The rate at which a biospecimen cools has been proven to have a dramatic impact on its long-term viability. Not only does cooling rate affect the rate of formation and size of both intracellular and extracellular ice crystals; it also can impact solution effects that occur during the freezing process.
In very general terms, rapid cooling minimizes solute concentration effects (because ice forms uniformly), but it maximizes intracellular ice formation (because water does not have time to migrate out of the cell). Slow cooling creates the opposite result. Slow cooling maximizes water loss from the cell and minimizes intracellular ice formation, but it increases solution effects.
Here are some of the other key facts biobankers need to know about cooling rates and cryopreservation:
Beware the latent heat of fusion. Most of the water present in cells will freeze at approximately -2°C to -5°C. Since this phase change from a liquid to crystalline form is an exothermic process, it releases energy, warming the sample until the equilibrium freezing point is reached, at which point temperature ice continues to form. To minimize the detrimental effects of this phenomenon (referred to as the latent heat of fusion), undercooling must be minimized by artificially inducing the formation of ice. This can be accomplished by seeding a suspension with ice or some other nucleating agent, or by rapidly dropping the temperature of the external environment to encourage ice crystal formation.
Different cell types have different optimal cooling rates. A uniform cooling rate of 1°C per minute from ambient temperature is generally regarded as effective for a wide range of cells and organisms. But because cells differ in size and water permeability, there are exceptions to this rule. For example, most bacteria and spore-forming fungi will tolerate less-than-ideal cooling rates and can be frozen by placing the material at -80°C for a period of time. By contrast, other bacteria and non-sporulating fungi require more uniform rates of cooling, and protists, mammalian cells and plant cells often need even greater control, including fastidious manipulation to minimize the detrimental effects of undercooling and the energy liberated during the latent heat of fusion. (As a general guideline: The larger the cells, the more critical slow, controlled-rate cooling becomes.)
Multicellular biospecimens present the most challenging cooling rate requirements. For optimal cryopreservation, multicellular biospecimens (embryos, tissues) require a more complicated cryopreservation process – usually a combination of controlled-rate freezing and vitrification.
You can achieve uniform, controlled cooling rates using a programmable rate cell-freezing apparatus. Basic cell-freezing units allow selection of only one cooling rate for an entire temperature range. More sophisticated units allow a selection of variable rates for different portions of the cooling curve.
Homemade freezing systems can be problematic. Even though a homemade freezing system may produce a cooling rate that averages 1°C per minute, the cells typically experience more rapid rates of cooling during some parts of the cooling curve. Variability like this makes cooling rates from homemade freezing systems difficult to reproduce. In addition, homemade processes can introduce contaminants. For instance, many homemade systems involve immersing sample vials in alcohol baths, but wicking of the alcohol on the exterior of the vials increases the risk of sample contamination – and also makes the vials slippery and difficult to handle!
Ice crystal formation and solution effects both play a role in cell damage and the long-term viability of cryopreserved biospecimens. However, if you determine – and then deliver – an optimum cooling rate for your sample, you will minimize the impacts of both phenomena.