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A431 Cells - In-Depth Analysis of Epidermoid Carcinoma Cells in Dermatological Research

A431 is a human epidermoid cancer cell line. It is widely used in the biomedical research field, mainly in cancer biology, immune-oncology, and toxicology studies. In addition, A431 cells overexpress EGFR (epidermal growth factor receptor), thus used as a valuable in vitro model to study the cell cycle and cancer-associated cell signalling pathways.

This article will explain the basics of the A431 cancer cell line. You will learn the following:

  1. A431 cells: General characteristics and origin
  2. A431 cell line: Culturing information
  3. Advantages & disadvantages of A431 cell line
  4. Research applications of A431 cells
  5. Research Publications Featuring A431 Cells
  6. Resources for A431 Cell line: Protocols, Videos, and More

1.      A431 cells: General characteristics and origin

This section of the article will help you know the general information about the A431 cell line that you need to know before working with it. Such as, what is the origin of What is the A431 cell line? What are A431 cell line characteristics? What is the morphology of A431 cells? Where do the A431 cells come from?

  • A431, a human skin cancer cell line, was obtained from the epidermis of an 85-year-old female epidermoid carcinoma patient [1]. It was established by D.J. Giard, et al., who developed many other cell lines from solid tumors.
  • A-431 cells possess epithelial morphology. They aggregate and form cell clusters.
  • The skin cancer cell line a431 is hypertriploid. The modal chromosome number for this cell line is 74, which occurs in around 36% of cells. Higher ploidies also exist in approximately 1% of a cell population.

Tumor growing under the skin and infiltrating the underlying tissues.

2.      A431 cell line: Culturing information

Knowing a cell line's cell culture requirements and procedures can make its handling trouble-free. This part of the article will help you learn key points for culturing the A431 cell line. Such as you will know, What is the doubling time of A431 cells? Is A431 cancer cell line adherent? What growth medium is used for A431?

Key Points for Culturing A431 Cells

Doubling Time:

The population doubling time for A431 cells ranges from 80 to 100 hours.

Adherent or in Suspension:

A431 is an adherent cell line.

Seeding Density:

1 x 104 cells/cm2 cell density is ideal for the A431 cell line. Cells take almost 4 days to get confluent at this density. Adherent cells are washed with PBS (1x) and incubated with Accutase passaging solution. Afterwards, cells were resuspended in a culture medium and centrifuged. Harvested cells were again resuspended and dispensed into new flasks for growth.

Growth Medium:

DMEM medium supplemented with 10% fetal bovine serum, 4.5 g/L Glucose, 1.0 mM Sodium pyruvate, 1.5 g/L NaHCO3, and 4 mM L-Glutamine is used to culture the A431 cell line. Media should be replaced every 2 to 3 days a week.

Growth Conditions:

A431 cancer cells are grown in a humidified incubator equipped with a 5% CO2 source at 37°C temperature.


The cell line can be stored in an electric freezer or vapour phase of liquid nitrogen at below -150°C temperature as it helps in protecting cell viability.

Freezing Process and Medium:

CM-1 or CM-ACF are the two highly recommended freezing media used for A431 cells. Cells are frozen by using a slow freezing method that allows the temperature to drop by only 1 degree per minute.

Thawing Process:

Frozen A431 cells are thawed in a 37°C water bath containing an antimicrobial agent for 40 to 60 seconds. When only a small ice clump is left, cells are added with culture media and centrifuged. Harvested cells are resuspended and poured into culture flasks for growth.

Biosafety Level:

Biosafety level one is recommended for handling and maintenance of A431 cultures.

A-431 spheroids at different cell densities.

3.      Advantages & disadvantages of A431 cell line

The A431 cell line possesses distinguishable characteristics and offers several advantages and disadvantages. Herein, we have summarized a few below.


The main advantages of the A431 cancer cell line are:

EGFR overexpression

A431 cells overexpress epidermal growth factor receptor (EGFR). They serve as a positive control for studying EGFR signalling and its implications in cancer. They are also used to evaluate new cancer therapies.


A431 cells are tumorigenic and have the ability to form tumours. They can be used to develop xenograft cancer models, which are valuable tools for studying tumour growth dynamics and assessing new cancer treatments.


The disadvantages of A431 cells are:

Genetic abnormalities

A431 is a cancer cell line comprising genetic mutations and alterations that may distinguish it from the original tumour characteristics.

Microbial contamination

A431 is prone to microbial contamination, primarily bacterial contamination. However, maintaining standard aseptic culture conditions can help prevent such contamination in A431 cell cultures.


4.      Research applications of A431 cells

The A431 cell line is extensively used in cancer research. A few promising applications of this skin cancer cell line are listed here.

