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CryoLetters 25(6) 375-388 (2004) © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPROTECTANTS: THE ESSENTIAL ANTIFREEZES TO PROTECT LIFE IN THE FROZEN STATE

Barry J. Fuller

University Department of Surgery, Royal Free & University College Medical School, London NW3 2QG, UK

Abstract

In the fifty years since the establishment of the cryoprotective effect of , banking by has become routine in many areas of and medicine. addition has become a rather mundane step within the overall protocol. However, for future advances in cryobiology and to meet new challenges in the clinical use of cryopreserved cells or tissues, it will be essential to have an understanding of the development and current status of the biological and chemical knowledge on (CPA). This review was undertaken to outline the history of CPA use, the important properties of CPA in relation to damage, and what can be learnt from natural freezing-tolerant . The conflicting effects of protection and resulting from use of CPA are discussed, and the role of CPA in enhancing ‘glassy’ states in the emerging field of are also set out. Keywords: review, cryoprotectant, cryoprotectant toxicity, natural cryoprotectants, nucleating agents, antifreeze , vitrification

INTRODUCTION

The application of cryopreservation to living cells and tissues has revolutionised areas of biotechnology, plant and animal breeding programmes, and modern medicine. The fact that cells from a multitude of prokaryotic and eukaryotic organisms can be recovered from down to almost two hundred degrees below the freezing point of water can be seen as a remarkable feat of resilience viewed from our current understanding of structural and molecular , but in most of these situations there is an essential necessary ingredient to achieve the trick – the presence of cryoprotectants. ‘Cryoprotectant’ (CPA) is the functionally-derived term coined to describe ‘any additive which can be provided to cells before freezing and yields a higher post-thaw survival than can be obtained in its ‘absence’ (46.44). There is considerable divergence across the classes of organic molecules that possess CPA activity; some have developed through to protect life in extreme environments and have been ‘re-discovered’ in laboratory experiments; some have been identified and put into practice from laboratory studies alone; and some have been extracted or modified from natural biological agents for applied use. This review will describe the history of our understanding of CPAs, the chemicals and their putative modes of action, and the most recent developments where new thoughts on CPAs are being translated into improved recoveries of cells after cryopreservation.

375 HISTORY OF CRYOPROTECTION

Some of the earliest concepts of cryoprotection were established by in the 19th and early 20th century who studied freezing, cold hardiness and resistance in the environment, most often in plants. For example, Hans Molisch in the 1890’s examined freezing in plants by direct using an early version of a cryomicroscope. In a recent translation of his original work (90), it is evident that Molisch was aware that the composition and concentration of substances in plant cell cytoplasm essentially dictated survival or death after freezing. He also discusses work by his contemporary, H. Muller- Thurgau, who reported the accumulation of sugars in cold hardened plants, although he did not specifically link sugars with cryoprotection in his monograph. The importance of sugars as CPA was clearly recognized by Maximov (61) in the early 1900’s (reviewed in 8). Similar observations on the of cold hardiness in plants and insects continued to be made over the next 30 years, but it was not until the pioneering work of Polge and colleagues on freeze storage of fowl that deliberate addition of a chemical to protect against freezing damage was made (71); in this case, glycerol was the additive. Following this work, other small molecular weight solutes with high aqueous solubility were shown to possess cryoprotectant properties (see Section 4. below), prompting the widespread application of cryopreservation in medicine, biotechnology, and plant and animal breeding. In the early years, these protective chemicals were called ‘cryophylatic agents’ (42) or ‘solute moderators’ (45), but, following from the lead set by the Society for Cryobiology in 1965, they were given their current designation of ‘cryoprotectants’.

