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Question 12 of 20 Intermediate Troubleshooting

A cell line suddenly grows more slowly after several passages. How would you investigate?

Short answer

Split the possibilities into contamination, culture conditions, reagents and the cells themselves. Test for mycoplasma, review medium lot, serum lot, incubator conditions and splitting practice, then compare against a fresh low-passage vial from the frozen bank. The frozen vial is the control that separates a changed line from a changed environment.

Why

Slow growth is a symptom with many causes. Mycoplasma inhibits proliferation and is invisible, so it is tested first. A new serum lot, a medium approaching its expiry, or glutamine that has degraded in storage all slow cells.

Incubator temperature, carbon dioxide calibration and humidity drift. Splitting too sparsely or letting cultures become overgrown both change behavior. The cells themselves also change.

ATCC notes that high-passage cells show altered morphology, stimulus responses, growth rate, protein expression and transfection efficiency compared with low-passage cells. Ben-David and colleagues went further, showing that cell lines evolve by clonal selection in culture, that 27 strains of MCF7 differed genetically and transcriptionally, and that at least 75 percent of compounds strongly inhibiting some strains were completely inactive in others. Returning to a frozen early-passage vial is therefore diagnostic as well as curative.

How to work it through

  1. Test the current culture for mycoplasma and inspect it for bacterial or fungal contamination.
  2. Record the medium, serum and supplement lot numbers and compare with the period when growth was normal.
  3. Verify incubator temperature, carbon dioxide and water pan, and check the plate position and seeding density.
  4. Thaw a fresh low-passage vial and grow it side by side in both the old and new medium lots.
  5. Count viable cells rather than judging by eye, and compute doubling time for each arm.
  6. Confirm identity by short tandem repeat profiling if the fresh vial behaves differently from the aged culture.

An example

A HEK293 culture at passage 38 doubles in 48 hours instead of 24. Mycoplasma PCR is negative. A fresh passage 12 vial doubles in 25 hours in the same medium, so the medium is fine and the old culture has changed.

The laboratory discards the high-passage culture, limits future work to 15 passages after thaw, and records the passage number in every experiment.

Where the answer stops holding

A frozen vial from the same drifted stock reproduces the same problem, so the bank must be early enough to be a useful reference. Some changes, such as selection in a particular serum lot, are reversible and some are not. Growth rate alone is a crude readout.

A line can keep normal doubling time while its drug response has already shifted.

A strong answer covers

  • Mycoplasma and contamination first
  • Reagent lots and incubator conditions
  • Seeding density and splitting practice
  • Fresh low-passage vial as the control
  • Cell lines evolve by clonal selection

Common mistakes

  • Changing several variables at once and losing the cause
  • Assuming a line is stable because it came from a repository
  • Recording no passage number so drift cannot be traced