General details of cell culturing
and sub-culturing

Contents
1. Preparing an aseptic environment
2. Preparation of cell growth medium
3. Creating the correct cell culture environment
4. Checking cells
5. Sub-culturing
6. Splitting
7. Sub-culturing loosely attached cell lines requiring cell scraping for sub-culture
8. Sub-culturing attached cell lines requiring trypsin
9. Sub-culturing of suspension cell lines
10. Changing media
11. Passage number1) Preparing an aseptic environment
Hood regulations(a) Close hood sash to proper position to maintain laminar air flow(b) Avoid cluttering
Autoclaving(a) Pipette tips (or can be purchases pre-autoclaved, DNAse/RNAse free)(b) Glass 9” Pasteur pipettes(c) 70% ethanol (Be sure to spray all surface areas)
All media, supplement, and reagents must be sterile to prevent microbial growthin the cell culture. Some reagents and supplements will require filter sterilization if
they are not provided sterile.

2) Preparation of cell growth medium
Before starting work check the information given with the cell line to identify what
media type, additives, and recommendations should be used.
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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 beadded if required (see table below).
Check which culture media and culture supplements the cell line you areusing requires before starting cultures. Culture media and supplements should
be sterile. Purchase sterile reagents when possible, only use under aseptic
conditions in a culture hood to ensure they remain sterile.
General example using DMEM mediaMediaMeasure

DMEM - Remove 50 ml from 500 ml bottle, add
other constituents.
10% FBS450 ml2 mM glutamine

5 ml

100 U penicillin / 0.1mg/ml streptomycin

5 ml

50 ml

3) Creating the correct culturing environment
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 asBAEC, flasks can be coated with 1% fibronectin.
1. Prepare 10mL of coating solution composed of 1% gelatin or 1% fibronectin bydiluting with distilled water, followed by filtration. This is efficient to coat about
5 flasks.
2. Pipette coating solution into flask. Rock back and forth to evenly distribute thebottom of the flask. Let sit in an incubator for 15-30 minutes.
3. Completely remove coating solution by aspirating before seeding.

4) Checking cellsCells should be checked microscopically daily to ensure they are healthy andgrowing as expected. Attached cells should be mainly attached to the bottom of theflask, round and plump or elongated in shape and refracting light around theirmembrane. Suspension cells should look round and plump and refracting light aroundtheir membrane. Some suspension cells may clump. Media should be pinky-orange incolor.
Discard cells if:-3

They are detaching in large numbers (attached lines) and/or look shrivelled
and grainy/dark in color.
They are in quiescence (do not appear to be growing at all).

5) Sub-culturing
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 becalculated 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 specificseeding 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 highsplit 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):-

1:2 split should be 70-80% confluent and ready for an experiment in 1 to 2 days
1:5 split should be 70-80% confluent and ready for an experiment in 2 to 4 days
1:10 split should be 70-80% confluent and ready for sub-culturing or plating in 4
to 6 days.

Split ratios are based on flask surface area, eg:
1 x 25 cm2 flask Split 1:3 would yield 3 x 25 cm2 flasks or 1 x 75 cm2Suspension cell lines should be maintained using cell line specific seeding densities(cells/mL):-

2e5 should be ready for an experiment in 3-4 days
1e6 should be ready for an experiment in 1-2 days

If cells are to be left unattended for longer periods (i.e. bank holiday weekends) it isrecommended to use a lower than normal seeding density/split ratio.

6) Adherent subculture protocol (using dissociation reagent)
When the cells are approximately 80% confluent (80% of the flask surface is coveredby cell monolayer), cells should still be in their log phase of growth and will require
sub-culturing. It is not recommended to allow cells to become over confluent as thismay negatively affect gene expression and cell viability.

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1. Remove cell culture media and dissociation reagent from the fridge andplace in a 37oC incubator and allow to come to temperate 37 oC.
- Do not leave media in the incubator for longer than is necessary as the media
components will degrade over time.
2. Switch on and perform a basic clean for your biological safety cabinet.
- Spray all media bottles, pipettes and centrifuge tubes with ethanol before
placing in the biological safety cabinet.
3. Under the biological safety cabinet, remove the conditioned media andgently wash the cell monolayer with room temperature DPBS.
- Carefully add DPBS to side of flask so not to forcefully dislodge adherent cells.
4. Remove the DPBS using a sterile serological pipette and add pre-warmed
dissociation reagent (Trypsin-EDTA) to the flask and place in an incubator for~2 mins (dissociation times can vary between cell lines). Check flask frequently
to ensure all cells have dissociated from flask surface.
- Not all cells will require trypsinization, and to some cells it can be toxic. Trypsin
can also induce temporary internalization of some membrane proteins, whichshould be taken into consideration when planning experiments. Other
methods such as gentle cell scraping or using a very mild dissociation reagent(Versene) can often be used as a substitute in these circumstances.

