Best Practices to Reduce Cell Loss in Cryopreservation

Cryopreservation is an essential process in cell culture laboratories, allowing for the long-term preservation of cells for future use. The success of cryopreservation is dependent on many factors; with cell selection, the choice of freezing medium, the cooling rate, and storage conditions all requiring careful consideration to ensure cell survival. And for sensitive cell types, this becomes even more important. This article gives some guidance and best practices to help reduce cell loss in cryopreservation.

Cell selection and harvesting

Choosing which cells, and when to freeze, is your first important choice in cryopreservation. Put simply, the healthier your cells are, and the more optimal concentration, the more likely they are to survive. “Your cells should have greater than 80% confluency before freezing,” explains Afrida Rahman-Enyart, Product Manager and Scientific Liaison at Proteintech, and optimal survival occurs when they near the end of the logarithmic growth phase. Make sure to carefully examine cells for signs of microbial contamination before harvesting and remove any dissociating agents, which can cause cell damage.

The cell concentration in your freezing vial is also key. While low concentration can reduce cell viability upon thawing, high concentrations can result in cell clumping. “The optimal concentration may vary depending on your cell type. You should try freezing the cells at multiple concentrations to determine which concentration gives the desired viability, recovery, and functionality upon thawing,” suggests Rahman-Enyart.

Using the correct medium

Once harvested, choosing the right medium for your cells is paramount to the success of cryopreservation. In addition to providing a suitable physiological environment, freezing media contains cryoprotectants like DMSO or glycerol that act to minimize cell injury from ice formation. While many commercial, ready-to-use solutions are available, Jamison Grailer, a Senior Research Scientist at Promega, emphasizes that you should always use what is best for your cells. “Exploration of different freezing medias, either commercially available or homebrew, can provide surprising results,” explains Grailer, so ensure you assess multiple formulations to choose the media that provides optimal cell survival.

Nilay Chakraborty, BioNexus Foundation Principal Scientist at ATCC, agrees that with sensitive cells the choice of medium requires even further consideration. “Some stem cells are more tolerant of cryopreservation methods than others,” explains Chakraborty so “when working with sensitive cells, start by ascertaining if the cells are highly sensitive to intracellular ice formation. Then determine the best protectant to select.” For stem cells, this often involves reducing DSMO or using animal by-product-free solutions. Finally, concludes Grailer, “the conditions for one cell type, or even subclone of the same cell line, do not always translate to future cells” so be prepared to repeat the optimization for each cryopreservation cycle.

Cooling rate

Cooling rates are critical to the survival of your cells. As Rahman-Enyart succinctly says, “The go-to rule is ‘freeze slowly and thaw quickly’.” Slow and uniform cooling maintains cell integrity and maximizes viability, freezing too rapidly leads to the formation of ice crystals that damage cell membranes. “The usual recommendation is to cool cells at a rate of about 1°C to 3°C per minute, until they reach -80°C,” advises Rahman-Enyart. Programmable cooling units are available, but many labs opt for the use of ultracold freezers as an inexpensive solution.

But as Grailer reminds us, the key is remembering to do what is best for your cells; “The freezing rate for your cultures should be optimized to ensure consistent freeze profiles and post-thaw performance.” It is recommended to test different cooling rates to determine the ones that provides optimal cell recovery. And if your cells are sensitive, this is all the more important."

Correct storage

Once the cells are frozen, you need to move fast to carefully transfer your stock to long-term storage. Any warming can cause cell damage, so transport vials on dry ice. When choosing storage, think the colder the better. As Grailer explains, “Higher storage temperatures will impact post-thaw performance, even of hardy cell lines so you should always store cells in liquid nitrogen vapor phase or in a -140°C freezer.”

Thawing and recovery

You have carefully selected your cells, chosen the right medium, and optimized the cooling rate and freezing conditions, now is the time to thaw your cells. But while you cooled your cells slowly, thawing cells should be done rapidly, explains Rahman-Enyart, to reduce the formation of intracellular ice crystals as cells rehydrate. For most cultures, placing vials in a 37°C water bath for 60 to 90 seconds is optimal.

Once thawed, you need to let your cells recover, and this means quickly removing the cryoprotective agents to prevent damage. And as with all steps in cryopreservation, how, and how quickly, this needs to be done depends on your cells. “It is imperative to tailor the cryopreservation media and process around post-thaw success. Think of it as a two-pronged approach: first, the preservation needs for the type of preserved cell are identified. Second, the cryoprotectants and cryopreservation process are optimized to ensure superior outcome," Chakraborty adds.

Conclusion

When completed successfully, cryopreservation can create lasting cell stocks for future research. But ensuring cell survival relies on a carefully crafted process; with cells needing to be frozen at the right time, in the right medium, and at the right rate. Even then, cell survival, especially for sensitive cells is not guaranteed. And with demand for cell research increasing, the need for less toxic cryoprotectants remains.

Key Takeaways

1. Cell selection and harvesting

• Choose healthy cells with over 80% confluency for freezing
• Freeze cells during the logarithmic growth phase for optimal survival
• Check for signs of microbial contamination and remove dissociating agents that can damage cells

2. Cell concentration

• Find the optimal concentration for freezing, as low concentrations reduce viability and high concentrations cause cell clumping
• Test multiple concentrations to determine the best viability, recovery, and functionality upon thawing

3. Choice of freezing medium

• Select a freezing medium that provides a suitable physiological environment
• Use cryoprotectants like DMSO or glycerol to minimize cell injury from ice formation
• Explore different freezing media to find the one that yields optimal cell survival

4. Cooling rate

• Freeze cells slowly and uniformly to maintain cell integrity and maximize viability
• Avoid rapid freezing, which can lead to the formation of damaging ice crystals
• Cool cells at a rate of about 1°C to 3°C per minute until they reach -80°C

5. Correct storage

• Transfer frozen cells to long-term storage quickly to avoid cell damage
• Transport vials on dry ice to prevent warming
• Store cells at the coldest possible temperature, such as in liquid nitrogen vapor phase or a -140°C freezer

6. Thawing and recovery

• Thaw cells rapidly to reduce the formation of intracellular ice crystals
• Place vials in a 37°C water bath for 60 to 90 seconds for optimal thawing
• Remove cryoprotective agents quickly to prevent damage, considering the specific needs of your cells