Mammalian Cell Tissue Culture

Aseptic Environment Preparation

To achieve an aseptic environment, all equipment and materials should be sterilized using autoclaving, dry heat, or chemical disinfectants. All cell cultures must be performed in a microbiologically safe cabinet and the work area should be disinfected with alcohol or other appropriate disinfectants. Technicians should wear gloves, lab coats, and face masks to prevent contamination by skin and respiratory droplets.

Tissue Culture Medium Selection

Cell culture media are the primary source of nutrients required for mammalian cell culture in vitro. There are many different formulations of tissue culture media obtainable. Experimenters must determine the appropriate medium for their needs based on the information provided by the cell line, either by using a commercially prepared medium or preparing it themselves. Commercially available media are often available in many different forms, such as sterile ready-to-use liquid, concentrated liquid, or powder form. In addition to providing nutrients for cell growth, the medium is often supplemented with antibiotics, fungicides, or both to prevent contamination. Most cell lines can be grown using RPMI medium or DMEM medium containing 10% fetal bovine serum (FBS).

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Sub-Culturing

Seeding densities or split ratios are used to ensure cells are prepared for an experiment or maintained for future use. Suspension cell lines are seeded based on volume (cells/mL), while adherent cell lines are seeded based on flask surface area (cells/cm²). It's important to check the guidelines for the specific cell line being used, as they may require specific seeding densities. Slow-growing cells may not grow well with a high split ratio, while fast-growing cells may require a high split ratio to prevent overgrowth.

As a general guideline, cells in one confluent flask should be split at a 1:2 ratio at 70-80% confluence and be ready for experiments in 1-2 days. A 70-80% confluent split ratio should be 1:5 and ready for experiments in 2-4 days. A 70-80% confluent split ratio should be 1:10 and ready for subculture or plating in 4-6 days. But this may depend on the growth rate of the cell line.

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Sub-culturing protocol

1. Make sure the cells are at least 80% confluent.
2. Warm fresh cell culture medium in a 37°C water bath or incubator for at least 30 min.
3. Make sure the flasks are properly labeled with the code number, cell line, split ratio, and date.
4. Prepare a waste container with bleach to hold approximately 100 mL of 10% sodium hypochlorite waste.

Sub-culturing protocol for loosely attached cells (cell scraping)

1. Carefully pour the media into the waste pot.
2. Add an equal volume of pre-warmed fresh medium to the flask.
3. Gently scrape the cells from the bottom of the flask into the medium using a cell scraper.
4. Make sure that all cells have been scraped off the flask.
5. Remove the required amount of cell suspension using a serological pipette. For example, for the 1:2 split, 1:5 split, and 1:10 split, remove 50 mL, 20 mL, and 10 mL from 100 mL into a new flask, respectively.
6. Add the required volume (considering the split ratio) to the pre-warmed fresh medium. For example, add about 5-10mL to a 25cm² flask and add about 10-30mL to a 75cm² flask.

Sub-culturing protocol for attached cell lines (trypsin)

1. Carefully pour the media from the flask into a waste container.
2. Pipette pre-warmed PBS into the flask by aseptic technique to wash the cells and remove any FBS in the residual culture media.
3. Shake the flask gently, rinse the cells with PBS, and pour the PBS back out into the waste container. Repeat 3 times. Removal of any residual FBS is important for efficient trypsinization.
4. After washing, add prewarmed Trypsin-EDTA to cover the cells. For example, add about 1mL for a 25cm² flask and about 5mL for a 75cm² flask.
5. Shake the flask gently to ensure that the trypsin contacts all cells.
6. Place the flask in a 37 °C incubator. Different cell lines require different trypsinization times. To avoid severe damage to the cells by excessive trypsinization, it must be checked every few minutes under the microscope while tapping the flask to dislodge the cells.
7. Once the cells are detached, add some medium to the flask. FBS in the medium will inactivate trypsin.
8. Count the cells and pipette the required volume of cells into a new flask. These flasks should then be topped up with medium to the desired volume.
9. Leave the cells overnight to recover and stabilize, and change the medium to remove any residual trypsin.

Sub-culturing protocol for suspension cells

1. Some suspension cell lines have recommended split ratios or subculturing cell densities. Please review the relevant content before starting an experiment.
2. Use a pipette to remove the required amount of cell suspension from the flask and place it into a new flask.
3. Add the required amount of pre-warmed cell culture medium to a fresh flask. For example, for a 1:2 split, take 50 mL of 100 mL of cell suspension, and add 50 mL of fresh medium.

Note: Passage number is the number of subcultures cells have undergone. The number of passages should be kept at a reasonable level and not too high. Cell lines with passage numbers greater than 30 were more likely to acquire genetic heterogeneity than lower passage cells.