**1.1 Different samples may have differing cryopreservation requirements**

For any cryopreservation protocol there are five key questions that govern the methodology and logistics of the freezing and storing process.


Reduction of temperature results in the retardation of metabolic processes and this can, in some circumstances, provide sufficient stability for the required period of storage. However, at temperatures below 0 °C the biological effects of cooling are dominated by the crystallization of ice: typically, water constitutes around 80 % of tissue mass. Freezing is the conversion of liquid water to crystalline ice but the term is commonly misused in circumstances where samples are cooled below their expected freezing point but without the formation of ice, for example by supercooling or by vitrification. The result of the freezing of water in a complex solution is that the concentration of the solutes in the remaining liquid phase increases and some solutes may precipitate if their concentration exceeds their solubility limits. This realisation provides two potential mechanisms of damage: direct mechanical effects of the formation of ice, and the rise in concentration of dissolved solutes.

In 1948 a method was discovered that permitted the freezing of many types of animal cells with good post-thaw recovery of living cells: Polge, Smith, and Parkes (1948) showed in a landmark paper that adding 10-20 % of glycerol enabled avian spermatozoa to survive freezing at -80 °C. Theories of freezing injury that were current at the time envisaged ice crystals damaging the cells and intracellular structures, and because glycerol increased the total solute concentration in the system, the amount of ice that formed was reduced. A little later, in the 1950s, Lovelock (1952) showed that the increase in concentration of salts as the volume of the suspending solution decreased was in fact the dominant damaging mechanism: salt concentration, rather than ice formation, was a major cause of freezing

Precision in Cryopreservation – Equipment and Control 509

water in the sample from liquid to crystalline. Crystalline water (ice) excludes the solutes previously dissolved in the water, resulting in two potentially dangerous mechanisms – direct effects of ice and secondary effects in the solute composition. At a sufficiently low temperature all biological activity is prevented and the physical state of the sample is preserved. In simple cases, where the only requirement is to preserve the physical state or where cellular structure is absent (viruses, DNA etc.), that is the end of the story; physical deterioration can be prevented at relatively high temperatures, and in many institutions worldwide this task is completed in the banks of -80 °C refrigeration units that proliferate in

The preservation of living cells and tissues and the post-thaw ability of cells to proliferate and thrive are determined by a number of factors: the laboratory techniques and the thermodynamic processes that a sample experiences during processing and freezing; the environment in which it resides between freezing and the ultimate use post-thaw. The potential of many samples is severely limited at this stage by the choices made by, or enforced upon, the technician regarding the freezing protocol. It may be that some stored samples lose significant value due simply to the omission of a few simple additional steps. The cryopreservation process has two main aims. The first is to reduce the temperature of the sample to a point where biological stability is achieved. The application of an external cryogenic environment will remove energy from the sample and create a very low-energy solid state within which biological and chemical activity are limited or prevented altogether. The second is that during the freezing process it is necessary to prevent the formation of intracellular ice crystals: such crystals damage the cellular structure and can lead to limited post-thaw recovery and post-thaw failure of the cell sample to function as required. Additionally, the protocol must take into account the stresses to which the cells are exposed during the freezing process (dehydration, hypothermia, chemical toxicity, and solute

The objective, therefore, is to create an environment in which, as the sample is cooled, the chemical composition inside the cell, is managed in such a way as to create an intracellular composition with a lower freezing point than the applied environment, whilst maintaining an external suspending composition that is able to solidify at the same temperature. The balance between the internal and external environment is managed chemically via the solutes in the micro-environment and thermodynamically via the application of an energy reducing (cooling) macro-environment. It is the combined action of these two factors that determines the success or otherwise of a cryopreservation protocol for the conservation of

The appropriate solute composition is created by including cryoprotective agents (CPAs) in the medium. These operate in one of two ways: either they modify the extracellular composition or alternatively they also replace some of the intracellular water. The first mechanism involves the addition of non-penetrating CPAs such as trehalose, polyethylene glycol (PEG) or Polyvinyl-pyrrolidone (PVP), to the medium. The second mechanism requires the addition of penetrating solutes that can traverse the cell membrane, such as glycerol, ethylene glycol and dimethyl sulphoxide (DMSO). Since water does not retain solutes when it freezes, a solution at equilibrium with ice will vary in osmotic potential as it freezes and because of this, the micro-environment of a cell will require either the cell to lose water to the environment or exchange water for CPA molecules, thereby maintaining

medical and biological research establishments.

vitality.

concentration) and the potential for an apoptotic response post-thaw.

injury to cells. Subsequently other cryoprotectant solutes were explored along with different rates of cooling, resulting in solidification of the stored samples but with a range of mixtures of ice and vitrified solid in the stored samples.

