**1.3 Long term cell survival and contamination**

All biological materials will, without intervention, naturally deteriorate, and if they are to be preserved it is necessary to utilize a method that will preserve both morphology and functionality while preventing any alteration of the fundamental nature of the material. The most common methodology available for this is cryopreservation. Biological materials, however, have widely different properties and in order to create a truly effective cryopreservation protocol, it is necessary to consider these properties as they affect the preservation of vital characteristics both during the freezing process and the subsequent environment in which the samples are to be stored long term.

Regarding the minimum storage temperature, no temperature is too cold. Once a sample is frozen and the residual liquid phase has vitrified, further cooling simply reduces molecular energy and vibration. It is possible for short-range structural changes to occur at a molecular level, but they do not affect post-thaw biological properties. It is worth noting that because the cell micro-environment within a frozen sample is chemically different from the majority of the frozen material, biological activity may continue, albeit slowly, at temperatures several degrees below the freezing point of the material.

The minimum melting point of the multi-dimensional phase diagram for typical cryopreservation media occurs at around -80 °C but the cell contents do not finally solidify to an amorphous state until around -120 °C. It is not sufficient simply to keep the samples frozen because, at a micro-environmental level, if the material retains the ability to diffuse it may also degrade, albeit at a much reduced rate. The glass transition temperature is therefore regarded as the "critical" temperature if truly long term storage is required

osmotic balance. The concentration of intracellular material lowers the effective freezing point of intracellular material and, provided the external temperature is correctly managed, prevents the formation of intracellular ice. As such, the creation of ice crystals within the cell is avoided. At temperatures below -130 °C (close to the glass transition temperature of the medium) the residual liquid has too little energy to orientate into long range molecular matrices and will form short range semi-solid structures; i.e. an amorphous solid or glass. At this point there is no possibility for significant chemical transport; biological activity, and

The options for control of this process are the chosen CPA and its concentration, and the cooling rate. Water and solute permeability are temperature dependent and nominally the higher the concentration of extracellular CPA, the less ice will form during cooling. With a very high applied concentration of CPA, very rapid cooling without the formation of ice may be possible – a process that is known as vitrification. At the other extreme, lower CPA concentrations that allow ice to form, require more precisely managed cooling rates which can be provided by programmable controlled rate freezers. The issue here is the toxicity of the applied CPA since high concentrations, even for short periods, can lead to excessive dehydration and high cell stress, whereas lower concentrations may involve prolonged cellular exposure to essentially toxic material. DMSO, for example, is an organic solvent and has been linked to cellular mutation. The choices made for the preparation and subsequent freezing of cells is a complex balance between thermodynamic and biochemical variables, the choice and management of which can have a profound effect on the post-thaw recovery

All biological materials will, without intervention, naturally deteriorate, and if they are to be preserved it is necessary to utilize a method that will preserve both morphology and functionality while preventing any alteration of the fundamental nature of the material. The most common methodology available for this is cryopreservation. Biological materials, however, have widely different properties and in order to create a truly effective cryopreservation protocol, it is necessary to consider these properties as they affect the preservation of vital characteristics both during the freezing process and the subsequent

Regarding the minimum storage temperature, no temperature is too cold. Once a sample is frozen and the residual liquid phase has vitrified, further cooling simply reduces molecular energy and vibration. It is possible for short-range structural changes to occur at a molecular level, but they do not affect post-thaw biological properties. It is worth noting that because the cell micro-environment within a frozen sample is chemically different from the majority of the frozen material, biological activity may continue, albeit slowly, at temperatures

The minimum melting point of the multi-dimensional phase diagram for typical cryopreservation media occurs at around -80 °C but the cell contents do not finally solidify to an amorphous state until around -120 °C. It is not sufficient simply to keep the samples frozen because, at a micro-environmental level, if the material retains the ability to diffuse it may also degrade, albeit at a much reduced rate. The glass transition temperature is

therefore regarded as the "critical" temperature if truly long term storage is required

hence deterioration, effectively ceases.

of living cells and hence the value of the sample.

**1.3 Long term cell survival and contamination** 

environment in which the samples are to be stored long term.

several degrees below the freezing point of the material.

Best practice dictates that freezers should maintain sample temperatures as far as possible below this critical temperature. By storing well below the critical temperature, transitory warming events above that temperature can be avoided during sample handling, retrieval, storage and in the event of any disruption to the availability of cryogen or power. Freezing a sample in such a way as to maintain maximum biological potential is not a trivial task, and the same care applied to this process should be brought to bear when designing and building storage environments.

