Why did my Cell Culture fail?
Cells fail to attach to the culture surface within the expected window (including epithelial lines that need collagen coating). Doubling time longer than expected for the authenticated line and media recipe. Visual or growth-pattern signs of microbial contamination in culture. Silent mycoplasma contamination suspected
Playbooks
- Cell Culture: poor attachmentCells fail to attach to the culture surface within the expected window (including epithelial lines that need collagen coating).
- Cell Culture: slow growthDoubling time longer than expected for the authenticated line and media recipe.
- Cell Culture: contaminationVisual or growth-pattern signs of microbial contamination in culture.
- Cell Culture: mycoplasma suspectedSilent mycoplasma contamination suspected from chronic underperformance without visible turbidity.
- Cell Culture: cell-line misidentificationWorking culture morphology, markers, or behaviour does not match the authenticated line.
- Cell Culture: low viabilityCell viability below expected threshold on counting.
- Cell Culture: clumpingCells aggregate instead of forming a single-cell suspension or even monolayer.
Calculators
FAQ — Cell Culture
- Poor attachment
- Cells fail to attach to the culture surface within the expected window (including epithelial lines that need collagen coating). Pre-coat plates with collagen I (or the cell-type-appropriate matrix) before seeding epithelial cells — Collagen coating → epithelial attachment is a common advance when untreated plastic fails. Replace coating reagent with a fresh, cell-type-matched lot — Rules out expired fibronectin/collagen/PDL as the silent material failure. Shorten trypsin exposure and neutralize immediately with serum-containing media — Preserves surface adhesion proteins during dissociation. Authenticate the line if attachment phenotype diverges from the stock description — Misidentified lines often show unexpected adherence behaviour.
- Slow growth
- Doubling time longer than expected for the authenticated line and media recipe. Prepare fresh complete media with new L-glutamine; use within 2 weeks — Glutamine and labile factors degrade in solution regardless of bottle expiry. Test for mycoplasma on this line and sister cultures in the same incubator — Silent mycoplasma commonly presents as slow growth only. Verify incubator temperature and CO₂ against a calibrated probe — Rules out shared environmental drift before discarding the line. Authenticate the cell line if growth rate diverges from the stock certificate — Misidentified lines often grow at unexpected rates.
- Contamination
- Visual or growth-pattern signs of microbial contamination in culture. Quarantine and discard the affected flask immediately — do not open near other cultures — Prevents cross-contamination of the rest of the incubator. Decontaminate the incubator shelf and biosafety cabinet; re-verify HEPA filter service date — Addresses the likely source before resuming culture. Thaw a fresh, early-passage vial from the verified cryostock — Restarts from a known-clean population rather than trying to rescue a contaminated line. Test for mycoplasma on all other active lines in the same incubator — Mycoplasma spreads silently between flasks sharing an incubator or media bottle.
- Mycoplasma suspected
- Silent mycoplasma contamination suspected from chronic underperformance without visible turbidity. Test for mycoplasma (PCR and/or luminescence) on the suspect line immediately — Confirms or clears the leading material cause before discarding stock. Quarantine positives; discard or treat only under lab policy — never share media bottles — Stops horizontal spread while decisions are made. Retest all active lines sharing the incubator; update lastMycoplasmaTestAt on Lab Index — Keeps Lab Index dates current so Diagnostics can raise this cause next time. Thaw a mycoplasma-negative authenticated vial and restart — Clean restart beats chronic underperformance from a silently contaminated stock.
- Cell-line misidentification
- Working culture morphology, markers, or behaviour does not match the authenticated line. Authenticate the cell line (STR / species PCR) and record lastAuthenticatedAt on Lab Index — Directly tests the leading material cause and updates Lab Index dates. Quarantine the suspect flask; thaw an authenticated early-passage vial in parallel — Stops spreading a wrong line while confirmation runs. Reconcile cryostock box map against vial labels and RRIDs before the next thaw — Addresses inventory mix-ups that present as “wrong phenotype”. If the unopened vendor vial also fails authentication, contact the vendor and stop using the lot — Upstream misidentification cannot be fixed by lab technique alone.
- Low viability
- Cell viability below expected threshold on counting. Pre-warm media and reagents to 37°C before every media change — Cold-shock is the most common cause of a sudden viability drop. Prepare fresh complete media with new L-glutamine, use within 2 weeks — Glutamine degrades in solution regardless of expiry date. Passage at 70–80% confluence, not later — Prevents nutrient depletion and contact-inhibition stress. Run a mycoplasma PCR/luminescence test on the line — Rules out silent contamination as the chronic driver.
- Clumping
- Cells aggregate instead of forming a single-cell suspension or even monolayer. Extend trypsin/Accutase incubation by 1–2 min and confirm single-cell suspension under the scope before plating — Directly fixes incomplete dissociation, the leading cause. Triturate 10–15× with a P1000 through a narrow tip, avoid bubbles — Mechanically breaks residual clumps after enzymatic dissociation. Pass the suspension through a 40 µm cell strainer before counting/plating — Removes remaining aggregates without affecting viable single cells. Prepare dissociation buffer in Ca²⁺/Mg²⁺-free PBS — Divalent cations support cell-cell adhesion and work against dissociation.