THE OF FREEZING DAMAGE IN RELATION TO CPA

The multi-factorial nature of freezing damage in cells has become clearer over the past 50 years following a number of detailed studies, and it is beyond the scope of this review to discuss these. Some areas of uncertainty and debate remain, but a distillation of the current understanding can be found in the following papers (44, 62, 63, 69). To briefly describe the main points for the purpose of this review, the challenge of successful cryopreservation is to be able to cool and recover cells from the ultra-low temperatures (below about -100ºC) at which no changes in and structure are possible over a time-scale of years. The biophysical changes brought about by the transition of water to ice during this cooling are the main causes of damage, rather than the low temperatures per se. As ice crystals grow (inevitably first in the extracellular medium surrounding the cells under normal cooling conditions), there is an effective osmotic stress as the solute concentration surrounding the cells is excluded into an ever decreasing solvent volume. This ‘freeze-’ was one of the first harmful consequences identified in cell cryobiology (56), later shown to cause a number of damaging events including changes in ultrastructure of cell membranes, loss or fusion of membrane bilayers and organelle disruption (63). Eventually, at a sufficiently low (below about -80ºC), the remaining highly concentrated, highly viscous solution within and outside the cells turns into a glassy matrix, which is the relatively stable form for long-term preservation. The second major damaging event identified during cell freezing was the propagation of intracellular ice crystals (54,57). The potential for such intracellular ice formation increases if the osmotic potential inside the cell becomes dislocated from that in the surrounding medium on a kinetic basis (usually during faster cooling when there is insufficient time for water to move down the chemical potential gradient from the (relatively) more dilute intracellular solution to the concentrated extracellular medium. The exact mechanisms of damage from intracellular ice remain unclear, but may include physical destruction of membranes, gas

376 bubble formation and organelle disruption (63,64). If the cells can be cooled under conditions which effectively inhibit ice crystal formation down to the region of low temperature glass, then successful preservation can be achieved (under special conditions in the process known as vitrification). From these descriptions, it will be seen that CPA indeed must perform a multi-faceted protection across a range of sites during cell freezing. This complexity is the main reason why some classes of organic chemicals are more successful as CPA than others, and why individual solutes within a class may show better activity than others with a similar (but not identical) molecular structure.

CRYOPROTECTANTS AND CELL SURVIVAL DURING FREEZING

The first recorded example of deliberately added CPA activity, that of glycerol in the works of Polge and colleagues (71,83), established some factors for success for cryoprotectant activity of a particular solute which were deceptively simple, but which still hold in a broad sense today. Glycerol is a small, poly-hydroxylated solute with a high solubility in water, and a low toxicity during short-term exposure to living cells. It can interact by hydrogen bonding with water (as indicated by its high heat of solution), and can permeate across the limiting plasma membrane of many different cell types, albeit at a relatively slow rate. Cells may tolerate exposure to glycerol in concentrations from between 1 to 5 mol/l, depending upon cell type and conditions of exposure. Lovelock considered these various properties and developed his theory of the colligative action of CPA (56). In this, because of the well-known molar depression of freezing point associated with mixtures of solutes in solution, he proposed that at any given temperature below the ice transition during cooling, the rise in salts (especially of sodium chloride as the main constituent of most cell media) would be ameliorated by the presence of the glycerol. This would prevent the attainment of the critical damaging salt concentration whilst the whole system was cooled sufficiently to achieve the ‘glassy matrix’ state. The increasingly high viscosity of glycerol during lowering of the temperature is another property that may also inhibit or retard ice crystal growth on a kinetic basis. During the subsequent two decades, a broad range of solutes (mostly alcohols, sugars, diols, and amides) were investigated for CPA activity, with a broad range of success. Solutes such as sucrose, 1,2 propanediol, ethanediol, and dimethyl sulphoxide (Me2SO) were shown to have high CPA activity in various systems (56,58; multiple authors reviewed in 46,47). Many other small molecular weight solutes, such as amino acids including alanine, glycine and proline, other sugars including , lactose and ribose, and amides including acetamide and formamide (56,98; multiple authors reviewed in 46,47) were all found to possess some CPA activity, but often only at a low efficiency. In his review, Karow (46) recorded 56 solutes with reported CPA activity. However, the large majority of these have not found their way into modern cryopreservation protocols because of this relative lack of efficiency. Indeed, in later review of the field, Ashwood-Smith (5) reported a list of only 5 CPAs which he considered to be ‘moderately or very effective’ in preserving nucleated cells. During this period and within the reviews discussed above, another major factor in the assessment of CPA activity came to light. In some cases, and under specific conditions, molecules of much larger molecular mass, up to the level of polymers of several thousand Daltons, could demonstrate CPA activity. As early as 1955, Bricka & Bessis (17) demonstrated the cryoprotection of human erythrocytes by polyvinylpyrrolidone and dextran. Doebbler & Rinfret (23) used hydroxyl ethyl starch as CPA, also for erythrocytes. The use of polymers in erythrocyte freezing has been studied in recent years by Sputtek and colleagues

