Long-term storage of tissues by cryopreservation: critical issues

Biomotrrials 17 (1996) 243-256 199fi Elsevier Science Limited Printed in Great Britain. All rights reserved 0

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0142-9612/96/$15.00

Long-term storage of tissues by cryopreservation: critical issues Jens O.M. Karlsson and Mehmet Toner Surgical Services, Massachusetts Genera/ Hospital, Boston, MA 02114 and the Shriners Burns Institute, Cambridge, MA 02139, USA; Department of Surgery, Harvard Medical School, Boston, MA 02114, USA The technique animation’ obstacles

of cryopreservation

at cryogenic

to the development

didactic

overview

with emphasis related issues

of the principles

on the processes

relevant

Keywords: Received

of injury

cells,

and mechanisms

Cryobiology, 18 November

in these

during

1994; accepted

tissue,

20 March

freezing

methods

for tissues

injury

unique

freezing

to tissues to tissue

and current

are reviewed.

of cryopreservation

during

intrinsic

of ‘suspended

applications

and thawing,

occurring

bulk systems,

of freezing

cryopreservation,

methods

changes

of cryopreservation

limitations

in a state

engineering

and the methodology

to cells

and biophysical

samples

use in tissue

cryopreservation

of cryobiology

to the application

ing heat and mass transfer

of biological

its potential

of effective

to the physicochemical

and cultured

(maintenance

temperatures),

A is given,

and how these

cryopreservation. are then addressed,

differences

between

are

Critical includisolated

systems.

injury

1995

protocols may be adequate for largely structural tissue (e.g. cartilage), for tissue in which function can be achieved even with a fraction of the initial cell population (e.g. sperm), or for tissue which can be repopulated from a small number of surviving progenitor cells (e.g. bone marrow), tissue engineering applications typically necessitate stricter requirements on post-thaw tissue viability. For example, in tissue with genetically modified cells, not only must cryopreservation yield high rates of cell survival, but continued expression of engineered phenotypes must be assured. Structural tissues, even though their function depends in large part on the properties of the extracellular matrix, have been shown to be more prone to post-implantation complication and failure if the cell population is non-viable’. Engineered tissues such as bioartificial livers, which may contain primary cells incapable of replicating in vitro, are typically designed with the smallest number of cells which can sustain tissue function, and therefore permit only minimal losses of cell viability during preservation. Even in bioartificial liver devices based on hepatomaderived cell lines, which can be repopulated subsequent to cell loss during freezing, such a restoration process may take several weeks, and may therefore not be a reasonable option for the end-user3. Likewise, it may not be technically feasible to restore the necessary three-dimensional architecture of certain engineered tissues if significant damage has been incurred during cryopreservation. Thus, freeze-thaw procedures for tissue products must result in recovery of both cell viability and tissue structure. Furthermore, in order for the use of engineered tissue

Given recent advances in the field of tissue engineering, and with the first engineered tissue substitutes concurrently undergoing clinical trials, the time when research and development in this field will begin to yield tangible fruit in the form of commercial products is rapidly approaching’. Consequently, the tissue increasingly engineering community is becoming concerned with the problems of bringing tissue products onto the market. One major such obstacle is the issue of preservation and storage of living biomaterials: manufacturers and/or distributors will be faced with the problem of maintaining large stocks in order to ensure a steady supply, while the unpredictable demand for specific tissues in clinical settings will make necessary the creation of tissue banks at hospitals. Simple preservation techniques, such as refrigeration or tissue culture, have drawbacks including limited shelf-life, high cost, risk of contamination or genetic drift. A more tenable option is cryopreservation, an approach based on the principle that chemical, biological and physical processes are effectively ‘suspended’ at cryogenic temperatures. Inasmuch as freezing of cells and tissue explants is not an uncommon procedure, one must be careful not to assume that current cryopreservation procedures can be applied with universal efficacy to engineered tissue. On the contrary, many freezing protocols have resulted in low recovery rates, or altogether non-viable tissue. Although suboptimal cryopreservation Correspondence to Dr M. Toner, Shriners Research Center, One Kendall Square, 14OOW. Cambridge. MA 02139,1JSA.

