Maintaining biological materials used in cell therapies at temperatures lower than -135°C is much more complicated than simply freezing samples.
The goal of cryopreservation is to prepare stocks of cells for preservation and storage, which prevents the need to maintain all cell lines in culture all the time. Cryogenic storage is especially valuable when dealing with cells presenting a limited lifespan. Even though the technique keeps cells and tissues at very low temperatures, advanced cryopreservation strategies are required to keep these cells viable after thawing. Cryopreservation techniques and equipment focus on bringing samples to very low temperatures without causing additional damage from the formation of ice and other effects of storing living matter at sub-zero temperatures.
Water is the Most Abundant Molecule in Cells, So Freezing is a Big Deal
Water is the primary component of all living cells, and the availability of water is essential for the chemical processes of life to occur, therefore cellular metabolism ceases when all the water of a system converts to ice.
The glass transition temperature of water is -135°C. Molecular movement ceases below this temperature and all biological activity is suspended. Storage at this temperature allows preservation of samples for months or years without significant damage.
Bringing samples to this thoroughly frozen state is advantageous in that it prevents deterioration of a variety of cells used for research and medicinal purposes, but low-temperature freezing and storage can damage biological materials. Samples stored at temperatures of -130°C and below have passed the glass transition phase and are therefore in a “glassy state,” which means the samples take on the properties of glass. Glass transition is typically associated with sudden changes in density that can result in high mechanical stress and material fractures. The extent of damage associate with freezing depends on the amount of free water in the sample and the ability of that water to crystallize as temperatures drop.
The formation of ice and osmotic imbalance
Ice forms in cells at different rates during the cryopreservation cooling process. Slow cooling leads to extracellular freezing before ice begins to form inside the cells. As ice forms outside the cell, it removes water from the extracellular environment to form ice; the removal of water increases salt concentrations. This osmotic imbalance crosses the cell membrane, causing water to leave the cell. This results in an increase of extracellular solute concentration, and even increase intracellular solute concentrate as water migrates out of the cell, both of which can be detrimental to cell survival. (My colleague Alex Esmon had discussed some of this in his recent blog - "The Physics of Ice: It All Begins with Nucleation")
Cooling rate has a significant effect on solute concentration effects. Rapid cooling causes uniform ice formation that minimizes the effects but often causes the formation of more intracellular ice because its quick cooling does not allow water to migrate out of the cell. In contrast, slow cooling causes greater intracellular water loss and less intracellular ice formation but often results in increased solution effects.
Cell permeability is also a factor, in that cells that are more permeable tolerate rapid cooling better than less permeable cells. Research suggests that solution effects and ice crystal formation each play a role in cell damage, and that use of an optimum cooling rate can minimize the negative effects of each. The preferred cooling rate is 1°C per minute for most cryopreservation applications, according to the Thermo Scientific Nalgene and Nunc Cryopreservation Guide published in ATCC.
If too much water remains inside the cell, damage due to ice crystal formation and recrystallization during warming can occur and is usually lethal.
Optimal Temperatures and Optimal Cooling Rates
Cell components, including DNA and RNA, may be stored at -20°C and bacteria at -80°C. Preservation of whole tissue, including sperm, stem cells, and bone marrow, usually requires temperatures of -130°C and colder. Determining the optimal storage temperature of each type of whole tissue sample can often be complicated.
Optimal cooling rates can vary widely between sample rates. Embryonic stem (ES) cells seem to do best at a cooling rate of 0.5°C/min while red blood cells should be preserved in the range of 200-1,000°C/min. Suboptimal cooling rates may negatively affect cell viability in thawed samples.
Samples begin to degrade at or above the solution’s glass transition temperature. Heart valve leaflets can retain protein synthesis capabilities for up to two years when stored below -130°C, for example, but begin to lose these capabilities when stored at temperatures warmer than -100°C.
Decades of intense research continues to reveal the complex phenomena involved in the freezing process, including the intricate processes that occur during the freezing of living cells and how to overcome adverse phenomena associated with cryopreservation, especially at very low temperatures.
For cryopreservation, glass transition point and optimal cooling rate are keys but there are several other areas to consider, such as epigenetic changes, tubes and equipment, protocols and approaches, are good ones to start with.
To learn more about an innovative way to cryopreserve your cells during transport, download our white paper Controlled-Rate Freezing of Cells During Ultra Cold Transit.