Identifying Problems:
Overview
Some general causes of cell death or other
problems:
• Contamination: Infections, chemical, or cell line
problems.
• Fixable things: Culture conditions/media components
• Less fixable things: Passage number, primary cell line
problems, messy lab mates, outside factors
• “Mystery stuff”
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Identifying Problems:
Contamination
What types of contaminants can be present?
Easily visible:
• Infectious: Yeast / bacteria / other fungus
• Other cells being cultured in the lab: fibroblasts, HeLa, etc.
Less visible:
• Infectious: Mycoplasma / viruses
• Chemical contaminants: Often sterilizing solution (ethanol
or bleach), but could be a bad batch of chemical used in
culture or other factor.
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Identifying Contamination
Identifying a yeast contamination:
• Rapid growth and consumption of media nutrients=rapid color
change in solution.
• Cloudy media
• “Baking bread” smell
• Dead/poorly spread cells
• Yeast morphology:
“string of pearls”
Treatment
1) Discard cells.
2) Keep an eye on other cell lines.
Image: http://img.photobucket.com/albums/v334/brensoft/yeast.gif
NBTC Cell Culture Minicourse
Identifying Contamination
Identifying bacterial contamination:
• Rapid growth and consumption of media nutrients: rapid color
change in solution.
• Cloudy solution
• Dead/poorly spread cells
• Morphology: Highly variable, depending on bacteria type.
Treatment
1) Discard cells.
2) Keep an eye on other cell lines.
Note: The source of contamination may be pre-existing in primary
cells, due to a patient’s infection prior to cell retrieval.
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Identifying Contamination
Identifying fungal contamination:
• Rapid growth and consumption of media nutrients: rapid color change in
solution.
• Cloudy/dark solution
• “Garbage” smell
• Dead/poorly spread cells
• Morphology:
fuzz or threads
Treatment
1) Discard cells.
2) Keep an eye on other cell lines.
3) Sticky mats/dehumidifiers in the labs may prevent the tracking in and
growth of mold spores.
Note: The source of contamination here may be pre-existing in primary cells
(prior exposure to mold spores).
Image: http://www.corning.com/lifesciences/technical_information/techdocs/cccontamination.asp
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Identifying Contamination
Contamination by other cell lines:
•
•
•
•
Change in growth rate (needs to be faster, in order for this contamination to
overtake the previous cell type)
Change in morphology
Mixed morphology
Change in response to stimuli
Treatment
1) Discard cells.
2) Keep an eye on other cell lines.
An intentional co-culture of HESCs and fibroblasts.
Note: The source of contamination here may be pre-existing in cell lines (ex: HeLa
contamination of many CDC cancer cell samples).
Note: Some culture techniques require intentional co-cultures.
Image: http://uem.testujeme.cz/article.asp?nArticleID=407&nLanguageID=2
NBTC Cell Culture Minicourse
Identifying Contamination
Less visible contaminants:
• Mycoplasma:
– May be seen by staining with DAPI
– Can be tested for (NBTC has kits)
– Results in changes in cell behavior and/or cell death
• Viruses:
Normal (L), Mycoplasma (R)
– May see some slight granularity (caused by viruses—viruses are not directly
visible by light microscopy).
– Usually results in rapid cell death.
• Chemicals:
– Not visible, but can result in poor cell morphology/response or apoptosis.
– Try replacing any recently-changed chemicals
– Make sure anything touching cells/culture has no residue on it.
Treatment for all these contaminations:
1) Discard cells.
2) Keep an eye on other cell lines.
Image : http://www.corning.com/lifesciences/technical_information/techdocs/cccontamination.asp
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Identifying Problems:
Fixable Things
• Incubator: low/high CO2, low/high temperature, low
humidity
– Check incubator temperature/CO2 level
• Food: Inappropriate nutrient mixture, cells not fed often
enough.
– Feed before significant color change occurs, compare media to
other protocols for similar cell lines.
• Cells: Over/under confluent when passaged, etc.
– Check on cells daily, if necessary.
Look back to the “What Cells Need” section. Look
familiar?
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Identifying Problems:
Less Fixable Things
• Over-passaged cells
– Primary cells can typically only be passaged 3-7 times before failing.
– Even “Immortalized” cell lines have genetic drift over time.
– Possible Solutions: Use cells from ~same passage for same experiments, go
back to an earlier, frozen-back passage, replenish primary supply.
• Genetic Drift: The overall profile of cell morphology and behavior can
change over time and many passages.
– Possible Solution: Go back to an earlier, frozen-back passage
• Problems with primaries: Age, disease, natural variation of donors.
• Mystery factors: Small unseen breaks in sterile packaging, insects, onetime error, variation in chemical lots, time of year, etc, etc.
– A frustratingly high percentage of cell problems that occur will fall under
this category. Check cells often, freeze back cell lines when possible, do
many experimental repeats to reduce effects of biological variation.
NBTC Cell Culture Minicourse
Identifying Problems: Review
• Lots of bad things can happen to cells, but many of
them are preventable if you can identify the problem.
• When in doubt, throw it out.
• When possible, freeze back stocks of cells that are
known to be high-quality.
If a consistent problem occurs in the NBTC: let us know
to help us identify any systemic issues.
Note in NBTC space: Read all signs.
NBTC Cell Culture Minicourse