Cryopreservation and Freeze-Drying Protocols

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Save to Library. Create Alert. Share This Paper. Citations Publications citing this paper. Cryopreservation and banking of mammalian cell lines Glyn Stacey , John R. Translating cryobiology principles into trans-disciplinary storage guidelines for biorepositories and biobanks: a concept paper. Erica E. Benson , F Betson , Barry J. Development of a new ultra-fast freezing procedure for zebrafish sperm cryopreservation Sandra Raquel de Melo Vieira Martins Rebocho.

Histological evaluation of the repair process of replanted rat teeth after storage in resveratrol dissolved in dimethyl sulphoxide. Long-term preservation of Leptospira spp. Srinivas Venkatnarayanan , P. References Publications referenced by this paper. The effect of seed oil content on viability assessment using tetrazolium: a case study using species. Wood , S. Miles , C. Rix , J. Terry , M. Development of a cryopreservation protocol for the microcyclic rust-fungus Puccinia spegazzinii.

Matthew J. Ryan , Carol A. Methods for the study of water relations under desiccation stress. We will now examine these mechanisms of cryoinjury and cryoprotection in a little more detail. Lovelock also demon- strated [3] that when glycerol was present, hemolysis started at the lower temperature at which the same critical concentration of salt was produced Fig. Correlation does not prove causation, but in this case, if the solution changes were not causative of freezing injury, then the correspondence would be a remarkable coinci- dence indeed.

It was these studies that led to the consensus that extracellular ice is harmless to cells and that freezing injury is caused by indirect effects of the formation of ice. Principles of Cryopreservation 7. Reprinted with permission from ref. However, the salt in the suspending medium is not the only solute to be concentratedthe cryoprotectant is concentrated to the same degree.

When red blood cells were frozen and thawed in the presence of a range of concentrations of glycerol, they demonstrated that the correspondence between the effects of salt exposure and of freezing was retained. This observation is important for two rea- sons: it shows that cryoprotectants are not innocuousoverall they are protective but at a price; second, the observation adds powerful support to the solution-effect theory. The optimum rate will be a trade-off between those two factors.

Of course, other cells have different water permeabilities and it has been shown by direct experiment that the cooling rate that produces intracellular freez- ing on a cryomicroscope corresponds with the cooling rate that produces significant intracellular supercooling [11]; Fig. Principles of Cryopreservation 9. The R values are the weight ratio of glycerol to NaCl in each solution. Reprinted with permis- sion from ref. The line labeled 0 is the equilibrium line. Reproduced with permission from ref. In fact, very small amounts of intracellular ice are compatible with recovery, and this is one reason why the warming rate has a profound effect.

This has been demonstrated to damage the cells in which it occurs; however, dur- ing rapid warming there is insufficient time for this to happen and the ice simply melts. Because the cooling rate influences the forma- tion of intracellular ice, while warming rate controls what happens to that ice subsequently, and because cells differ in their water per- meability and probably also in their susceptibility to intracellular ice, then it follows that cells will differ in their cooling and warm- ing requirements and cooling rate will interact with warming rate.

Experiment has shown that the proportion of red blood cells sus- pended in 2. The increase in hemolysis as the hematocrit is increased is amelio- rated by increasing the glycerol concentration. These observations cannot be accounted for by the classical mechanisms of cryoinjurysolution effects and intracellular freez- ing.

The most likely explanation is that densely packed cells are more likely to be damaged by mechanical stresses when the chan- nels within which they are sequestered change shape. This is a result of recrystallization of the ice that forms their boundaries. Principles of Cryopreservation Freezing involves changes in the concentra- tion and composition of aqueous solutions and this also produces driving forces for the movement of water and solutes.

Biological systems contain numerous barriers to the free diffusion of solutes membranes , and these can result in transient, and sometimes equilibrium, changes in compartment volumes; if excessive, these changes can be damaging. Hence, the processes of diffusion and osmosis are very important for cryopreservation. Fortunately, the quantitative description of mass transfer processes is well devel- oped [13, 14]. The driving force for flow is pressure. Thus, the flow of water, Jv, through a membrane is given by.

J is given the subscript v to signify volumetric flux. When the driv- ing force for the flow of water through a membrane is osmotic pressure rather than hydrostatic pressure, flow can be described by the same equation if osmotic pressure, , is substituted for hydro- static pressure, thus,. The constant k has the same value in the two equations, providing only that the membrane and the solvent, water in this case, remain the same. Thus, both hydrostatic and osmotic pressure differences can be incorporated into a single equation. This equation states that flux across unit area of membrane is proportional to the solute permeability s, and the difference in con- centration of the solute across the membrane.

The convention for the direction of flux is that outsideinside is positive. A somewhat more complex formalism was elaborated by Kedem and Katchalsky [13] in and their equations are often used in cryobiology where they are usually referred to as the KK equations. This led to modification of the equations for Jv and Js, as shown.

The equation for Js has an addi- tional term that represents solvent drag on the permeant solute, which is present in the membrane at concentration cs. Clearly, the KK is more complex and curve fitting routines can lead to uncertain results because of the lack of independence of the parameter,.

