Cell culture is the basis for many experiments. Below are the general procedures for cell culture.

Tips

• Cell culture must be performed in a microbiologically safe cabinet using aseptic techniques to ensure sterility.

Procedures

1. Prepare a sterile environment

• Provisions for protective hoods
  Drop the hood curtains to the proper position to maintain laminar air flow
  Avoid cluttering
• Autoclaving
  Pipette tips (or pre-autoclaved, DNAse/RNAse-free ones can be purchased)
  9-inch glass Pasteur pipettes
  70% ethanol. Be sure to spray all surface areas

Tips

• All culture media, supplements, and reagents must be sterile to prevent the growth of microorganisms.
• In cell culture, some reagents and supplements need to be filter sterilized if they are not sterile.

2. Preparation of cell growth medium

• Before starting work, check the given cell line information to determine the type of medium, additives, and recommended medium to be used.
• Most cell lines can use DMEM medium or RPMI medium with 10% fetal bovine serum (FBS), 2 mM glutamine, and antibiotics if necessary.

• Check which medium and culture supplements are required for the cell line you are using before starting the culture.
• Culture media and supplements should always be sterile. Whenever possible, purchase sterile reagents and use them only under sterile conditions in the culture hood to ensure that they remain sterile.

3. Creating the right culture environment

Most cell lines will grow on culture flasks and do not require special substrates and others. However, some cells, especially primary cells, need to be grown on special substrates, such as collagen, to promote cell attachment, differentiation, or cell growth. We recommend reviewing the relevant literature for further information on the cells you are culturing.

Here is an example of endothelial and epithelial cells.

For human cells, coat the flask with 1% gelatin. Alternatively, for other cell types, such as BAEC, the flask can be coated with 1% fibronectin.

• Prepare 10 ml of a coating solution consisting of 1% gelatin or 1% fibronectin, dilute with distilled water, and filter. This is effective for coating approximately 5 flasks.
• Use a pipette to transfer the coating solution into the flasks. Shake back and forth to distribute it evenly across the bottom of the flask. Allow standing in the incubator for 15–30 minutes.
• Remove the coating solution completely by suction before seeding.

4. Examining the cells

• Cells should be examined microscopically daily to ensure that they are healthy and growing as expected. Attached cells should be predominantly attached to the bottom of the flask, round and plump or elongated in shape, and refract light around their membrane. Suspended cells should appear round and plump and refract light around their membranes. Some suspension cells may clump together. The color of the culture medium should be pinkish orange.
• Discard cells in the following cases.
  The cells are heavily shed (attachment lines) and/or look dried out and granular/dark in color.
  They are in a quiescent state (do not appear to be growing at all).

5. Sub-culturing

Tips

• Sub-culturing should always be performed under sterile conditions using aseptic techniques.
• Split ratios can be used to ensure that cells are ready for experiments on a given day or simply to keep cell cultures running for future use or as a backup. Suspension cell lines usually have a recommended sub-culture seeding density. Always check the procedures for the cell line being used. Some slow-growing cells may not grow if a high split ratio is used. Some fast-growing cells may require a higher split ratio to ensure that they do not overgrow. Note that most cells should not be split more than 1:10 because the seeding density is too low for the cells to survive.
  As a general procedure, take cells from a confluent flask:
  1:2 split cells should be 70–80% confluent and ready for experimentation within 1–2 days.
  1:5 split should be 70–80% confluent and ready for experiments within 2–4 days.
  1:10 split should be 70–80% confluent and ready for passaging culture or plating within 4–6 days.
• If the cells are less than 70–80% confluent, but you wish to pass them on to culture before the weekend (e.g., Friday), then they should be split at a lower split rate in order to seed the cells at a high enough density to survive, e.g. using a 1:2 or 1:5 split.

6. Splitting

• When the cells are about 80% confluent (the cell monolayer covers 80% of the flask surface), they should still be in the log phase of growth and need to be sub-cultured. (Do not allow the cells to over-confluence as it will cause cell death which is unreversible).
• To sub-culture, first heat fresh medium in a 37 °C water bath or incubator for at least 30 minutes. Then perform one of the following appropriate procedures.
• Make sure the flasks are labeled with the cell line, passage number, split ratio, date, operator initials, and cell flask number. Place the flask directly into the 37 °C CO₂ incubator. Write down the details of the sub-culturing in the culture record log sheet. There should be a separate log sheet for each vial of cells resuscitated and in use.

