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Thawing Cells - Fast

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to thaw cells that were frozen down in the liquid nitrogen or -80C Freezers. After thawing, cells should be ready to use in culture or experiments. This is the Fast Variant of this protocol.

Reagents and Materials

  • Thaw Media
  • Frozen down Cell Aliquots
  • 50mL Centrifuge Tube

Things to Note before starting

Estimated Timing

As this is the fast variant, the cells should be ready for culture in ~5-10 minutes. 1

Procedure

  1. Remove Cell Aliquots from their long term storage in the -80C Freezer or the Liquid Nitrogen Tank.
  2. Using the floating foam tube rack, float the Cell Aliquot in the Hot Water Bath. 2
  3. Monitor the Cell Aliquot every 3 minutes. When the Aliquot has a tiny crystal of ice remaining at the bottom, remove it from the hot water bath. 3
  4. Using a p1000, transfer the Cell Aliquot into a 50mL Centrifuge Tube containing the Thaw Media.
  5. Invert the 50mL Centrifuge Tube 3 times to mix.
  6. Using a p1000, wash the Cell Aliquot Tube with the 1X Thaw Media to ensure all cells were transferred.
  7. Incubate at Room Temperature for 4 minutes.
  8. Spin down at 800 x g for 5 minutes. A small pellet should be seen at the bottom of the tube. Aspirate to the pellet.
  9. Resuspend the cells in the appropriate media for cell culture.

Next Steps

The cells recovery can be quantified by assessing viability with the Countess. After assessing viability, the cells can be placed into culture or used immediately for an experiment.

Appendix

Recipe's for Buffers

Thaw Media

This buffer is used to recover cells from liquid nitrogen storage. The recipe below is buffer to thaw 1 sample. Make each aliquot of this media in it's own 50mL Centrifuge Tube. This buffer should be kept on ice while being used.

  • 31.5mL of Heat Inactivated FBS
  • 3.5mL of DNase I

Footnotes

  1. What are the negatives of the fast protocol? There has been anecdotal evidence that the fast nature of this protocol causes shock to some cells. The immediate exposure to the large volume of chilled media might be toxic to some cells - causing reduced viability. No scientifically significant viability loss has been demonstrated at the time of writing this protocol. However, if you are working with precious cells and need the best recovery chances, I would recommend using the slower variant. Speak with CB Wolf or anyone in the Mouse Core for the up to date recommendations. ↩

  2. The floating foam tube racks can be located in the top shelf underneath the hot water bath. If you are having difficulty finding it, speak to someone in the Mouse Core (Heather, CB, or Mitch at the time of writing this protocol) ↩

  3. The goal of this step is to not over thaw the Cell Aliquot with the hot water bath. Taking the Cell Aliquot out when there is only a tiny sliver of ice allows us to be confident that the Cell Aliquot was not sitting in the Hot Water Bath for longer than necessary. ↩