Thawing Frozen Cells

The thawing procedure is stressful to a frozen culture. Using good technique and working quickly ensures that a high proportion of the 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.

• Thaw frozen cells rapidly (< 1 minute) in a 37°C water bath.
• Dilute the thawed cells slowly before you incubate them, using pre-warmed growth medium.
• Plate thawed cells at high density to optimize recovery.
• Always use proper aseptic technique and work in a laminar flow hood.
• Always wear personal protective equipment, including a face mask or goggles.  Cryovials stored in liquid-phase present a risk of explosion when thawed.
• Some freezing media contain DMSO, which is known to facilitate the entry of organic molecules into tissues. Handle reagents containing DMSO using equipment

• Cryovial containing frozen cells
• Complete growth medium, pre-warmed to a temperature of 37°C
• Disposable, sterile centrifuge tubes
• Water bath at a temperature of 37°C
• 70% ethanol
• Tissue-culture treated flasks, plates, or dishes

The following protocol describes a general procedure for thawing cryopreserved cells.  For detailed protocols, always refer to the cell-specific product insert.

1. Remove the cryovial containing the frozen cells from 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 it into a laminar flow hood.  Before opening, wipe the outside of the vial with 70% ethanol.
4. Transfer the desired amount of pre-warmed complete growth medium appropriate for your cell line dropwise into the centrifuge tube containing the thawed cells.
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.