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MCF10A Cell Line: Unraveling Breast Cancer Biology in Non-Tumorigenic Contexts

The MCF10A cell line is a pivotal tool in breast cancer research, representing an immortalized yet non-tumorigenic human mammary epithelial cell model. This cell line is extensively utilized to explore the intricacies of normal breast cell function, transformation processes, and the underlying mechanisms of breast biology, including cellular behaviors, signaling pathways, and gene expression patterns. Furthermore, MCF10A cells serve as a crucial resource for delving into breast tumor development, understanding its progression, and evaluating potential therapeutic strategies.

Origin and General Characteristics of MCF10A Cells

Delving into the MCF10A cell line, researchers prioritize understanding its origins and distinguishing features, which shed light on its application and utility in research. The MCF10A cell line, derived from the mammary gland of a 36-year-old Caucasian female with fibrocystic breast condition in 1984, is renowned for its non-tumorigenic profile, making it an exemplary model for studying normal human breast tissue in vitro.

Key characteristics of the MCF10A cell line include:

  • Epithelial Morphology: Typically growing in monolayers, MCF10A cells can also form dome-like structures in confluent cultures, highlighting their dynamic growth patterns.
  • Cell Size: The size of MCF10A cells varies between 14.5 μm and 26.2 μm, accommodating a range of experimental setups.
  • Karyotype: MCF10A cells exhibit a karyotype with 47 chromosomes, offering insights into genetic studies and chromosomal research in breast epithelial cells.

MCF10AT1: A Pre-malignant Derivative

The MCF10AT1 cell line, developed by transfecting MCF10A cells with the HRAS gene, represents a pre-malignant stage capable of forming ductal structures and lesions akin to Atypical Ductal Hyperplasia (ADH) and Ductal Carcinoma in Situ (DCIS) when introduced into immunocompromised mice. This transformation underscores the cell line's utility in modeling early-stage breast cancer developments and studying the transition from benign to malignant states.

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

MCF10A Cells: Cell Culture Information

MCF10A, a widely utilized cell line in breast cancer research, necessitates precise handling and maintenance to ensure its viability and utility in experimental settings. This guide outlines the essential considerations for the effective culturing of MCF10A cells, touching upon their doubling time, preferred media, seeding density, and adherence properties.

Key Points for Culturing MCF10A Cells

  • Population Doubling Time: The MCF10A cell line typically has a doubling time of around 20 hours, indicative of its robust growth rate under optimal conditions.

  • Adherence Characteristics: These cells exhibit an adherent growth pattern, necessitating a solid substrate for attachment and proliferation.

  • Sub-cultivation Practices: For sub-culturing, a split ratio of 1:2 to 1:4 is recommended. The protocol involves washing the cells with PBS, detaching them with Accutase, and then transferring them to a new flask after centrifugation and resuspension in fresh media. It's advisable to refresh the culture media two to three times per week to support healthy growth.

  • Growth Medium: MCF10A cells thrive in MEGM, a specialized medium that should be fortified with 100 ng/ml cholera toxin to optimize cell growth and function.

  • Optimal Growth Conditions: Cultures should be maintained in a humidified incubator set at 37°C with a 5% CO2 atmosphere to replicate physiological conditions closely.

  • Storage Guidelines: For long-term storage, cells should be kept in the vapor phase of liquid nitrogen or at temperatures below -150°C in an ultra-low temperature freezer.

  • Freezing and Thawing Procedures: The recommended freezing medium for MCF10A cells is either CM-1 or CM-ACF. Employ a slow freezing technique to minimize thermal shock. Thawing should be done gently in a 37°C water bath until a small ice clump remains. Subsequently, cells should be mixed with fresh culture media, centrifuged, and the cell pellet resuspended in new media before transferring to a culture flask.

  • Biosafety Considerations: MCF10A cell cultures can be safely handled within Biosafety Level 1 laboratory settings, ensuring straightforward maintenance and compliance with safety standards.

Adhering to these guidelines will facilitate the successful culturing of MCF10A cells, enabling their continued contribution to the advancement of breast cancer research.

