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Cryoinjury is the freezing injury that cells, tissues or other organelles suffer during the cooling processing of cryopreservation, which will lead to unsuccessful recovery of the sample. The actual mechanism of cryoinjury is unclear, but there are different theories that try to explain the cause, which we are going to introduce below.

Theory #1: The Mechanical Effects

Ice crystals form during the cooling process can piece or tease apart the cells, destroying them by direct mechanical action. In addition to the form of ice crystals, change in liquid composition can also exert mechanical force on the cell and cause cryoinjury [1].

Theory #2: The Solution Effects

Some scientists believe that, the ice-forming process is accompanied by change of solute concentration inside the cell, and therefore, the change of salt concentration is the cause of cryoinjury [2]. As it is widely understood in physics, as solution freezes, the remaining liquid will have increasing concentration and therefore has a lower freezing point. Therefore, cells will remain in this supercool condition for longer time and get damaged by the ultra-salty environment.

Theory #3: A Combination of the Two Above

Some scientists believe that both of the causes above matter, and the more “deciding” effect might depend on cell type, cooling rate, warming rate, as well as the cryoprotectants. For examples, just as we mention in Theory #2 that, cells can be damaged by the increasingly salty solution, some argue that if the freezing process is slow enough to allow cells to lose water, the solution-effects can be minimized, because no intracellular ice can be formed. This can be done by slow-cooling and using the right type of cryoprotectant.

In almost a century, although cryopreservation became an inseparable part of life lab practice, there is no conclusion on what causes cryoinjury, or how to completely eliminate it. One fact that adds to the complexity is that, even with the same protocol and reagent, different types of cells can have very different viabilities. For example, according to a research done by Leibo in 1977, in an controlled environment, the optimal viability of OVA cell was achieved by cooling at around 0.2°C/min, while HeLa was around 20°C/min, and RBC was about 100°C/min [3]. However, for all these cell types, the best viability was achieved when intracellular ice was at its minimum level. Despite of this, with empirical evidence, many scientists come to the concensus that slow-cooling can be an almost universal step for most cells to achieve reasonably high viability. ATCC recommends optimal cooling rate of -1°C/min to -3°C/min, which is a protocol followed in many labs.

[1] Day, John G., and G. Stacey. Cryopreservation and Freeze-drying Protocols. Hannover: Humana Press, 2015.

[2] Mazur P(1963) Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J Gen Physiol 47:347 - 369

[3] Leibo SP(1977) Fundamental cryobiology of mouse ova and embryos. In: The freezing of mammalian embryos. Ciba Foundation symposium 52(new series). Elsevier, Amsterdam, The Netherlands, pp 69-92.