Cryopreservation of articular cartilage. Part 3: The liquidus-tracking method.

Although it is relatively straightforward to cryopreserve living isolated chondrocytes, at the present time there is no satisfactory method to preserve surgical grafts between the time of procurement or manufacture and actual use. In earlier papers we have established that the cryoprotectants dimethyl sulphoxide or propylene glycol do penetrate into this tissue very rapidly. Chondrocytes are not unusually susceptible to osmotic stress; in fact they appear to be particularly resistant. It appears that damage is associated with the formation of ice per se, even at cooling rates that are optimal for the cryopreservation of isolated chondrocytes. We then showed that current methods of cartilage cryopreservation involve the nucleation and growth of ice crystals within the chondrons rather than ice being restricted to the surrounding acellular matrix. This finding established the need to avoid the crystallization of ice-in other words, vitrification. Song and his colleagues have published a vitrification method that is based on the use of one of Fahy's vitrification formulations. We confirmed the effectiveness of this method but found it to be very dependent on ultra rapid warming. However, we were able to develop a 'liquidus-tracking' method that completely avoids the crystallization of ice and does not require rapid warming. The ability of cartilage preserved in this way to incorporate sulphate into newly synthesized glycosaminoglycans (GAGs) approached 70% of that of fresh control cartilage. In this method the rates of cooling and warming can be very low, which is essential for any method that is to be used in Tissue Banks to process the bulky grafts that are required by orthopaedic surgeons. Work is continuing to refine this method for Tissue Bank use.

The relevance of ice crystal formation for the cryopreservation of tissues and organs.

This paper discusses the role of ice crystal formation in causing or contributing to the difficulties that have been encountered in attempts to develop effective methods for the cryopreservation of some tissues and all organs. It is shown that extracellular ice can be severely damaging but also that cells in situ in tissues can behave quite differently from similar cells in a suspension with respect to intracellular freezing. It is concluded that techniques that avoid the formation of ice altogether are most likely to yield effective methods for the cryopreservation of recalcitrant tissues and vascularised organs.

Principles of cryopreservation.

Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal and this chapter seeks to analyze some of the mechanisms involved and to show how cooling can be used to produce stable conditions that preserve life. The biological effects of cooling are dominated by the freezing of water, which results in the concentration of the solutes that are dissolved in the remaining liquid phase. Rival theories of freezing injury have envisaged either that ice crystals pierce or tease apart the cells, destroying them by direct mechanical action, or that damage is from secondary effects via changes in the composition of the liquid phase. Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity. Many compounds have such properties, including glycerol, dimethyl sulfoxide, ethanediol, and propanediol. In fact, both damaging mechanisms are important, their relative contributions depending on cell type, cooling rate, and warming rate. A consensus has developed that intracellular freezing is dangerous, whereas extracellular ice is harmless. If the water permeability of the cell membrane is known it is possible to predict the effect of cooling rate on cell survival and the optimum rate will be a trade-off between the risk of intracellular freezing and effects of the concentrated solutes. However, extracellular ice is not always innocuous: densely packed cells are more likely to be damaged by mechanical stresses within the channels where they are sequestered and with complex multicellular systems it is imperative not only to secure cell survival but also to avoid damage to the extracellular structure. Ice can be avoided by vitrification-the production of a glassy state that is defined by the viscosity reaching a sufficiently high value (~10(13) poises) to behave like a solid, but without any crystallization. Toxicity is the major problem in the use of vitrification methods. Whether freezing is permitted (conventional cryopreservation) or prevented (vitrification), the cryoprotectant has to gain access to all parts of the system. However, there are numerous barriers to the free diffusion of solutes (membranes), and these can result in transient, and sometimes equilibrium, changes in compartment volumes and these can be damaging. Hence, the processes of diffusion and osmosis have important effects during the introduction of cryoprotectants, the removal of cryoprotectants, the freezing process, and during thawing. These phenomena are amenable to experiment and analysis, and this has made it possible to develop effective methods for the preservation of a very wide range of cells and some tissues; these methods have found widespread applications in biology and medicine.

The relevance of ice crystal formation for the cryopreservation of tissues and organs.