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Kleinhans [14] has discussed these problems in detail and moreover he has argued that the KK formalism is often invalid because of the presence of separate channels for water and solute. In practice, the simpler for- malism is adequate for the current needs of cryobiologists. The two equations are solved simultaneously by numerical methods and pro- grams to carry out these calculations can be run on an ordinary PC. Several methods are available for the determination of perme- ability parameters in cryobiology. If the solute under study can be radiolabeled, the time-course of isotope uptake is easily measured but the calculation of concentration requires the additional measurement of water content at each time-point.

Permeating sol- utes can be extracted after known times of exposure and high per- formance liquid chromatography methods are often suitable for their assay. The Karl Fischer method, using a back titration scheme, is a convenient method for water [15]. If the compound under study has a distinctive nuclear magnetic resonance spectrum, nuclear magnetic resonance can be used to determine the time- course of both solute and water content simultaneously, so this technique yields concentration directly. We will now consider in more detail some situations in which these permeability parameters are relevant to cryopreservation.

If the cryoprotective compound per- meates, the cells then increase in volume, water entering along with the cryoprotectant until the cells reach their final volume. The extent of shrinkage and the rate of change in cell volume are determined by the permeability parameters.

The final equilibrium volume depends on the concentration of impermeant solutes in the solution and is the same as the normal volume only if the concentration of imper- meant solutes is isotonic in molar per liter terms. This is because of the fact that the cryoprotectant occupies space within the cells and the volume of water must therefore be lower than the physiological water content if the total volume is to be normal [16]. The rate of change of volume, and particularly the equilibrium volume, are both important and must be optimized in cryopreservation procedures.

They then shrink as the cryoprotectant moves out, accompanied by suf- ficient water to maintain osmotic equilibrium; they return to physi- ological volume only if nonpermeating solute has neither been lost nor gained during the process. Because cells are generally more sensitive to swelling than to shrinkage, removal of cryoprotectants tends to be more hazardous than their addition. Again, both the rate of change of volume and the final volume must be considered when designing protocols for the recovery of cryopreserved cells.

This phenomenon is an extremely important determinant of intracellular freezing. The final extent of shrinkage depends on the cryoprotectant concentration. In reality there is always an intercept on the y-axisthe so-called nonosmotic water volume or Vinf. This probably represents a physically distinct portion of the cell water that is so structured that it does not participate in solution phenom- ena. Alternatively, it could reflect the nonideal behavior of the intra- cellular solutes such that osmolality increases with concentration more than in linear proportion. Experimentally, the collection of such data can usefully be combined with determining the upper and lower vol- ume limits that the cells will tolerate without damage.

Examples include the long-term preser- vation of spermatozoa of many species, including cattle, laboratory animals, and man, very early embryos and ova, red and white blood cells, hemopoietic stem cells, tissue culture cells, and so on. For each type of cell there is a set of conditions that is optimal for pres- ervation, determined by the interaction of the particular properties of the cell in question with the cryobiological factors that have been discussed.

If the characteristics of the cell are known, it is usually possible to predict with reasonable precision the conditions that will provide effective cryopreservation. Cell survival is still Systems required, of course, but tissues and organs contain a heterogeneous collection of cells, which may have quite different optimum require- ments for preservation, unlike the situation in cell preservation where one is usually dealing with a single type of cell.

Yet it is neces- sary to find a method that will secure adequate survival of all the cells that are important for the function of that tissue. Fortunately, the use of high concentrations of cryoprotectant results in a flatten- ing of the bell-shaped survival curve and a broadening of its peak: with sufficiently high concentrations of cryoprotectant it is possible to secure overlapping survival curves for many different cells. Ice that forms outside the cells when a cell suspension is frozen is outside the system that it is desired to preserve, and it can damage the cells only by indirect means solution effects or by exerting a shear or compressive force on them externally.

The situation is quite differ- ent for organized tissues; here, extracellular ice is still within the system that is to be preserved and can disrupt the structure of the tissue directly. Structural studies using freeze substitution showed that ice formed within the muscle bundles [20]. If cooling was slowed to 0. This showed that extracellular ice damaged this tissue, but the extent of such damage was dependent on the site at which the ice formed. Damaging effects of extracellular ice have also been demonstrated in kidneys and livers, where it has been shown to cause rupture of the capillaries.

The Principles of Freeze-Drying

Rubinsky and Pegg [10] have proposed a mechanism for this effect; ice forms within the vessel lumens, drawing in water from the surrounding tissue until the volume of intraluminal ice exceeds the elastic capacity of vessel and rupture ensues. In organs and tis- sues that require an intact vasculature for function, vascular rup- ture is lethal, even if many cells survive, and this mechanism provides the major barrier to effective cryopreservation of such systems. The avoidance of freezing, or at least limitation of the amount of ice to very small quantities in the least susceptible loca- tions, seems to be the only way to avoid this problem.

Attempts to cryopreserve complex multicellular systems simply by adapting techniques from single-cell systems have generally been unrewarding. In the medical field, the situation may be more favorable with tissues that can be transplanted without revascular- ization; it all depends on the precise requirements for surgical acceptability.