243

Biomaterials

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Cryopreservation

244 become practical in the clinical setting, the cryopreservation process must require minimal postthaw processing by the end-user. Therefore, cryoprotective chemicals should be used sparingly, since their removal from tissue can require complex dilution procedures, especially when higher concentrations are employed4. Also, evaluation of the success of cryopreservation must take into account the time required for tissue stabilization after thawing, since long post-thaw recovery times requiring days or weeks of tissue culture may be unacceptable to the end-user”. It is clear, then, that the requirements on cryopreservation protocols for tissue storage are formidable, at best. The difficulty of developing high-viability cryopreservation procedures becomes apparent when one considers the hostile environment to which cells and tissues are subjected during the freezing process: the temperature drops from t37 C to -196 C, loss of over 95% of cell water can be incurred, the electrolyte concentration inside and outside the cells can increase by several orders of magnitude relative to isotonic conditions, concentrated organic solvents in the freezing media permeate the cells, ice crystals intercalate the tissue and mechanically deform cells, and ice may form inside cells, disrupting intracellular due to an increased structures. Nonetheless, understanding of the physicochemical processes occurring during cryopreservation, aided by the use of theoretical models to predict the freezing response of cells, it has been possible to develop successful cryopreservation protocols for a number of cell types’. Although experimental studies specifically addressing the problem of cryopreserving engineered tissue”, 7~11 and theoretical models of tissue freezing’“,” are sparse, we stand to gain valuable insight into the issues important in developing suitable cryopreservation methods for bioartificial tissue products from the available information on cell freezing. Whereas the freezing of tissues and tissue equivalents represents a relatively new application of cryopreservation methods, we do not provide an exhaustive literature survey on this topic, but instead attempt to enumerate and discuss the critical issues which must be addressed in order to make the cryopreservation of engineered tissues a reality. Thus, we first provide a didactic overview of the significant principles of cryobiology, then review the basic methodology of cryopreservation, and finally address a number of issues which are specific to the problem of cryopreserving tissue samples. to

PRINCIPLES OF CRYOBIOLOGY At temperatures below - -0.6C, biological water under isotonic conditions becomes thermodynamically unstable, and will favour the crystalline state. Because of the abundance of water in biological systems, the water-ice phase transition in biomaterials is a phenomenon of critical importance in cryopreservation. Here, we summarize the current understanding of the biophysical and biological effects of ice in the extracellular environment and inside the cells, as well as the mechanism and Biomaterials

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kinetics mitigate

of tissue:

J.O.M.

Karlsson

of ice formation, and methods this phase transition.

Physicochemical

and M. Toner

to suppress

or

aspects of ice formation

By definition, the water-ice phase transition in an solution is thermodynamically favourable aqueous only at temperatures below the equilibrium melting point. When ice forms in such a supercooled solution, solutes are rejected from the growing ice lattice, and the unfrozen fraction of the solution becomes increasingly concentrated. Whereas solutes depress the equilibrium melting temperature of the unfrozen solution, the degree of supercooling will decrease concomitantly. When the unfrozen fraction is no longer supercooled, crystallization ceases, as the ice phase is in thermodynamic equilibrium with the liquid solution. The final equilibrium concentration of the unfrozen fraction is a function of temperature only, and is determined by the phase diagram of the solution. On the other hand, the total amount of ice that forms at a given temperature is a function of the initial composition of the solution, and can be determined from phase diagram information using the lever rule. (The lever rule states that the mole fraction of ice is given by (x, ~ x:,)/(1 ~ xk), where x, is the initial mole fraction of water and x1, is the final equilibrium mole fraction of water in the unfrozen the thermodynamic equilibrium fraction.) Thus, and water serves as a regulatory between ice mechanism for both the volume of solution which remains unfrozen and the chemical composition of this solution. Under special circumstances, the amount of ice formed is governed by kinetic rather than thermodynamic Ice formation in supercooled constraints. solutions is initiated by a nucleation process, i.e. the molecules stochastic of water into aggregation thermodynamically stable clusters from which ice crystals can grow’“. Thus, the rate of crystallization depends on the kinetics of both nucleation and the subsequent crystal growth. Because both of these processes slow down if the solution viscosity is increased, the rate of ice formation decreases with temperature. At the so-called glass transition temperature, the viscosity is sufficiently high to effectively preclude molecular diffusion, thus stopping all phase transitions: the unfrozen solution remains in a metastable state, with an amorphous, non-crystalline structure. Inasmuch as ice formation can occur at any temperature below the equilibrium melting point but above the glass transition temperature, the total amount of ice formed during freezing can be modulated by controlling the time allowed for cooling the solution from the melting point to the glass transition point. For example, by using very high cooling rates, the amount of ice forming before the glass transition temperature is reached can be significantly reduced. Vitrification is said to occur if the total fraction of the sample which crystallizes remains below lo-” (Ref. 13). Because increased concentrations of solutes or polymers in the solution will increase the solution viscosity, thus reducing the rates of nucleation and crystal growth,

