Lessons Direct

Why speed matters

Most cryopreserved cells are frozen in medium containing DMSO, which protects cells during freezing but is toxic to cells once they are thawed, especially at room temperature or 37 °C. For this reason, the thawing process should be quick and deliberate, and DMSO should be diluted and removed as soon as possible after thawing.

Materials

• Cryovial of frozen cells
• Dry ice and bin, if there will be delay between tube retrieval and thaw
• Correct complete growth medium, pre-warmed to 37 °C
  • Use the exact medium formulation appropriate for the specific cell type
• 37 °C water bath
• Biosafety cabinet (BSC)
• Sterile 15 mL conical tubes
• Centrifuge
• Appropriate culture vessel (flask or plate)

Before you begin

1. Confirm the correct growth medium for the cell type and warm sufficient volume to 37 °C.
2. Label conical tubes and prepare the culture vessel with fresh warm medium.
3. Ensure all materials are ready so cells are not left sitting in freezing medium longer than necessary.

Preparation is important—once the cells are thawed, there is little time to pause.

Thawing and Diluting the Cells

Label a conical tube. Add warm media to the conical tube. The size and volume will depend on the cell type, but 20 mL of media in a 50 mL conical tube usually works well.

1. Remove the cryovial of cells from the -150 degrees C freezer. If delaying thawing, place the cells on dry ice. Otherwise, immediately hold the cells in a 37 °C water bath.
2. Gently swirl the vial in the warm water bath until thawed, stopping when only a small ice crystal remains. This usually takes approximately 40-60 seconds.
   • Thawing should be rapid.
   • Do not allow the vial to sit while fully thawed.
3. Disinfect the outside of the vial and transfer it to the biosafety cabinet.
4. In a labeled conical tube, add warm complete medium. The size and volume will depend on cell type. We recommend at least 20 mL in a 50 mL conical tube.
5. Slowly transfer the thawed cell suspension (in the cryo vial) to the warm medium (in the conical tube).
   • Set pipettor to low to medium-low speed.
   • Aspirate 2 mL of warm media into a 5 mL serological pipet.
   • Aspirate the thawed cell suspension.
   • Add the thawed suspension to the conical of warm media.
   • This step quickly dilutes the DMSO and reduces its toxic effects on the cells.

Removing Freezing Medium (DMSO)

In most cases—especially for primary cells or sensitive cell lines—the freezing medium should be removed after dilution.

6. Centrifuge the diluted cell suspension according to your lab’s standard practice for that cell type.
7. Carefully aspirate the supernatant without disturbing the cell pellet.
   • This step removes most of the DMSO.
8. Gently resuspend the pellet in fresh, warm complete growth medium.

Some robust immortalized cell lines may be plated directly after dilution without a centrifugation step, but this should only be done if consistent with established lab protocols.

Plating the Cells

9. Transfer the resuspended cells to the prepared culture vessel.
10. Place the vessel in a properly set cell culture incubator.
11. Gently move the vessel to evenly distribute cells, then allow them to attach undisturbed.

After Thawing

• Cells are often stressed immediately after thawing; reduced attachment or viability during the first 24 hours is common.
• For adherent cells, many labs perform a medium change after 12–24 hours to remove residual DMSO and dead cells.
• Monitor cells closely during the first few days of recovery.

Key Takeaways for New Lab Members

• Work quickly once cells are thawed—DMSO is toxic at warm temperatures.
• Always use the correct pre-warmed medium for the specific cell type.
• Follow lab-specific practices for removing freezing medium and plating cells.

Reflective Questions:

1. Why is it important to use warm media when thawing cells (instead of room temperature or cold media)?
   
2. You need to thaw 1 million cells to start a culture. After counting your cells, you realize you have 5 million cells. What should you do with the excess 4 million cells?
   
3. You finished thawing the cells and placing them in the appropriate flask. What information should you write on your flask?
   
   
4. You finished using the cryovial that contained your cells. What should you do with the sticker on the cryovial that has all of your cells’ information on it?
   
   
5. Why is it important to move quickly and deliberately when thawing cells?
   
   
6. Why is it better to use a warm water bath instead of a warm bead bath for thawing cells?
   
   
7. If DMSO is toxic to cells, why do scientists add it to freeze media?