH-7 cells
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.
Genetic Insights and Molecular Profiling Through HuH7 Cells: The distinct HuH7 cell lines, including various derivatives and variants, have enabled a deeper understanding of gene expression and the role of specific genomic markers in disease. Techniques such as Raman spectroscopy, championed by researchers like Evgeny Gladilin, have been applied to HuH7 cells for molecular and cellular profiling, revealing insights into the cellular response to viral infection. The potential of Raman spectroscopy, combined with methods like quantitative proteomics, has facilitated the exploration of host cell responses, including the interferon response and changes in lipid droplets, which are critical for understanding HCV replication and the effect of SARS. The expression of different oncogenic markers can be investigated to understand their behavior in response to any treatment, such as in a study that used HuH7 cells to investigate 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 been conducted to investigate 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.
HuH7 Cells: A Focal Point in Viral Research and Cellular Analysis HuH7 cells have become a cornerstone in the study of viral infection and infectious diseases, particularly hepatitis C virus (HCV) and their interactions with coronaviruses like SARS-CoV-2, responsible for coronavirus disease 2019 (COVID-19). The development of experimental HCV cell culture systems (HCVcc) using HuH7 cells has significantly advanced our ability to study HCV's intricate replication process and host factors essential for HCV infection. This includes examining the efficiency of HCV RNA replication and the production of infectious virions. The distinct lines of HuH7 cells, including senescent and infected variants, provide a diverse panel for investigating the multiplicity of infection, viral propagation, and inhibition mechanisms against viruses like SARS.
In summary, the study of HuH7 cells encompasses a broad spectrum of scientific inquiries, from the molecular mechanisms underpinning viral infections to the detailed cellular responses elicited by these pathogens. Through the lens of HuH7 research, the scientific community continues to uncover the complex dynamics of host-virus interactions, contributing to the advancement of antiviral strategies and our understanding of cellular determinants in viral diseases.
HuH-7 publications
There is a plethora of publications using the HuH7 cell line for different purposes. Here, we have mentioned a few recent and significant publications.
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).
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.
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.
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.
- Rhenium Perrhenate (188ReO4) Induced Apoptosis and Reduced Cancerous Phenotype in Liver Cancer Cells
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.
This study is published in the International Journal of Nanomedicine. The study used a HuH7 cells to explore the antiviral activity of curcumin-containing chitosan nanoparticles against HCV genotype 4a.
HuH-7 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.
Cell culture protocols
The following resources can help you get started with HuH7 cultivation and maintenance.
HuH-7 Cell Culture Info and Resources: This website has a lot of useful information, including the subculturing protocol of the HuH7 cell line, its storage, and its handling.
- HuH7 cell culture protocol
Transfection protocols of HuH7
HuH7 cells are transfected in many ways. The following resources provide a bulk of information regarding transfection protocols, required reagents and chemicals, useful tips, and precautions.
- HUH-7 Transfection: This website gives details about transfection reagents for the HuH7 cell line. Moreover, it also provides the following links for the simple Huh7 transfection protocol and the Huh7 CRISPR/Cas9 transfection protocol.
Videos related to HuH7 cell line
There are many educational videos available on basic subculturing, plating and transfection protocols, and more.
- Passaging Cells: Cell Culture Basics: This video is about the basic culturing protocol for the cell line.
We expect that the information provided in this article will help you gain basic and advanced knowledge about the HuH7 cell line before working on it. If you want to use these cells in your research work, you can order from us.