MCF10A Cells: Unraveling Breast Cancer Biology in Non-Tumorigenic Contexts

MCF10A is an immortalized human mammary epithelial cell line. It is non-tumorigenic and used as an invaluable cell model to study the functioning and transformation of normal breast cells [1]. Moreover, researchers use these cells to investigate breast biology, including cell behaviour, signalling pathways, and gene expression. Besides, MCF10A cells are also used to study breast tumour development, progression, and therapeutic interventions.

This article will help you learn all the necessary information about MCF10A cells that you must know before working with them. You will go over the following:

Origin and general characteristics of MCF10A cells
MCF10A cells: Cell culture information
Advantages & limitations of MCF10A cell line
Research applications of MCF10A cell line
MCF10A cells: Research publications
Resources for MCF10A cell line: Protocols, Videos, and More

1. Origin and general characteristics of MCF10A cells

The first thing a researcher wants to know about a cell line is its origin and general characteristics that distinguish it from other cell lines and define its use. This section of the article will help you learn the origin and general characteristics of the MCF10A cell line. You will know: What is MCF10A human breast epithelial cell line? What is the origin of MCF10A cells? What type of cells are MCF10A? What are the characteristics of the MCF10A cell line? What is MCF10A cell size?

MCF10A, a human breast epithelial cell line, originated from the mammary gland of a 36 years old Caucasian female diagnosed with fibrocystic breasts in 1984. It was deposited by Michigan Cancer Foundation. MCF10A is non-tumorigenic and the most widely used in vitro normal human breast model.
MCF10A cell line has an epithelial morphology. Generally, they grow into monolayers; however, they can form domes in confluent cultures.
The MCF10A cell size ranges between 14.5 μm and 26.2 μm.
MCF10A cells represent a karyotype of 47 chromosomes [2].

MCF10AT1

MCF10AT1 is a cell line established by transfecting MCF10A breast cells with constitutively active HRAS. These pre-malignant cells have the ability to form ducts and lesions when transplanted to immunocompromised mice similar to human Atypical Ductal Hyperplasia (ADH) and Ductal carcinoma in situ (DCIS) breast cancers [3].

Doctor checks mammography x-ray. Mammography diagnosis for the prevention of breast cancer.

2. MCF10A cells: Cell culture information

MCF10A is a commonly used cell line in breast cancer research laboratories. It is imperative to know about the handling and maintenance of its cultures. This section will explain all the key points for efficient culturing of MCF10A cells. You will learn: What is the MCF10A doubling time? What is the MCF10A media? What is the seeding density of MCF10A? Is MCF10A breast cancer cell line adherent?

Key Points for Culturing MCF10A Cells

Population doubling Time:	The MCF10A doubling time is approximately 20 hours.
Adherent or in Suspension:	MCF10A breast cells are adherent.
Sub-cultivation ratio:	The split ratio recommended for MCF10A cells is 1:2 to 1:4. Adherent MCF10A cells are rinsed with 1 x PBS and incubated with Accutase at ambient temperature for 8 to 10 minutes. After cell detachment, culture media is added and cells are centrifuged. Harvested cells are resuspended and poured into the flask for growth. Media should be replaced 2 to 3 times a week.
Growth Medium:	MEGM is used as MCF10A media. It is supplemented with 100 ng/ml of MCF10A cholera toxin for ideal cell line growth.
Growth Conditions:	MCF10A cell cultures are maintained in a humidified incubator connected with a 5% CO2 supply at 37°C temperature.
Storage:	Frozen cells are stored in the vapour phase of liquid nitrogen or at below -150°C temperature in an ultra-low temperature freezer for the longer term.
Freezing Process and Medium:	The freezing media recommended for the MCF10A cell line is CM-1 or CM-ACF. Cells are frozen via a slow freezing method that permits a gradual 1°C fall in temperature and prevents cells from any shock.
Thawing Process:	Cryopreserved cells are kept in a water bath at 37°C for almost 60 seconds or until a small ice clump is left. Thawed cells are mixed with fresh culture media and centrifuged. After centrifugation, old freezing media is removed, and the cell pellet is again resuspended in the culture medium. Cells are dispensed into the new flask containing culture media for MCF10A growth.
Biosafety Level:	Biosafety level 1 laboratory settings are required to handle and maintain MCF10A cultures.

MCF10A cells growing together in adherent clusters at 20x and 10x magnification.

3. Advantages & limitations of MCF10A cell line

The advantages and limitations associated with MCF10A cells are discussed in this section.

Advantages

The significant advantages of MCF10A cells are mentioned below:

Non-tumorigenic	MCF10A cells are non-tumorigenic, meaning they do not cause tumors when transplanted into immunodeficient mice. Thus, they are ideal to study normal breast cell behaviour and biology.
Formation of 3D structures	MCF10A cells can form three-dimensional structures when grown in specific media, such as collagen. They spontaneously form acinar structures that resemble normal breast epithelium. This makes them useful to study breast cell organization and behavior in a three-dimensional context.

Limitations

The limitation associated with MCF10A cells are:

Phenotypic plasticity

MCF10A cells exhibit phenotypic and behavioural changes when grown in different culture conditions. This may affect the reproducibility of experiments and introduce variability in results.

