**2. Types of technique**

512 Current Frontiers in Cryobiology

increased vulnerability to inadequate amounts of liquid nitrogen between refills. Modern high-efficiency liquid nitrogen cooled vessels now allow storage in the vapour phase without these problems. These vessels are vacuum insulated and the surface area that is not insulated by the vacuum is minimised ensuring that the evaporation rate of liquid nitrogen is kept low. Restricting the amount of energy entering the vessel ensures that the temperature in the vapour phase is maintained close to the liquid nitrogen temperature. The upper region of the refrigerator, close to its access point where the temperature would otherwise be higher, can be efficiently cooled if it is ensured that the heat exchange surface extends right from the bottom to the top of the refrigerator: gas phase temperatures of

The weak point in the process of maintaining safe low temperatures for samples focuses on the time in transport to and from its storage. Small samples of low thermal mass, such as vitrified straws, can warm at the rate of thousands of degrees Celsius per minute and regulatory inspections requiring the removal of samples for identification can be another

Under most regulatory environments, a rigorous sample tracking system is a key and mandatory component of compliance. It is vital that the individual location of any sample is recorded accurately, and that the sample is labelled with a unique identifier such that the identity of a sample at any location can be verified. For many research and therapy provision operations it is also necessary to have all processing, analytical and, if relevant,

Labelling can be a challenge as sample containers can be small and the cryogenic environment hostile; however commercially available cryogenic-proof labels and label

Sample location databases should be organized hierarchically, such that the location of any individual sample can be readily identified; for example: Room / Freezer / Shelf; or Segment / Rack / Position of Box in Rack / position of Vial in Box; or Room / Freezer /

Most regulatory environments require the label to include both machine and human readable identifiers (bar code plus text) and where a sample is stored in a secondary container (such as a blood bag in a cassette) it is vital that both the external container and the primary sample container be correctly labelled; see for example the European Directives

Concomitant with good identification procedures are good location and retrieval methods and there are a number of commercial software systems available with varying degrees of sophistication to accommodate larger or smaller numbers of stored samples. However an often overlooked part of the storage process is the logging, monitoring and associated alarms. Recording the parameters of storage is sometimes seen only as a regulatory obligation but liquid nitrogen levels or temperatures and the performance of mechanical freezers is of front-line importance. Alarms that work in practice rather than in theory are

printing systems are readily available. RFID tags are also a promising solution.

around -190 °C can be achieved.

patient data linked in a central database.

2004/23/EC and 2006/86/EC

Canister / Cane / Goblet / Straw position in Goblet.

vital additions to a comprehensive storage environment.

weak point.

**1.4 Traceability** 

There are various options to consider when choosing the methodology and equipment for the cooling process. In conventional cryopreservation, where the intention is to control the rate of formation of ice in the material, it is necessary either to vary the rate of application of a cryogen when working against a constant warm environment, or to provide insulation or energy while maintaining a constant external cold environment. If insulation is used, the cooling rate at any point is approximately proportional to the difference in temperature between the sample and the environment as modified by the insulation and the change in specific heat of the sample as cooling proceeds. Thus, during the process, the cooling rate asymptotically approaches zero as thermal equilibrium is achieved. Applying variable energy to a sample in a cold environment allows the rate of cooling to be modified during the process. The aim is to maintain a composition within the cells that varies as cooling proceeds such that its freezing point remains below the applied environmental temperature. Alternatively, if the concentration of cryoprotectant is high enough, it may be possible to cool the sample sufficiently rapidly that ice cannot form – an approach called vitrification. The required cooling rate will depend on the cryoprotectant and its concentration, the latter being dependant on the concentration that the cells will tolerate. In general, very rapid but uncontrolled cooling is used. The new technique of liquidus tracking allows slow cooling and vitrification.