377 (85,86). They produced the only systematic study so far reported on cryopreservation of red blood cells for autologous transfusion using the polymeric hydroxyl ethyl starch (HES) as the sole CPA (41). In general, such high molecular weight CPA have been found to be most effective in cryopreservation, and they possess limited activity for preservation of nucleated cells when used as sole CPA (18). However, they may make a significant contribution to success of freezing when used in combination with other CPA (see also Cryoprotectants and the Glassy State, below). In such situations, the addition of the polymeric agent can assist in reducing the required concentration of the permeating CPA, helping to avoid toxicity. In fact, this principal is used in the clinical cryopreservation of autologous haemopoietic cells (87), which is one of the more routine applications of cell cryopreservation in current medicine. The protection afforded by polymeric CPA has been linked to their non- ideal behaviour at high concentrations aqueous solution. At high concentrations (in excess of 5% w/v), these agents exert appreciable effects on freezing point depression of the system (18,49), in excess of that predicted from their molar concentration (which will be very small). The high probability of hydrogen bonding between the multitude of hydrophilic side chains of polymers such as HES, and their increasing viscosity during freeze concentration of the solution combine to restrict ice crystal growth on a kinetic basis during cryopreservation, as demonstrated by differential scanning calorimetry (49). These agents may also suppress or inactivate ice nuclei in the extracellular spaces (36). For these effects to be best exploited, relatively fast cooling rates are essential, and success with erythrocytes has been optimised using rates of between -200 and -500ºC/min (85,86,87). It is interesting to consider the activity in cryopreservation of different cell systems in the early years of applied cryobiology, and that at the onset of the 21st century. The choice of cryoprotectants is to some extent dictated by different cells under study and pragmatic factors such as ease of handling, or approval for clinical use. For comparison, Table 1 sets out the reported research publications between 1950 and 1969, and those in 2000. It will be seen that, on a per annum basis, activity has increased 20-fold from approximately seven publications in the early years to 140 in 2000.

Table 1. Activity in cryopreservation of different cell types in the early years of cryobiology (1952-1969) compared to that in 2000. For simplicity, broad categories have been chosen; (blood cells includes erythrocytes, white blood cells, stem cells; and oocytes includes ovarian tissues; bacteria/fungi includes all ).

Cells and tissues cryopreserved Years 1952 -1969 Year 2000 % Activity1 % Activity2

Blood cells 54 9

Sperm 6 45 Embryos/Oocytes 2 42 Cell cultures 17 2 Tissues (segments) 12 6 Organs (vascularised) 5 3 Plant cell cultures N.D. 8 Insect cells N.D. 1 Bacteria/Fungi N.D. 2 Parasites 4 N.D.

Total papers published 125 142

The data were compiled from 1Karow (46) with minor additions, and 2ISI Web of Science data base.