4. Research applications of MCF10A cell line

Here are some significant applications of MCF10A cell lines in research.

Study of normal breast biology: MCF10A cells serve as an in vitro model to study normal breast epithelial cell behaviour and processes, including cell adhesion, morphogenesis, and signalling pathways. Like researchers studied the transcriptomics of MCF7 breast cancer cells and MCF10A normal breast epithelial cells and compared it to luminal A breast cancer and normal samples, the findings suggested that both cell lines limit their ability to fully represent the cancer-related processes in clinical luminal A samples [4].
Drug screening: MCF10A cell line is commonly used in drug screening and therapy development research. It evaluates the cytotoxicity and efficacy of anti-breast cancer drugs and treatment strategies. For instance, research conducted in 2021 used MCF10A cells as normal breast epithelial cells and studied the cytotoxicity of Senna alata plant extract and its fractions [5].
Tumorigenesis: MCF10A cells are non-tumorigenic. However, they are used to investigate the development and progression of breast cancer. Briefly, these cells are either used in combination with other cell lines or genetically manipulated to study breast cancer development-associated genetic mutations and signalling pathways. For instance, MCF10A brca1 and MCF10A Her2 cell lines are developed by genetically manipulating MCF10A cells. A study genetically manipulated MCF10A cells and knockdown a gene called PHLDA1 (pleckstrin homology-like domain, family A, member 1) involved in several cellular processes, including cell death. Researchers found that the knockdown of the PHLDA1 gene resulted in morphological changes in MCF10A cells and promoted their migration and invasion. This indicates that PHLDA1 is an important drug target to combat breast cancer [6].

5. MCF10A cells: Research publications

Some significant and most cited research publications featuring the MCF10A cell line are mentioned here.

TGF-β-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells

This research article in the International Journal of Oncology (2004) studied the TGF-β signalling pathway in MCF10A cells. The findings suggest that TGF-β treatment induces migrative and invasive phenotypes in MCF10A cells.

Cytotoxic, necrotic, apoptotic, and autophagic properties of venom sac extract of Vespa orientalis in T47D and MCF10A breast cell lines

This study published in Toxin Reviews (2023) evaluated the necrotic, cytotoxic, and autophagic activity of venom sac extract of Vespa orientalis hornet on MCF10A cells.

Leptin promotes expression of EMT-related transcription factors and invasion in a Src and FAK-dependent pathway in MCF10A mammary epithelial cells

This research is published in the Cells (2019). This study proposed that leptin, an adipokine enhances the expression of epithelial to mesenchymal transition-related transcription factors and invasion of MCF10A cells in a FAK and Src-dependent manner.

Connexin 32 induces pro-tumorigenic features in MCF10A normal breast cells and MDA-MB-231 metastatic breast cancer cells

This research was published in 2020 in Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. The study proposed that the connexin-32 protein exerts tumorigenic properties in MCF10A cells.

Potential Effect of Pseudevernia furfuracea (L.) Zopf Extract and Metabolite Physodic Acid on Tumour Microenvironment Modulation in MCF-10A Cells

This article was published in the Biomolecules (2021). The study evaluated the effect of Pseudevernia furfuracea (L.) Zopf extract and its secondary metabolite physodic acid on tumour microenvironment modulation in the MCF10A cell line.

6. Resources for MCF10A cell line: Protocols, Videos, and More

The following are a few online resources for MCF10A cells.

MCF10A transfection: This link will provide a detailed protocol for the transfection plasmid DNA into MCF10A cells.
Cell culture protocols: This video will explain the basic protocol for passaging, freezing, and thawing adherent cells.

The MCF10A cell culture protocol is listed here.

MCF10A cell culture protocol: This document contains a step-by-step protocol for passaging MCF10A cells.
MCF10A cells subculturing: This link will help you learn the protocol for subculturing of MCF10A breast epithelial cells.
MCF10A cell line: This website will help you learn all the basic MCF10A cell culture protocol, including protocols for subculturing and handling of proliferative and cryopreserved cultures.

References

Qu, Y., et al., Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells. PLoS One, 2015. 10(7): p. e0131285.
Marella, N.V., et al., Cytogenetic and cDNA microarray expression analysis of MCF10 human breast cancer progression cell lines. Cancer Res, 2009. 69(14): p. 5946-53.
So, J.Y., et al., Differential Expression of Key Signaling Proteins in MCF10 Cell Lines, a Human Breast Cancer Progression Model. Mol Cell Pharmacol, 2012. 4(1): p. 31-40.
Goh, J.J.H., et al., Transcriptomics indicate nuclear division and cell adhesion not recapitulated in MCF7 and MCF10A compared to luminal A breast tumours. Sci Rep, 2022. 12(1): p. 20902.
Modarresi Chahardehi, A., et al., Low cytotoxicity, and antiproliferative activity on cancer cells, of the plant Senna alata (Fabaceae). Revista de Biología Tropical, 2021. 69.
Bonatto, N., et al., PHLDA1 (pleckstrin homology-like domain, family A, member 1) knockdown promotes migration and invasion of MCF10A breast epithelial cells. Cell Adh Migr, 2018. 12(1): p. 37-46.