For example, the primary requirement for heart valve grafts is that the collagen structure is intact, and it is unclear whether the survival of donor fibroblasts has any useful effect. Similarly, human skin can be cryopreserved by methods similar to those used for cell suspensions and will then retain significant num- bers of viable keratinocytes, although it is questionable whether these influence the clinical results when skin grafts are used as a temporary covering on seriously burned patients.

For other tissues, such as small elastic arteries, satisfactory methods have only been developed relatively recently [21]. For corneas and for whole vas- cularized organs there are no effective methods. The intersection of the melting curve and the glass transition curve at Tg indicates the lowest concen- tration of glycerol that, in theory, will vitrify. In practice, the lower temperatures on the melting curve are unlikely to be reached owing to the high viscosity pre- venting the crystallization of ice.

Luyet devoted a great deal of effort to the search for conditions that would produce a vitreous or glassy state with biological systems and that living cells could survive. In conventional cryopreservation, the concentration of solute in the remaining liq- uid increases during progressive freezing, and a temperature Tg is eventually reached with many systems where the residual liquid vit- rifies in the presence of ice Fig.

Both the formation of the nuclei and the subsequent growth of ice crystals are temperature dependent. Nucleation is unlikely just below the equilibrium freezing point hence the phe- nomenon of supercooling , but it becomes more probable as the temperature falls, reaches a maximum rate, and then decreases as the movement of water is limited by viscosity.

However, the growth of ice crystals is maximal just below the freezing point and is pro- gressively slowed, and eventually arrested, by cooling. The interac- tion of these two processes creates three possibilities for a cooled sample Fig. Upon warming, however, there are only two possibilities; if heated sufficiently rapidly it will escape both nucleation and freez- ing during warming; the alternative is that the trajectory passes through both the nucleation and the ice crystal growth zones and, therefore, it will nucleate if it is not already nucleated and the ice crystals will then grow before eventually melting.

Therefore, unless a sufficient concentration of cryoprotectant has been used to ensure that no ice can form under any circumstances, there is a risk that freezing will occur during warming. For small samples it is more feasible to cool rapidly, as was demonstrated by the successful vitrification of Drosophila melanogaster embryos [24]. These were complex organ- isms comprising some 50, cells with advanced differentiation into organ systems, and they cannot be preserved by conventional freezing methods.

The successful method required careful permea- bilization of the waxy vitelline membrane to allow penetration of the cryoprotectant, exposure to 8. The extremely high rate of warming was far more critical than the rate of cooling, which is consistent with the crucial importance of maintaining the vitreous state. The demonstration that ice forming in tissues produces so much damage has created renewed interest in the possibility of using vitrification with very high concentrations of appropriate cryoprotectants to avoid the formation of ice completely.

Current research aims to identify materials that will inhibit the formation of ice crystals during warming [25, 26], and one interesting possibility is the antifreeze proteins that some polar fish and overwintering insects have evolved to avoid freezing in nature.

One effect of such compounds is to reduce the warming rate required to prevent ice crystallization to more manageable rates. This approach is being used in conjunction with electromagnetic heating [27, 28] to achieve more rapid and more uniform heating. However, despite progress in the design of vitrification cocktails with reduced toxicity, the major problem remains cryoprotectant toxicity.

One approach to this problem is to increase the concentration of cryoprotectant progressively during cooling so that the tissue concentration fol- lows the liquidus curve: ice does not form but the cells do not experience any greater concentration of cryoprotectant than occurs during freezing. This has recently proved to be practical and very effective for the cryopreservation of articular cartilage, an otherwise recalcitrant tissue [29].

The same method may potentially be effec- tive for other resistant tissues and perhaps even for organs. References 1. Pegg DE Cryobiology. In: Proceedings of spermatozoa after vitrification and dehydra- of the fourth international cryogenic engineer- tion at low temperatures. Nature ing conference, Eindhoven. IPC Science and 2. Pegg DE Cryobiologya review. Lovelock JE The mechanism of the engineering. Plenum Publishing Corporation, protective action of glycerol against haemolysis NewYork, NY, pp by freezing and thawing. Biochim Biophys 7. Pegg DE Mechanisms of freezing dam- Acta age.

Mazur P Kinetics of water loss from cells and animal cells. Symposia XXXXI of the soci- at subzero temperatures and the likelihood of ety for experimental biology. The Company of intracellular freezing. J Gen Physiol Biologists Ltd. Pegg DE Organ storagea review. In: tissue stored at 21C or 60C. Cryobiology Maxwell Anderson J ed The biology and sur- gery of tissue transplantation.

Blackwell Pegg DE, Diaper MP On the mecha- studies to elucidate the pattern of ice formation nism of injury to slowly frozen erythrocytes. J Microsc Biophys J Leibo SP Fundamental cryobiology of In: The freezing of low temperatures. Biodynamica, Normandy, MO mammalian embryos. Ciba Foundation sympo- Elsevier, Amsterdam, vitrification: basic principles. Kluwer Academic rate on the packing effect in human erythro- Publishers, Dordrecht, Netherlands, pp cytes frozen and thawed in the presence of 2M Cryobiology Mahowald AP Cryobiological preserva- Science analysis of the permeability of biological mem- branes to non-electrolytes.