The physical nature of the sample dictates the thermal transfer characteristics of the cooling process for that specific sample and either the physical size or cell-type will affect the appropriate cooling rate and other parameters of the cryopreservation protocol. Similarly, the physical type and ultimate intended use of the sample (for example dose requirement in the case of future therapeutic use) will determine the size of the individual packaging. An additional layer of packaging may be necessary to prevent microbiological contamination – so-called 'double bagging'. Likewise the ultimate destination of the sample will also dictate the care required during the freezing process and the conditions necessary for long-term storage. Some tissues and most larger biological samples are currently difficult or impossible to cryopreserve successfully and new techniques, such as Liquidus Tracking (discussed later in this chapter) may address some of the problems associated with cryopreservation of these types of sample.

It is sometimes the case that the ultimate use of the samples stored is not known at the time of the initial collection and storage and sometimes the significance of particular samples may change with time. However, in many cases, the potential of the stored samples is fixed or limited at the time of selecting the cryopreservation and storage methods. The importance of these choices will be covered later; however it is pertinent to note here that the storage process may have an important impact on the value of samples when they are recovered from storage; changes in the properties of the recovered samples may be irreversible and this is therefore a key to maximising the sample's potential.

The term "viability" is frequently used in the context of cell and tissue banking. Strictly speaking it means the potential to exhibit the signs of life at some future stage, whereas it is often misused to mean the extent to which a sample demonstrates attributes of life at the present time. But that is "vitality" not "viability." However, it is also the case that not all the attributes of life are exhibited by all living things, and the possession of one attribute does not imply the presence of them all. In fact, few of the properties that characterise "life" can be measured quantitatively. The term is best avoided; functional measurements should be named to describe what they actually measure: membrane integrity; a specified metabolic function; ability to reproduce. In addition there are obvious cases where the tissue does not have to be alive in order to function; in bone for example. But equally, in many cases fully functional survival is paramount; the haemopoietic stem cells in cord blood will not graft in the recipient if the cell concentration is lower than a threshold value. In such cases a low total recovery of living cells in the thawed sample will limit the use of the thawed sample. Another common situation is where samples are stored in order to ensure that a supply of identical cells will be available throughout a long-term study. Although it is possible to regrow new cell batches from recovered samples, repeating this process can lead to progressive degradation due to mutations.

#### **1.2 The physics of freezing**

The process of freezing is ultimately simple; it is merely the application of an environment that removes energy from the sample over a period of time and changes the physical state of

injury to cells. Subsequently other cryoprotectant solutes were explored along with different rates of cooling, resulting in solidification of the stored samples but with a range of mixtures

The physical nature of the sample dictates the thermal transfer characteristics of the cooling process for that specific sample and either the physical size or cell-type will affect the appropriate cooling rate and other parameters of the cryopreservation protocol. Similarly, the physical type and ultimate intended use of the sample (for example dose requirement in the case of future therapeutic use) will determine the size of the individual packaging. An additional layer of packaging may be necessary to prevent microbiological contamination – so-called 'double bagging'. Likewise the ultimate destination of the sample will also dictate the care required during the freezing process and the conditions necessary for long-term storage. Some tissues and most larger biological samples are currently difficult or impossible to cryopreserve successfully and new techniques, such as Liquidus Tracking (discussed later in this chapter) may address some of the problems associated with

It is sometimes the case that the ultimate use of the samples stored is not known at the time of the initial collection and storage and sometimes the significance of particular samples may change with time. However, in many cases, the potential of the stored samples is fixed or limited at the time of selecting the cryopreservation and storage methods. The importance of these choices will be covered later; however it is pertinent to note here that the storage process may have an important impact on the value of samples when they are recovered from storage; changes in the properties of the recovered samples may be

