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The following is a general guideline for culturing of cell lines. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility.
?All media, supplement, and reagents must be sterile to prevent microbial growth in the cell culture. Some reagents and supplements will require filter sterilization if they are not provided sterile.
Watch our aseptic technique video protocol? for detailed guidelines on avoiding contamination.
Before starting work check the information given with the cell line to identify what media type, additives, and recommendations should be used.
Most cell lines can be grown using DMEM culture media or RPMI culture media with 10% Foetal Bovine Serum (FBS), 2 mM glutamine and antibiotics can be added if required (see table below).
Check which culture media and culture supplements the cell line you are using requires before starting cultures. Culture media and supplements should be sterile. Purchase sterile reagents when possible, only use unders aseptic conditions in a culture hood to ensure they remain sterile.
??General example using DMEM media
|DMEM - Remove 50 ml from 500 ml bottle, add other constituents.||450 ml|
|10% FBS||50 ml|
|2 mM glutamine||5 ml|
|100 U penicillin / 0.1 mg/ml streptomycin||5 ml|
Most cell lines will grow on culture flasks without the need for special matrixes etc. However, some cells, particularly primary cells, will require growth on special matrixes 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.
The following is an example for endothelial and epithelial cells:
For human cells, coat flasks with 1% gelatin. Alternatively, for other cell types such as BAEC, flasks can be coated with 1% fibronectin.
Cells should be checked microscopically daily to monitor health, grow rates and confluency (% surface area covered with cell monolayer).
Adherent cells should be mainly attached to the bottom of the flask, show an adherent morphology (cell line dependant) and refract light around their membrane (refer to Abcam cell line data sheet images).
Suspension cells should show a circular morphology and refract light around their membrane. Some suspension cells may clump (dissociation reagents such as Pluronic PF68 could be added to promote clump removal).
Media that includes phenol red should be pink/orange in color (media color may change depending on CO2 environment). For imaging application media without phenol red can be used and will avoid interference with imaging acquisition. A pale yellow colour of media would indicate a acidity and decrease of pH which is often associated with contamination or unhealthy cells.
Discard cells if:
Also referred to as cell splitting and cell passaging.
Split ratios or seeding densities can be used to ensure cells are ready for an experiment on a particular day or maintain cell cultures for future use or as a backup. Suspension cell lines are seeded based on volume so seeding densities will be calculated as cells/mL, whereas adherent cell lines are seeded based on flask surface area so will be calculated as cells/cm2. Cell lines often require specific seeding densities so always check the guidelines for the cell line in use. Slow growing cells may not grow if a high split ratio is used. Fast growing cells may require a high split ratio to make sure they do not overgrow.
Adherent cell lines can be split using cell line specific split ratios or seeding densities (cells/cm2):
Split ratios are based on flask surface area, e.g.:
1 x 25 cm2 flask Split 1:3 would yield 3 x 25 cm2 flasks or 1 x 75 cm2
Suspension cell lines should be maintained using cell line specific seeding densities (cells/mL):
If cells are to be left unattended for longer periods (i.e. bank holiday weekends) it is recommended to use a lower than normal seeding density/split ratio.
When the cells are approximately 80% confluent (80% of the flask surface is covered by cell monolayer), cells should still be in their log phase of growth and will require subculturing. It is not recommended to allow cells to become over confluent as this may negatively affect gene expression and cell viability.
Note – not all cells will require trypsinization, and to some cells it can be toxic. It can also induce temporary internalization of some membrane proteins, which should be taken into consideration when planning experiments. Other methods such as gentle cell scraping, or using very mild detergent can often be used as a substitute in these circumstances.
This may be repeated another one or two times if necessary (some cell lines take a long time to trypsinize and these will need more washes to get rid of any residual FBS to help trypsinization)
If cells have been growing well for a few days but are not yet confluent, then they will require a media changing to replenish nutrients and keep correct pH. Cells produce positive growth promoting factors which are secreted into their media so it can be beneficial to perform a half media change replenish nutrients provided by the media and also maintain these positive growth factors.
To change media, warm up culture media at 37°C using a water bath or incubator for at least 30 min. Aspirate old media from the flask and replace the media with the necessary volume of fresh pre-warmed culture media and return to incubator.
The passage number is the number of sub-cultures the cells have gone through. Passage number should be recorded and not get too high. This is to prevent use of cells undergoing genetic drift and other variations.