Human breast cancer cell culture

                                                     MCF-7

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  MCF-7 is a breast cancer cell line isolated in 1970 from a 69-year-old White woman. MCF-7 is the acronym of Michigan Cancer Foundation-7, referring to the institute in Detroit where the cell line was established in 1973 by Herbert Soule and co-workers. The Michigan Cancer Foundation is now known as the Barbara Ann Karmanos Cancer Institute. Prior to MCF-7, it was not possible for cancer researchers to obtain a mammary cell line that was capable of living longer than a few months.

The patient, Frances Mallon died in 1970. Her cells were the source of much of current knowledge about breast cancer. MCF-7 and two other breast cancer cell lines, named T-47D and MDA-MB-231, account for more than two-thirds of all abstracts reporting studies on mentioned breast cancer cell lines, as concluded from a Medline-based survey.

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    MCF-7 cells have the following characteristics: they are a cell line derived from infiltrating ductal carcinoma of the breast with pleural effusion, exhibiting a phenobarbital epithelial phenotype and expressing progesterone and estrogen receptors. HER2/neu protein with overexpression, but without amplification of ERBB2 gene. When estrogen is present, MCF-7 cells will proliferate. In addition, MCF-7 cells have tumorigenicity in mice, but estrogen supplementation is only necessary when implanted in subcutaneous fat or breast fat pads. If implanted within a catheter, it has tumorigenicity in mice without estrogen supplementation.

    This cell line retains several characteristics of breast epithelial tissue that have undergone cell differentiation, including the ability to process estradiol through cytoplasmic estrogen receptors and the ability to form domes. It is currently known that tumor necrosis factor alpha can inhibit the growth of MCF-7 cells, and the use of anti estrogen during treatment can regulate the secretion of insulin-like growth factor binding protein. Studies have shown that omega-3 fatty acids and six fatty acids (such as eicosapentaenoic acid, docosahexaenoic acid, and arachidonic acid) can inhibit the growth and proliferation of MCF-7 cells. In addition, some studies have found that MCF-7 cells have PIK3CA spiral mutations and low activation rates of protein kinase B (AKT).

Cell Culture Method

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1. Preparation of culture media

1. 89% MEM Media
2. 10% Fetal Bovine Serum (FBS)
3. 1% Penicillin-streptomycin (P/S)
4. Other cellular additives

2. Cell recovery

1. Thaw: soak the freezer tube in 37℃ water bath with tweezers; shake repeatedly and thaw quickly;
2. Centrifugation: After spraying with 75% alcohol, open the frozen storage bottle in a sterile environment, move the cells together with the frozen storage liquid to a 10 mL centrifuge tube containing 1 mL of complete medium with a pipette, centrifugation at room temperature at 1200 rpm for 3-5 min, and sink the cells at the bottom of the centrifuge tube;
3. Culture: The supernatant was gently discarded, added 2 mL complete medium, gently suspended the cells, and then transferred all cell suspension to a T25 culture flask containing 3 mL complete medium in a 37℃, 5% CO ² incubator;
4. Observation: Observe the cell growth status (medium color, cell adhesion status, morphology and density, etc.) daily to take photos and video recording.

3. Passage

        Cell density of up to 80% -90% was started by passage

1. Wash: remove the medium aseptically and wash once with 37℃ preheated PBS;
2. Digestion: add 1 mL of 0.25% trypsin digestion solution, turn the culture bottle to infiltrate all the cells, digest at room temperature (or 37℃), most of the cells are observed to be round and fall off under the microscope, quickly take back the super clean table, tap the culture bottle and add 1 mL of complete medium to stop the digestion;
3. Centrifugation: gently blow well with a pipette and then transfer the suspension to a 10 mL centrifuge tube, centrifugation at 1200 rpm for 3-5 min;
4. Culture: sterile discard supernatant, add 1 mL of complete medium and move to T25 vial; add complete medium at 1:2-1:3 ratio, gently blow well with pipette, divide into vial and grow in 37℃, 5% CO ² incubator.

4. Notice

    Because MCF-7 cells grow in three-dimensional clusters, they will reattach to three-dimensional islands after resuscitation and passage (there will be clusters that do not reattach). Growth eventually spreads from the islands, and over time the cells slowly become monolayers, when the cells are basically in logarithmic growth.

MCF-7 human breast cancer cells are a slow-growing cell itself, subcultured with cell density. When the cells are cultured to the confluence of 70~80% of the cells, they can be divided into bottles. Generally, it is recommended to pass T25 bottles by 1:2, which can be passaged 2~3 times a week. If the cell growth rate is slower than normal, the serum concentration can be appropriately increased.

After MCF-7 cells, cells will have some floating state, and these floating cells are normal. General recovery, subculture after 3 days of floating cells will continue to adhere to adhesion, until growth spread from the island and spread in the bottle, usually in the liquid when we will add medium, if must change fluid, suspension cells can be collected by centrifugation, and suspension cells inoculation into the new bottle.

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