Identifying cell culture problems LECTURE OF SUBJECT : Dr. sharafaldin Al-musawi College of Biotecholgy LECTURE: 7 SUBJECT: Animal Tissue culture LEVEL: 4 Identifying cell culture problems

Some general causes of cell death or other problems: 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

Identifying Problems: What types of contaminants can be present? Contamination What types of contaminants can be present? Easily visible: • Infectious: Yeast / bacteria / 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.

Identifying yeast contamination: Identifying Contamination Identifying 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.

Yeast Yeast Yeast Yeast Cells Cells Yeast

Yeast Cells

Identifying bacterial contamination: 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.

Identifying fungal contamination: 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).

Fungal contamination

CELLS Fungal contamination

Contamination by other cell lines: 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. 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.

Contamination by other cell lines

Less visible contaminants: 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 Treatment: 1) Discard cells. 2) Keep an eye on other cell lines.

Normal Mycoplasma

Less visible contaminants: Identifying Contamination Less visible contaminants: Viruses: May see some slight granularity (caused by viruses-viruses are not directly visible by light microscopy). Usually results in rapid cell death. Treatment: 1) Discard cells. 2) Keep an eye on other cell lines.

Uninfected cells Virus infected cells

Less visible contaminants: Identifying Contamination Less visible contaminants: 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: 1) Discard cells. 2) Keep an eye on other cell lines.

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.

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.

Identifying Problems Less Fixable Things 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.

Identifying Problems Less Fixable Things • Mystery factors: Small unseen breaks in sterile packaging, insects, onetime error, variation in chemical lots, time of year, 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.