5. When all cells are detached, neutralise the dissociation reagent with serum
containing growth medium appropriate to the cell line in culture.
6. Transfer cell suspension to a centrifuge tube. Using sterile media, wash flask
and transfer to centrifuge tube, ensuring all cells have been harvested fromflask.
7. Centrifuge the cell suspension for 5min @ 1000rpm, room temperature.
8. Discard the supernatant and gently flick the cell pellet (to break up pellet),then resuspend cells in sterile media to a suitable volume for counting.
9. Consult Abcam counting cell using a hemocytometer protocol.
10. Based on count and viability data, seed cell suspension for an appropriate
flask and density, e.g. T175, 30mL at 2e4 cells/cm2.
- Label culture flask with all necessary info e.g. Cell Line, passage number, etc.
11. Immediately incubate the newly seeded cultures in a 37oC/5% CO2 airhumidified incubator.

7) Sub-culturing loosely attached cell lines requiring cell scrapping for subculture
1. When ready, carefully pour off media from flask of the required cells into
waste pot (containing approximately 100 ml of 10% sodium hypochlorite)
taking care not to increase contamination risk with any drips.
2. Replace this immediately by carefully pouring an equal volume of prewarmed fresh culture media into the flask.
3. Using a cell scraper, gently scrape the cells off the bottom of the flask into themedia. Check all the cells have come off by inspecting the base of the flask
before moving on.
4. Take out required amount of cell suspension for required split ratio using aserological pipette.
eg for 1:2 split from 100 ml take 50 ml into a new flask
1:5 split from 100 ml take 20 ml into a new flask
1:10 split from 100 ml take 10 ml into a new flask
5. Top the new flasks up to required volume (taking into account split ratio) with
pre-warmed fresh culture media
eg in 25 cm2 flask approximately 5-10 ml
75 cm2 flask approximately 10-30 ml
175 cm2 flask approximately 40-150 ml8) Sub-culturing attached cell lines requiring trypsin
Note – not all cells will require trypsinization, and to some cells it can be toxic. It canalso induce temporary internalization of some membrane proteins, which should betaken 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 thesecircumstances.
1. When ready, carefully pour off media from flask of the required cells into
waste pot (containing approximately 100 ml 10% sodium hypochlorite) taking
care not to increase contamination risk with any drips.
2. Using aseptic technique, pour/pipette enough sterile PBS into the flask to givecells a wash and get rid of any FBS in the residual culture media. Tip flask
gently a few times to rinse the cells and carefully pour/pipette the PBS backout into waste pot.
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)
3. Using a pipette, add enough trypsin EDTA to cover the cells at the bottom of
the flask.
eg in 25 cm2 flask approximately 1 ml5

75 cm2 flask approximately 5 ml175 cm2 flask approximately 10 ml
4. Roll flask gently to ensure trypsin contact with all cells. Place flask in 37°C
incubator. Different cell lines require different trypsinization times. To avoid
over-trypsinization which can severely damage the cells, it is essential to checkthem every few minutes.
5. As soon as cells have detached (the flask may require a few gentle taps) add
some culture media to the flask (the FBS in this will inactivate the trypsin)
6. Using this cell suspension, pipette required volume of cells into new flasks atrequired split ratio. These flasks should then be topped up with culture media
to required volume
eg in 25 cm2 flask approximately 5-10 ml
75 cm2 flask approximately 10-30 ml
175 cm2 flask approximately 40-150 ml
Leave cells overnight to recover and settle. Change media to get rid of any
residual trypsin.

9) Sub-culturing of suspension cell lines
1. Check guidelines for the cell line for recommended split ratio or sub-culturing
cell densities.
2. Take out required amount of cell suspension from the flask using pipette andplace into new flask.
eg For 1:2 split from 100 ml of cell suspension take out 50 ml
For 1:5 split from 100 ml of cell suspension take out 20 ml
3. Add required amount of pre-warmed cell culture media to fresh flask.
eg For 1:2 split from 100 ml add 50 mls fresh media to 50 ml cell suspension
For 1:5 split from 100 ml add 80mls fresh media to 20 ml cell suspension10) Changing media
1. If cells have been growing well for a few days but are not yet confluent (eg ifthey have been split 1:10) then they will require media changing to replenish
nutrients and keep correct pH. If there are a lot of cells in suspension(attached cell lines) or the media is staring to go orange rather than pinkyorange then media change them as soon as possible.
2. To change media, warm up fresh culture media (section 5.1) at 37°C in water
bath or incubator for at least 30 min. Carefully pour of the media from the flask
into a waste pot containing some disinfectant. Immediately replace themedia with 100 ml of fresh pre-warmed culture media and return to CO2 37°Cincubator.

11) Passage number
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 ofcells undergoing genetic drift and other variations.

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