This paper discusses the role of ice crystal formation in causing or contributing to the difficulties that have been encountered in attempts to develop effective methods for the cryopreservation of some tissues and all organs. It is shown that extracellular ice can be severely damaging but also that cells in situ in tissues can behave quite differently from similar cells in a suspension with respect to intracellular freezing. It is concluded that techniques that avoid the formation of ice altogether are most likely to yield effective methods for the cryopreservation of recalcitrant tissues and vascularised organs.

Reduction of cryoprotectant toxicity in cells in suspension by use of a sodium-free vehicle solution.

Propane-1,2-diol (PD) possesses physico-chemical properties that make it a useful biological vitrifying agent but, although of relatively low toxicity, it still has substantial damaging effects on cells. This study aimed to identify possible toxic mechanisms using primary cell cultures from vascular tissue: these were exposed to the cryoprotectant at room temperature to avoid any possibility of hypothermic injury. Toxicity was evaluated by measuring the ability of the cells to divide in culture after exposure to the cryoprotectant. A variety of interventions, which addressed either possible consequences of PD exposure, or known mediators of other types of cell injury, were utilized in an attempt to inhibit PD toxicity. Some comparative studies with dimethyl sulphoxide (Me2SO) exposure were also made. Replacing sodium in the vehicle solution with choline was the only intervention that reduced PD toxicity. It did so both in smooth muscle cells, where the loss of functional capacity was reduced from 56% to 13%, and in endothelial cells. where the reduction was from 40% to 18%. Similar observations were also made in smooth muscle cells exposed to Me2SO. We failed to find evidence for a role of pH regulation, for oxidative injury and/or an involvement of redox-active iron as a mediator of the injury. The results strongly suggest that the influx of sodium into the cell provides one mechanism whereby both PD and Me2SO exert their toxic effects. We suggest that the use of choline-based vehicle solutions in cryopreservation would be beneficial.

Vitrification of rabbit tissues with propylene glycol and trehalose.

A previous study had suggested the use of a mixture of propanediol and trehalose for the preservation of tissues by vitrification. In this paper, we describe experiments in which stepwise procedures were developed for adding these cryoprotectants to high final concentrations in two rabbit tissues-carotid artery and cornea. The tissue concentration of the additives was measured at the end of each step so that the temperature of the next step could be chosen to reduce toxicity but avoid freezing. This process was arrested when a concentration had been reached that should permit vitrification if the tissues were cooled rapidly to -175 degrees C. They were stored at that temperature; warmed rapidly by conduction; the cryoprotectants removed by stepwise dilution; and appropriate active functions measured. These were contraction and relaxation for arteries and endothelial integrity and ability to control stromal swelling for the corneas. In control experiments the exposure and functional assays were carried out without vitrification. It was shown that the tissue concentration of propanediol was 33%w/w in artery and 30% in cornea. These permitted cooling to -175 degrees C without freezing but devitrification occurred during the warming of the arteries, though not of the corneas, despite the lower tissue concentration reached in the cornea. The function of the vitrified arteries was severely reduced but the endothelium of the corneas was substantially intact although we were unable to demonstrate any ability to control stromal swelling during normothermic perfusion. It appears that concentrations of cryoprotectants sufficient to prevent freezing in these tissues during cooling were well tolerated so long as appropriate stepwise means of addition and removal were used. Devitrification during warming remained a major problem with arteries, but not with corneas. We suggest that the composition of the aqueous phase in the tissue with respect to components other than the vitrifying agents may be crucial here and that the search for agents that will suppress devitrification is an important avenue for further study.

Principles of cryopreservation.

Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal and this chapter seeks to analyze some of the mechanisms involved and to show how cooling can be used to produce stable conditions that preserve life. The biological effects of cooling are dominated by the freezing of water, which results in the concentration of the solutes that are dissolved in the remaining liquid phase. Rival theories of freezing injury have envisaged either that ice crystals pierce or tease apart the cells, destroying them by direct mechanical action, or that damage is from secondary effects via changes in the composition of the liquid phase. Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity. Many compounds have such properties, including glycerol, dimethyl sulfoxide, ethanediol, and propanediol. In fact, both damaging mechanisms are important, their relative contributions depending on cell type, cooling rate, and warming rate. A consensus has developed that intracellular freezing is dangerous, whereas extracellular ice is harmless. If the water permeability of the cell membrane is known it is possible to predict the effect of cooling rate on cell survival and the optimum rate will be a tradeoff between the risk of intracellular freezing and effects of the concentrated solutes. However, extracellular ice is not always innocuous: densely packed cells are more likely to be damaged by mechanical stresses within the channels where they are sequestered and with complex multicellular systems it is imperative not only to secure cell survival but also to avoid damage to the extracellular structure. Ice can be avoided by vitrification--the production of a glassy state that is defined by the viscosity reaching a sufficiently high value (approximatly 10(13) poises) to behave like a solid, but without any crystallization. Toxicity is the major problem in the use of vitrification methods. Whether freezing is permitted (conventional cryopreservation) or prevented (vitrification), the cryoprotectant has to gain access to all parts of the system. However, there are numerous barriers to the free diffusion of solutes (membranes), and these can result in transient, and sometimes equilibrium, changes in compartment volumes and these can be damaging. Hence, the processes of diffusion and osmosis have important effects during the introduction of cryoprotectants, the removal of cryoprotectants, the freezing process, and during thawing. These phenomena are amenable to experiment and analysis, and this has made it possible to develop effective methods for the preservation of a very wide range of cells and some tissues; these methods have found widespread applications in biology and medicine.

Further work on the cryopreservation of articular cartilage with particular reference to the liquidus tracking (LT) method.

The cryopreservation of articular cartilage with survival of living cells has been a difficult problem. We have provided evidence that this is due to the formation of ice crystals in the chondrons. We have developed a method in which the concentration of the cryoprotectant dimethyl sulphoxide (Me(2)SO) is increased progressively, in steps, as cooling proceeds so that ice is never allowed to form, but the very high concentrations of Me(2)SO required at low temperatures are reached only at those low temperatures. In this paper, we describe some new experiments with discs of ovine articular cartilage similar to those used in our previous studies and we show that continuous stirring throughout the process resulted in a significant increase in the rate of (35)S sulphate incorporation into glycosoaminoglycans (GAGs), now reaching 87% of the corresponding fresh control values. We confirmed that the method is also effective for human knee joint cartilage, which gave 70% of fresh control ability to synthesise GAGs; continuous stirring was also used in this experiment. We then extended the method to ovine knee joint osteochondral dowels and showed that, again with continuous stirring, the method produced tissue concentrations of Me(2)SO that were sufficient to prevent freezing in dowels too, and to permit cell function at 60% of control. The most important mechanical property (instantaneous compressive modulus) was unaffected by the process. Finally, we experimented with some technical variations to facilitate clinical use-a more rapid process for warming and removal of Me(2)SO was developed and a method of short-term storage before or after cryopreservation was developed. Finally, pilot experiments were carried out to provide proof of principle for a closed, continuous flow method in which both temperature and Me(2)SO concentration were computer-controlled.

Analysis of the permeation of cryoprotectants in cartilage.

Some tissues, such as cartilage and cornea, carry an internal fixed negative charge, leading to a swelling pressure that is balanced by tensile stress in the tissue matrix. During the addition and removal of cryoprotectants the changes in osmotic pressure will cause the tissue to deform. Because of the fixed charge and osmotic deformation, the permeation process in such tissues differs from ordinary diffusion processes. In this paper a biomechanical multi-solute theory is introduced to describe this process in cartilage tissue. Typical values for the physiological and biomechanical properties are used in the simulation. Several parameters - the aggregate modulus, the fixed charge density and the frictional parameter - are analyzed to show their impact on the process. It is shown that friction between water and cryoprotectant has the greatest influence but the fixed charge density is also important. The aggregate modulus and the frictional parameter between the cryoprotectant and the solid matrix have the least influence. Both the new biomechanical model and the conventional diffusion model were fitted to published experimental data concerning the time course of mean tissue cryoprotectant concentration when cartilage is immersed in solutions of dimethyl sulphoxide or propylene glycol: in all cases and with both models a good fit was obtained only when a substantial amount of non-solvent water was assumed.

