How to Thaw Cryopreserved Cells

Vaccinated PBMCs, Antigen-Specific T Cells

Download PDF

Thaw Cryopreserved Vaccinated PBMCs and Antigen-Specific T Cells

Thawing cryopreserved cells properly is crucial to ensure the viability and functionality of the cells. Using good technique and working quickly ensures that a high proportion of the cells survive the procedure.

Thawing cells is simple. However, speed is key to the optimum cell recovery.

As with other procedures, HemaCare scientists recommend that you closely follow the steps provided to ensure best results.


  • Vial of frozen cells
  • 37°C water bath
  • Tissue culture medium containing 5-10% serum or other protein
  • Centrifuge


  • The cell suspension may form clumps after standing at room temperature. This can be avoided by preparing and using the cells promptly, or by adding DNase to the suspension at a final concentration of 10 units per mL.
  • Always wear personal protective equipment and use universal precaution when working with human-derived biological materials and liquid nitrogen.


  1. Place vial of cells in 37°C water bath and agitate until thawed. It is important to thaw the cells quickly. Do NOT allow thawed cells to remain in freezing media any longer than necessary.
  2. Slowly add thawed cells to 9 mL of medium containing serum. Invert tube 2 or 3 times to mix, or mix gently by pipetting up and down several times.
  3. Centrifuge for 10 minutes at 200 x g.
  4. Aspirate or decant the supernatant and gently resuspend the cell pellet in 10 mL of medium.
  5. Remove an aliquot for cell count and proceed with experimental manipulations.

For Technical Support dial 877.944.4362 or email