Tips for Quickly Master MCF7 Cell Culture and Gene Editing !

Tips for Quickly Master MCF7 Cell Culture and Gene Editing!

MCF7 cells are established from pleural effusion of a metastatic breast cancer patient and exhibit the molecular characteristics of estrogen receptor (ER) positive and progesterone receptor (PR) positive. They are commonly used as ER-positive breast cancer cell models. However, compared to other breast cancer cell lines, MCF7 cells have the characteristics of slow proliferation rate and slow adherence after thawing or passage. Improper handling can affect gene editing or the following experiments. Today, let’s explore how to culture MCF7 cells effectively and master some gene editing tips!

Figure 1: Cells grow normally (100×)

Appearing in the form of a loose three-dimensional cluster, growing in island shape in the early stage. As time goes by, gradually forming a flat monolayer, usually in regular shapes such as polygons or triangles.

Figure 2: Poor cell growth (100×)

Poor cell growth leads to morphological changes, collapsing cells, and signs of aging.

1. Cell Thawing

• 1. Preparation:Prepare 7mL complete culture medium in a centrifuge tube for use;
• 2. Thawing:Remove the cells from dry ice, holding the lid with forceps, quickly thaw cells in a 37°C water bath by gently swirling the vial (Note: keep the cap out of the water). In about 1 minute, it would completely thaw;
• 3. Centrifugation:Transfer the thawed cell suspension to a centrifuge tube and centrifuge at 1100 rpm for 4 minutes, discard the supernatant;
• 4. Resuspension and Seeding:Resuspend the cells in complete culture medium and seed them in culture flasks with appropriate size;
• 5. Culture:Place the culture plate or flask in a 37℃ incubator and observe cell status and adhesion after 24 hours.

2. Cell Passaging (using T25 flask as an example):

• 1. When the cell confluence reaches 80-90%, it is ready to passage. Inside the ultra-clean bench, discard the medium from the culture flask and briefly rinse the cells 1-2 times with 5mL PBS;
• 2. Add 1mL of trypsininto the flask, gently swirl it to ensure trypsin covers the cells, and then put it into the incubator for 1-2 minutes, monitoring under a microscope until most cells appear round and bright. When the majority of the cells have detached, immediately stop digestion;
• 3. Add 2mL of complete culture medium to stop digestion, transfer to a 15mL centrifuge tube;
• 4. Centrifuge at 1100 rpm at room temperature for 4 minutes, discard the supernatant, and resuspend the cells in complete culture medium;
• 5. Passage at a ratio of 1:2-1:3, observe cell status the next day.

3. Cell Cryopreservation:

• 1. Collecting Cells:Same as procedures of cell passaging, digest the cells and collect the digested cells into a centrifuge tube;
• 2. Centrifugation: Centrifuge at 1100 rpm for 4 minutes, discard the supernatant.
• 3. Resuspension and Cryopreservation: Resuspend cells in freezing medium, allocating them to freezing tubes with 1×10^6 cells per 1mL of freeze solution.Label with name, passage number, and date, etc.
• 4. Cooling and Storage: Place freezing tubes in a programmed cooling box overnight at -80℃, then transfer to a liquid nitrogen for storage.

Cell Culture Tips

1. 1. MCF7 cells are slow to adhere after thawing or passaging, it is recommended to observe and continue the experiments after 48-72 hours.
2. 2. Control digestion time to ensure cells to be singlecells but avoid over-digestion.
3. 3. Seeding density should not be too low; a passage ratio of 1:2-1:3 is recommended.
4. 4. Some viable cells may float after passaging or thawing; During medium change, centrifuge to collect and continue culture.
5. 5. If cell proliferation is slow, consider increasing serum concentration for cell culture.
6. 6. Black spots in the background may be cell metabolites and fragments, which do not affect cell growth. If there are many black spots, gently rinse with pre-warmed PBS during medium change.

How to Adjust Poor Cell Condition?

1. 1. Mediumand serum: Ensure the correct basal medium and appropriate serum concentration; adjust serum concentration based on cell condition.
2. 2. Cultureenvironment: Ensure that culture temperature, humidity, and air conditions are normal.
3. 3. Avoid using expired or long-stored media; fresh complete medium is best used within two weeks.
4. 4. Cell passaging: During cell passaging, pay attention to digestion time and trypsin concentration to avoid cell damage.

How to Adjust Cell Aging?

1. 1. Ensure that culture conditions and environment are correct.
2. 2. Avoid excessive trypsin exposure: use appropriate trypsin concentration and digestion time, and avoid harsh pipetting to cells.
3. 3. Increase serum concentration or select high-quality fetal bovine serum.
4. 4. Adjust cell density: Too high density can lead to nutrient deprivation and waste accumulation, speeding up aging; keep density appropriate.
5. 5. Add growth factors to promote cell proliferation and differentiation, which may help improve aging conditions.
6. 6. Utilize low passage cells for experiments.
7. 7. Use methods like single-clone selection to isolate cells with growth advantages from aging populations for purification.

Notes on Transfecting MCF7 Cells:

1. 1. Ensure cells are in good condition and in the logarithmic growth phase, in which the cells have high activity and vigorous division and it’sideal for enhancing transfection efficiency, with cell density generally between 70-90%.
2. 2. Monitor digestion time to avoid over-digestion, which can harm cells.
3. 3. Ensure cells are in single-cell form during the experiment, avoiding clumping.
4. 4. Aim for a cell viability of ≥80% during experiments

Electroporation transfection:

1. 1. Control the amount of cells during electroporationand then seeding into suitable culture plates.
2. 2. Ensure that adhesion rate is ≥70%after electroporation
3. 3. Control experimental time; the entire electroporation process should not be excessively prolonged

Lentivirus transduction:

1. 1. Control the cell confluenceto 30-40% before infection , avoiding excessively high density.
2. 2. Conductpreliminary experiments to determine the optimal MOI before formal experiments; add the transduction reagent Polybrene before transduction.
3. 3. Ensure transduction reagents are well mixed to guarantee uniformity beforeexperiments.

Figure 3: Lentiviral transduction of MCF7 (100×)

Figure 4: MCF7 Single Clones (40×)

Notes on MCF7 Single Cloning Experiments:

• 1. Ensure cells are normal in condition with minimal aging; aim for a confluence around 70%.
• 2. Ensure cell viability is ≥85% when isolating for single cloning.
• 3. Conduct preliminary experiments to find the appropriate cloning gradient to avoida too-low proportion of single clones.
• 4. Ensure even distribution of cells when seeding in 96-well plates.
• 5. After cell dilution, the best results shouldbe within the range of 1×10^6 - 2×10^6

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