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Troubleshooting: Cell Culture


Possible Cause

Suggested Solution

Rapid pH shift
in medium
Incorrect carbon dioxide (CO2)

Increase or decrease percentage of CO2 in the incubator based on concentration of sodium bicarbonate in medium. For sodium bicarbonate concentrations of 2.0 to 3.7 g/L, use CO2 amounts of 5% to 10%, respectively.

Switch to CO2-Independent Medium.

  Overly tight caps on tissue culture flasks Loosen caps one-quarter turn. 
  Insufficient bicarbonate buffering Add HEPES buffer to a final concentration of 10 to 25 mM. 
  Incorrect salts in medium  Use an Earle’s salts-based medium in a CO2
environment and a Hanks’ salts-based medium in atmospheric conditions.
  Bacterial, yeast, or fungal contamination  Discard culture and medium.

Try to decontaminate culture.

Precipitate in medium, no change
in pH
Residual phosphate left over from detergent
washing, which may precipitate powdered medium components
Rinse glassware in deionized, distilled water
several times, then sterilize.
  Frozen medium Warm medium to 37°C and swirl to dissolve. If precipitate remains, discard medium. 
Precipitate in medium, change in
Bacterial or fungal contamination  Discard medium.

Try to decontaminate culture.

Cells not adhering to culture vessel  Overly trypsinized cells
Trypsinize for a shorter time, or use less
  Mycoplasma contamination  Segregate culture and test for mycoplasma infection. Clean hood and incubator. If culture is contaminated, discard.
  No attachment factors in medium For serum-free formulations, be sure they contain attachment factors.
Decreased growth of culture  Change in medium or serum  Compare media formulations for differences in glucose, amino acids, and other components.

Compare the old lot of serum with the new lot in a growth experiment.

Increase initial cell inoculum.

Adapt cells sequentially to new medium.


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