Cell Culture: low viability

1. Ranked causes

   1. Media past its use-by date / glutamine degradedSlow decline in viability across the week, not a sudden drop.
   2. Osmotic or thermal shock during thaw or passageViability drop coincides with a recent thaw or media temperature change.
   3. Over-confluent culture before passage (contact inhibition / nutrient depletion)Viability drops correlate with delayed passaging.
   4. Mycoplasma or other low-level contaminationChronic, unexplained decline across multiple independent cultures.
   5. Incubator CO₂ / temperature driftMultiple lines in the same incubator decline together.

2. Recovery steps

   1. Pre-warm media and reagents to 37°C before every media changeCold-shock is the most common cause of a sudden viability drop.
   2. Prepare fresh complete media with new L-glutamine, use within 2 weeksGlutamine degrades in solution regardless of expiry date.
   3. Passage at 70–80% confluence, not laterPrevents nutrient depletion and contact-inhibition stress.
   4. Run a mycoplasma PCR/luminescence test on the lineRules out silent contamination as the chronic driver.

3. Controls first

   • Did untreated / vehicle control wells also show the same viability drop?If yes: Media, incubator, or contamination — not a treatment-specific effect.
   • Did the drop follow a thaw or fresh passage within 24 h?If yes: Thermal/osmotic shock — adjust thaw/passage technique before troubleshooting media or contamination.

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