378 It should also be noted that in the early years, many of the cryopreservation studies were made using an end-storage temperature of –79ºC, since was not as readily available as it now is. Also, this information is on research publications and takes no account of the massive applied use of cryopreservation in biotechnology and medicine, such as storage of patients’ embryos for clinical infertility treatment or maintenance and marketing of type cultures of a host of different cell types by agencies such as European Collection of Cell Cultures. Nevertheless, some trends can be seen from Table 1, including the large increase in activity in cryopreservation of reproductive cells to account for more than 70% of the reports by 2000, whilst blood cells (for which many methods can now be counted as high success and routine) have shown a fall in interest to 8% of activity, compared to 55% in the early period. Also, non-mammalian systems feature more in the current activity than in the early years of applied cryobiology. The mode of action of solutes during cryopreservation is likely to be multi-factorial, and, as yet, has not been comprehensively explained. Nash (65) attempted to explain cryoprotectant action on a combination of parameters based around the ability to modulate hydrogen bonding, interact with water molecules (achieving high aqueous solubility), and the volume occupied by a molecule of the solute. By computing these factors, he was able to derive a ‘protection coefficient’ (Q), which, for the solutes he investigated, could provide an indication of good CPA activity (Q>1). However, he also recognised that biological toxicity (at that time, indicated mostly by the oil/water partition) would have to be included for a more comprehensive designation of CPA activity. It was also difficult to expand the calculation of Q to take into consideration the known CPA effects of high molecular weight polymers. Since Nash’s work, there appear to be few other attempts to produce a more sophisticated, predictive model (but see Fahy et al., 34 below) to describe CPA activity of given solutes, which, even now, leaves a large gap in our understanding. Other secondary actions of CPAs may play a minor, but nevertheless important, role in success of cryopreservation. Nash himself mentioned the mild anti-bacterial effects of many CPA, and also alludes to the importance of the solubility of salts in the CPA (since exposure to high salt concentrations during freezing were known to be damaging). More recently, the ability to scavenge oxygen free radicals by some CPAs (such as Me2SO) has been suggested as an enhancing factor for CPA action (9,10). Additional modes of action of CPA have been suggested relating to inter-molecular interactions between the agents and biologically important macromolecules. Arakawa, Timasheff and colleagues have presented a series of arguments in which they describe the propensity for solutes to interact with proteins either by preferential binding or preferential exclusion from the surface (3,4). Agents that are preferentially excluded, act to stabilise proteins thermodynamically under conditions where other stresses (such as dehydration during freezing) occur (reviewed by Crowe et al., 19). CPAs such as Me2SO may interact elecrostatically with phospholipid bilayers (2). Disaccharide sugars, notably sucrose and trehalose, have also been shown to stabilise membranes during hypertonic exposure as ice crystals grow, by interacting with polar head groups of phospholipids (78). In fact, Crowe’s group have made the case that freezing and dehydration at higher temperatures have very similar biological consequences (19), and agents which protect against one stress will often show comparable protection in the other. There is one other important aspect of CPA application. In his work, Nash (65) described the property of CPA used in sufficiently high concentrations to produce complete inhibition of ice formation during cooling, in the process termed vitrification. Based on his own observations and those of his contemporaries (57), he felt that this was a theoretical property, which could not be achieved in applied cryopreservation. However, over the next twenty years, occasional reports alluded to the achievement of a ‘glassy state’ during cooling

379 experiments. For example, Elford and Walter (28) attempted to increase, in a step-wise fashion, the concentration of CPA surrounding smooth muscle strips at successively lower temperatures, and in this way inhibited ice formation. Later workers (74) employed high concentrations of a mixture of CPA, and rapid cooling in small volumes, to successfully recover mouse embryos from deep sub-zero temperatures. These observations spurred a number of biophysical investigations into the low temperature ‘glassy’ state and its stability (59,60). The current knowledge on vitrification has recently been reviewed (93). In summary, applied vitrification requires very high (in excess of 45% weight for weight CPA concentrations) often in mixtures. High cooling rates are generally necessary, and there are biophysical issues concerning the mechanical stresses in the glassy matrix at low temperatures, which can produce ice crystal growth during warming. Nevertheless, it is an expanding area of interest in applied cryopreservation (84), which will be discussed further below.

NATURAL CRYOPROTECTANTS : REDISCOVERING NATURE’S STRATEGIES

As described above, the study of survival in the frozen state in natural habitats has a long history in the biological sciences, and the importance of applied cryobiology has led to a re- evaluation and fruitful interplay between the various disciplines. Many species of terrestrial arthropods, plants inhabiting extreme environments, polar marine fish species and some other lower have all been shown to have evolved molecular strategies for survival at low temperatures. It is not possible to review this diverse field here, but detailed information can be found in (24,35,52,68,88,97). However, a few salient points are relevant to the current discussion. The methods for surviving effects of freezing can be classified under three main headings; ice nucleators, anti-freeze proteins, and compatible solutes.

