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 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” NBTC Cell Culture Minicourse
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.
NBTC Cell Culture Minicourse
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. NBTC Cell Culture Minicourse
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
NBTC Cell Culture Minicourse
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
NBTC Cell Culture Minicourse
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? NBTC Cell Culture Minicourse
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