The key considerations when looking at a cryopreservation process were listed at the beginning of this chapter. Clearly, the process should be able to maximise the potential for use after processing and storage. Because the future use may be unclear, the preservation and storage procedures should be designed to provide the best possible opportunity for future exploitation. The storage of cells without either adequate care during the initial cryopreservation process or at too high a temperature during subsequent long-term storage are key problems that should be avoided and when the purpose of storage is to maintain biological potential, it is vital that the mechanism of freezing injury be considered.

As the liquid in which the cells are suspended begins to freeze, any solutes in the unfrozen solution become more concentrated and this results in a depression of the freezing point of the remaining solution. The result of this, when the temperature is reduced, is that the cells are exposed to a solution of progressively higher concentration. The increasing concentration increases the osmotic gradient across the cell membrane which results in water leaving the cell in order to maintain balance. Hence, controlling the cooling rate provides a mechanism for controlled dehydration of the cells. Eventually the aqueous phase is so viscous that there is insufficient energy available for the water molecules to form a crystalline solid and the solution becomes an amorphous solid or glass. The temperature at which this condition is reached is known as the "glass transition" temperature (Tg). Once the sample is below this temperature, diffusion within and without the cell stops and the sample is biologically inert. At temperatures below Tg the sample can be maintained indefinitely; other physical interactions, such as background radiation, may have an impact on extremely long-term storage but such effects are probably without any significance in practice (Glenister et. al., 1984).

Unlike freezing, the glass transition is not based upon a thermodynamically defined phase change but rather on the observed dramatic change in viscosity that occurs in cryoprotectant solutions typically at around -120 °C. It is important to ensure that samples are maintained below this temperature throughout the storage term. A temperature of -150 °C is typically stated to be the critical storage temperature for cell products since this temperature provides a reasonable safety margin to ensure that that samples remain below the glass transition temperature during transitory events such as handling, but in practice, storage in liquid nitrogen at -196 °C is a convenient and reliable way to meet this requirement; moreover the additional safety margin provides even greater sample security.

However, storage in liquid nitrogen is not without its disadvantages which include the risk of explosion during warming should liquid nitrogen have entered the vials. Microbiological cross-contamination is another hazard of storage in liquid nitrogen (Byers, 1999) and may lead to the application of a secondary enclosure ('double bagging'). Storage in the gas phase has been advocated to avoid these problems. In the past, the temperature gradient in the vapour phase of liquid nitrogen refrigerators has been a problem, and there may have been

Precision in Cryopreservation – Equipment and Control 513

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

Passive cooling uses insulation to moderate the cooling rate of samples that have been equilibrated with low concentrations of cryoprotectant and then placed inside traditional electromechanical refrigerators at -20 °C, -40 °C, -80 °C or even at lower temperatures. The cells are dessicated slowly during the cooling process. This method can be used for most robust cells but even under the best circumstances the post-thaw recovery rates may not be ideal. In addition, in most cases no instrumentation monitoring or recording of the process is provided. The variation of temperature within mechanical refrigerators is well known with one study reporting values of -43.5 °C to -90 °C in upright freezers (Su et. al., 1996). Since there is no active control during the process, it follows that the poor repeatability of the process can affect the cooling rate and hence the efficiency of the whole procedure. Variability might be improved if the local environment were more stable and protected from instantaneous variation due to external factors such as door openings etc. It is generally preferable to use a liquid nitrogen gas phase freezer for this approach since the internal

This approach to cooling and freezing is increasingly being used for material provision in pharmaceutical drug screening programmes as they move from supply by continuous culture towards a "cells-as-reagents" concept. In this approach, the cells are insulated in polystyrene containers as they are cooled initially to -80 °C and then transferred for cryogenic storage into liquid or vapour phase nitrogen. The need for rapid use of the cells for drug assays following cryopreservation, places an increased importance on the postthaw quality of the cells. In recent work carried out at LGC (Teddington,UK) this has been shown to be compromised by this type of freezing where the cooling rates are not actively controlled but rely on the passive characteristics of the system. In particular, temperature fluctuations within the polystyrene container and the storage time at -80 °C can significantly

temperature variation is small and the environment disturbed less frequently.

impact the post-thaw recovery of the cells and their biological function.

**2. Types of technique** 

and vitrification.

**2.1 Freezing in mechanical freezers** 

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 around -190 °C can be achieved.

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 weak point.
