1. Cell Culture
Part 2

2. Recovery of Cells from Storage
Cells were stored in liquid nitrogen for a long time and need to be recovered for experimental use
The vial should be immersed in a 37oC water bath and gently shaken until thawed
The contents of the vial should be transferred into a 15 ml sterilized centrifuge tube containing about 10 ml pre-warmed complete medium and centrifuged at 1000 rpm for 5 min
The supernatant should then be discarded and cell pellet should be resuspended in 15 ml complete medium
Finally, the cell suspension is transferred to a sterile 75 cm2 tissue culture flask and incubated at 37oC in a humidified atmosphere of 95% air and 5% CO2

4. Subculturing Cells
In order to maintain cell cultures in optimum conditions it is essential to keep cells in the log phase of growth
Adherent cells will continue to grow in vitro until either:
they have covered the surface available for growth
or they have depleted the nutrients in the surrounding medium
Cells kept for prolonged periods in the stationary phase of growth will lose plating efficiency and may become senescent, lose viability and other characteristics, or even die

5. Subculturing Adherent Cells
The frequency of subculture is dependent on a number of factors and will vary between cell lines, the factors include:
inoculation density,
growth rate,
plating efficiency
a measure of the number of colonies originating from single cells,
and saturation density
The maximum cell number attainable, under specified culture conditions, in a culture vessel
This term is usually expressed as the number of cells per square centimeter in a monolayer culture
or the number of cells per cubic centimeter in a suspension culture

6. Subculturing Adherent Cells

7. Subculturing Adherent Cells
Examine the condition of the cell monolayer using an inverted microscope and ensure that the cells are healthy and sub-confluent
The medium is discarded from the vessel and the cells monolayer should be washed twice with warm PBS (magnesium & calcium free)
The cells are detached using a warm solution of Trypsin/EDTA solution and the plate is left at 37oC for ~ 5 minutes or until the cells are detached with monitoring of cells under inverted microscope
Cells should only be exposed to trypsin long enough to detach the cells
Prolonged exposure can damage surface receptors on cells
Sub-confluent
Overgrown

8. Subculturing Adherent Cells
Attached
Detached
Gently tap the flask with the palm of the hand a couple of times to release any remaining detached cells
A warm complete medium is added to the cells to inactivate trypsin and the cells are pipetted several times to break clumps
Remove the cell suspension from the flask and place in to a sterile container
Centrifuge typically at 1000 rpm for 5 min to sediment the cells
Pellet

9. Subculturing Adherent Cells
Pour off the supernatant from the container and resuspend the pellet in complete medium
Perform a viable cell count and reseed a flask with an aliquot of cells at the required density
The size of culture flask used depends on the number of cells required
An appropriate volume of complete medium is added to the flask
A cell count may not always be necessary if the cell line has a known split ratio
Label each flask with cell line name, passage number, and date

10. Counting of Cells
For the majority of manipulations using cell cultures, such as cytotoxicity tests, transfections, cell fusion techniques, cryopreservation and subculture routines it is necessary to quantify the number of cells prior to use
Trypan blue is the most commonly used vital dye in microscopy for cell counting and to measure cells viability
The principle of trypan blue is based on the fact that the chromophore is negatively charged and does not interact with the cell unless the membrane is damaged
Equal volumes of cell suspension and 0.4% trypan blue are mixed by pipetting and left for 5 minutes at RT
Prepare a clean hemocytometer chamber and fill it with cell suspension

11. Counting of Cells
Count the number of cells in the four large corner squares:
viable (seen as bright cells)
and non-viable cells (stained blue) Use the following formula to calculate viable number of cells 1 2 4 3

12. Subculturing Suspension Cells
Examine the cultures microscopically for signs of cell deterioration and high-density growth
Cells in the exponential phase of growth will appear bright, round, and refractile, whereas dying cultures show cell lysis and shrunken cells
Method 1:
Remove a volume of the cells for a viable cell count
Transfer the cells to a sterile container and centrifuge the cells at 1000 rpm for 5 min to form a pellet
Discard the supernatant and resuspend the pellet with fresh media and split as desired

13. Subculturing Suspension Cells
Method 2:
Take out required amount of cell suspension from the flask using pipette and place into new flask
e.g. For 1:2 split from 20 ml of cell suspension take out 10 ml
For 1:5 split from 20 ml of cell suspension take out 4 ml
Add required amount of pre-warmed cell culture media to fresh flask.
e.g. For 1:2 split from 20 ml add 10 ml fresh media to 10 ml cell suspension
For 1:5 split from 20 ml add 16 ml fresh media to 4 ml cell suspension

14. Changing Media
If cells have been growing well for a few days but are not yet confluent 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 starting to go orange rather than red then media should be changed as soon as possible
To change media, warm up fresh culture media at 37oC in water bath or incubator for at least 30 mins
Carefully pour of the media from the flask into a waste pot
Immediately replace the media with fresh pre-warmed culture media and return to CO2 37oC incubator

