**3.2 Alcohol bath freezers**

These commonly used laboratory units are essentially refrigerated circulators. A reservoir of cooling medium (normally an alcohol) is passed through a cooling system and re-enters a reservoir, reducing the temperature. The degree of refrigeration applied and the flow rate through the cooling coils determine the derived temperature of the reservoir. The relatively large volume of cooling liquid creates two noticeable effects: temperatures are very stable due to the large heat capacity of the available fluid and cooling rates can be controlled very accurately for a similar reason. The corollary to this however is that the rates achievable are very low and so rapid (> 1 °C/minute) rates are very hard to achieve. In addition, alcohol bath freezers are normally limited to temperatures above -80 °C.

Precision in Cryopreservation – Equipment and Control 519

film strip to hold the sample. These open systems typically expose the sample directly to the liquid nitrogen which assists in achieving the very high cooling rates. Of course exposing the sample directly to liquid nitrogen in this manner raises questions of potential contamination from the cryogen. Other approaches, such as the Cryologic Vitrification Method, still use an open device at the stage of vitrification but cool the sample by touching on a liquid nitrogen cooled aluminium block. This means that the sample is not directly exposed to the liquid nitrogen and the block avoids the Leidenfrost effect. Alternative approaches use closed straws. These avoid the contamination issues but at the expense of the cooling rate. By definition, the vitrification stage of the process is difficult to measure, monitor or document, so validation and on-going quality control are qualitative exercises

Originally conceived in 1816 by the Reverend Stirling, the Stirling engine converts heat energy into mechanical work. The principal also works the other way round to convert mechanical energy to heat, when the Stirling engine forms a heat pump able to move heat 'uphill' from a cold place to a warmer one. This gives the Stirling engine an application as a

Most refrigerators operate on the Rankine cycle which depends on refrigerants existing with appropriate boiling points. Triple stage Rankine machines are at the limit of the technology and achieve roughly -140 °C. Although the Stirling cycle is less efficient than Rankine cycle machines, it is capable of cooling to lower temperatures and therefore comes into its own below -140 °C; miniature cryo-coolers based on Stirling engines are now quite common. Due to relative inefficiency, these Stirling based cryo-coolers can normally freeze only quite small samples of a few tens of grams maximum and cannot compete with liquid nitrogen powered machines for cooling capacity. On the other hand, they excel in clean rooms where it is not possible to obtain a supply of liquid nitrogen and it is only desired to freeze very small

Because liquidus tracking is a relatively novel technique, there is little choice of equipment to assist with research into its use. Planer plc do manufacture a Liquidus Tracking controller that can be used for research into this approach. The equipment comprises a conventional slow-rate chamber coupled with a liquidus tracking controller

The controller cools the sample in a similar manner to the conventional slow-rate freezing process. The cooling profile is typically a simple linear ramp. During the cooling of the sample, the controller monitors the current chamber temperature and adjusts the speeds of the two pumps to dynamically alter the concentration of cryoprotectant surrounding the samples. In the ideal process the concentration of cryoprotectant is maintained just above the liquidus curve. As the temperature decreases, the concentration of cryoprotectant is therefore increased. However, as the temperature of the sample decreases, the toxicity of the cryoprotectant decreases and this allows the sample to tolerate the ever increasing

only.

**3.6 Stirling engines** 

refrigeration unit.

samples.

**3.7 Liquidus tracking equipment** 

and two peristaltic pumps.

concentrations; see figure 1.
