Cell Culture Troubleshooting Guide
The table below addresses some of the common problems encountered when culturing cells, along with their possible causes and suggested solutions.
|Problem||Possible Cause||Suggested Solution|
|Rapid pH shift in medium||Incorrect carbon dioxide (CO2) tension||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. (See Decontaminating Cultures with Antibiotics and Antimycotics.)
|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 pH
||Bacterial or fungal contamination
Try to decontaminate culture. (See Decontaminating Cultures with
Antibiotics and Antimycotics.)
|Cells not adhering to culture vessel
||Overly trypsinized cells||Trypsinize for a shorter time, or use less trypsin. (See Dissociation of Cells from
||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.