Biochim Biophys Sutton RL Critical cooling rates to Acta avoid ice crystallization in solutions of cryopro- Kleinhans FW Membrane permeability tective agents. Cryobiology CryoLetters high concentrations of glycerol or propylene Cryobiology Biomed Eng Cell cryoprotectant and sample shape on unifor- Biophys mity of heating. Phys Med Biol Pegg DE Ice crystals in tissues and organs.

Plenum Cryopreservation of Articular Cartilage 3. The Press, NewYork, pp liquidus tracking method. Abstract Vitrification is an alternative approach to cryopreservation that enables hydrated living cells to be cooled to cryogenic temperatures in the absence of ice. Vitrification simplifies and frequently improves cryo- preservation because it eliminates mechanical injury from ice, eliminates the need to find optimal cooling and warming rates, eliminates the importance of differing optimal cooling and warming rates for cells in mixed cell type populations, eliminates the need to find a frequently imperfect compromise between solu- tion effects injury and intracellular ice formation, and enables cooling to be rapid enough to outrun chilling injury, but it complicates the osmotic effects of adding and removing cryoprotective agents and introduces a greater risk of cryoprotectant toxicity during the addition and removal of cryoprotectants.

Vitrification is therefore beginning to realize its potential for enabling the superior and convenient cryopreservation of most types of biological systems including molecules, cells, tissues, organs, and even some whole organisms , and vitrification is even beginning to be recognized as a successful strat- egy of nature for surviving harsh environmental conditions. However, many investigators who employ vitrification or what they incorrectly imagine to be vitrification have only a rudimentary understanding of the basic principles of this relatively new and emerging approach to cryopreservation, and this often limits the practical results that can be achieved.

A better understanding may therefore help to improve present results while pointing the way to new strategies that may be yet more successful in the future. To assist this understanding, this chapter describes the basic principles of vitrification and indicates the broad potential biological relevance of vitrification.

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Key words Vitrification, Freezing, Intracellular ice formation, Devitrification, Recrystallization, Chilling injury, Cryoprotective agents, Cryoprotectant toxicity, Osmotic limits, Protein denaturation, Biobanking, Glass transition, Glassy state, Optimal cooling rate, Organ preservation. The industrial significance of vitrification is well understood and long-standing. Beyond the manufacturing of famil- iar glassy items such as porcelain and windows, for example, obsid- ian, which is a vitrified form of lava [3], was used to make artifacts.

Fahy andBrianWowk. On the other hand, the potential biological significance of vitrification has been reasonably well appreciated for less than 80years. The possibility of vitrifying water was postulated as long ago as [6]. In , the successful vitrification of a 0. But the successful use of vitrification to preserve biological viability or molecular stability in the vitreous state, which is the focus of this chapter, was most unambiguously achieved even earlier, in , when human eryth- rocytes were vitrified in a rapidly cooled aqueous solution of 8.

But vitrification or something very close to it can also be achieved in nature or in the laboratory by drying, and some organisms [11, 12] and many proteins [13] can be pre- served successfully in this way. The ability of vitrification to preserve molecules, cells, tissues, whole organs, and even some whole organisms has many current and future agricultural, medical, scientific, and ecological ramifica- tions. The application of vitrification to cryopreservation has been growing exponentially since the early s [] and Fig. Given the broad potential biological relevance of vit- rification, which is illustrated in detail in the remaining contribu- tions to the present volume, an understanding of the basic principles of vitrification is becoming increasingly important.

TG is usually defined on the basis of a change in heat capacity detected by, for example, differ- ential scanning calorimetry DSC. Additional discussion of the nature of the glass transition is given below. TG can be measured during either cooling or warming, but there is no generally accepted word that describes the reverse of vitrification, i.

The terms vitromelting or vitrofusion were once suggested to describe this transition [19, 20], but they have not been adopted. Principles of Vitrification The data are cen- sored prior to to avoid extraneous references, but are not censored thereafter. TG is a theoretical temperature reached when freezing is able to concentrate the unfrozen liquid portion of a solution until its melt- ing point becomes equal to its glass transition temperature [21, 22].

Such extreme freeze concentration is rarely, if ever, achieved in real life in the case of aqueous solutions of the low-molecular-weight glass-forming solutes typically used for cryopreservation by vitrifi- cation [23] Fig. No further comment is made about the somewhat controversial concept of TG in this review. Freezing is the reorganization of water molecules into ice crystals [30].

Although freezing is often used to mean arresting motion or change, the use of this sense of the word in the context of vitrification, in which the object is to avoid ice crystallization, is misleading and inappropriate. Thawing is the melting of ice. Continuing cooling then leads to vitrification of the concentrated unfrozen solution. The thin line, denoted by Tm, is the solution melting tem- perature. The thick shaded line denoted by TG is the glass transition temperature.

