The Greener Option

The cooling and subsequent storage of frozen cells is a vital step in providing a continuous source of tissue and genetically stable living cells for procedures such as bone marrow transplant, IVF, and organ donation. Cryopreservation works by inhibiting all biochemical activity, including apoptotic chemical reactions, to prevent any damaging cell deterioration. Various cell types can therefore be effectively preserved, although cell damage during the freezing process must also be minimized in order to maintain viability upon thawing.

As a major component of all living cells, water must be present for most chemical reactions to occur effectively. Consequently, the extent of freezing damage is extremely dependent upon the amount of free water within the living cells. During freezing, cellular metabolism is halted and, as ice forms in the extracellular environment, an osmotic imbalance is created, causing water to move out of the cell. As a result, cellular dehydration and shrinkage can occur. There are a number of ways in which these detrimental effects can be minimized, such as effectively using cryoprotective agents, using an appropriate controlled-rate freezer to control the cooling rate, maintaining an appropriate storage temperature, and controlling the thawing rate.

Maintaining cell viability

Cryoprotective agents, such as glycerol or dimethyl sulphoxide (DMSO), can be used to prevent the formation of ice both within the cell and in the extracellular environment. These agents protect slowly frozen cells via a range of different mechanisms. These include suppressing high salt concentrations to reduce cell shrinkage at a given temperature, reducing the fraction of the solution frozen, and minimizing intracellular ice formation. There are two types of cryoprotectants available, one that acts intracellularly and one that acts extracellularly. Intracellular cryoprotectants permeate the cell membrane to effectively minimize cell damage, whereas extracellular cryoprotectants work by inducing vitrification (the solidification of water due to increased viscosity rather than crystallization), which does not have the same biologically damaging effects as freezing.

Thermo Scientific CryoMed controlled-rate freezers can be tailored to specific protocols, including those for stem cells and cord blood.

Controlled-rate freezing before long-term sample storage is a vital step in the cryopreservation procedure, since the rate at which cells are cooled has an extremely significant influence on cell survival. A uniform cooling rate, ideally of 1°C per minute, needs to be implemented in order to minimize the associated risks of thermal shock. Once nucleation (the initial formation of ice crystals) has occurred, the state of the sample shifts from a liquid to a crystalline phase. However, as freezing is an exothermic reaction, heat (the latent heat of fusion) emitted during ice formation must be released away from the materials being frozen. Controlled-rate freezers can provide a temperature compensation for the latent heat of fusion, enabling the user to initiate nucleation at a given temperature for uniform freezing. Ice formation in slowly cooled systems is usually initiated in the extracellular solution, causing the solute concentration outside of the cell to increase. This results in cell shrinkage due to osmosis. Optimal slow cooling conditions are defined by the cooling rate that permits some cell shrinkage without significant amounts of intracellular ice being formed, resulting in maintained cell viability.

Providing an optimized environment

Systems for long-term storage of frozen cells provide a stable, low-temperature environment to optimize the life span of the samples. Typically, lower storage temperatures enable a longer viable storage period. Most cells need to be maintained at temperatures of –130°C or below in order to completely halt the chemical reactions responsible for cellular degradation. Initiation of this process (formation of cellular fractures) depends on the interaction of several factors, including the mechanical properties of the samples, solute concentration, temperature gradients, overall temperature, and the rate of temperature change. As such, providing an optimized environment to prevent apoptotic processes and subsequent cell damage from occurring is extremely important.

Cryopreservation is an extremely complex procedure with many variables that need to remain under strict regulation. The lengthy process of slow-rate freezing and the subsequent long-term storage of these valuable cells can often be costly, consuming large amounts of energy to accurately maintain such low temperatures. Therefore, implementing the use of equipment that consumes minimal energy without emitting any waste products, such as noise or heat, to the surrounding environment provides the user with cost-effective and environmentally friendly cryopreservation.

