THE HISTORY OF CRYOGENIC STORAGE

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1 Labtec Services AG 5612 Villmergen T F info@labtec-services.ch THE HISTORY OF CRYOGENIC STORAGE Labtec Services AG 1 von 6

2 DEVELOPMENTS IN CRYOGENIC STORAGE Roel van den Bosch, CBS, Translation Labtec Services AG History Cryogenics developed in the 19 th century as a result of efforts by scientists to liquify permanent gases. Of the known permanent gases, oxygen and nitrogen being the primary constituents of air, received the most attention. In the liquification of gases a Scottish chemist James Dewar ( ) played am important role. He was also the one who invented in the year 1892 the first insulating bottle for the storage and transport of liquid gases. This is the type of vacuum vessel which is today still widely in use and some of them are still named Dewars, after the name of the inventor. James Dewar Cryogenics Cryogenics is the science of producing and studying low-temperature conditions. The word Cryo comes from the Greec word cryos, meaning cold. In Cryogenics as a scientific discipline, the term cryogenic applies to temperatures from approximately -100C, down to absolute zero (-273C). Cryopreservation Is a process where f.i. cells or whole tissues are preserved by cooling down to cryogenic temperatures such as -196C (the boiling point of Liquid Nitrogen) At these temperatures, any biological activity is effectively stopped. However, when the material being preserved, is not sufficiently protected against extracellular ice formation, dehydration and intracellular ice formation, it is often damaged during the freezing proces and/or during the thawing proces, which will have a negative effect on the viability. Cryoprotectants, Rate freezing, vitrification and controlled thawing can be means and mechanism to reduce the freezing damage. Liquid Nitrogen (LN2) Liquid Nitrogen is a relatively inexpensive cryogenic liquid and is for this reason widely used for different cryogenic purposes. Nitrogen gas (N2) makes up the major portion of the amosphere (78.03%) At atmospheric pressure Nitrogen gas (N2) condenses (liquifies) at a temperature of - 196C and freezes at -210C. Liquid Nitrogen (LN2) is inert, colourless, non corrosive, non flammable and extremely cold. Very small amounts of liquid vaporise in large amounts of gas. One liter of LN2 becomes 700 liter of N2 (gas) Liquid Storage Since James Dewar invented the vacuum Dewar in 1892, this type of storage container has been and is still widely used for longterm storage in Liquid Nitrogen. Cryopreservation in Liquid nitrogen at a temperature of -196C has for decades been the preferred method of cryogenic storage. Notwithstanding the fact that submerging and retrieving material from Liquid Nitrogen is a risky and unpleasant activity, it was for many years the only available method to store material at a temperature well below the recristallization point (Tg) of water. This Tg is laying around -135C. Labtec Services AG 2 von 6

3 frequently overlooked or underestimated is the possible leakage of Liquid Nitrogen into the vials, when submerged. Under proper conditions modern vials may be perfectly capable of immersion without leakage, but the manufacturer of the vials has no control over the handling of the cap-vial connection. This is also the reason that vial manufacturers now demand that their vials should not be submerged into Liquid Nitrogen. At that time it was already known that cells will remain viable and unchanged during longterm storage at the LN2 temperature of - 196C, a safe 60 degrees away from the recristallization point, above which, ice crystals could bring unrepairable damage to the stored material. In addition, storing at a constant -196C was giving the best possible temperature gradient whereby damage caused by temperature fluctuations could also be eliminated. Cross-contamination and contagious diseases Like all liquids, is also Liquid Nitrogen a possible transporter of contaminants. During long term cryopreservation ice sedements accumulate in storage dewars and this ice can be a risk of microbial contamination to stored samples. Ice accumulates in LN2 through ice forming in the atmosphere above an open dewar and falls in the Liquid Nitrogen in the vessel or ice forming on cold surfaces of the dewar or the inventory system that enters the LN2 in the vessel. These ice crystals aggregate and entrap other materials such as bacteria, fungal spores and general debris present within the Liquid Nitrogen. But besides the above, there is also the risk that Liquid Nitrogen can be a transmitter of biological contamination between samples. There has been a clear demonstration of the transmission of Hepatitis B between frozen bone marrow samples in a liquid nitrogen storage tank, which resulted in clinical infection on transplantation into patients (Teddar et al., 1995; Fountain et al., 1997). It is clear that adequate aseptic techniques and other lab protocols are the basis of safeguarding the samples in storage, but what is So, even with the most modern vials, there is the possibility of leakage. Besides the fact that cryogenic temperatures are certainly hard on the plastic vials and its cap material, the biggest threat is coming from the differential pressure that is developed when the vials are cooled 200 degrees celcius. The state of an amount of gas is determined by its pressure, volume and temperature, according to the equation: pv=nrt. When the temperature is reduced by submerging into Liquid Nitrogen, the pressure inside the vial will reduce as well, creating a difference in pressure forcing Liquid Nitrogen into the vial if the cap is not properly screwed on, has a deformed gasket or is screwed on too tight. Besides the cross-contamination risk, the ingress of Liquid Nitrogen into the vial will possibly cause an explosion when the vial is taken into the lab environment, due to the rapid boil off of the trapped nitrogen (remember when LN2 turns into N2 it expands 700 times). Hilfsmittel für die Kryolagerung When in the 1980 s HIV/AIDS was first recognized, the possible cross-contamination risk when storing samples in Liquid Nitrogen, became an increasing concern. Later these concerns have been demonstrated (Bie- Labtec Services AG 3 von 6