It tracks the melting temperature until increasing viscosity prevents sufficient ice growth to attain equilibrium. Below the TG line, the sample consists of a mixture ice and glass. Rewarming, or the warming of a previously cryopre- served system, whether frozen or vitrified, is a more accurate term than thawing in the context of vitrified systems. Devitrification is not the reverse of vitrification.

Instead, it is the formation of ice during warming after previous vitrification [] and is explained in detail below. Recrystallization is the transfer of water molecules from small ice crystals to larger crystals for an early discussion, see [34]; for particularly illustrative photos, see [35]. This can happen under iso- thermal conditions, during which no net change in the total quan- tity of ice may occur, or during warming, in which case the quantity of ice may change even as recrystallization proceeds.

Recrystallization tends to be damaging because it results in the conversion of a large number of relatively innocuous small ice crystals into a smaller num- ber of larger and more damaging ice crystals. As noted below, recrys- tallization appears to be more important than devitrification per se in governing the fate of previously vitrified systems.

The ability of ice to form during either cooling or warming depends on how much time is available for ice nucleation and growth. The critical cooling rate is the cooling rate above which appreciable ice formation is not observed [36], and the critical warming rate is the warming rate that completely or sufficiently suppresses ice formation during warming [36]. The critical cooling [37] and warming [38] rates for a given system depend very strongly on the total solute content of the system as well as on the chemical nature of the solute.

The solutes used for vitrification are generally the same as or similar to those used to pro- tect against freezing injury and are generally referred to as cryopro- tective agents CPAs or cryoprotectants []. Glycerol, which has a molec- ular mass of To cross cell membranes, pCPAs must not possess a net charge. Although it has been argued that when cryoprotectants are used to enable vitrification they should be called vitrificants rather than cryoprotectants [42], cryoprotectants are defined to be agents that reduce or prevent freezing injury, so the term cryo- protectants remains proper in the context of vitrification because in fact these agents continue to prevent freezing injury, even if they do so by preventing ice formation altogether.

However, the term vitrificants, while not widely used, is also correctly descriptive of agents that facilitate vitrification. A relatively new type of cryoprotectant is the ice blocker, which is a molecule that is capable of undergoing specific inter- actions with ice or ice nucleating agents so as to reduce or pre- vent ice nucleation, ice growth, or both [16, ]. Antifreeze proteins are proteins that can adsorb to the surface of ice crys- tals and prevent them from growing even when the temperature is lowered below the thermodynamic melting point of the ice [41, 48].

Although antifreeze proteins, or AFPs, were the first natural examples of ice blockers, ice blockers are usually thought of as being lower in mass and either synthetic or non-protein- aceous natural products. A vitrification solution [49] is a solution of cryoprotectants suf- ficiently concentrated to enable extracellular and intracellular vitrifica- tion of the system at hand under the intended cooling conditions. A carrier solution is the physiological support medium in which CPAs are dissolved to enable cells to be exposed to CPAs without injury beyond the injury associated with the CPAs themselves.

Chilling injury is injury caused by cooling per se. Although chilling injury is most conspicuous in the absence of ice, there has been some speculation and some strong evidence that it can occur also during freezing in specific cases []. Thermal shock or cold shock is injury caused by rapid cooling but not by slow cooling, whereas chilling injury is observed during slow cooling and may even be outrun by very rapid cooling if the system is not subject to injury from thermal shock. Anhydrobiosis [55, 56] is the survival of life in a desiccated state.

It is relevant to vitrification in the sense that sufficiently con- centrated cytoplasm undergoes a glass transition that contributes to the survival of organisms, cells, or seeds that are adapted to or prepared for preservation by drying [57, 58]. Although it is of considerable potential applied and ecological significance [], this chapter focuses primarily on low-temperature vitrification rather than on high-temperature anhydrobiosis or aestivation. Most molecular constitu- Temperatures ents of cells are reasonably stable under low-temperature condi- tions in situ even without special precautions, although there are exceptions.

Generally speaking, neither freezing and thawing nor cooling per se causes the formation or breakage of covalent chemi- cal bonds. The reversible formation of S-S cross-links in frozen thiogels [61], one particular protein but not others extracted from freeze-killed cabbage [62], and one of five SH groups in F-actin [63] has been reported, but no change in S-S or S-H con- tent was found in lethally frozen sea urchin eggs [64], and an increase of S-H content in frozen-thawed bull spermatozoon membranes was observed [65]. Temperature reduction inhibits most chemical reactions e. Exothermic phase changes such as the crystal- lization of water or the formation of the liquid crystalline gel state [67, 68] or HEXII state [69] of membrane lipids are favored over limited temperature ranges, but these phase changes do not destroy but only rearrange the participating molecules and in most cases are reversible.

Protein cold denaturation, discussed in more detail below, may or may not be spontaneously reversible, but usually does not involve covalent modification of the protein. Additional reviews of the history of biological vitrification are avail- able elsewhere [15, 16, 42, ]. Cryopreservation by vitrification was apparently first intro- duced conceptually although without much clarity or influence by Stiles [73] in Apparently independently, the idea was rein- troduced actively, clearly, and influentially by Luyet [31] in The original concept was that if the water in living systems could be cooled rapidly enough, there would be insuffi- cient time for crystals to form before reaching the glass transition temperature of water, and the living system could therefore be trapped in the vitreous state [31].