The preservation of tissues for transplantation.

This paper is a written version of a lecture given during the celebration of Professor Rudolf Klen's 90th birthday. Dr. Klen played by far the major part in the introduction and the development of Tissue Banking in Europe. His concept of a tissue bank envisaged the storage of all types of cell, tissue and organ that physicians and surgeons might need for the treatment of their patients. There has been much progress towards this goal, but still the final objective remains elusive. This review of the current position starts with the recognition that some tissues are required to comprise or include cells that exhibit all the formal characteristics of life if they are to function as grafts, whereas other tissues do not. For some tissues, the preservation of mechanical properties is crucial: for others it is not. These considerations are crucial for the design of preservation methods for specific tissues: bone tendon and skin can provide useful grafts in the absence of living cells and this may even be true of cardiac valves: the crucial requirement here is that the mechanical properties remain intact. Simply freezing at around -80 degrees C may be sufficient. In contrast, many cell systems, and all metabolizing organs do require healthy cells to function. Cryopreservation is often an effective remedy for isolated cells, for example haemopoietic stem cells, but the damaging effects of the formation of ice are sufficient to rule out this approach for whole vascularised organs and for some tissues too. The damaging mechanisms are discussed, and it is concluded that the site of ice crystallization is crucial. Cartilage has hitherto been recalcitrant, but we have recently developed a method that permits this tissue to be stored at liquid nitrogen temperatures without any ice and with the recovery of living cells and intact mechanical properties after storage. Thus, many methods are available to help develop tissue banking originally envisioned by Dr. Klen.

Optimising cryopreservation protocols for haematopoietic progenitor cells: a methodological approach for umbilical cord blood.

Current cryopreservation protocols for haematopoietic cells have developed largely empirically and there is no consensus on an optimal method of preservation. These protocols, though providing sufficient cells to permit engraftment, can lead to cell loss of the order of 50 percent. In the context of umbilical cord blood such losses are unacceptable. Whilst an empirical approach can provide an acceptable level of recovery, the cryopreservation process can only be optimised by adopting a methodological approach. This paper provides an overview of just such an approach as illustrated by a study on CD34 cells from umbilical cord blood. It involves firstly the determination of membrane permeability parameters that can then be used to model safe addition and elution protocols for the chosen cryoprotectant, in this case dimethyl sulphoxide. This in turn permits cryoprotectant toxicity to be evaluated free from the confounding effect of osmotic damage caused by inappropriate addition and elution protocols. Finally, non-toxic concentrations of cryoprotectant may be investigated in a cooling rate study to provide an optimal cryopreservation protocol. Using the model, the effect on CD34 cells of current addition and elution protocols was also examined.

Cryopreservation of articular cartilage. Part 2: mechanisms of cryoinjury.

Although isolated chondrocytes can be cryopreserved by standard methods, at the present time there is no satisfactory method that will preserve living chondrocytes in situ in surgical grafts, between the time of procurement or manufacture and actual use; survival of living chondrocytes in situ is inadequate at best and is also very variable. The first step in identifying the cause of this discrepancy was to establish that the cryoprotectants we had chosen to use, dimethyl sulphoxide and propylene glycol, do actually penetrate into the tissue rapidly. They do. Moreover, chondrocytes were shown to tolerate 10 or 20% Me2SO and were not unusually susceptible to osmotic stress. An experiment in which the effects of freezing with 10% Me2SO to -50 degrees C were separated from the effects of the concomitant rise in solute concentration showed that injury was associated with the formation of ice as such. Freeze substitution microscopy showed that large ice crystals were formed within the chondron, some at least within chondrocytes, even when the cooling rate was optimal for isolated chondrocytes. It is proposed that the nucleation and preferential growth of ice within the chondron (rather than the surrounding acellular matrix) is responsible for the very poor survival of chondrocytes in situ when current methods of cartilage cryopreservation are used.

Cryopreservation of articular cartilage. Part 3: the liquidus-tracking method.

