The thawing procedure is stressful to frozen cells. Using good technique and working quickly ensures that most cells survive the procedure. As with other cell culture procedures, we recommend that you closely follow the instructions provided with your cells and other reagents for best results when freezing and thawing.

Video: Thawing cells

This video presents the best way to thaw cells without harming them in this stressful process. Our scientists demonstrate how to carefully transfer cells from storage in liquid nitrogen to the incubator.


Materials for thawing cells

Prepare to thaw your cells by gathering the following materials:


Thawing cells: protocol

The following protocol describes a general procedure for how to thaw cells. For detailed protocols, always refer to the cell-specific product insert. When thawing, always use proper aseptic technique and work in a laminar flow hood. Remember to wear personal protective equipment, including a face mask or goggles.

NOTE: Use caution when thawing cryovials stored in liquid-phase, they present a risk of explosion.

Some freezing media contain DMSO, which facilitates the entry of organic molecules into tissues. Handle reagents containing DMSO using equipment and practices appropriate for the hazards posed by such materials.

  1. Remove the cryovial containing frozen cells from the liquid nitrogen storage and immediately place it into a 37°C water bath.
  2. Quickly thaw the cells (<1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial.
  3. Transfer the vial into a laminar flow hood. Before opening, wipe the outside of the vial with 70% ethanol.
  4. Transfer the thawed cells dropwise into a centrifuge tube containing the desired amount of prewarmed complete growth medium appropriate for your cell line.
  5. Centrifuge the cell suspension at approximately 200 × g for 5–10 minutes. The actual centrifugation speed and duration varies depending on the cell type.
  6. After the centrifugation, check the clarity of supernatant and visibility of a complete pellet. Aseptically decant the supernatant without disturbing the cell pellet.
  7. Gently resuspend the cells in complete growth medium and transfer them into the appropriate culture vessel and into the recommended culture environment. Plate thawed cells at high density to optimize recovery.

Note: The appropriate flask size depends on the number of cells frozen in the cryovial, and the culture environment varies based on the cell and media type.


Troubleshoot thawing cells

The purpose of thawing cells is to effectively revive them from a frozen state with minimal damage. Proper techniques and troubleshooting common issues like slow recovery or contamination are essential for maintaining cell viability.

ConcernRecommendation

Cells were stored incorrectly

  • Obtain new stock and store in liquid nitrogen. Keep the cells in liquid nitrogen until thawing.

Homemade freezer stock is not viable

  • Freeze cells at a density recommended by the supplier.
  • Use low-passage cells to make your own freezer stocks.
  • Follow procedures for freezing cells exactly as recommended by the supplier.
  • Obtain new stock.

Cells were thawed incorrectly

  • Follow procedures for thawing cells exactly as recommended by the supplier. Note the thawing procedure recommended by this page is a general procedure provided as a guideline only.
  • Make sure that you thaw the frozen cells quickly but dilute them slowly using pre-warmed growth medium before plating.

Thawing medium is not correct

  • Use the medium recommended but the supplier. Make sure the medium is pre-warmed.

Cells are too dilute

  • Plate thawed cells at high density as recommended by the supplier to optimize recovery.

Cells not handled gently

  • Freezing and thawing procedures are stressful to most cells. Do not vortex, bang the flasks to dislodge the cells (except when culturing insect cells), or centrifuge the cells at high speeds.

Glycerol used in the freezing medium was stored in light (if applicable)

  • If stored in light, glycerol gets converted to acrolein, which is toxic to cells. Obtain new stock.

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