Ice nucleating agents Whilst there are dramatic effects of ice formation for single cells (discussed above), there are even more problems to consider if intact tissues and organs are to survive freezing. Random ice crystal growth can be lethal in tissues (such as cardiac muscle), where integrated cell co-operation is essential for normal function, by a process of mechanical disruption. It is thus important to define where ice will form in the body, if low temperature exposure cannot be avoided. Also, by achieving ‘deliberate’ ice growth in certain sites, generalised super- cooling can be avoided, which reduces the likelihood of damaging intra-cellular ice formation (11). Over-wintering stages of many insects posses ice-nucleating agents (INA) in the haemolymph (6,27,51). There may also be a ‘symbiotic’ relationship between plants and insects in their natural habitat, and bacteria which possess ice-nucleating activity in their outer coat structures (52,53,96). In recent years, the structures of some of these protein INA have been determined (66), but much still remains to be understood. Whilst the ability to control ice nucleation in applied cryobiology and biotechnology is an obvious advantage, the application of natural INA has not yet made a major impact due to issues of safety and quality control (55), but it will remain an area of active research for the future.

Anti-freeze proteins Antifreeze proteins (AFP), also known as thermal hysteresis proteins, are proteins which lower the non-equilibrium freezing points of water, whilst not changing the melting point. AFP have been identified in a range of species including fungi and bacteria, plants, insects, and polar marine fish (40). The magnitude of the difference in temperature between freezing and melting may be about 1.5ºC in fish, but as high as 3 – 6ºC in insects. The proteins have a

380 regular repeat sequence (in insects these are 12-13 amino acid residues (26), with some conserved regions). AFP activity was first most extensively investigated in polar marine fishes (22) and, so far, in fish a greater diversity of AFP has been recorded - Types I ( 25), II (82), III ( 37) and IV (20). AFP may have a range of functions, including stabilisation of cell membranes, but the major consensus is that they act by adsorbing to the surface of small ice crystals, inhibiting ice crystal growth in the preferred low curvature planes and thus making ice crystal growth thermodynamically unfavourable until lower temperatures are encountered (22,35). This activity also aids inhibition of ice re-crystallisation (48), which normally proceeds towards the growth of energetically favourable large (and structurally more damaging) ice crystals. The use of AFP as complementary agents in applied cryopreservation is attractive, again for the reasons of control of ice crystal growth and possible damaging re-crystallisation during warming. However, the use of AFP may be more complicated than originally thought (21), and under some conditions addition of AFP can increase, rather than reduce, freezing damage (38). In the specific case of vitrification, AFP inhibition of ice growth during warming may have an important role (67). Also, the concept of ‘ice blocking’ has led to a search for synthetic compounds which will perform this function. Derivatives of polyvinyl alcohol (100) and cyclohexanediols (93) have shown promise, and may become important in the future.

Compatible solutes Compatible solutes are metabolites synthesised and accumulated by a variety of organisms in response to environmental stress. The importance of solutes such as sugars and glycerol in colligative protection during applied cryopreservation, and kinetic inhibition of ice crystal growth by high viscosity, has been described above, but it is now becoming clear that many naturally cold-resistant species have already evolved such strategies. In many extreme cold environments, environmental dehydration accompanies the low temperatures, and such solutes seem to be equally important for protection against water loss at the cellular level, whether it is from ice formation or evaporation (11). Sugars have been known for over a century to be important in the development of over-wintering strategies in a range of plants (61). Trehalose is a sugar first identified in drought-resistant existing at relatively high temperatures, but the same sugar has been found during winter-hardening in a range of cold and freezing-resistant insect species (11). Other ‘laboratory’ identified CPA such as glycerol have also been found at high levels in over-wintering insect stages (76,77). Many insects synthesise a range of polyols during hardening (39,89), which are thought to assist in cold survival. The challenge for the future in harvesting the potentially additive benefits from such a ‘multiple agent’ strategy is understanding how such mixtures optimally interact with biological structures under different, defined freezing regimes.

