Published: 2023  |  Last reviewed: May 2026

Reviving Cells After Cryopreservation

Cells, tissues, and even some species can be stored for an extended period and then recovered for further study. This article looks at the various procedures used for thawing cells in cryopreservation and provides guidance for reviving and ensuring the cells' viability. However, it is important to closely follow the instructions given by your cell culture provider.

Key Takeaways

• Thawing cells is a crucial step in the cryopreservation process, which involves the gradual warming of frozen cells to room temperature.
• For thawing cancer cells and stem cells it is recommended to thaw the vials slowly because it reduces the risk of cell damage.
• During thawing, the thawing rate, temperature, and exposure to cryoprotectant are all factors that can cause cell damage.
• Reviving cells requires providing them with the nutrients and minerals they need for growth.
• After reviving cells and before using them in experiments, it is necessary to check the cells for damage and viability, as well as monitor their growth and proliferation.
• To ensure successful thawing and reviving of cells, it is essential to adhere to recommended guidelines and protocols, handle cells with care, and maintain accurate records.

Thawing protocol

1. Rapidly thaw cells in a 37°C water bath for 1 – 2 minutes until only a small ice clump is left in the cryovial.
   • From this point forward, all procedures must be performed under aseptic conditions.
2. Transfer the cryovial to a sanitized sterile flow cabinet.
3. Carefully open the vial and use a 2 mL serological pipette to determine the total number of cells by measuring the total volume of the cell solution. Then, remove 20 µL of cells for counting as an aliquot.
4. Place the cell suspension in a 15 ml centrifuge tube that contains 8 ml of culture medium (room temperature).
5. Suspend the cells with care.
6. Centrifuge for 3 minutes at 300 x g and discard the supernatant.
   • The centrifugation step may be omitted and the cells be placed in a cell culture flask immediately, but make sure to change medium 24 hours later.
7. Place cells in 10 ml of fresh cell culture medium (pre-warmed to room temperature or 37 °C in an incubator) immediately to provide growth nutrients and dilute the cryoprotectant, such as DSMO. As a cryoprotectant, DMSO protects cells from damage caused by freezing by preventing the formation of ice crystals, but it can be toxic to cells at high concentrations. Two T25 cell culture flasks should be filled with cells.
8. Place the cells in an incubator and change the medium the next day.
9. Continue incubating the revitalized cells as instructed for this particular cell line.
10. Before performing your experiments, allow the thawed cells to recover in culture medium so that the cells can recover from the thawing stress. Also, check the cells for damage and viability by microscopic observation, or by conducting a viability test, and monitor the cells’ growth.

Note: Don’t forget to look up and set the incubation temperature and CO₂ suited to your cells! Most cells are incubated at 37 °C but some insect cells for example require lower temperatures.

Why is thawing stressful to cells?

The thawing process exposes the cells to potentially harmful environmental changes as described below:

• Temperature changes: Cells can be damaged during transition from cryogenic to warmer temperatures. Thermal shock during thawing can damage membranes, enzymes, and other cells.
• Osmotic changes: Osmotic pressure fluctuations during thawing can damage the cell membrane and other structures. Toxic substances can enter cells that have been cryopreserved with DMSO or glycerol.
• Removal of cryoprotectant: Cryoprotectants are added to cells before freezing to prevent ice crystal formation, but they are toxic if not removed after thawing.
• Changes in pH: pH changes during thawing can stress cells. Temperature changes or the release of metabolic waste products can cause pH changes.
• Mechanical stress: As cells are pipetted and centrifuged during defrosting, mechanical stress can occur. This may damage cells and reduces their viability.

Thawing can stress cells and damage their viability and function. Cryoprotectants and a good thawing protocol can minimize this damage.

5 Tips for Successful Cell Thawing and Reviving

1. Use a protocol for thawing cells specific to the type of cells being thawed, such as a slow thawing technique for cancer cells and stem cells.
2. Reduce exposure to high temperatures and prolonged thawing time to minimize the risk of cell damage.
3. During thawing and revival, handle the cells with care to avoid damage.
4. Inspect cells for damage and viability and track their growth before using them in experiments.

Stick to recommended protocols and guidelines for thawing and reviving cryopreserved cells.