Mammalian cells are important research tools in biological research, and good cell culture habits are required to ensure cell stability. Creative Biolabs offers comprehensive cell culture services to make your cells more stable. Also, we can develop stable cell lines to help your research according to your experimental project needs.

Below are the general procedures for mammalian cell culture.

Preparation

1. A sterile environment

• Hood regulations
  ① Close the hood sash in a proper position to maintain laminar airflow
  ② Avoid clutters
• Autoclaving
  ① Pipette tips (or purchase pre-autoclaved, DNAse/RNAse-free ones)
  ② 9-inch glass Pasteur pipettes
  ③ Spray all surfaces with 70% ethanol

2. Cell growth medium

• Before starting, check the information provided with the cell line. Determine the type of medium, additives, and recommendations based on specific information.
• Most cell lines can use DMEM medium or RPMI medium containing 10% fetal bovine serum (FBS), with the addition of 2 mM glutamine and antibiotics if desired (see table below).

Table 1. General example using DMEM media

3. The correct culturing environment

• Most cell lines are cultured in culture flasks without special matrixes. However, some cells, especially primary cells, need to be cultured in special matrixes such as collagen to promote cell attachment, differentiation, or cell growth. We recommend that you review the relevant literature for more information about the cells you are culturing before you begin.
• Take endothelial and epithelial cells as examples:
  For human cells, coat the flask with 1% gelatin. For other cell types, such as BAEC, flasks can be coated with 1% fibronectin.
  ① Prepare and filter a 10 mL coating solution containing 1% gelatin or 1% fibronectin that is diluted by distilled water. This is effective to coat about 5 flasks.
  ② Pipette the coating solution into the flask. Rock back and forth to distribute it evenly across the bottom of the flask. Leave in the incubator for 15-30 minutes.
  ③ Before seeing the cells, aspirate the coating solution and rinse with sterile dH2O.

4. Cells checking

• Cells should be examined daily under the microscope to monitor health, growth rate, and confluency (percentage of surface area covered by cell monolayers).
• Adherent cells should attach primarily to the bottom of the flask, show an adherent morphology (cell line-dependent), and refract light around their membrane.
• Suspension cells should be in circular shapes and refract light around their membrane. Some suspension cells may clump together (dissociation reagents can be added to facilitate clump removal).
• Media containing phenol red should be pink/orange in color (media color may change due to the CO2 environment). For imaging applications, a phenol red-free media can be used to avoid interference with imaging acquisitions. A pale-yellow color of the medium indicates a decrease in acidity and pH, which is usually associated with contaminated or unhealthy cells.
• Cells should be abandoned once the following conditions occur
• They are in a static state (seem not to grow at all).
• They are heavily detached (attached lines) and/or look shriveled and grainy/dark in color.

Tips

All media, supplements, and reagents must be strictly sterile to prevent the growth of microorganisms in cell cultures. Reagents and supplements that are not sterile need to be sterilized by filter sterilization.

All operations should be aseptic and performed in a microbiological safety cabinet to ensure sterility.

Procedures

1. Sub-culturing

• Split ratios or seeding densities can be used to ensure that cells are ready for experiments on a specific date, or to retain cell cultures for future use or as a backup.
• Before starting work, always check the information accompanying the cell line to determine the seeding density required for the cell line.
• Suspension cell lines are seeded based on volume, so seeding density will be calculated as cells/mL, while adherent cell lines are seeded based on flask surface area, so the density will be calculated as cells/cm².
• Slow-growing cells may not grow if a high fractionation ratio is used. Fast-growing cells may require high shunt ratios to ensure that they do not overgrow.
  Adherent cell lines can use cell line-specific division ratios or seeding densities (cells/cm²). Split ratios are determined based on the flask surface area. 1 x 25 cm² flask Split 1:3 would yield 3 x 25 cm² flasks or 1 x 75 cm².
  ① 1:2 split should be 70-80% confluent for experiments within 1-2 days.
  ② 1:5 split should be 70-80% confluent for experiments within 2-4 days.
  ③ 1:10 split should be 70-80% confluent for sub-culturing or plating within 4-6 days.
  Suspension cell lines should be maintained by using the specific seeding density of cell lines (cells/ml).
  ① 2e5 should be ready for an experiment in 3-4 days.
  ② 1e6 should be ready for an experiment in 1-2 days.

Tips

If cells are left unattended for a long time, it is recommended to use a lower seeding density/split ratio than that of the normal seeding density/split ratio.