15. Storage of Cell Lines (Cryopreservation)
Because an established cell line is a valuable resource and its replacement is expensive and time consuming, it is vitally important that they are frozen and preserved for long-term storage
If a cell line can be expanded sufficiently, preservation of cells by freezing will allow:
secure stocks to be maintained without aging
and protect them from problems of contamination
incubator failure
or medium and serum crises
Ideally, 1×106–1×107 cells should be frozen in 10 ampoules

16. Factors Affectining Survival After Freezing
Factors favoring good survival after freezing and thawing are:
High cell density at freezing (1×106–1×107 cells/ml)
Presence of a preservative, such as glycerol or dimethyl sulfoxide (DMSO) at 5–10%
Cryoprotective agents reduce the freezing point of the medium and also allow a slower cooling rate, greatly reducing the risk of ice crystal formation, which can damage cells and cause cell death
Slow cooling, 1oC/min, down to −70oC and then rapid transfer to a liquid nitrogen freezer
Rapid thawing
Slow dilution, ∼20-fold, in medium to dilute out the preservative

17. Factors Affectining Survival After Freezing
Reseeding at 2- to 5-fold the normal seeding concentration
For example, if cells are frozen at 5×106 cells in 1 ml of freezing medium with 10% DMSO and then thawed and diluted 1:20, the cell concentration will still be 2.5×105 cells/ml at seeding, higher than the normal seeding concentration for most cell lines, and the DMSO concentration will be reduced to 0.5% which most cells will tolerate for 24 h
Changing medium the following day (or as soon as all the cells have attached) to remove preservative

18. Quality Control
All cell culture laboratories should be run according to Good Laboratory Practice Guidelines
A number of organizations provide accreditation of clinical laboratories, and although most cell culture is probably done within research laboratories, similar principles should be applied
The major issues are infection and contamination which cause inaccurate results

19. Culture Contamination
If a culture is contaminated this must be discovered as soon as possible, either to:
Discard the culture before the contamination can spread to other cultures
or to attempt decontamination
The latter should only be used as a last option
Decontamination is not always successful and can lead to the development of antibiotic-resistant organisms

20. Culture Contamination
Most bacterial, fungal, and yeast infections are readily detected by regular careful examination with the naked eye (e.g., by a change in the color of culture medium) and by using the microscope
However, one of the most serious contaminations is mycoplasma, which is not visible by routine microscopy
Any cell culture laboratory should have a mycoplasma screening program in operation
Those collecting tissue for primary culture are particularly at risk

21. Culture Contamination
The precautions that should be observed are as follows:
Check frequently for contamination by looking for:
a rapid change in pH (usually a fall, but some fungi can increase the pH)
cloudiness in the medium
Extracellular granularity under the microscope
or any unidentified material floating in the medium
If a contamination is detected, discard the flask unopened and autoclave
If in doubt, remove a sample and examine by phase microscopy, Gram's stain, or standard microbiological techniques

22. Bacterial or Fungal Culture Contamination
Bacterial or fungal infections of cell cultures are usually obvious
The phenol red, if present, will turn yellow as the infection uses up available nutrients and acidifies the medium
Under the inverted microscope hyphae, yeast, or colonies of bacteria can be observed
However, it is common practice to add antibiotics to cultures and this can mask low level infection for a considerable time

23. Mycoplasma Culture Contamination
The most common and most missed infection in cell culture laboratories is probably Mycoplasma
Several species are involved and their effects are insidious
The indicators that there might be a problem include:
reduced growth rate,
morphological changes,
chromosomal aberration, and altered metabolism
There are several ways of testing for Mycoplasma and many manufacturers provide kits
It is possible to treat Mycoplasma infection with antibiotics, but avoidance is the best policy
Most laboratories simply dispose of infected cultures and start again
Fluorescence of the eukaryotic nuclei and the extranuclear prokaryotic DNA

24. Viruses
Viral infection is also insidious, some cultures contain viruses
Viruses are either integrated into their genome, or as endogenous non-lethal infections
In many cases, these are not regarded as infections, and viral transformation of cells is an age-old method of producing continuous cell lines

25. Cross-Contamination
If there are other cell lines in use in the laboratory, they can cross-contaminate even a primary culture, or misidentification can arise during subculture or recovery from the freezer
Precautions must be taken to avoid cross-contamination:
Do not handle more than one cell line at a time
If this is impractical, do not have culture vessels and medium bottles for more than one cell line open at one time
Never be tempted to use the same pipette or other device for different cell lines

26. Cross-Contamination
Do not share media or other reagents among different cell lines
Do not share media or reagents with other people
Keep a panel of photographs of each cell line, at low and high densities, above the microscope, and consult this regularly when examining cells during maintenance
This is particularly important if cells are handled over an extended period, and by more than one operator
If continuous cell lines are in use in the laboratory, handle them after handing other, slower-growing, finite cell lines