Problem	Possible Reason	Suggested Solution
Lack of viable cells upon thawing	Incorrect storage	Obtain new stock stored in liquid nitrogen that have not been thawed
	Incorrect thawing	Thaw cells quickly but gradually, dilute frozen cells drop-wise with prewarmed medium, handle cells gently, and only centrifuge at low speeds
	Glycerol exposure to light	If the cryopreservative agent glycerol was used and exposed to light, its by-product acrolein may be toxic for the cells
Lack of cell attachment to culture vessel after subculturing	Residual digestive enzyme activity	Thoroughly wash cells in prewarmed medium before replating
	Excessive digestion of cells	Reduce digestion time and block enzymatic activity using inhibitor (FBS for Trypsin)
	Mycoplasma contamination	Perform routine Mycoplasma PCR tests on cultures
Slow cell growth	Incorrect growth medium	Growth medium must fit the requirements of the culture cell line and (if applicable) contain serum that has been screened
	Depletion or breakdown of essential cell culture components	Ensure presence of growth-promoting factors and substitute unstable components such as glutamine with GlutaMax
	Incorrect storage of medium and supplements	Follow manufacturer’s instructions closely
	Passage number is too high	The proliferation rate of cell cultures may cease with continuous subcultures—cells should be replaced with low-passage stocks
	Confluency is too low	Enhance concentration of initial plating density
	Confluency is too high	Cells should be passaged in their log-phase (at around ~80% confluency)
	Mycoplasma contamination	Perform routine Mycoplasma PCR tests on cultures
Rapid shift in medium pH	Incorrect CO2 tension	Adjust CO2 concentration of incubator based on HCO3− concentrations of medium: 2.0 g/L requires CO2 levels of 5%, while 3.7 g/L rely on 10% supplementary CO2
	Lack of gas exchange	Loosen caps of tissue culture flasks
	Insufficient bicarbonate buffering in medium	Add HEPES (10–25 mM)
	Bacterial contamination	Investigate cultures under light microscope
Cell death	Lack of CO2 or fluctuating temperature	Monitor CO2 and temperature levels of incubator and do not leave cells outside the incubator for extended periods of time
	Accumulation of toxins	Regularly replace cell culture medium
	Incorrect osmotic pressure	Review osmolality of cell culture medium and potential effects of added drug compounds or HEPES