4 lanski, et al., 2000) that when Liquid Nitrogen is contaminated with a virus, embryos stored in unsealed containers in this nitrogen can become contaminated by these viruses. We can conclude that HIV/AIDS and other contagious diseases have changed the traditional storage in Liquid Nitrogen to storage in the Vapour of Nitrogen. Vapour Phase Storage Storing samples in the vapour of Liquid Nitrogen is eliminating the possible risk of cross-contamination through Liquid Nitrogen. This way of storage is mostly performed in the same refrigerators (dewar type or autofill type) that are used for ordinary liquid storage. In the case of vapour storage the level of LN2 in the vessel is reduced to 1/3 and the racks with the samples are either separated from the liquid by the use of a platform, or the lowest positions of the racks (the positions that stand in the liquid) are not used for sample storage. When storing in vapour there is no sample exposure to LN2, but besides this big advantage, there are also downsides such as 1)Loss of storage capacity, as the part where the LN2 is, cannot be used, 2)The vapour temperature depends too much on the level of LN2, 3)Large vertical temperature gradients possible. Unless biological material is stored at temperatures below the glass transition of aqueous solutions (approx to -140C; Mazur, 1970), the viability decreases during longterm storage. This may be appropriate for material wherby large numbers are frozen and some loss of viability may not cause practical problems, but for other material this could cause practical problems. The usual goal is to maintain samples at a temperature well below this glass transition point, as the bigger the buffer between Tg and actual storage temperature, the better. Unfortunately are many conventional storage systems showing large vertical temperature gradients, whereby it is not unlikeky that samples will be exposed to temperatures above the glass transition point. In addition are some materials even sensitive to temperature changes in the range between -130C and -190C. For instance Hybridoma s have the reputation to be very sensitive to any temperature changes. A stabile storage temperature, with the largest possible buffer to the glass transition point, with the best possible gradient is the ideal solution. Conventional vapour phase freezers cannot meet these ideal requirements. Some products are retrofitted with heat shunts to increase the evaporation of LN2, in order to decrease the vapour temperature, but besides the fact that such units increase the LN2 consumption and may increase the ice formation around the lid, they still do not reach the stability and temperature submerging in LN2 was giving. Latest Developments = -190C Dry Storage In fact are all previous ways of cryogenic storage based upon the invention of James Dewar, meaning that this type of storage is in use for well over 100 years. In the meantime there have been many developments when it comes to f.i. culturing cells. Think of all the new media and new techniques, but at the end most material still ends-up in a dewar with LN2. Lots of things have been done to increase the viability of the material stored, but very little has changed when it comes to cryogenic storage. It was in the year 2000 that an American manufacturer( Custom Biogenic Systems) of Cryogenic Storage Freezers launched the first -190C Dry Storage system, in fact the first major innovation in cryogenic storage since James Dewar. Dampfphasenlagerung in Standard Behälter Labtec Services AG 4 von 6

5 through LN2 and unsafe manipulation by the operator) European Directives The risks of cross-contamination, as well as the risks of viability reduction by large vertical temperature gradients during cryogenic storage, are a serious concern of all European governments. Especially concerning human tissues and cells, the European Community has issued Directive 2004/23/EC, which is a Directive on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. Terms of complying were in April 2006 This new storage system is using the same vacuum vessel as conventional freezers, but instead of having the pool of Liquid Nitrogen in the tank, the LN2 is in the walls of the storage freezer. Besides the fact that they were reducing the risk of cross-contamination through LN2 in this way (there is no LN2 in the tank), they were also able to create an air circulation in the freezer that gives in combination with the convection from the bottom and the walls a very stabile temperature of about -190C from top to bottom. This in combination with the fact that actually the entire interior of the storage freezer can be used for storage, makes this the preferred central cryogenic storage method for the future. There are of course alo mechanical freezers that are able to reach cryogenic temperatures. Companies like Thermo and Sanyo are bringing this type of freezers on the market and as also in these freezers there is no liquid contact possible, these freezers also belong to the dry storage category. The only difference with these mechanical dry storage systems and the non-mechanical ones, is the fact that the first have a lowest storage temperature of -152C compared to -190C of the others. Article 20.3 of this Directive addresses the fact that establishments shall include in their standard operating procedures special provisions in order to prevent the contamination of other tissues or cells. If your human tissues and cells are still submerged in LN2 it maybe time to reconsider this. Article 21.3 of this Directive addresses the fact that establishments shall establish and apply procedures that prevent any situation arising that might adversely affect the functioning or integrity of tissues and cells. If you are still storing your tissues and cells in a vapour temperature that is too warm or has a too large gradient, it maybe time to do a risk analyses. No doubt that the -190C dry storage systems have a great future. They give the advantages of LN2 (stabile temperature about 60C away from the glass transition point) and not the disadvantages (crosscontamination Labtec Services AG 5 von 6

6 Conclusions In 100 years cryogenic storage was synonimus with storage in Liquid Nitrogen. The increasing number of contageous diseases has lead to a shift from storing in liquid to storage in vapour. Whilst developments in techniques, media and cryoprotectants have contributed to improved viability, the shift to storage in vapour with large vertical temperature difference caused on its turn a reduction in viability. As a result of the above, as well as risk analysis resulting from the new European Directives, there is now a shift towards the new - 190C dry storage systems, cryogenic freezers that have been specifically designed to operate without Liquid Nitrogen in the storage area. Summary Developments in Cryogenic Storage are moving from Liquid Storage to Vapour Storage to -190C Dry Storage Labtec Services AG Nordstrasse Villmergen T F Labtec Services AG 6 von 6