This approach in principle limited vitrification to samples that could be cooled and warmed very rapidly, but it was nevertheless pursued energetically by Luyet and his associates for 21years [70]. In , evidence emerged indicating that Luyets primary indica- tion of vitrification, optical transparency of thin films or thin living systems after cooling followed by opacification or continued trans- parency on warming, had been misleading and had deceived Luyet into believing he had attained complete vitrification when in fact what he had attained was partial vitrification involving predomi- nantly the formation of ice crystals known as spherulites that were too small or too thin to scatter visible light enough to be visible to the naked eye [77, 78].

This setback apparently chastened Luyet, who never again advocated or claimed cryopreservation by vitrification. Instead, he turned his focus onto ice and studied its formation and morphol- ogy under an extensive array of conditions [70]. Eventually, he and his colleagues learned that the presence of very high concentra- tions of cryoprotectants, including both small molecules [27, 28, 79, 80] and larger polymers [26], could in fact enable vitrification even at low cooling rates [70, 81]. This is the key observation that has enabled most modern methods of vitrification, but ironically, Luyet himself never proposed using high concentrations of cryo- protectants to cryopreserve living systems by vitrification.

The ability of cryoprotectants to enable vitrification only began to be elucidated and disseminated by Luyet and his colleagues as of the end of [80] and continued until [, 79]. At least in part for that reason, in , a historical opportunity was missed.

As later pointed out by Fahy etal. However, in , it was still assumed that cooling below 79C would lead to ice formation [82]. As noted above, the first definitive report of the successful vit- rification of a living cell was published in by Rapatz and Luyet, who showed that erythrocytes cooled at high rates in the presence of 8. Before this, it is possible that some of Luyets successes in recovering life after very rapid cooling and warming in the presence of cryoprotectants which were used to achieve dehy- dration prior to cooling so as to reduce the volume of water that required vitrification [] might have included some level of useful vitrification, but this is difficult to infer from available knowl- edge.

Ironically, the vitrification of red cells by Rapatz and Luyet, which might have been regarded as the culmination of Luyets life work, was noted little, if at all, outside of Luyets laboratory for many years and is still almost never cited. In [87] and [88], Rapatz reported successfully cooling frog hearts to 79C and rewarming them with good recovery using 11M ethylene glycol EG and variations on Farrants [82] pioneering method.

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He presumably could have vit- rified and successfully recovered these hearts in a viable condition and, unlike Farrant, must have understood that this was possible, but he reported no attempts to do so. This is likely to be because, as he reported from the podium during his presentation [87] but did not mention in his published abstract [87] , when hearts loaded with EG were transferred into liquid nitrogen, they shat- tered, as he put it a general problem that is discussed in detail below.

He later reported that 10M EG was the minimum con- centration allowing recovery of frog hearts from 79C, but that rat hearts could not tolerate more than 5M EG and therefore could not be successfully preserved [89]. Nevertheless, establish- ing that frog hearts, at least, can theoretically be vitrified and recovered remains one of the most outstanding achievements in biological vitrification.

From [90] to [91], Elford similarly worked out a method for preserving strips of intestinal smooth muscle in a supercooled state at 79C using variations of Farrants method. Pegg, personal communication that some muscle strips cooled in liquid nitrogen the ones that had not experienced the same kind of shattering or fracturing observed by Rapatz recovered after warming and would therefore have been the first successfully and definitively vitrified organized tis- sues. However, once again, there was no suggestion that vitrifica- tion as opposed to deep supercooling might be used as a method of cryopreservation.

The first intimation that high concentrations of cryoprotec- tants might in theory be used to enable cryopreservation by vitrifi- cation came in , when Pierre Boutron, a physicist who turned to cryobiology partly on the basis of the phase diagram work of Luyet and colleagues after previously having studied the structure of amorphous solid water [92], published a landmark paper that contemplated vitrification in a new way. This paper was the first to thoroughly describe the kinetics of ice formation in vitrifiable aqueous cryoprotectant solutions containing glycerol, Me2SO, and mixtures of the two in view of the theoretical possibility of completely vitrifying cell suspensions using mixtures of cryopro- tectants to facilitate vitrification and reduce toxicity [36].

It also pioneered the combined use of X-ray diffraction and differential scanning calorimetry DSC to investigate ice formation and glass transitions in aqueous solutions, introduced the concepts of the critical cooling rate and the critical warming rate, and introduced mathematical models of the kinetics of ice formation relevant to vitrifiable solutions [36].

However, Boutrons aim was to find a very stable amorphous state of the whole solution even for diluted solutions emphasis added , whereas the accumulated work of Luyet and his colleagues had clearly established that amorphous solutions can only be stable against ice formation when they are concentrated, not when they are dilute. Undeterred, Boutron and Kaufmann went on to study, in , the stability of the amorphous state in aqueous solu- tions of ethanol [93], glycerol plus ethylene glycol [94], glycerol plus ethanol [95], and, most significantly, propylene glycol PG, or 1,2-propanediol [96].