The term "viability" is frequently used in the context of cell and tissue banking. Strictly speaking it means the potential to exhibit the signs of life at some future stage, whereas it is often misused to mean the extent to which a sample demonstrates attributes of life at the present time. But that is "vitality" not "viability." However, it is also the case that not all the attributes of life are exhibited by all living things, and the possession of one attribute does not imply the presence of them all. In fact, few of the properties that characterise "life" can be measured quantitatively. The term is best avoided; functional measurements should be named to describe what they actually measure: membrane integrity; a specified metabolic function; ability to reproduce. In addition there are obvious cases where the tissue does not have to be alive in order to function; in bone for example. But equally, in many cases fully functional survival is paramount; the haemopoietic stem cells in cord blood will not graft in the recipient if the cell concentration is lower than a threshold value. In such cases a low total recovery of living cells in the thawed sample will limit the use of the thawed sample. Another common situation is where samples are stored in order to ensure that a supply of identical cells will be available throughout a long-term study. Although it is possible to regrow new cell batches from recovered samples, repeating this process can lead to

The process of freezing is ultimately simple; it is merely the application of an environment that removes energy from the sample over a period of time and changes the physical state of

irreversible and this is therefore a key to maximising the sample's potential.

of ice and vitrified solid in the stored samples.

cryopreservation of these types of sample.

progressive degradation due to mutations.

**1.2 The physics of freezing** 

water in the sample from liquid to crystalline. Crystalline water (ice) excludes the solutes previously dissolved in the water, resulting in two potentially dangerous mechanisms – direct effects of ice and secondary effects in the solute composition. At a sufficiently low temperature all biological activity is prevented and the physical state of the sample is preserved. In simple cases, where the only requirement is to preserve the physical state or where cellular structure is absent (viruses, DNA etc.), that is the end of the story; physical deterioration can be prevented at relatively high temperatures, and in many institutions worldwide this task is completed in the banks of -80 °C refrigeration units that proliferate in medical and biological research establishments.

The preservation of living cells and tissues and the post-thaw ability of cells to proliferate and thrive are determined by a number of factors: the laboratory techniques and the thermodynamic processes that a sample experiences during processing and freezing; the environment in which it resides between freezing and the ultimate use post-thaw. The potential of many samples is severely limited at this stage by the choices made by, or enforced upon, the technician regarding the freezing protocol. It may be that some stored samples lose significant value due simply to the omission of a few simple additional steps.

The cryopreservation process has two main aims. The first is to reduce the temperature of the sample to a point where biological stability is achieved. The application of an external cryogenic environment will remove energy from the sample and create a very low-energy solid state within which biological and chemical activity are limited or prevented altogether. The second is that during the freezing process it is necessary to prevent the formation of intracellular ice crystals: such crystals damage the cellular structure and can lead to limited post-thaw recovery and post-thaw failure of the cell sample to function as required. Additionally, the protocol must take into account the stresses to which the cells are exposed during the freezing process (dehydration, hypothermia, chemical toxicity, and solute concentration) and the potential for an apoptotic response post-thaw.

The objective, therefore, is to create an environment in which, as the sample is cooled, the chemical composition inside the cell, is managed in such a way as to create an intracellular composition with a lower freezing point than the applied environment, whilst maintaining an external suspending composition that is able to solidify at the same temperature. The balance between the internal and external environment is managed chemically via the solutes in the micro-environment and thermodynamically via the application of an energy reducing (cooling) macro-environment. It is the combined action of these two factors that determines the success or otherwise of a cryopreservation protocol for the conservation of vitality.

The appropriate solute composition is created by including cryoprotective agents (CPAs) in the medium. These operate in one of two ways: either they modify the extracellular composition or alternatively they also replace some of the intracellular water. The first mechanism involves the addition of non-penetrating CPAs such as trehalose, polyethylene glycol (PEG) or Polyvinyl-pyrrolidone (PVP), to the medium. The second mechanism requires the addition of penetrating solutes that can traverse the cell membrane, such as glycerol, ethylene glycol and dimethyl sulphoxide (DMSO). Since water does not retain solutes when it freezes, a solution at equilibrium with ice will vary in osmotic potential as it freezes and because of this, the micro-environment of a cell will require either the cell to lose water to the environment or exchange water for CPA molecules, thereby maintaining

Precision in Cryopreservation – Equipment and Control 511

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

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

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

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

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

additional safety margin provides even greater sample security.

biological potential, it is vital that the mechanism of freezing injury be considered.

building storage environments.

practice (Glenister et. al., 1984).

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 hence deterioration, effectively ceases.

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 of living cells and hence the value of the sample.