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

Advantages & Limitations of MCF10A Cell Line

Exploring the MCF10A cell line provides a nuanced understanding of both its beneficial attributes and inherent constraints, crucial for its effective application in breast cancer research.


  • Non-tumorigenic Nature: A hallmark of MCF10A cells is their non-tumorigenic characteristic, allowing researchers to study normal breast cell behavior and biology without the complication of tumor formation in immunodeficient mice.

  • 3D Structure Formation: MCF10A cells possess the unique ability to form three-dimensional acinar structures resembling normal breast epithelium when cultured in specific media, such as collagen. This capability is instrumental in studying breast cell organization and behavior within a 3D context, offering insights closer to in vivo conditions.


  • Phenotypic Plasticity: Despite their advantages, MCF10A cells display variability in phenotype and behavior under different culture conditions, potentially impacting the consistency and reproducibility of experimental results.

Research Applications of the MCF10A Cell Line

The MCF10A cell line is a cornerstone in multifaceted research paradigms, particularly in the field of mammary cell biology and oncology. Herein, we delineate its diverse applications:

Normal Mammary Epithelial Function

MCF10A cells are instrumental in vitro for elucidating the intricacies of normal mammary epithelial cell functions, encompassing cell-cell adhesion mediated by proteins like E-cadherin, morphogenetic processes, and intricate signaling cascades. Although invaluable, juxtaposition with malignant counterparts such as MCF7 cells occasionally underscores the cell line’s inability to completely recapitulate the cancer-associated milieu observed in vivo.

Pharmacological Profiling

As a preeminent model, MCF10A cells are exploited in pharmacological profiling to discern the cytotoxicity and therapeutic potential of nascent anti-breast cancer compounds. For example, these cells have been pivotal in determining the efficacy of bioactive constituents from botanicals such as Senna alata, thereby substantiating their contribution to novel therapeutic strategies.

Carcinogenesis Research

Despite their non-tumorigenic origin, MCF10A cells provide a malleable template for studying breast tumorigenesis. Utilized in concert with tumorigenic cell lines or modified via genetic engineering, they facilitate exploration into the molecular genesis and progression of breast cancer. Such applications are exemplified by research that manipulates genes, including PHLDA1, within MCF10A cells to examine their influence on cellular migration and invasion, thereby spotlighting new potential targets for intervention.

Three-Dimensional Culture Models

MCF10A cells thrive within three-dimensional (3D) culture systems, such as mixed Matrigel environments, which mimic the in vivo conditions, fostering our understanding of the spatial and mechanical context of cell behavior. This 3D approach is instrumental in delineating the pathways that govern mammary cell differentiation and the morphological evolution of early neoplastic lesions.

Metastatic Potential Assessment

Investigations into the mechanisms underlying metastasis leverage MCF10A cells to simulate the epithelial to mesenchymal transition, a pivotal event in metastatic dissemination. Researchers observe these transitions within various cell models, utilizing markers such as E-cadherin, to gain insights into cellular dynamics during breast carcinoma progression.

Mammosphere Formation and Progenitor Cell Studies

The ability of MCF10A cells to form mammospheres when cultured in non-adherent conditions makes them an invaluable resource for studying mammary progenitor cells and their role in breast cancer biology, from initiation to the acquisition of invasive characteristics.

The remarkable versatility and fidelity of MCF10A cells to human breast epithelium fortify their status as an indispensable asset in the ongoing quest to unravel the complexities of breast cancer, underscoring their perpetual value in cutting-edge research.

Unlock the potential of your research with our MCF10A cells

MCF10A Cells: Research Publications

Highlighted here are some of the most notable and frequently cited research studies that have utilized the MCF10A cell line, contributing significantly to the field of breast cancer research.

  • TGF-β Signaling Pathway Insights: A pivotal study published in the International Journal of Oncology (2004) delved into the TGF-β signaling pathway in MCF10A cells, revealing that TGF-β treatment can induce migratory and invasive phenotypes, underscoring the complexity of cellular responses to TGF-β.

