Apart from the suggested supplements, EGF, cholera toxin, hydrocortisone, and even insulin are usually required for the MCF10 cells to grow and survive in EMEM media since optimized media for epithelial cells is DMEM. By closely monitoring the attachment, morphology and proliferation rate, you would be able to find at which stage the cells are dying and add the additives accordingly.

Referring to the ATCC database, when culturing MCF10 cells, additional cytokines need to be added on the basis of the basal medium. At present, the culture conditions for directly culturing in the basal medium EMEM have not been found.

MCF10 cells are divided into two types. One is MCF10A, which is derived from human breast fibrocystic lesion tissue, and the other is MCF10F, which is derived from normal human breast ductal epithelial cells (breast tissue). The culture conditions can be referred to at https://www.atcc.org/products/crl-10317

https://www.atcc.org/products/crl-10318.

Taking MCF10A as an example, the following is a reference for the culture method:

Initial Cultivation

Use a T25 or T75 culture flask and add at least 10 mL or 15 mL of complete medium (Mammary Epithelial Basal Medium, MEBM) respectively.

There is no need to change the medium immediately after the cells are thawed. Just let them stand for recovery.

Medium Requirements

The basal medium is MEBM, to which the following components need to be added:

• 100 ng/mL cholera toxin.
• EGF provided by the kit (it cannot be filtered, and avoid filtering the medium after adding it).
• 1% penicillin-streptomycin. Do not use GA-1000 in the kit.

Cell Characteristics

The cells have weak adhesion. In the initial stage, there will be some suspended cells, all of which are viable cells and should be retained.

The suspended cells can be recovered by low-speed centrifugation (125 ×g for 10 minutes) and then added back to the adherent cells.

Subculture Steps

• Remove the old medium and wash the cells with PBS.
• Add 3 mL of 0.05% trypsin-0.02% EDTA and incubate at 37°C for 10-15 minutes until the cells become round.
• Neutralize the trypsin with 3 mL of 0.1% soybean trypsin inhibitor.
• Centrifuge (125 ×g for 10 minutes) to completely remove the digestion reagents, and resuspend the cells in fresh complete medium.

The recommended subculture ratio is 1:3 to 1:4.

Precautions

• Trypsin must be strictly removed or neutralized in the serum-free medium.
• The cells have weak adhesiveness. Avoid excessive pipetting or discarding the suspended cells.

Without knowing your exact experimental background, I would recommend using the medium described below with the listed supplements for optimal cultivation of MCF-10A cells:

RPMI 1640

• 5% FCS (Fetal Calf Serum)
• 1% P/S (Penicillin/Streptomycin)
• 20 ng/ml EGF (Epidermal Growth Factor)
• 1.4 × 10⁻⁶ M Hydrocortisone
• 100 ng/ml Cholera Toxin
• 10 µg/ml Insulin

In addition, the cells require optimal incubation conditions at 37°C and 5% CO₂. Cell confluency is also important—when passaging, the cells should not be seeded too densely or too sparsely.