  • Cancer biology: The skin cancer cell line A431 is a great research tool to investigate cellular and molecular mechanisms driving cancer growth, development, metastasis, and invasion. Researchers have extensively used this cell line to intensely study the cell signalling pathways involved in different cancer cell processes. For instance, a study conducted in 2018 explored that PI3K/AKT/mTOR signalling pathway is involved in cancer cell proliferation, and inhibition of this pathway induces apoptosis in skin tumour cells, A431 [2]. Besides, another study also reported PI3K/AKT/mTOR pathway contribution in skin cancer cell metastasis and epithelial to mesenchymal transition (EMT), indicating its crucial role in cancer development and progression [3].
  • Drug testing and evaluation: A431 cells are used to evaluate the efficacy and effectiveness of potential anti-cancer drug candidates. Researchers determined the effects of drugs on cancer cell proliferation, metastasis, invasion, and apoptosis by conducting several lab experiments—research conducted by Nurhidayah Ab. Rahim and colleagues utilized A431 cells and assessed the therapeutic potential of a biogenically synthesized Alstonia angustiloba plant silver nanoparticles in 2022. The findings revealed that angustiloba-AgNPs exerted antiproliferative effects on skin squamous carcinoma cells [4].
  • Tumor Xenograft models: A431 cell line is tumorigenic and can form tumours when inoculated into a mouse model. Thus, it is a helpful tool for developing mouse xenograft models to investigate cancer biology in vivo. Several studies have used the A431 cancer cell line to develop tumour xenograft models. Such as Yu Jin Lim and his team developed a skin tumour xenograft mouse model using A431 cells to assess the anti-tumour and radiosensitizing effects of exogenous epidermal growth factor (EGF) in vivo [5].

5.      Research Publications Featuring A431 Cells

Here are some significant research publications featuring the A431 skin carcinoma cell line.

Efficacy of biopolymeric PVA-AuNPs and PCL-Curcumin loaded electrospun nanofibers and their anticancer activity against A431 skin cancer cell line

This article was published in Materials Today Communications in 2020. The study evaluated the anticancer potential of poly-ε-caprolactone curcumin-loaded electrospun nanofibers and polyvinyl alcohol-AuNPs against A431 cancer cells.

miRNA-221 promotes cutaneous squamous cell carcinoma progression by targeting PTEN

This study published in the Cellular & Molecular Biology Letters (2019) proposed that microRNA-221 plays an oncogenic role in cutaneous squamous cell carcinoma by targeting the PTEN gene.

Fungal vincristine from Eutypella spp-CrP14 isolated from Catharanthus roseus induces apoptosis in human squamous carcinoma cell line-A431

This research in BMC Complementary Medicine and Therapies (2016) suggests that vincristine, a secondary metabolite isolated from a fungus, Eutypella spp – CrP14, induces apoptosis in A431 cells.

Overexpression of CDC42SE1 in A431 cells reduced cell proliferation by inhibiting the Akt pathway

This study was published in 2019 in the Cells journal. This research proposed the CDC42SE1 gene as an important biomarker of skin cancer progression as its downregulation facilitates tumorigenesis.

Metformin inhibits the proliferation of A431 cells by modulating the PI3K/Akt signaling pathway

This research was published in Experimental and therapeutic medicine in 2015. It proposed that metformin suppressed the proliferation of the skin cancer cell line a431 by regulating PI3K/Akt signalling pathway.

6.      Resources for A431 Cell line: Protocols, Videos, and More

Here are some online resources featuring the A431 skin cancer cell line.

The following link contains cell culture information for A431 cells. 

  • A431 cancer cell line: This link contains necessary cell culture information about the A431 cell line. It includes culture media, cell density, handling of cryopreserved and proliferative cultures, etc.
  • A431 cell culturing: This website contains a brief protocol for culturing the A431 cell line.



  1. Quadri, M., et al., Investigating Cutaneous Squamous Cell Carcinoma in vitro and in vivo: Novel 3D Tools and Animal Models. Front Med (Lausanne), 2022. 9: p. 875517.
  2. Zeng, N., et al., Anticancer activity of caffeic acid nbutyl ester against A431 skin carcinoma cell line occurs via induction of apoptosis and inhibition of the mTOR/PI3K/AKT signaling pathway Retraction in/10.3892/mmr. 2021.12011. Molecular medicine reports, 2018. 17(4): p. 5652-5657.
  3. Rahaman, A., et al., Eucalyptol targets PI3K/Akt/mTOR pathway to inhibit skin cancer metastasis. Carcinogenesis, 2022. 43(6): p. 571-583.
  4. Rahim, N.A., et al., Investigation of antiproliferative mechanisms of Alstonia angustiloba-silver nanoparticles in skin squamous cell carcinoma (A431 cell line). Journal of Molecular Structure, 2022. 1250: p. 131814.
  5. Lim, Y.J., et al., Tumor growth suppression and enhanced radioresponse by an exogenous epidermal growth factor in mouse xenograft models with A431 cells. Cancer Research and Treatment: Official Journal of Korean Cancer Association, 2015. 47(4): p. 921-930.