Thawing Cells

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.

Prepare to thaw your cells by gathering the following materials:

• Cryovial containing frozen cells
• Complete growth medium: basal medium, serum, and supplements pre-warmed to 37°C
• Disposable, sterile centrifuge tubes
• Water bath or Lab Armor beads warmed to 37°C
• 70% ethanol
• Tissue-culture treated flasks, plates, or dishes

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.

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.

Learn more about seeding, subculturing, and maintaining cells
See the protocol for subculturing adherent cells
See the protocol for subculturing suspension 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.