7. Subculture of loosely attached cell lines that require scraping of cells for subculture

• Carefully pour the medium from the flask containing the desired cells into the waste pool (containing approximately 100 ml of 10% sodium hypochlorite), and be aware of not allowing any drops to increase the risk of contamination.
• Replace immediately by pouring an equal volume of the pre-warmed fresh medium into the flask.
• Gently scrape the cells from the bottom of the flask into the medium. Check that all cells have been shed by checking the bottom of the flask before moving on.
• Use a serological pipette to remove desired cell suspension to achieve the desired separation ratio.
  For example, to separate from 100 ml at a ratio of 1:2, take 50 ml into a new flask.
  Separate from 100 ml at a ratio of 1:5 by taking 20 ml into a new flask.
  Separate from 100 ml in a ratio of 1:10 and take 10 ml to a new flask.
• Add pre-warmed fresh medium to the new flask to get desired volume (taking into account the splitting ratio).
  For example, about 5-10 ml in a 25 cm² flask
  Approximately 10–30 ml in a 75 cm² flask
  Approximately 40–150 ml in a 175 cm² flask

8. Sub-culturing attached cell lines requiring trypsin

Tips

• Not all cells require trypsinization, which may be toxic for some cells and can also induce temporary internalization of some membrane proteins and should be carefull considered when planning experiments. In such cases, other methods, such as gentle cell scraping, or the use of very mild detergents, are often taken as alternatives.
• Repeat once or twice if necessary (some cell lines take a longer time to trypsinize and they require more washes to remove any residual FBS to aid in trypsinization).
• Carefully pour the medium for the desired cells from the flask into the waste pool (containing approximately 100 ml of 10% sodium hypochlorite), and be aware of not allowing any drops to increase the risk of contamination.
• Using aseptic technique, pour/pipette enough sterile PBS into the flask so that the cells get washed and get rid of any FBS in the remaining medium. Gently decant the flask several times to rinse the cells and carefully pour/pipette the PBS into the waste tank.
• Using a pipette, add enough trypsin EDTA to cover the cells at the bottom of the flask.
  For example, approximately 1 ml in a 25 cm² flask
  Approximately 5 ml in a 75 cm² flask
  Approximately 10 ml in a 175 cm² flask
• Gently roll the flask to ensure that the trypsin is reaching all cells. Place the flask in a 37 °C incubator. Different cell lines require different trypsin treatment durations. Check every few minutes to avoid excessive trypsinization that can severely damage the cells.
• Once the cells are detached (the flask may need to be tapped for a few times), add some medium that has FBS in it which will inactivate the trypsin to the flask.
• Using this cell suspension, the desired volume of cells is transferred into new flasks in appropriate split ratio. These flasks are then topped up with culture medium to the desired volume.
  For example, about 5–10 ml in a 25 cm² flask
  About 10–30 ml in a 75 cm² flask
  Approximately 40–150 ml in a 175 cm² flask
• Allow the cells to recover and settle overnight. Replace the medium to remove any residual trypsin.

9. Sub-culturing of suspension cell lines

• Check the cell line guidelines for the recommended split ratio or subculture cell density.
• Use a pipette to remove the desired cell suspension from the flask and place it in a new flask.
  For example, remove 50 ml of cell suspension from 100 ml divided in a 1:2 ratio.
  Divide 1:5 from 100 ml of cell suspension and remove 20 ml.
• Add proper amount of pre-warmed cell culture medium to the new flask.
  For example, for a 1:2 ratio dispensing from 100 ml, add 50 ml of fresh medium to 50 ml of cell suspension.
  For a 1:5 ratio from 100 ml, add 80 ml of fresh medium to 20 ml of cell suspension

10. Changing media

• If cells have been growing well for several days but are not yet confluent (e.g., if they have been split 1:10), then they will need to be replaced in the medium to replenish nutrients and maintain the correct pH. If there are excessive cells (attached cell lines) or the medium turn orange instead of pink, then the medium needs to be changed as soon as possible.
• When changing the medium, heat the fresh medium (Section 5.1) in a 37 °C water bath or incubator for at least 30 minutes. Carefully pour the medium from the flask into a waste basin containing some disinfectant. Immediately replace the medium with 100 ml of fresh pre-warmed medium instead and return to the CO₂ incubator at 37 °C.

11. Passage number

• Passage number is the number of sub-cultures the cells have undergone. The passage number should be recorded and should not be too high to prevent the use of cells that have undergone genetic drift and other mutations.

Customized Cell Culture Services

Creative Biolabs can customize the appropriate cell culture service according to your experimental needs, including 2D and 3D cell culture.

SIAT 2D Cell Culture^®

Disclaimer

This procedure should be used only as a guide. Please note that Creative Biolabs does not guarantee the client's cell culture status.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.