Cryogenic preservation is a technique that allows cells to be stored at very low temperatures, usually in liquid nitrogen, for long periods of time without losing their viability or functionality. Cryogenic preservation is useful for preserving valuable cell lines, avoiding genetic drift or contamination, and reducing the cost and labor of maintaining cells in culture. However, cryogenic preservation also poses some challenges, such as the risk of cell damage due to ice crystal formation, osmotic stress, and chemical toxicity. Therefore, it is important to follow some best practices to ensure successful revival and recovery of cryogenically preserved cell lines.
In this blog post, I will discuss some of the main considerations and tips for reviving cryogenically preserved cell lines.
Consideration 1: Choosing the appropriate thawing method
The first consideration is to choose the appropriate thawing method that ensures rapid and gentle warming of the frozen cells. The optimal thawing method depends on the type and volume of the cryovials, but generally involves placing them in a 37°C water bath until they are about 80% thawed. This should take no longer than 1 minute.
The thawing method has a significant impact on the survival and performance of the cells after cryogenic preservation. Thawing too slowly or too quickly can cause cell damage due to ice recrystallization and osmotic shock. Therefore, it is essential to follow some best practices for thawing cryogenically preserved cell lines, such as:
- Use a water bath that is set at 37°C and has a lid to prevent contamination.
- Use sterile gloves and a face shield to handle the cryovials and avoid direct contact with liquid nitrogen.
- Wipe the top of the cryovial with 70% ethanol before opening it inside a laminar flow hood.
- Transfer the contents of the cryovial to a centrifuge tube containing pre-warmed culture medium as soon as possible.
- Avoid exposing the cells to room temperature or air for prolonged periods.
Consideration 2: Removing the freezing medium
The second consideration is to remove the freezing medium that contains a cryoprotectant agent, such as dimethyl sulfoxide (DMSO) or glycerol, which can be toxic to cells at high concentrations. The freezing medium also contains salts and other components that can cause osmotic imbalance and pH changes in the cells.
The removal of the freezing medium can be achieved by diluting and washing the cells with fresh culture medium. This will also provide the cells with the nutrients and growth factors they need to survive and proliferate.
The removal of the freezing medium has a significant impact on the viability and functionality of the cells after cryogenic preservation. Leaving the freezing medium in contact with the cells for too long or removing it too abruptly can cause cell damage due to chemical toxicity and osmotic stress. Therefore, it is essential to follow some best practices for removing the freezing medium from cryogenically preserved cell lines, such as:
- Use culture medium that is pre-warmed to 37°C and has the appropriate pH and osmolarity for your cell type.
- Dilute the cells in culture medium at least 10 times to reduce the concentration of the cryoprotectant agent.
- Centrifuge the cells at low speed (~200 x g) for 5 minutes to pellet them and remove the supernatant.
- Resuspend the cells in fresh culture medium and wash them at least once more to remove any residual freezing medium.
Consideration 3: Seeding the cells into a culture vessel
The third consideration is to seed a desired number of cells into a culture vessel with fresh culture medium. This will allow the cells to attach, spread, and grow in optimal conditions.
The seeding of the cells into a culture vessel has a significant impact on the recovery and performance of the cells after cryogenic preservation. Seeding too few or too many cells can affect their growth rate, morphology, differentiation potential, and responsiveness to stimuli. Therefore, it is essential to follow some best practices for seeding cryogenically preserved cell lines, such as:
- Count the number of viable cells using a hemocytometer or an automated cell counter after staining them with trypan blue or another dye that excludes live cells.
- Seed according to your cell type and application, usually ranging from 1 x 103 to 1 x 106 cells per cm2 using a sterile pipette.
- Distribute the cell suspension evenly over the surface of the culture vessel by gently swirling or rocking it.
- Transfer the culture vessel to an incubator set at 37°C with 5% CO2 and humidified atmosphere.
Conclusion
Reviving cryogenically preserved cell lines is a vital skill for any life science researcher who works with cell-based assays or experiments. Cryogenically preserved cell lines are cells that have been frozen at very low temperatures to maintain their viability and functionality for long-term storage. However, reviving them requires careful preparation and execution to ensure optimal cell survival and performance. By following the considerations and tips outlined in this blog post, you can improve your chances of successful revival and recovery of your cryogenically preserved cell lines. Happy thawing! ❄️