2. Adherent subculture protocol using dissociation reagent

• When the cells are approximately 80% confluent (80% of the flask surface is covered by cell monolayer and the cells are in their logarithmic phase of growth), subculturing is required. Allowing cells to over-confluence is not recommended, as this may negatively impact gene expression and cell viability.
  ① Remove the cell culture medium and dissociation reagent from the refrigerator, place them in a 37℃ incubator, and allow to come to a temperature of 37℃. (Do not leave the medium in the incubator longer than necessary, as the medium components will degrade over time.)
  ② Open and perform basic cleaning of your biosafety cabinet. (Spray all media bottles, pipettes, and centrifuge tubes with ethanol before placing them in the biosafety cabinet.)
  ③ In the biosafety cabinet, remove the conditioning medium and gently wash the cell monolayer with room-temperature DPBS. (Carefully add DPBS to the side of the flask to avoid forcible removal of the adherent cells.)
  ④ Remove DPBS by using a sterile serological pipette, add pre-warmed dissociation reagent (Trypsin-EDTA) to the flask, and put it in the incubator for about 2 minutes (dissociation time varies by cell line). Check the flask frequently to ensure that all cells have been dissociated from the flask surface. (Not all cells require trypsin digestion and it may be toxic for some cells. Trypsin can also induce the temporary internalization of some membrane proteins, which should be considered when scheduling experiments. In these cases, other methods such as gentle cell scraping or the use of a very mild dissociation reagent can often be used as alternatives.)
  ⑤ When all cells are detached, neutralize the dissociation reagent with a serum-containing growth medium suitable for the cultured cell line.
  ⑥ Transfer the cell suspension to a centrifuge tube. Use a sterile medium, wash the flask, and transfer the medium to a centrifuge tube. Please ensure that all cells have been harvested from the flask.
  ⑦ Centrifuge the cell suspension for 5 minutes with 1000 rpm at room temperature.
  ⑧ Discard the supernatant, gently flick the cell pellet (break up the pellet), and resuspend the cells in sterile medium to reach the appropriate volume for counting.
  ⑨ Count cells with the hemocytometer.
  ⑩ Seed cell suspensions in appropriate flasks and densities based on counting and viability data, i.e. T175, 30mL, 2e4 cells/cm². (Label the culture flasks with all necessary information such as cell line and passage number.)
  ⑪ Immediately incubate the newly seeded cultures in a humidified incubator at 37℃/5% CO2 air.

3. Sub-culture of loosely adherent cell lines that require scraping cells for sub-culture

① When ready, carefully pour the medium for the required cells from the flask into the waste pool (containing approximately 100 ml of 10% sodium hypochlorite), taking care not to allow any drops to increase contamination risk.
② Immediately replace it by pouring an equal volume of the pre-warmed fresh medium into the flask.
③ Gently scrape the cells from the bottom of the flask with a cell scraper into the medium. Check that all cells have come off by observing the bottom of the flask before moving on.
④ Use a serological pipette to remove the desired amount of cell suspension to achieve the desired division ratio.
At a 1:2 split ratio, take 50 ml from 100 ml into a new flask
At a 1:5 split ratio, take 20 ml from 100 ml into a new flask
At a 1:10 split ratio, take 10 ml from 100 ml into a new flask
⑤ The new flask is capped to the desired volume with prewarmed fresh medium according to the split ratio
25 cm² flask approx. 5-10 ml
75 cm² flask approx. 10-30 ml
175 cm² flask approx. 40-150 ml

4. Adherent subculture protocol using trypsin

Tips

Not all cells require trypsinization, and for some cells, it may be toxic. It can also induce temporary internalization of some membrane proteins, which should be taken into account when planning experiments. In these cases, other methods, such as gentle cell scraping, or the use of very mild detergents can often be used as an alternative.
① When ready, carefully pour the medium for the desired cells from the flask into the waste pool (containing approximately 100 ml of 10% sodium hypochlorite), taking care not to increase the contamination risk by any drops.
② Use an aseptic technique, pour/ pipette enough sterile PBS into the flask to allow the cells to be washed, and remove the FBS from the residual medium. Gently decant the flask several times to rinse the cells and carefully pour/ pipette the PBS into the flask. (Repeat once or twice if necessary. Some cell lines take a long time to trypsinize, and these cell lines require more washes to get rid of any residual FBS to aid trypsinization.)
③ Use a pipette, and add enough trypsin EDTA to cover the cells at the bottom of the flask.
Approximately 1 ml in a 25 cm² flask
Approximately 5 ml in a 75 cm² flask
Approximately 10 ml in a 175 cm² flask
④ Gently roll the flask to ensure that the trypsin reaches all cells. Place the flask in a 37°C incubator. Different cell lines require different trypsin treatment times. Importantly, check every few minutes to avoid excessive trypsinization that could severely damage the cells.
⑤ Once the cells are detached (the flask may need a few gentle taps), add some medium to the flask (the FBS in it will inactivate the trypsin).
⑥ Use this cell suspension, and transfer the desired volume of cells into a new flask in the desired split ratio. Then add medium to these flasks to the needed volume
Approximately 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
Allow the cells to recover and settle overnight. Replace the medium to remove any residual trypsin.

5. Sub-culturing of suspension cell lines

① Check the information on the cell line used for the recommended division ratio or subculture cell density.
② Use a pipette to remove the desired amount of cell suspension from the flask and place it in a new flask.
For a 1:2 split ratio, remove 50 ml from a 100 ml cell suspension.
For a 1:5 split ratio, remove 20 ml from 100 ml of cell suspension.
③ Add the desired amount of pre-warmed cell culture medium to a new flask.
Add 50 ml of fresh medium from 100 ml to 50 ml of cell suspension in a 1:2 ratio.
Add 80 ml of fresh medium from 100 ml to 20 ml of cell suspension in a 1:5 ratio.

6. Changing media

If the cells have grown well for several days but have not yet aggregated, then a medium change is needed to replenish the nutrients and maintain the correct pH. Cells produce positive growth-promoting factors that are secreted into their medium, so it is beneficial to perform a half-medium change to replenish the nutrients provided by the medium and to maintain these positive growth factors.

To replace the medium, the medium needs to be warmed at 37°C for at least 30 minutes in a water bath or incubator. Aspirate the old medium from the flask, replace the medium with the necessary volume of pre-warmed fresh medium, and place it back in the incubator.

7. Passage number

Passage number is the number of subcultures the cells undergo. High cell passage numbers are prone to genetic drift and other variations, so passage numbers should be strictly recorded during the experiment to ensure scientific validity.

Disclaimer

This procedure should be used only as a guide. Please note that Creative Biolabs cannot guarantee the status of the client's mammalian cell tissue culture.

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