Thawing Frozen Cells
- Thaw frozen cells rapidly (< 1 minute) in a 37°C water bath.
- Dilute the thawed cells slowly, 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 37°C
- Disposable, sterile centrifuge tubes
- Water bath at 37°C
- 70% ethanol
- Tissue-culture treated flasks, plates, or dishes
||Video 5 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.|
The following protocol describes a general procedure for thawing cryopreserved cells. For detailed protocols, always refer to the cell-specific product insert.
- Remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37°C water bath.
- 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.
- Transfer the vial it into a laminar flow hood. Before opening, wipe the outside of the vial with 70% ethanol.
- Transfer the desired amount of pre-warmed complete growth medium appropriate for your cell line dropwise into the centrifuge tube containing the thawed cells.
- Centrifuge the cell suspension at approximately 200 × g for 5–10 minutes. The actual centrifugation speed and duration varies depending on the cell type.
- After the centrifugation, check the clarity of supernatant and visibility of a complete pellet. Aseptically decant the supernatant without disturbing the cell pellet.
- Gently resuspend the cells in complete growth medium, and transfer them into the appropriate culture vessel and into the recommended culture environment.
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.