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Question 19 of 20 Advanced Practical

How would you build a cell bank for a line that will be used for years?

Short answer

Expand an authenticated early-passage culture into a small master bank, use one master vial to make a larger working bank, and take every experiment from the working bank. Test identity and mycoplasma at each stage, record passage numbers, and store vials in more than one location.

Why

A two-tier bank limits how far any experimental culture has drifted from the original. Because cell lines evolve by clonal selection, as Ben-David and colleagues showed across 27 MCF7 strains with widely different drug responses, every experiment starting from an unbroken continuous culture is working on a slowly changing population. Going back to a working vial resets the clock.

ATCC's guidance emphasizes that high-passage cells differ in morphology, stimulus response, growth rate, protein expression and transfection efficiency, and that passage numbers belong in published methods. Testing is tied to the bank rather than to each experiment, which makes it affordable. Storage in two independent freezers or sites protects against a single failure, which is not a hypothetical risk.

How to work it through

  1. Start from the earliest authenticated material available and confirm identity by short tandem repeat profiling and freedom from mycoplasma.
  2. Expand with as few passages as possible and freeze a master bank of around 10 to 20 vials in controlled-rate conditions.
  3. Thaw one master vial, expand it and freeze a working bank of 50 or more vials, then retest identity and mycoplasma on that bank.
  4. Record for every vial the line, source, passage number, date, medium, operator and freezer position.
  5. Set a maximum number of passages after thaw for experimental use and record the passage number in every experiment.
  6. Keep part of each bank in a second freezer or a second site, and check viability of a vial periodically.

An example

A laboratory receives a line at passage 8, confirms its profile and freezes 12 master vials at passage 10. One master vial is expanded to 60 working vials at passage 13. Experiments use working vials and are discarded by passage 28, giving 15 passages of use.

When a result cannot be reproduced two years later, a fresh working vial at passage 13 reproduces it, which points to drift in the culture rather than to the protocol.

Where the answer stops holding

A bank preserves what was frozen, so if the starting culture was already drifted or misidentified the bank locks that in. Freezing and thawing select for cells that survive it, which can itself change the population. Liquid nitrogen storage carries a small risk of cross-contamination between vials, so sealed storage and the vapor phase are preferred.

A bank does not remove the need for periodic retesting, since contamination can enter during expansion.

A strong answer covers

  • Master bank and working bank in two tiers
  • Authenticate and test mycoplasma at each tier
  • Record passage number and set a maximum after thaw
  • Full traceability of lot, date and freezer position
  • Split storage across freezers or sites

Common mistakes

  • Running a single continuous culture for years without returning to frozen stock
  • Freezing a bank before confirming identity and mycoplasma status
  • Keeping the entire bank in one freezer