The types of equipment available

Precious frozen cells can be stored in one of two types of equipment: a mechanical freezer or a liquid nitrogen dewar. Traditional laboratory freezers are unable to attain temperatures low enough for cryopreservation, so the auto-cascade freezer was developed. The auto-cascade freezer is a unique refrigeration system that employs an orbital compressor and multiple mixed refrigerants to effectively remove heat from the chamber. These mechanical freezers for cryopreservation are powered, like conventional laboratory freezers, from the supplied electricity to maintain a uniform temperature throughout the chamber. However, they often have a large footprint and will therefore occupy a significant amount of space within the laboratory. Furthermore, auto- cascade freezers are in constant use over long periods of time, so associated electricity costs can easily mount up. These high costs are often not accounted for in initial budgets, and the constant use of auto-cascade freezers, which also create waste heat and noise pollution, is not environmentally friendly. In contrast, liquid nitrogen can maintain equally low temperatures independent of electricity. At atmospheric pressures, liquid nitrogen is a naturally cryogenic fluid that can cause rapid freezing of living tissue. Therefore, it can maintain low temperatures based on the naturally occurring properties of a liquefied atmospheric gas.

‘Going green’ with liquid nitrogen

The use of liquid nitrogen is an effective long-term method for storing viable samples while maximizing energy efficiency and providing an environmentally friendly approach to cryopreservation. This innovative freezing method ensures that cells remain viable, and indefinite storage is possible.

As “green” methods of cryopreservation, both liquid and vapor phase liquid nitrogen dewars do not consume excessive amounts of electricity. Unlike mechanical freezers, which need a constant supply, these storage dewars use only 0.8 amps of energy every two to three days to support features such as auto-filling, monitoring and alarms. With the ability to consume so little energy, liquid nitrogen dewars employ a highly cost-effective and economical method of low-temperature storage, minimizing the unnecessary use of electricity. These systems can therefore operate for long periods of time with virtually no deleterious environmental effects, such as the production of ozone. A combination of small size and zero heat or noise pollution makes them suitable for convenient placement under the laboratory bench; this enables researchers to easily carry out any maintenance and complete quick and effective checks on temperature or liquid nitrogen levels.

Liquid nitrogen storage can occur in either the liquid or vapor phase, each with different associated costs and benefits.

Liquid Phase

Liquid phase storage accurately maintains a uniform temperature of –196°C. The dewar is filled with liquid nitrogen, into which the inventory racks are submerged. The cells are contained in cryovials, so that they do not come into direct contact with the liquid nitrogen itself, minimizing the risk of cellular fractures while still benefiting from its extreme cooling effects. However, there is always the minute possibility that these vials may leak and allow liquid nitrogen to enter the package, bringing with it a range of potential contaminants.

Locator Plus cryogenic storage includes indexed rack and boxed systems for quick and efficient sample retrieval.

Vapor Phase

In contrast, vapor phase storage eliminates the possible contamination issues associated with liquid phase storage. This is due to the fact that the samples are not submerged in the liquid nitrogen but instead benefit from the cooling effects of the nitrogen vapors. However, using the vapor phase to maintain low temperatures can often result in a temperature gradient, which needs to be closely monitored. The temperature throughout the chamber must be kept below –130°C to ensure that all metabolic activity remains arrested. Maintaining a raised liquid level or adding a conductive liner material such as aluminum will significantly minimize this gradient. Also, filling the dewar with the maximum number of racks and using a temperature or liquid level monitoring device will reduce fluctuations within the storage chamber.

Since liquid nitrogen dewars do not require electricity to maintain low temperatures, users have the added assurance that even during situations out of their control, such as a power failure, temperatures will be maintained and no precious samples will be damaged in an unscheduled thawing.

Thawing

The thawing process, much like the freezing process, must be closely monitored to ensure that cell viability is maintained. In most cryopreservation procedures, the cooling rate is optimized for rapid rewarming, and in these circumstances slow rewarming can reduce survival rates. However, for some cells, such as mammalian embryos, slow warming is essential for survival. During freezing, cells can become heavily loaded with solutes, resulting in osmotic lysis upon thawing. Slow rewarming allows sufficient time for cell rehydration and a gradual loss of accumulated solutes, to minimize the risk of such lysis.

Conclusion

Today’s need to minimize carbon emissions and reduce ozone damage means it is important to ensure that any laboratory is as environmentally friendly as possible. Therefore, providing equipment for long-term low-temperature storage that requires minimal energy without emitting any noise or heat pollution enables researchers to concentrate on the complexities of the freezing process with the knowledge that their cryogenic experiments are extremely environmentally friendly. Liquid nitrogen dewars enable convenient in-lab placement with no noise or heat pollution. Furthermore, they maintain low temperatures for very long storage periods without consuming electricity. This cost-effective method not only saves on laboratory resource, but also is extremely friendly to the environment.