Although it is relatively straightforward to cryopreserve living isolated chondrocytes, at the present time there is no satisfactory method to preserve surgical grafts between the time of procurement or manufacture and actual use. In earlier papers we have established that the cryoprotectants dimethyl sulphoxide or propylene glycol do penetrate into this tissue very rapidly. Chondrocytes are not unusually susceptible to osmotic stress; in fact they appear to be particularly resistant. It appears that damage is associated with the formation of ice per se, even at cooling rates that are optimal for the cryopreservation of isolated chondrocytes. We then showed that current methods of cartilage cryopreservation involve the nucleation and growth of ice crystals within the chondrons rather than ice being restricted to the surrounding acellular matrix. This finding established the need to avoid the crystallization of ice--in other words, vitrification. Song and his colleagues have published a vitrification method that is based on the use of one of Fahy's vitrification formulations. We confirmed the effectiveness of this method but found it to be very dependent on ultra rapid warming. However, we were able to develop a 'liquidus-tracking' method that completely avoids the crystallization of ice and does not require rapid warming. The ability of cartilage preserved in this way to incorporate sulphate into newly synthesized glycosaminoglycans (GAGs) approached 70% of that of fresh control cartilage. In this method the rates of cooling and warming can be very low, which is essential for any method that is to be used in Tissue Banks to process the bulky grafts that are required by orthopaedic surgeons. Work is continuing to refine this method for Tissue Bank use.

Cryopreservation of articular cartilage. Part 1: conventional cryopreservation methods.

There is increasing interest in the possibility of treating diseased or damaged areas of synovial joint surfaces by grafts of healthy allogeneic cartilage. Such grafts could be obtained from cadaver tissue donors or in the future they might be manufactured by 'tissue engineering' methods. Cartilage is an avascular tissue and hence is immunologically privileged but to take advantage of this is the graft must contain living cells. Preservation methods that achieve this are required to build up operational stocks of grafts, to provide a buffer between procurement and use, and to enable living grafts of a practical size to be provided at the right time for patient and surgeon. Review of the literature shows that it has been relatively straightforward to cryopreserve living isolated chondrocytes, but at the present time there is no satisfactory method to preserve cartilage between the time of procurement or manufacture and surgical use. In this paper, we review the relevant literature and we confirm that isolated ovine chondrocytes in suspension can be effectively cryopreserved by standard methods yet the survival of chondrocytes in situ in cartilage tissue is inadequate and extremely variable.

The role of vitrification techniques of cryopreservation in reproductive medicine.

Traditional cryopreservation methods allow ice to form and solute concentrations to rise during the preservation process: both ice and high solute concentrations can cause damage. Cryoprotectants are highly soluble, permeating compounds of low toxicity; they reduce the amount of ice that crystallises at any given temperature and thereby limit the solute concentration factor. Vitrification methods use cryoprotectant concentrations that are sufficient to prevent the crystallisation of ice altogether: the material solidifies as an amorphous glass and both ice and solute concentration are avoided. However, the concentrations of cryoprotectant required are very high indeed and therefore are potentially, and often actually, harmful to cells. Optimisation of the temperature and the rate of introduction and removal of such high cryoprotectant concentrations are critical. The necessary concentration can be lowered if very rapid cooling, and even more rapid warming, are used. This paper draws on experience in other fields of cryobiology to discuss these basic phenomena and to consider the place of vitrification techniques in the cryopreservation of human gametes, embryos and gonads.

Use of peracetic acid to sterilize human donor skin for production of acellular dermal matrices for clinical use.