Perhaps because such meth- ods would not be applicable to systems much larger than single cells and because single cells can generally be preserved well by freezing using much lower and hence less toxic concentrations of cryoprotectants, Boutrons method did not seem to inspire much attention at the time.

In , Fahy [83, ] proposed a different approach to vitrification that, in principle, is both definitive and universally applicable to almost all biological systems. Inspired by his desire to overcome mechanical injury from ice in whole organs [15, 49, 72, 83, 97], his method relies on the fact that at suffi- ciently high concentrations, both the critical cooling rates and the critical warming rates needed to suppress ice formation become low enough to enable, in principle, even the vitrification of objects as large as human organs.

Unlike Boutrons approach of attempt- ing to sidestep toxicity by using lower concentrations, Fahy elected to attack the problem of high-concentration toxicity head on [83, ] so as to avoid the need for high cooling and warming rates. Also, unlike the approaches of Farrant, Elford, and Rapatz, which required introducing cryoprotectant at temperatures as low as 55C [87], Fahys approach sought to enable the use of cryo- protectants at much higher temperatures that were more compat- ible with organ perfusion and the very temperature-dependent rate of passage of pCPAs across cell membranes.

This approach was shown to work when applied to mouse embryos by Rall and Fahy in [49]. This demonstration, which showed that vitrification was at long last a feasible general method for cryopreservation, inspired much subsequent research on a variety of living systems using a variety of cryoprotectant solu- tions and methods Fig. The emphasis since has been largely on refining the basic but complex parameters of cryoprotec- tant selection; the concentration, temperature, timing, and osmotic effects of each step of cryoprotectant introduction and removal; methods and equipment for cooling and warming; and methods for avoiding fracturing.

About this book

These methods have been applied to a wide variety of living systems see, e. The number of papers devoted to these topics since is beyond the scope of this introductory review, but their contents are reflected by the scope of the other contributions to this volume. To under- andDisadvantages stand the advantages and disadvantages of vitrification, it is there- ofVitrification fore necessary to understand something about freezing injury. Conventional cryopreservation by freezing involves, by definition, the formation and dissolution of ice during cooling and warming, respectively.

The effects of ice formation are in part due to this concen- trating action, which increases both the osmotic concentration of the cellular environment and the individual concentrations of dissolved solutes such as electrolytes, buffers, etc.

source link If cooling proceeds sufficiently slowly, ice formation begins extra- cellularly [, ], there is time for cells to lose water down the transmembrane osmotic gradient established by the extracellular ice, and the cells will consequently shrink. If shrinkage proceeds too far, osmotic injury may result [].

If cooling proceeds more rapidly, the rate of water subtraction from the cell fails to keep up with the rate of water subtraction from the extracellular environment, leaving the cell interior significantly more dilute than the extracellular solution [].

This means that the thermo- dynamic freezing point of the cell fails to fall as rapidly as the prevailing temperature, i. This defines a state of supercooling cooling below the freezing point without ice formation , and as supercooling increases, the risk of ice formation within the cytosol increases.

In summary, cells cooled too slowly are liable to injury related to shrinkage and changes in solution composition solution effects injury , whereas cells cooled too quickly are liable to injury related to intracellular ice formation IIF []. Between these two ends of the spectrum, there is an optimum cooling rate that minimizes both sources of injury []. These considerations are relevant to vitrification for three rea- sons.

First, the existence of an optimum cooling rate is problem- atic. The optimum cooling rate can only be determined experimentally for every cell type of interest, which is inconvenient, particularly for multicellular tissues, which may contain not only multiple cell types but also cells in different relationships to each other and to the extracellular environment, all of which affect the optimal cooling rate [].

Moreover, given the existence of a dif- ferent optimal cooling rate for different cells, finding a compro- mise rate that gives high recoveries of all cells may be difficult and has been proposed as a limiting factor for cryopreserving complex systems []. And finally, the use of cryoprotective agents to increase sur- vival at the optimal cooling rate also changes the optimal cooling rate itself [], again in a way that will be cell type dependent. The second point of relevance to vitrification is that, as noted, pCPAs must generally be used to obtain high survivals after freez- ing and thawing, since they mitigate solutions effects injury.

Although relatively low concentrations of pCPAs are needed to prevent solution effects injury in many cells, the concentrating effect of freezing on dissolved solutes pertains just as much to pCPAs as it does to other solutes, the result being that pCPA con- centrations may be driven high enough in the frozen state to induce toxic effects of their own [].

Interestingly, the concentrations generated by freezing actually exceed the concen- trations required for the vitrification of even large living systems [83, , ], so the advantage of using lower concentrations for freezing is not necessarily as large as it at first appears. The third reason the constraint of an optimum cooling rate is relevant to vitrification is that some important living systems such as oocytes are subject to chilling injury see below , and attempts to cool more rapidly than the kinetics of chilling injury proceed are precluded if the result is death secondary to IIF intracellular ice formation.

