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Protocol for Freezing Cells

All work must under sterile conditions.

Use only healthy, Mycoplasma free, growing cells to make banks. Do not use 'old' cultures or cultures which show signs of poor growth.

  1. Label vials with cell line name (full name), passage number at time of handling, # of cells/vial, thaw to T... (insert size of flask to thaw to), the date, your initials. If the bank is for R&D purposes, additional information such as a notebook reference may be desired. Printed labels are very handy as they are easier to read. Be sure to use labels that will withstand LN2 temperatures.
  2. To freeze cells, you must have a suspension. Therefore, trypsinize adherent cells. Be sure to add adequate media with serum to stop the Trypsin as the cells will remain in this solution for a time prior to freezing.
  3. Remove a sample and count the cells.
  4. Calculate the needed volume of cell suspension as follows:
    desired # of cells per vial = 2.5 x10^6 cells
    Y = # of vials needed +1 (for viability testing) + 1 (so you don't come up short)
    Z = total number of cells needed = Y x 2.5x106
    Z / # of cells /mL in stock = volume of suspension needed to make bank.
  5. Remove the calculated volume to a sterile conical tube and spin cells at ~1000 rpm for 5 minutes. Remove the supernatant. Send 10ml of supernatant for Mycoplasma testing.
  6. Add 90% of the desired final volume of serum to the tube (Y x 1mL x 0.90).
  7. Add 10% of the desired final volume of sterile DMSO to the tube (Y x 1mL x 0.10). Resuspend the cells.
  8. Put 1mL in each labeled vial. Discard any excess.
  9. Put the vials in the -80°C freezer.
  10. After the bank is in the freezer, seed the vessels that were harvested at a normal split ratio.
  11. Record the making of a bank and the number of vials made on the culture sheet. Include an exact copy of what the vials were labeled.
  12. The next day, get one vial and thaw it. (See thawing protocol)
  13. Observe daily for sterility, viability, normal morphology, and normal growth.
  14. It is not uncommon for the cells to require a feed prior to confluence being attained. Cells should be fed two or three times a week if required. If the cells take more than twice the usual time between splits to reach confluence post thaw, consider making a fresh bank.
  15. If freeze is not sterile, is of poor viability or is otherwise of undesirable quality, discard it and make a new one.
  16. Once freeze is confirmed 'good', transfer the cells to the LN2 freezer and log them in.
  17. Do not let cells stay at -80°C any longer than is absolutely necessary. A week is a good rule of thumb.
  18. Record all information on culture sheet, include mycoplasma results.
  19. Carry the original culture vessels as backups until the freeze is confirmed 'good'.

Other numbers of cells may be frozen in a vial, but make sure it is listed on the vial. Do not exceed 10^7 cells/mL, they do not survive well beyond that density.

Additional cells may be put in a vial by increasing the volume of suspension in the vial. Be sure to label the vials accurately, ie '1x10^7 cells/ml, 2 ml/vial' would be a vial with 2x10^7 cells in 2 ml in the vial. It could also be expressed as '2x10^7 cells/vial at 1x10^7 cells/ml'.

Do not overfill vials as they may burst as they freeze.

Cells may also be frozen in 90% growth media + 10% DMSO. Or 80% growth media + 10% serum + 10% DMSO. Other combinations are possible.

Always add the DMSO last.

After the addition of DMSO, it is important to freeze the cells as soon as possible as DMSO is toxic.

The purpose of the DMSO is to prevent the formation of ice crystals which will rupture the cells as they thaw.

Freeze the lowest possible passage.

It is important to have current mycoplasma results for your cells. The easiest way to do this is to test at the time of banking.

You can also make a bank using the following calculation:
Total # of cells available / 2.5x10^6 = # of vials you can make.

This will usually yield a whole number with a fraction, so take the whole # and run the calculation again, use the remaining cells to seed your backup.