how cold?

Water is the major component of all living cells, and must be present in order for chemical reactions to occur within a cell. During cryopreservation, when water changes to ice, all cellular metabolism ceases. During this process, as the ice forms, the cells become dehydrated, leading to changes in the concentrations of salts and other metabolites that are present. This osmotic imbalance can be highly detrimental to cell recovery. However, cell survival is strongly influenced by a number of factors, most notably the cooling rate. Each cell type has a characteristic optimum-cooling rate, which reflects the highest percentage of survival. This rate can be modified by the use of a cryoprotective agent. Cell survival also depends on the rewarming rate and the storage temperature.

However, the duration of storage is not indefinite and the storage temperature will directly influence the time during which the samples can be recovered without damage. Lower storage temperatures are associated with extended viability of the preserved samples. While many samples are stored at -80°C, it should be noted that at this temperature metabolic activity has not ceased, it has only slowed down (due to small amounts of unfrozen water). By reducing sample temperatures to below the glass transition phase of water (-132°C), all metabolic activity comes to a halt. Storage below -130°C in liquid nitrogen therefore offers the most secure form of preservation.

Since it is clear that storage in liquid nitrogen containers represents the best long-term option for sample preservation, the question that then needs to be addressed is whether storage should be in the liquid or vapour phases. In liquid phase, samples are completely submerged in liquid nitrogen at -196°C. However, there are a number of risks associated with direct storage in the liquid phase that need to be highlighted. Storage of samples in glass ampoules is not advised, as during the transition from liquid nitrogen to room temperature, the rapid conversion to a gas phase may cause it to explode. While the use of plastic screw-cap cryotubes minimises this potential for explosion, during warming, the liquid may still spray from the interface between the cap and the tube. For this reason it is advisable to open cryotubes within a contained area. The alternative to direct storage in the liquid phase is to store samples above the liquid nitrogen in the vapour phase at -150°C. As this is well below the glass transition phase of -132°C (where all metabolic activity ceases), storage in the vapour phase is therefore both an excellent and safe means of storing your samples.