Vitrification eliminates that obstacle by eliminating IIF at high cooling rates and has often been pursued for that reason [52, 54, , ]. Beyond changes in solution composition and IIF, freezing can result in injury in at least two additional ways, both of them being mechanical in nature. First, the physical displacement of structures in organized tissues by the simple growth of extracellular ice can cause considerable damage to both the vascular bed and to paren- chymal structures [98, ].

In fact, it was the observation that dog kidneys frozen to 30C and stored for a week using 3M glycerol could perfuse normally and respond well to pressors invitro but urinated whole blood and stopped perfusing within min of being transplanted Fahy, Goldman, and Meryman, unpublished results that inspired the proposal to investigate vitri- fication as a more promising approach to organ cryopreservation. Fortunately, Taylor and his colleagues have provided extensive microscopic evidence using freeze substitution methods that vitri- fiable solutions successfully prevent tissue distortion by ice [16, , ].

Second, even single cells can be injured by intracellular [] and extracellular [, ] recrystallization. Both forms of mechanical injury secondary to ice formation argue for vitrification as a potentially less damaging preservation method, particularly for complex organized tissues and organs. Vitrification does have significant disadvantages as well, how- ever [, ]. First, the need to tolerate very high concentra- tions of CPA requires relatively sophisticated methods of adding and removing these agents and careful selection of the right CPA blend for the living system at hand.

Finally, although rapid warming is generally beneficial for frozen systems, it is even more important for vitrified ones due to the need to avoid injury from devitrification and subse- quent recrystallization. A potential disadvantage of certain techniques of vitrification comes from the use of container-free cooling methods to acceler- ate the cooling and warming rate and thereby enable the use of minimal concentrations of cryoprotectant.

The lack of a container may result in contamination of the sample being preserved []. The need for such methods is questionable, however, and it seems likely that closed-system vitrification will eventually remove the risk of contamination. A number of insects, for example, survive the winter by freeze avoidance [57, 58, , ], achieved by suppressing the presence of ice nucleating substances, synthesizing high con- centrations of cryoprotectants such as glycerol, and producing antifreeze proteins that bind to ice and prevent it from grow- ing see Subheading2.

In one case, that of the larval Alaskan red flat bark beetle Cucujus clavipes puniceus [], more than half of the individuals tested supercooled to below 60 to 70C and none showed exotherms indicative of freezing when cooled to C in a DSC. Two large larvae had a second small TG at 96 or 98C. When unselected larvae were cooled to In any case, at least some larvae appear able to survive cooling to below even the lowest observed TGs.

Further, the coldest tem- peratures recorded in nature This suggests that some organisms have actually sur- vived low temperatures in a vitreous state under natural condi- tions using endogenous cryoprotectants similar in both molecular weight and concentration to those being used for artificial vitri- fication in cryobiological laboratories to those being used for artificial vitrification in cryobiological laboratories in C. Species whose moisture content varies with the ambi- ent humidity are said to be poikilohydric, and this water loss can be sufficient to induce cytoplasmic vitrification [, , , ] and may even make vitrification under natural conditions more com- mon than survival by freezing tolerance [, ].

As one example, soil nematodes dried to below 0. However, vitrification by dehydration enables survival at high temperatures as well as at low temperatures [11, 12]. The lowest common terrestrial temperatures are typically between about 30 and 60C [, ], but the cells of many species have been shown to have glass transition tempera- tures above 50C [57, , ]. Twigs of Populus balsam- ifera were shown to undergo a glass transition at about 45C and are known to be able to survive immersion in liquid nitro- gen []. Highly frost-hardy plants in general, according to Sakai, survive in conjunction with their ability to form intracel- lular glasses [].

Similarly, freeze concentration of the extracellular solution and concomitant osmotic reduction of cell volume see also Subheading 1. The solution may initially ofAqueous Solutions supercool before the first ice crystal forms, but thereafter the con- centration of the remaining unfrozen solution follows the melting temperature line Tm with continued cooling. Increasing solution viscosity during cooling eventually inhibits ice growth, causing a small departure from thermodynamic equilibrium [23]. Final cool- ing then continues with little change in concentration until the glass transition temperature TG is reached [83].

It does so while retain- ing the random molecular arrangement of a liquid. A solid with the same unstructured molecular arrangement as a liquid is called a glass [1]. This is the basis of cryopreservation by vitrification. During cryopreservation by vitrifi- cation, the entire sample volume remains substantially free of ice during cooling. As shown in Fig. Heat transfer limitations neces- sitate the use of high solute concentration and slow cooling rates when vitrifying large volumes, such as tissues and organs.

As hinted at in Fig. The curve labeled Th designates the homo- geneous nucleation temperature, which is further described in Subheading2. Th sets the limit beyond which the solution cannot be supercooled without ice nucleation. Unlike freezing, with vitrification the solution concentration remains constant during cooling because cooling is too rapid for ice to form or grow appreciably. Unstable vitrification requires cooling at thousands of degrees per minute, or more, due to high ice nucleation and growth rates associated with homogeneous nucleation.

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