We previously reported methods for sterilizing human skin for clinical use. In a comparison of gamma-irradiation, glycerol, and ethylene oxide, sterilization with ethylene oxide after treatment with glycerol provided the most satisfactory dermis in terms of structure and its ability to produce reconstructed skin with many of the characteristics of normal skin. However, the use of ethylene oxide is becoming less common in the United Kingdom due to concerns about its possible genotoxicity. The aim of this study was to evaluate peracetic acid as an alternative sterilizing agent. Skin sterilized with peracetic acid was compared with skin sterilized using glycerol alone or glycerol with ethylene oxide. The effect of subsequently storing peracetic acid sterilized skin in glycerol or propylene glycol was also examined. Acellular dermal matrices were produced after removal of the epidermis and cells in the dermis, processed for histological and ultrastructural analysis, and the biological function was evaluated by reconstitution with keratinocytes and fibroblasts. Results showed that sterilized acellular matrices retained the integrity of dermal structure and major components of the basement membrane. There were no overall significant differences in the ability of these matrices to form reconstructed skin, but peracetic acid alone gave a lower histologic score than when combined with glycerol or propylene glycol. We conclude that peracetic acid sterilization followed by preservation in glycerol or propylene glycol offers a convenient alternative protocol for processing of human skin. It is suggested that this sterile acellular dermis may be suitable for clinical use.

Banking of non-viable skin allografts using high concentrations of glycerol or propylene glycol.

The aims of this study were to investigate the kinetics of the current glycerol banking method for the preservation of non-viable skin allografts; to improve it with respect to efficiency and microbial safety; and to investigate the possibility of using propylene glycol in place of glycerol to provide a more rapid process. Skin grafts were preserved in 98% v/v glycerol (GLY) according to the method used in the Sheffield Skin Bank. During the addition and removal processes, the amounts of GLY and water in the skin were determined using the Karl Fischer method and HPLC respectively. Propylene glycol (PG) was investigated as an alternative to glycerol with the object of shortening the process. To avoid the need for prolonged storage in glycerol to disinfect the tissue, and to improve the effectiveness of disinfection, exposure to peracetic acid (PAA) was included and its influence on the kinetics of the preservation process was evaluated. The histological and ultrastructural appearances of skin that had been banked by these methods was also investigated. It was found that the permeation of GLY in skin probably involves two processes: diffusion and binding; the rate of transport was attenuated as the GLY concentration in the skin increased. The current incubation time could be shortened, but an inconveniently prolonged washout process was required. The substitution of PG for GLY accelerated the whole process, particularly the removal process, making the method more convenient for the emergency use of skin grafts in the clinic. The penetration of PG also involved diffusion and binding, but there was no attenuation of transport as the concentration increased. The addition of PAA sterilisation did not alter the transport of GLY or PG. Structural integrity was also maintained with the new banking treatments. An improved banking method can now be proposed; it can be completed in only one working day and the risk of disease transmission is reduced.

Vitrification of ECV304 cell suspensions using solutions containing propane-1,2-diol and trehalose.

In this paper, we report on the suitability of solutions containing propane-1,2-diol (propylene glycol, PD), sugars, and salts for the vitrification of the human cell line, ECV304. Cooling (at 10 degrees C/min) and rewarming (at 80 degrees C/min) were at rates that are practicable for the tissues to be studied later. Under these conditions, 45% PD in phosphate-buffered saline (PBS) sometimes froze during cooling and always devitrified during rewarming but both events were avoided if the PBS salts were replaced by an osmotically equivalent concentration of sucrose or trehalose. The effect of such solutions on cells was evaluated using a cell culture assay in which the number of cells recovered after 3 days of culture was divided by the number cells plated, giving a cell multiplication factor or CMF. In the absence of PD the cells tolerated a low-salt concentration in solutions that were made isotonic with sugars, but they recovered poorly when 45% PD was also present. Trehalose gave significantly better recovery than sucrose. When 39% PD and 15% trehalose were included in a low-salt vehicle solution (LSV) that contained approximately 5% of the total salt concentration of PBS (this solution was designated LSV/39/15), the cells exhibited approximately 40% of untreated control CMF following exposure for 9min. LSV/39/15 vitrifies with a glass transition temperature of -102 degrees C, does not devitrify when warmed at 80 degrees C/min, and has suitable dielectric properties for uniform and rapid dielectric heating. An improved method for adding and removing LSV/39/15 gave a CMF of approximately 55% of untreated controls. Using this method, 1.0ml suspensions of ECV304 cells was cooled to, and stored briefly at, -120 degrees C and then rewarmed by immersion in a 37 degrees C water bath ( approximately 75 degrees C/min). The CMF of the cooled samples was similar to that of the exposure-only controls, approximately 50% of the untreated control CMF in both cases.