Cryopreservation vitrification concentrated

of tissue: J.O.M. Karlsson and M. Toner

can occur solutions.

at

lower

cooling

rates

245 in

Biophysical effects of ice formation As a biological sample is cooled to temperatures below its equilibrium melting point, ice forms in the extracellular liquid either spontaneously, due to breakdown of the metastable state of the supercooled solution, or by deliberate ‘seeding’ of ice, typically by touching the sample with a chilled needle. This extracellular ice phase plays a major role in the biophysical response of cells to the cryopreservation process, as it alters the chemical environment of the cells, mechanically constrains and deforms the cells, and can induce ice formation inside the cells. One of the most fundamental consequences of the presence of ice in the external medium is the concomitant regulatory effect on the composition of the unfrozen fraction of the extracellular solution. Because the extracellular solution becomes increasingly concentrated in solutes as temperature decreases and the ice phase grows, a chemical potential imbalance between the cytosol and the unfrozen external solution results, giving rise to a driving force for diffusion of solutes into the cell and for a water flux out of the cell. However, because the permeability of the plasma membrane to water is typically significantly larger than the corresponding solute permeability at low temperatures, the plasma membrane effectively behaves as a semipermeable membrane (on time scales relevant to cryopreservation)14. Consequently, the cell responds to the increased tonicity of the external milieu during freezing by expressing water via osmosis. Mazur’” was the first to mathematically model the kinetics of cell during freezing. Mazur’s seminal dehydration modelling work’” and subsequent modified versions of that the transport of this model*fi.17 have assumed water from the intracellular to the extracellular rate-limited by transmembrane compartment is transport, and not by diffusion to and from the membrane. The validity of this assumption has been verified, and shown to break down only for cells with high permeabilities membrane very (e.g. erythrocytes)l’ or at very low temperatures’“. Thus, the rate of water efflux is proportional to the magnitude of the driving force [i.e. the osmotic pressure difference across the membrane) and the permeability of the plasma membrane to water. The membrane permeability is strongly dependent on and is commonly described by an temperature, Arrhenius relationship as followszo: L, (T) = Lpg rxp

[-%

(Gi)]

where L, is the membrane water permeability; T, the at the reference temperature; L,,, the permeability temperature ( Tr,f); EL,,, the activation energy for water constant. The cell and R, the gas transport; dehydration kinetics during freezing are thus sensitive to the values of L,, and EL,,. Because these parameters considerably between different cell vary may a corresponding variability in the response typesy7,“,

of various cells to the same freezing protocol is expected. The ice matrix surrounding the cells also acts as a mechanical constraint, and can cause cell deformation as an increasing proportion of the extracellular medium solidifies. However, because the ice crystal morphology is extremely sensitive to experimental conditions, the mechanical effects of ice on cells have been difficult to characterize quantitatively. Nonetheless, because cells are typically sequestered into channels of unfrozen solution between ice crystals, the fraction of the extracellular solution which remains unfrozen has been used as a measure of the amount of liquid volume available to the cells?. Rapatz et al.‘” have directly measured the width of the unfrozen liquid channels between ice crystals, and observed that channel diameters decreased with temperature and that cells confined in the channels became deformed as the channel width decreased to cell dimensions. Tondorf et ~1.‘~ found that the force of adhesion between cell-sized liposomes and a planar ice interface is significant. Even though a similar investigation of the adhesion between oocytes and ice did not find support for an adhesive forceZ5, the results of Tondorf et ~1.‘~ indicate the potential for significant direct mechanical interaction between ice and cells. In addition to influencing the chemical and mechanical environment of the cell, the extracellular ice is thought to be directly involved in the initiation of ice formation inside cells. Experimental comparison of ice formation in cells frozen in the presence and in the absence of extracellular ice has shown that intracellular ice formation occurs at significantly higher temperatures when extracellular ice is present, suggesting that external ice induces or catalyses intracellular ice formation17.2fi. Although the exact mechanism of intracellular ice formation and the role of the external ice in this process are not known, several hypotheses have been advanced to explain ice formation in cells, and have served as frameworks for mathematical models to predict intracellular ice models have been used formationz7. Phenomenological models have by Mazur’a and Pitt16, while mechanistic been developed by Toner et 01.~” and Muldrew and McGann”‘. Here, we will use the approach of Toner et al.‘“, which assumes that ice forms inside cells by nucleation on intracellular catalytic sites. A mathematical model based on this theory has been successful in predicting intracellular ice formation in a wide range of diverse cell types17. The model uses classical nucleation theory” to obtain an expression for the rate of formation of intracellular ice nuclei, the ice-like clusters of water molecules from which ice crystals grow. The rate of nucleation is strongly dependent on the degree of cytoplasmic supercooling, as is evident from its mathematical form: j(T)