THE TWO FACES OF CPA: PROTECTION VERSUS TOXICITY

The discussion so far has concentrated on the beneficial aspects of CPA modulation of freezing damage. It was recognised early on, however, that CPA are chemicals (in the essentially protective concentrations) not normally encountered by living organisms. A marked toxicity (both osmotic and chemical) can be detected if the exposure to CPA is not optimised (5). The osmotic toxicity can be readily understood in that CPA, when added to cells in the required concentrations (1 mol/L upwards) cross biological membranes only relatively slowly compared to water, so there is a well-documented rapid water efflux from the cells, with associated volume collapse (54). At the end of any cryopreservation procedure, the reverse is true (washing cells with normal isotonic media whilst they are still loaded with

381 CPA), leading to rapid ‘over-swelling. Cells can only tolerate moderate excursions in cell volume without significant damage, and a great deal of effort has been placed on designing steps in cryopreservation which avoid transgressing these limits (69). The nature of chemical from CPA are inevitably complex, given the ranges of different molecular structures of the commonly used agents. Although most CPA (neutral solutes and polyols) are relatively innocuous compared to, for example, exposure to equivalent concentrations of salts, there is an appreciable time and temperature-dependent effect in most cases. At a practical level, attempts have been made to avoid these effects by minimising the exposure time to CPA before and after freezing, and by using lower temperatures of exposure. However, this selection of lower exposure temperatures in itself may cause problems, because the passive permeation of the CPA into cells is also slowed, so it takes longer to achieve the sufficient concentrations. Early reports of toxicity of agents such as Me2SO were reviewed by Fahy (31) who discussed evidence that suggested, in selected cases, there was an indication of an underlying chemical toxicity for several CPA, beyond the protection afforded by their colligative action on reducing salt concentrations during freezing. He pointed out that in some studies, damage during cryopreservation could be simulated without freezing by exposing cells and tissues at low temperatures to CPA concentrations equivalent to those experienced during freezing. In some cases, using high concentrations of CPA, more injury was noted than could be accounted for on the basis of the calculated increase in the salt concentration. Fahy (31) conceded that during freezing, it is very difficult to uncouple the effects of osmotic or chemical toxicity, but concluded that ‘the detrimental effects of cryoprotectants are almost as relevant to cryobiology as are their cryoprotective effects’. Kruuv and colleagues (50), noted that certain chemicals acted as ‘cryosensitisers’ and could enhance freezing damage when present in low concentrations during ice formation, and different CPA had different abilities to reverse this effect. Fahy and colleagues undertook more detailed studies of chemical toxicities of a range of CPA mixtures (33), but were unable to formulate a definitive explanation. In the same study (33), the authors attempted to identify chemicals that could act as ‘counteracting solutes’ or ‘toxicity neutralisers’, and found a weak positive response to some agents, notably formamide. Indeed, a unifying concept for CPA chemical toxicity in cells remains beyond our grasp. Studies (on mammalian oocytes) determined that CPA can have direct effects on structures such as microtubules and microfilaments, causing disassembly which could be reversed if the CPA exposures were of relatively brief duration, or at lower temperatures. Certain CPA, such as butanediol, appear to produce a ‘chaotropic’ effect leading to membrane blebbing (94), whilst CPA effects on proteins, with possible enhanced ‘disulphide bridge’ formation remains a concern (33). The area of CPA toxicity remains one of considerable uncertainty, and is ripe for re-evaluation using modern molecular techniques. More recently, a new approach to quantifying CPA toxicity, based on the average water hydrogen bonding of the polar groups within the molecular structures (or qv*) has been proposed (34). This has the advantage that it can be used to quantify the ‘water binding’ variable within multi-component mixtures of CPA, such as used in vitrification solutions. Initial studies have supported the use of qv* to design less toxic CPA mixtures with enhanced post-thaw recoveries in a limited number of model systems, including mouse oocytes (34.), but more detailed work will be necessary to confirm this. As cryopreservation becomes increasingly important in banking of genetic resources, there is a growing interest in investigating phenotypic and genotypic responses to the techniques, and the role for CPA toxicity (if any) in these has not yet been clearly identified.

