Must samples be cooled at a controlled rate to below -132°C - the "glass transition temperature of water"?
The glass transition of pure water is -132°C. What this means is that when water is cooled ultrarapidly (> 1 x 1012 °C sec-1) to below -132°C it will vitrify, ie a glass will form rather than crystalline ice. It also means that if vitreous water is warmed above -132°C it will de-vitrify, i.e. turn from a glass to a crystalline solid. In much product literature it is stated that during controlled rate cooling samples should be cooled in a controlled rate to a temperature below -132°C and that long-term storage should be at temperature below -132°C. This is a confusion and would only apply if cells were suspended in water and vitrified.
In practice, cells are processed in complex mixture of cryoprotectant, salts and in many cases proteins, which during conventional freezing become freeze concentrated. This freeze concentrated material will have a glass transition temperature, which may be determined by differential scanning calorimetry etc. For example the freeze concentrated solution formed during the freezing of glycerol and isotonic salts will have a glass transition temperature of approx -64°C. During freezing cells are suspended in such solutions and the intracellular environment is at a similar osmotic concentration. Electron microscopy shows that cells are located in the freeze concentrated material and generally there is no physical contact of cells with ice crystals in the extracellular fluid..
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