**2.3 Vitrification**

The process of vitrification usually uses the highest concentration of cryoprotectant that the cells in the tissue will tolerate and follows this by very rapid but uncontrolled cooling, usually by plunging the sample into liquid nitrogen. The crucial element is exposure of the cells or tissue to potentially toxic levels of CPA: too low a level or too short a time and ice will form killing the cells. If the levels are too high or the process time is too long, the chemicals employed will prove toxic to the cell and post-thaw viability will be limited. Since this process depends on rapid cooling, vitrification has only ever proven applicable for

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

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

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

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

The process of vitrification usually uses the highest concentration of cryoprotectant that the cells in the tissue will tolerate and follows this by very rapid but uncontrolled cooling, usually by plunging the sample into liquid nitrogen. The crucial element is exposure of the cells or tissue to potentially toxic levels of CPA: too low a level or too short a time and ice will form killing the cells. If the levels are too high or the process time is too long, the chemicals employed will prove toxic to the cell and post-thaw viability will be limited. Since this process depends on rapid cooling, vitrification has only ever proven applicable for

**2.2 Controlled freezing, protocols and seeding** 

normally several degrees below the nominal freezing point.

liquid region of the phase diagram. This process is called "seeding".

cell dehydration in the sample.

consequences for the cells

**2.3 Vitrification** 

samples with very small volumes, ideally those with very high surface area-to-volume ratios; for example cryogenic straws can fit this description. It is important to be aware of the Leidenfrost effect where a sheath of vapour will surround a warm sample when plunged into liquid cryogen, essentially insulating the sample for a short period of time. For many vitrification protocols however, even this short additional time period before the sample is vitrified has proven fatal to the cells due to increased toxic exposure to the CPA and decreased cooling rates. In conventional vitrification, very high cooling rates are achieved by exposing small samples directly to the liquid nitrogen. The sample is surrounded with as little physical material as possible to achieve the maximum cooling rates. With large samples, however, such high cooling rates are impracticable.

Although vitrification is normally associated with cooling rates in the tens of thousands of degrees Celsius per minute, slower techniques have been reported such as the S3 vitrification technique for blastocysts (Stachecki & Chen, 2008); this uses rates <200 °C/minute. But in fact vitrification does not necessarily require rapid cooling at all. It all depends on the dependence of the critical cooling rate required to prevent freezing on the concentration of the cryoprotectant. (Sutton, 1991). As the following section describes. vitrification can be produced at really low cooling rates.