382 CRYOPROTECTANTS AND THE GLASSY STATE

Since a major damaging role has been assigned to ice formation during cryopreservation (whether it be the total quantity of ice formed, the presence of ice inside cells or the relationships between ice and high densities of cells in fixed geometries in tissues), the possibility of achieving low temperature storage which avoids ice formation has long been a dream. Luyet (57) is credited with the first serious attempts to achieve the glassy state in biological systems, applying the concept of cooling sufficiently quickly to avoid ice crystal formation on a kinetic basis, until such low temperatures were reached such that ice crystals would not grow. Luyet and his colleagues worked for several years on this approach, and although did not achieve a robust technique for allowing recovery of living cells, nevertheless set the groundwork for later studies (reviewed in 32). The physical concepts of aqueous vitrification are complex, and depend on an understanding of nucleation events (both heterologous and homologous nucleation), material properties and stability of the glassy state, and interaction of organic solvents (for our purpose, the CPA) at the glass transition temperature (59). However, in simplistic terms, it can be described as solidification of a liquid into an amorphous state whilst maintaining essentially the same molecular orientations that existed in solution before the glass transition; i.e. ‘a solid which is like a snapshot of the liquid state’ (32). Through several important studies, using physical techniques such as differential scanning calorimetry, it has been established that different classes of CPA have different critical concentrations to achieve the glassy-forming tendency (GFT) at biologically achievable cooling rates, and exhibit different critical warming rates to avoid devitrification (12,14,15,16,60). Dilute aqueous solutions can be vitrified when cooled at extremely rapid 6 rates (in excess of 10P ºCP /sec; (32,60) in very small volumes, but this is not applicable to typical cryopreservation requirements. The glassy state can be achieved at more manageable 2 cooling rates (around 10P °P C/min) if high concentrations of CPA can be employed (74). For most neutral solutes conventionally used as CPA, concentrations in excess of 50% weight/volume are required to achieve biologically applicable vitrification, but such concentrations impose severe problems of potential osmotic and chemical toxicities to cells. In aqueous systems, it has been proposed that an ‘unstable glassy state’ can be achieved over the same range of cooling rates using slightly lower concentrations of CPA (32), in which there is a potential for devitrification and ice crystal growth during warming. This obviously necessitates use of warming rates fast enough to avoid this ice growth, but the benefits in reduced toxicity from the lower CPA concentration make this approach practically attractive in applied uses. Vitrification techniques were first studied in detail in animal preservation (74), but variations of the method were soon applied to plant cells and tissues, especially plant shoot apices (7,81). In general, the mixtures of CPAs used to achieve GFT are similar, although there are differences in relative concentrations of the various components, especially sugars. Plant vitrification has become a growing area of interest in conservation of plant genetic resources and refinement to the technologies specific to plant requirements have steadily been developed (7,80). These include ‘dehydration-encapsulation’, whereby an air dehydration stage is included, which increases the effectiveness of both naturally abundant and added CPAs, including sugars (7,30,80). The important interactions of CPAs which enhance formation of the glassy state are similar to those which are important to the modulation of ice formation in conventional cryopreservation. Beyond the colligative actions of the agents, the ability to interact with water molecules by hydrogen bonding, there are possible effects on masking nucleation sites. It has also been established that steric conformations play a role in differently affecting the glass-forming tendency, and different isomers of the same solute have different effects (13,60). Recently, the number and orientation of OH groups in sugar and polyalcohol CPAs

