Thawing Cryopreserved Cells

Youssef Farhat, Written 7/9/12 and last updated 5/6/14

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This protocol was developed for cryopreservation vials that contained 10 million tendon fibroblasts (tenocytes) frozen in 1 mL of media.  The same procedure can potentially be generalized and used for other cell types.


  • Water bath set to 37°C
  • Pipette tips and micropipettors (one set to 1000 mL and the other set to 50 mL) under the culture
  • 50-mL centrifuge tube
  • Several T-175 culture flasks
  • If counting cells, use Trypan Blue Stain 0.4% (Gibco, #15250), a hemocytometer and a glass cover slip


1. Thaw the cells

1)    Warm culture media in the water bath to 37°C.

2)    When the media is warm, take some warm water into a clean beaker.

3)    Take a vial of cryopreserved cells out from the -80°C or -130°C freezer and drop the vial into the warm water in the beaker.  Swirl the beaker so that it warms evenly.

4)    Meanwhile, place the warm culture media under the hood, spray and wipe it with 70% EtOH.

5)    Once the cells in the vial appear to have thawed, take the vial out of the water, place it under the hood, spray and wipe it with 70% EtOH.

6)    Add 1 mL (i.e. 1000 mL) of warm media to the vial and suspend the cells by pipetting up and down several times.

2. Remove the cryopreservation media from the cells by centrifugation

1)    Transfer the cells to an empty 50 mL centrifuge tube.

2)    Add 3 mL of media to the 50 mL centrifuge tube.

3)    Centrifuge the cells down to a pellet (1500 rpm, 5 min at 4°C)

4)    Return the centrifuge tube to the hood, spray and wipe with 70% EtOH.

5)    Remove the media from the cell pellet using a vacuum or pipettor.

6)    Resuspend the cells in 10 mL of media (as a rule of thumb, suspend in about 1 mL for each million of cells in the vial).

3. Count the cells under the microscope (optional)

1)    Take 50 mL of the cell suspension and place it into a 1.5 mL Eppendorf tube.

2)    Add 50 mL of Trypan Blue and mix by pipetting up and down.

3)    Set up the microscope (10X objective recommended) with the hemocytometer and cover slip.

4)    Pipette 10 mL into the hemocytometer and count the number of live and cells.

4. Seed cells into the tissue culture flasks

1)    After counting the cells, determine how much cell suspension must be added to each T-175 flask to seed with 500,000 cells (if the cells were not counted, assume that it is 500 mL).

2)    Pipette 20 mL of media into each flask to be used.

3)    After mixing the cells to resuspend them, pipette the amount needed into each flask.

4)    Label the flasks with your name, the date, the cell type, and the passage number of the cells and place them in the incubator.

Citing this Protocol

Farhat, Y. “Thawing Cryopreserved Cells.” The Protocol Place. July 2012. Accessed on mm/dd/yyyy. <>

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