  • Venom Sac Extract Study: Research featured in Toxin Reviews (2023) explored the effects of Vespa orientalis hornet venom sac extract on MCF10A cells, examining its cytotoxic, necrotic, apoptotic, and autophagic properties, thus opening new avenues for understanding cell response to natural toxins.

  • Leptin's Role in Cell Invasion: A study in Cells (2019) proposed that leptin, a well-known adipokine, promotes the expression of EMT-related transcription factors and enhances invasion in MCF10A cells through a pathway dependent on Src and FAK, highlighting the intricate interplay between adipokines and cancer cell behavior.

  • Connexin 32's Tumorigenic Features: Published in Biochimica et Biophysica Acta (BBA) - Molecular Cell Research (2020), this study posited that connexin-32 protein may impart pro-tumorigenic characteristics to MCF10A cells, suggesting a potential role for connexin-32 in the early stages of breast cancer development.

  • Effect of Pseudevernia furfuracea Extract: An article in Biomolecules (2021) assessed the impact of Pseudevernia furfuracea (L.) Zopf extract and its metabolite physodic acid on the tumor microenvironment modulation in MCF10A cells, offering insights into the potential therapeutic applications of natural compounds in modulating tumor-stromal interactions.

These publications underscore the versatility and applicability of the MCF10A cell line in advancing our understanding of breast cancer biology, from exploring cellular signaling pathways to evaluating the potential therapeutic effects of natural and synthetic compounds.

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.

Exploring MCF10A Cells: A Comprehensive FAQ on Their Role in Breast Cancer Research and Cellular Biology

MCF 10A cell lines are immortalized, non-tumorigenic epithelial cells derived from human breast tissue. They are extensively used as in vitro models to study breast tumor progression due to their close mimicry of normal breast epithelium and their ability to undergo oncogenic transformation.

The MCF 10A cell line expresses E-cadherin, a critical protein in cell-cell adhesion and maintenance of epithelial integrity. Alterations in E-cadherin expression in MCF 10A cells allow researchers to study its role in breast cancer tumorigenesis, particularly how its downregulation may lead to epithelial to mesenchymal transition, a key step in metastasis.

MCF 10A cells are capable of forming mammospheres in suspension culture, which is indicative of the presence of mammary progenitor cells. Mammosphere culture is a technique used to enrich for these progenitor cells and to study their role in mammary cell biology and cancer.

Mixed Matrigel matrices provide a three-dimensional scaffold that closely resembles the extracellular matrix in vivo, promoting the growth and differentiation of MCF 10A cells into mammospheres. This 3D environment is crucial for studying the cells’ phenotype in 3D culture and their behavior during tumorigenesis.

Immunofluorescence staining of MCF 10A cells can reveal the expression and localization of specific proteins, providing insights into the molecular mechanisms underlying the transition from a normal to an invasive breast carcinoma phenotype. Such studies can also elucidate the role of genomic signaling in this process.

The MCF 10A model serves as an effective in vitro system to study the EMT by allowing researchers to induce EMT markers and observe the resultant phenotypic changes. This helps in understanding the progression from a non-invasive to an invasive phenotype in cancer.

EGF is a vital component of the culture media for MCF 10A cells, especially in 3D culture models. It acts as a mitogen and is essential for the cells' proliferation and survival. Its absence or presence can significantly affect the cells' phenotype and behavior.

MCF10A sublines, which possess specific genetic modifications, and soybean trypsin inhibitor, a component used to inhibit trypsin activity during cell passage, are tools employed by the breast cancer research community to explore various aspects of cancer biology, including resistance mechanisms and treatment responses.

Immunohistochemistry and immunofluorescence staining are essential techniques for characterizing the phenotype of MCF 10A cells within mammospheres. They allow for the visualization of specific proteins and their distribution, facilitating the study of cell differentiation and the identification of stem-like cells within mammospheres.

The expression of EMGFP-tagged E-cadherin in MCF 10A cells allows real-time visualization of E-cadherin-mediated cell signaling. This enhances the understanding of how E-cadherin contributes to cell adhesion, signaling pathways involved in cell growth, and the dysregulation of these processes in cancer development.


  1. Qu, Y., et al., Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells. PLoS One, 2015. 10(7): p. e0131285.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.

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