Last Updated on June 22, 2022

What Is Cryopreservation?

Cryopreservation is the process of preserving cells or tissues under low-temperature conditions to avoid cell injury or death. It is a way to preserve cells for future use. Cryopreservation usually occurs at a temperature between -80˚C to -196˚C. Cells are traditionally frozen with liquid nitrogen, directly in a freezer, or with dry ice (carbon dioxide).

Viability is the measure of living cells that are capable of dividing and multiplying when cultured as well as maintaining cellular function. This article will discuss cell viability after cryopreservation. There have also been studies on how long cells can survive after the freeze-down process, but the conclusion remains inconclusive due to vast differences in study methods and results.

Cell Viability After Cryopreservation

The most important part of cryopreservation is cell viability after thawing. Cells can remain viable for years in a state of suspended animation under cryopreservation. When cells are cryopreserved, if they are frozen too fast, ice crystals can form, which can damage the membrane and cause the cell to die. Once they are cryopreserved, cells may be thawed and revived to function normally.

The freezing and thawing of cells cause a series of mechanical and metabolic stresses that can compromise their viability. In addition, the process of freezing and thawing causes the release of intracellular metabolites, leading to cell death or sub-optimal viability.

Moreover, the time and temperature at which a cell is frozen down (freeze velocity) can have a significant impact on its viability.

Intracellular ice crystals can be the cause of cell death or destruction when they are formed during freezing processes. Sometimes, this damage is caused by extensive cell dehydration (the “solution effect”). Other times, it’s the result of intracellular ice crystal formation (“mechanical damage”), which in turn causes a mechanical strain on cells. Or a combination of both.

This can cause a decrease in cell viability and lead to cell death. At sub-zero temperatures, ice crystals and nucleation sites form in the cytoplasmic fluid. This can cause additional mechanical stress on the cells, leading to various levels of cell damage. The extent of damage caused by freezing depends on the duration of exposure, the temperature, and the concentration of ice crystals. These effects can be minimized by freezing cells slowly at low temperatures and maintaining them at a constant level of ice crystals throughout the freezing process.

How Long Cells Survive After Cryopreservation

Cells have an amazing ability to recover following cryopreservation. Once thawed, the cells start recovering from the trauma of freezing and re-adapting to their original homeostasis.

The duration of recovery depends on the type of cryopreservation and the species of the sample. The length of time that cells spend in recovery will largely depend on the length of time that the sample was frozen. Many researchers agree that the vast majority of cells are most likely able to recover completely after about two weeks. There are a few reports of increased recovery over time and other reports of lesser recovery over time.

The length of time and the number of times that the sample is thawed can also have a significant bearing on the recovery time. In addition, thawing samples at different temperatures can also have an impact. The rationale for this is that cells that have been exposed to high temperatures may be more damaged and may have a shorter recovery time.

 

Controlled-Rate Freezer

The controlled-rate freezer, CytoSensei, is a system that can cryopreserve your cells by simply plugging it into a standard wall outlet and connecting to the designated laptop with a USB cable. The system does not require liquid nitrogen, which avoids the risk of contamination.

A custom freezing protocol can be easily programmed with the included software before samples are loaded into an aluminum freezer block to then be cryopreserved.

The powerful Stirling engine cools the cells to -80°C without the use of liquid nitrogen. It has 3 temperature sensors that can be read in an automatically generated PDF report. The built-in heater restores the freeze block back to room temperature before proceeding with the next cryopreservation protocol; this increases efficiency by shortening the waiting time to introduce another set of samples to the sample rack.

 

Cell viability after cryopreservation
Cell Freezer

There are 3 critical stages to increase viability when using cryopreservation.

  1. Before freezing down, the sample must be kept at 4°C after transferring it to the culture media with a cryoprotectant like DMSO or glycerol, in order to avoid damage to the cells.
  2. The temperature change needs to be on a declining slope. The best “Goldilocks” freeze rate is typically around 1C/min, but the optimal rate is cell-specific based on the surface area, cell permeability, and other factors.
  3. At the end of the freeze down process, it needs to be transferred to cell deep storage transporting it in dry ice first is ok).

Conclusion

Cryopreservation is the process of preserving cells or tissues under low-temperature conditions to avoid decay. It is a way to preserve cells for future use. Once they are cryopreserved, cells may be thawed and revived to function normally. The length of time that cells spend in recovery will largely depend on the length of the time that the sample was frozen. To increase viability and efficiency, the CytoSensei is an excellent tool.

Find out more about our controlled-rate freezer
and how it can help you in your research.