**2.2 Controlled freezing, protocols and seeding**

Liquid nitrogen may be applied via a pressurised supply and cryogenic valve to create a very accurate cooling profile of temperature over time. This methodology offers the most options for optimization since the cooling rate can be varied at multiple stages in the process. As freezing proceeds the concentration of solutes in the medium increases causing cell dehydration in the sample.

As described in the opening section, cooling protocols are designed to manage the intracellular solute concentration. The key point is the nucleation temperature of the suspending medium that is, the temperature at which ice starts to form. The ice is extracellular, resulting in an increase in the extracellular solute concentration and and hence an osmotic pressure difference between the intracellular and the extracellular solutions that leads to the withdrawal of water from the cells. It is important to recognize that under normal circumstances, solutions do not freeze at their freezing point; they freeze at their nucleation temperature, which is variable and depends on the availability of nucleation centres in the sample. The nucleation temperature is normally several degrees below the nominal freezing point.

Once the extracellular fluid begins to freeze, two major events occur. First, as explained above, the concentration of CPA increases in the fraction of the extracellular fluid that has not at this point frozen, and this causes the cells to dehydrate. Secondly, the temperature of the suspension where freezing has commenced rises towards the nominal freezing temperature and remains at or close to this temperature until the freezing process is complete. This is followed by a drop in temperature as the sample catches up with the temperature of the surrounding medium, but if the cooling rate is too rapid the intracellular CPA concentration may be insufficient to prevent intracellular freezing – with severe consequences for the cells

In order to avoid this hazard, the control program may be designed to allow equilibration of the sample and its suspending medium at a temperature marginally below the calculated freezing point and at this temperature the sample forced to begin to freeze by applying either a physical nucleation point via a cold instrument placed on the external wall of the sample container, or via a sudden, short-lived introduction of cryogen into the environment. This causes the sample to commence freezing. As the sample was originally held only marginally below the nominal fusion temperature, the cell experiences a much more moderate reduction in temperature when the fusion is complete and the temperatures re-equilibrate. After this, the cooling processes is started and continues with a temperature program that is designed to effect the necessary concentration changes to maintain the intracellular composition in the liquid region of the phase diagram. This process is called "seeding".
