**3.6 Stirling engines**

518 Current Frontiers in Cryobiology

Storage of important biomaterial in liquid nitrogen at -196 °C is widely practised. This method allows for a 70 °C plus safety zone when considering the -120 °C threshold for longterm storage; the significance of -120 °C, the glass transition temperature, has been previously discussed. Liquid nitrogen storage does provide the greatest safety zone. However, it also presents a number of problems, including personal safety and potential

Storage in the vapour stage is felt to address these issues but it does come with its own set of problems. The vapour is not as cold as the liquid nitrogen itself and as such the 70 °C safety margin is diminished. However, modern vapour storage vessels use carefully designed vacuum insulation to minimise the heat leakage from the environment into the vessel. This allows the vessel to maintain a vapour temperature at around -190 °C resulting in samples still being maintained at a safe distance from the glass transition temperature. Efficient designs also result in very low liquid nitrogen usage and temperatures can be maintained for up to a month without additional filling; temperatures are even maintained with the lid

As described previously, up to present times, the controlled rate freezer offers the widest control options for a freezing protocol. With a truly variable application system for cryogen, most sample sizes can be easily accommodated and rates from the very slow (< 0.1 °C/minute) to in excess of 50 °C/minute are both achievable and controllable. Sample size, container dimension, cell volume, membrane permeability etc. are all variable factors. As the controlledrate freezer allows complex, fully controlled temperature versus time profiles to be created, protocols can be designed that are appropriate to the cell type and cryoprotectant concentration. Additional steps such as pauses for manual 'seeding' or rapid plunges to initiated freezing can be added to the profile. Transition to different rates can be triggered from the chamber temperature or representative sample; triggering the transition from the

sample temperature can help remove variability introduced by different sample loads..

**3.5 Equipment for conventional (high cooling rate) vitrification** 

From an instrumentation standpoint, the programming and record-keeping intrinsic within the system meet most external compliance standards and optional software packages are generally available to enhance this aspect beyond the current requirements of any legislative authority. The fact that it is possible to optimise processes for every unique cell type together with the compliance aspect lend great versatility to this type of instrument in most

Such protocols call for extremely rapid solidification of the sample, typically by plunging it directly, and in a somewhat uncontrolled manner, into liquid nitrogen. Intracellular ice formation is avoided by the application of very high concentrations of CPA. Equipment such as the VitMaster (IMT ltd.) can be used to increase the cooling rate. This uses negative pressure to depress the freezing point of liquid nitrogen to below -205 °C thereby increasing the cooling rate. Several open techniques have been developed to minimise the sample volume and achieve high cooling rates; for example the Cryotop method which uses a thin

**3.3 Liquid nitrogen vessels: Liquid and vapour** 

removed for short periods.

application areas.

**3.4 Controlled rate liquid nitrogen freezers** 

microbiological cross-contamination via the liquid nitrogen.

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 refrigeration unit.

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 samples.
