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Freezing Down Living Cells

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to take a solution of cells (either from animal samples or from a cell line) and prepare them to be frozen down in liquid nitrogen. Once in liquid nitrogen, these cells can be stored for years and be functionally recovered using the Thaw Protocol.

Reagents and Materials

  • Cell Suspension - with known number of cells
  • Mr.Frosty
  • IPA
  • Freeze Media
  • CryoVials
  • Cryo-Baby Labels

Things to Note before starting

Freeze Media Choice

The choice of Freeze Media depends the cells you are freezing down. Common choices are 90% FBS + 10% DMSO or Cryostar 10.

Estimated Timing

This is a quick buffer change procedure. However, the samples have to be stored in the Mr. Frosty within a -80C Freezer overnight before they can be transferred into liquid nitrogen.

Procedure

  1. Take the total number of cells and divide it by the number of aliquots you want to create. That is how many cells will be present in each aliquot. 1
  2. Spin down the cell suspension at 800 x g for 5 minutes. Aspirate to the pellet.
  3. Resuspend the cell pellet in 1mL per aliquot you want to freeze down.
  4. Pipette up and down with a p1000 to ensure the solution is mixed completely.
  5. Aliquot 1mL of solution into CryoVials. 2
  6. Place CryoVials into a room temperature Mr. Frosty. 3
  7. Place the Mr. Frosty overnight in a -80C Freezer
  8. The next day, remove samples from the Mr. Frosty and leave the Mr. Frosty at room temperature to cool down before it is used again.
  9. Place the cells into liquid nitrogen. Make sure you record the location of the samples.

Next Steps

Usually when these cells are frozen down, they will eventually be thawed.

Appendix

Recipe's for Buffers

Freeze Media

This buffer is used to freeze down cells for liquid nitrogen storage. The recipe below is for the 1X solution. Scale the recipe up depending on how many samples you have. Always make more than strictly necessary to account for pipetting errors.

  • 900mL of Heat Inactivated FBS
  • 100mL of DMSO

Footnotes

  1. I would recommend freezing between 1e6 to 10e6 cells per 1mL aliquot. The maximum I would imagine working would be 20e6. ↩

  2. CryoVials are important to use because of the threaded nature of their cap. A regular eppendorf tube might explode due to the pressure differential of air when thawed out of liquid nitrogen. ↩

  3. The Mr. Frosty allows the samples to gradually approach chilled temperatures rather than being shocked immediately. To ensure this, make sure the Mr. Frosty's are filled with IPA up to their fill line. ↩