Cell Culture: slow growth

1. Ranked causes

   1. Media past use-by / glutamine or growth-factor depletedGrowth slowed gradually across a week after media bottle opened.
   2. Serum lot or concentration suboptimal for this lineSide-by-side with previous FBS lot restores expected doubling.
   3. Mycoplasma or other low-level contamination slowing proliferationChronic underperformance without visible turbidity; mycoplasma test overdue or never run.
   4. Over-passaged or misidentified line with a different growth rateDoubling time matches a known contaminant line, not the authenticated stock.
   5. Incubator CO₂ / temperature driftMultiple lines in the same incubator slow together.

2. Recovery steps

   1. Prepare fresh complete media with new L-glutamine; use within 2 weeksGlutamine and labile factors degrade in solution regardless of bottle expiry.
   2. Test for mycoplasma on this line and sister cultures in the same incubatorSilent mycoplasma commonly presents as slow growth only.
   3. Verify incubator temperature and CO₂ against a calibrated probeRules out shared environmental drift before discarding the line.
   4. Authenticate the cell line if growth rate diverges from the stock certificateMisidentified lines often grow at unexpected rates.

3. Controls first

   • Did untreated / vehicle control cultures of the same line also grow slowly?If yes: Media, incubator, contamination, or identity — not a treatment-specific effect.
   • Is media visibly turbid or has it shifted colour rapidly?If no: Consider mycoplasma testing and media freshness before assuming genetics.

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