The Guide to MCF 10A Culture: From Key Characteristics to Practical Tips

MCF 10A is a landmark cell line in breast cancer research. Its spontaneously immortalized non-tumorigenic characteristics make it an invaluable tool for studying normal breast physiology and the mechanisms underlying carcinogenesis. In this issue of the Cell Culture Academy, we’ll introduce you to MCF 10A and provide essential culture tips, helping you get started quickly and successfully carry out your experiments with confidence.

I. Introduction to MCF 10A

MCF 10A is a normal human breast epithelial cell line established by Herbert D. Soule and colleagues at the Michigan Cancer Foundation in 1984. It was derived from benign fibrocystic breast tissue obtained via subcutaneous mastectomy from a 36-year-old female patient. This tissue, designated MCF-10M, contained dense stroma and parenchyma. After processing, epithelial cells were isolated for primary culture.

• Under different calcium concentrations, MCF-10M gave rise to two immortalized subtypes:

MCF 10A: Grows as an adherent epithelial monolayer with a cobblestone-like arrangement. It serves as a model for normal breast epithelial cells and is widely used to study early events in carcinogenesis.

MCF 10F: Grows in clusters in suspension, making it suitable for investigating anchorage-independent growth and characteristics associated with metastasis.

The immortalization of both subtypes is driven by a chromosomal translocation, t(3;9) (p14;p21), which results in the homozygous deletion of the CDKN2A/CDKN2B locus, encoding the tumor suppressors p16 and p15. This deletion allows the cells to resist calcium-induced senescence and proliferate indefinitely while maintaining a near-diploid karyotype.

• Differences in culture conditions between the two subtypes:

MCF 10A: Differentiates at physiological calcium levels (1.05 mM) and proliferates at low calcium levels (0.04 mM).

MCF 10F: Maintains its suspension growth at low calcium (0.04 mM), while high calcium levels cause morphological changes.

II. Culture Information of MCF 10A

Growth characteristics: Epithelial-like, adherent growth

Culture system: DMEM/F12 supplemented with 5% Donor Equine Serum, 20 ng/mL EGF, 0.5 μg/mL Hydrocortisone, 10 μg/mL Recombinant Human Insulin Solution, 1% Non-Essential Amino Acid Solution (NEAA) , 1% Penicillin-Streptomycin Solution

Recommended subculturing ratio: 1:2-1:4

Recommended medium renewal: 2-3 times per week

Freezing medium: 55% basal medium + 40% FBS + 5% DMSO

Freezing temperature: Liquid nitrogen

II. Culture Characteristics of MCF 10A

MCF 10A cells are epithelial-like and grow as a single-layer adherent monolayer with a characteristic cobblestone pattern. During culture, the cells gradually form patches as they aggregate. The cell doubling time is typically 3-4 d, but actual doubling rates are dependent on the culture environment. After subculturing, cell attachment to the surface is relatively slow. Cells generally begin to adhere within approximately 4 h, but full attachment and spreading takes longer. By 24 h, the cells are fully spread and begin to proliferate noticeably. After 36 h, they aggregate further, forming a tightly connected, sheet-like structure (Fig. 1).

0h	4h	8h
24h	36h	60h

Fig. 1. Morphological changes of MCF 10A after subculturing

IV. Tips for Culturing MCF 10A

1. Monitor the digestion process

MCF 10A cells adhere strongly to the surface, making digestion relatively slow. During subculturing, digestion time can be extended to 10-15 min (Fig. 2). Alternatively, fractional digestion can be used to ensure complete cell detachment. During digestion, gently tap the culture flask to help cells round up and detach, and then promptly terminate the digestion. This method allows for better control and helps prevent cell damage caused by overdigestion.

1 min	3 min	5 min
7 min	10 min	15 min

Fig. 2. Digestion process of MCF 10A

2. Choosing the culture medium

While the DMEM/F12 culture system is the original culture condition for MCF 10A, its preparation can be cumbersome. For convenience, it is recommended to use a specialized MCF 10A Cells Complete Medium. This medium not only simplifies preparation but also better supports cell growth, maintaining cells in optimal health.