383 have been suggested to be of significance in successful vitrification of plant shoot apices (95.87), although stability of the glassy state was not reported. In earlier studies, mixtures of CPA to achieve a generic total solute concentration for GFT were advocated, as a way of reducing risks of chemical toxicity of any one particular CPA (74), but later studies showed that it was possible to use single CPAs to achieve successful vitrification (75). As the understanding of solute properties increased, attempts have been made to enhance glass- forming tendencies by chemical modifications of such agents. For example, Wowk and colleagues (99) reported on methoxylation of CPA such as ethylene and propylene glycols, which produced a marked change (reduction of the critical cooling rate by an order of magnitude) in GFT. Sugars, especially disaccharides such as sucrose and trehalose can also be effective contributors in aqueous solution towards producing GFT (91). For example, addition of 6% w/v disaccharides were shown to reduce the concentration of CPA (in this case, 2,3- butanediol) needed to achieve the glassy state at a given critical cooling rate by approximately the same degree (about 6% w/v). This trade-off does not sound impressive in biophysical terms, but in avoidance of chemical toxicity to cells of the diol CPAs, it can be significant. Recently, Wusteman and colleagues (101) developed a vitrification protocol in which the disaccharide (trehalose) was used to substitute for a large part of the ionic component of the medium (in these mammalian cell studies, mainly the sodium chloride), so increasing the effective sugar concentration whilst minimising the osmotic stress associated with exposure to the vitrification solution. The issue of the low permeabilities of many mammalian cells to disaccharides such as trehalose has been addressed in recent studies from Toner’s group (79). Greater biological stability may require the sugars to interact not only with the plasma membrane, but also with those of internal organelles, such as mitochondria. Increased permeability to disaccharides has been sought by attempts to bio-engineer a switchable membrane defect or ‘pore’ (29), and by direct microinjection into large cells such as oocytes. A similar approach has been taken for other CPA in refractory cells such as zebrafish embryos (43). High molecular weight CPAs also have a rather specific role in GFT. Apart from the solute:water interactions existing between multiple hydrogen bonding sites on polymer side chains (in solutes such as polyvinyl pyrrolidone), the high molecular weight agents tend to have increasingly high viscosities are low temperature, which the solution effectively becomes too viscous to allow water molecules to join growing ice crystals (91,92). Also, the specific ability of some proteins (AFP, PN and the novel agents discussed above) has a potential application in the formulation of vitrification solutions (93). Synthesis of a polymerised form of glycerol has been found to yield an agent capable of high activity against ice nucleation produced by bacterial ice nucleators (100), and this may have a potential role in vitrification technology in the future. An additional area of study, particularly where vitrification is applied to larger structures such as tissue grafts, is that of low temperature materials science. The tissue and glassy matrix produced by these cooling techniques is subject to thermo-mechanical stresses, which can lead to fracture (72). These stresses are influenced by CPA (73), and little is yet understood about the models required to determine which CPA and at what concentrations such stresses can be minimised (93). It may seem that vitrification in applied cryopreservation is an exotic, technical approach dependent on a mixture of laboratory-based and organic chemistry. However, as with other aspects of CPA science, there may be parallel cryobiological events that occur in nature. For example, a similar state may be reached in freeze tolerant insect tissues during over-wintering (11).

384 Vitrification as currently recognised was not used during the early years of cryopreservation (c.f. Table 1). However, by the year 2000, a total of 74 reports were recorded in the ISI databases. Of these, the vast majority (60%) concerned use in storage of embryos and oocytes. The second highest group of interest was that of plant cell and tissue preservation (25%). This compares to only 8% of recorded activity for plant cells in 2000 in conventional cryopreservation, and indicates that vitrification has led to an important expansion in low temperature technology for applied plant breeding and biotechnology. Uses of vitrification may expand into other areas of biotechnology in the future. Research is already underway into production of the glassy state using sugar mixtures at supra-zero temperatures (1).

CONCLUSION

Applied low temperature technology has progressed significantly since the early years to occupy a central role in many modern scientific endeavors. Whilst much has been learnt about the role of CPA and their mode of action, there still remain significant gaps in our understanding about their molecular interactions with cell components and potential toxicities. In the coming decades, the push towards therapy by manipulation and tissue will highlight these remaining uncertainties and demand answers of a greater depth. Cryobiologists will be required to embrace and collaborate with new physical and molecular sciences to meet this challenge.

Acknowledgments: The author would like to acknowledge the many supportive suggestions and discussions from a host of colleagues, including Sharon Paynter, Paul Watson, Erica Benson, Nick Lane, and colleagues in the UNESCO Chair of Cryobiology.

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Accepted for publication 6/11/04

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