= Uexp-ti

Tm”ATm2]

(2)

where J is the nucleation rate; AT, the supercooling; R and K, the kinetic and thermodynamic coefficients, respectively. The coefficients 62 and K are different for each cell type17. Nucleation is a stochastic process, and Equation 2 can be used to calculate the probability of ice formation in a given cell population undergoing Biomaterials

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246

cellP. In assuming identical cryopreservation, practice, freezing conditions which result in intracellular ice formation typically have an associated characteristic nucleation temperature at which the probability of ice formation rises sharply from 0 to 1. However, nucleation theory is not sufficient to fully predict the extent of intracellular crystallization, since it predicts only the onset of ice formation. Recently, a mathematical model of the growth of intracellular ice nuclei into macroscopic crystals was developed, assuming diffusion-limited crystal growthl”. In this model, the crystal growth velocity is proportional to of intracellular water and the the diffusivity cytoplasmic supercooling, which is the driving force for crystallization. Thus, the radius of an intracellular ice crystal as a function of time is given by the following equation: 12 4 z(t)‘D(t) dt (3) r,: (t) = 1 [I 0 where t is the time after appearance of the ice nucleus; x, the non-dimensional crystal growth parameter (which is proportional to the cytoplasmic supercooling AT); D, the effective diffusivity of intracellular water. Because the cytoplasmic supercooling and the diffusion constant for intracellular water depend on the instantaneous properties of the intracellular solution, the dynamics of ice formation inside cells are highly modulated by the concurrent dehydration process. Due to water transport during freezing, the composition and volume of the cytosol are continually changing, thus affecting the rates of nucleation and crystal growth’“. This physical coupling between the processes of ice formation and freeze-induced dehydration is characteristic of the freezing of cells, and is absent in the freezing of non-cellular matter. Intracellular water which becomes supercooled during freezing may attain thermodynamic equilibrium either by leaving the cell and forming extracellular ice, or by forming a new ice phase inside the cell. The fate of the cell water during cryopreservation thus depends on the relative magnitudes of the rate of water transport and the rate of nucleation. When cells are cooled slowly, the rate of water efflux is sufficiently high to prevent excessive levels of supercooling, and thus cell dehydration is favoured over intracellular ice formation. Conversely, at rapid rates of cooling, water exosmosis is slow compared to the rate at which the intracellular solution becomes supercooled, and intracellular ice formation is favoured. Using a mathematical model of the water transport process, together with models of intracellular nucleation and crystal growth, it is possible to predict what proportion of a cell population will form intracellular ice during freezing and what fraction of the intracellular liquid will crystallizel”. Biological

effects of ice formation

As demonstrated above, cells are subjected to a range of thermal, chemical and mechanical forces during cryopreservation, which can profoundly affect their biological function. Although the suspension of metabolic and other reactions at cryogenic

of tissue:

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and M. Toner

temperatures is beneficial for long-term storage of biomaterials, the processes of freezing and thawing are often deleterious. Due to the complexity of biological systems, the exact mechanisms of cell damage during cryopreservation have not yet been elucidated. However, characteristic survival signatures generated by measuring cell survival as a function of cooling rate appear to be qualitatively similar for all cell types. Figure 1 shows a typical survival signature for a hypothetical cell: cell survival is low at very slow and very fast cooling rates, and there is an intermediate cooling rate yielding optimal survival. Even though the optimal cooling rate and the width of the curve can vary by orders of magnitude for different cell types, the qualitative behaviour appears to be universal. Based on such observations, Mazur et al.“’ proposed the ‘two-factor hypothesis’ of freezing damage, according to which there are two independent mechanisms of damage during freezing, one active at slow cooling rates, the other at fast cooling rates. Cell injury at fast cooling rates is attributed to intracellular ice formation, although membrane rupture due to osmotic fluxes has also been proposed as a damage mechanism at fast cooling rates’“‘.“2. At slow rates of cooling, cell injury is thought to be due to the effects of exposure to highly concentrated intra- and extracellular solutions or to mechanical interactions between cells and the extracellular ice. There is significant evidence for a correlation between cell injury and intracellular ice formation during rapid cooling”, It is generally believed that injury results from mechanical forces due to the intracellular iceZ8; possible sites of damage include the plasma membrane”” and the membranes of intracellular organellesi4. Various non-mechanical modes of injury have also been proposedZ7, for example, the induction of gas bubble formation by intracellular ice”“,“f’, or osmotic effects due to the melting of intracellular ice during warming”‘. Despite the strong evidence for involvement of intracellular ice formation in cell damage during freezing, observed cases of innocuous ice formation in cells indicate that the presence of intracellular ice per se does not cause cell death, especially when the extent of intracellular crystallization is limited”s~“H-40. The high concentrations of electrolytes and other solutes in the extracellular medium and the resulting cell dehydration during slow cooling have been proposed as a source of cell damage’“,4’. A number of specific mechanisms have been suggested to explain these so-called ‘solution effects’. Lovelock provided evidence that hypertonic salt solutions caused denaturation of lipoproteins, and that this process could induce haemolysis in red blood cells. Other theories have focussed on the potentially damaging effect of cell shrinkage as a response to a highly extracellular solution. Meryman4” concentrated proposed the existence of a critical minimum cell volume, shrinkage beyond which was presumed to be deleterious, while Steponkus et a1.44 showed that plasmalemma lipid can be deleted from the membrane during dehydration, osmotic and suggested that damage occurs during rehydration if there is insufficient membrane material for the cell to return to

Cryopreservation

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Karlsson

and M. Toner

its isotonic volume. The osmotically induced flow of water through the cell membrane has also been proposed as a potential source of damage3’. In addition to solution effects, mechanical interactions between the extracellular ice and cells have been implicated as a possible source of cryoinjury at suboptimal cooling rates. Nei4” demonstrated that, at temperatures above -lO”C, erythrocyte haemolysis cannot be attributed to increased electrolyte concentrations alone, and concluded that mechanical effects are a significant factor in cell damage. Mazur and coworkers experimentally varied the fraction of solution which remained unfrozen during a given freezing measuring protocol, the resulting survival of erythrocytes and mouse embryos, and found that for values of the unfrozen fraction below some critical level, there was a significant inverse correlation between the unfrozen fraction and cell damage’~22’4fi. Ashwood-Smith et ~1.~~ showed that contact between extracellular ice crystals and oocytes or embryos could predispose these cells to damage at the contact site, while electron microscope studies by Fujikawa and codirect evidence for ultrastrucworkers47,4R provided tural changes in the plasma membrane caused by mechanical stress due to the formation of extracellular ice. Directional solidification techniques, which allow ice crystal morphology to be controlled without varying the thermal or chemical history experienced by cells, have been used by Hubel et ~1.~” to investigate possible mechanical effects of the extracellular ice. Hubel and co-workers found that cell damage at slow rates of cooling appeared to be correlated with the presence of secondary branches on ice dendrites, and suggested mechanical shear forces due to dendrite coarsening as a possible damage mechanism. The inherently complex nature of cells and the inability to decouple the effects of the diverse insults suffered by cells during cryopreservation have made it exceedingly difficult to determine conclusively what mechanisms are responsible for cell injury during freezing and thawing. Further research is clearly necessary, particularly on the modes of damage during slow freezing.

METHODOLOGY

OF CRYOPRESERVATION

There are two basic approaches to cryopreservation of cells and tissues: freeze-thaw procedures and vitrification. In freeze-thaw preservation techniques, the extracellular solution is frozen, but steps are taken to minimize the probability of intracellular ice formation; in vitrification procedures, there is an attempt to prevent ice formation throughout the entire sample. The former approach takes advantage of the regulatory properties of the extracellular ice, whereas the latter seeks to avoid the potentially damaging effects of intraand extracellular ice. The cryopreservation protocols for both techniques typically involve loading the with cryoprotective chemicals and then sample cooling the sample to the temperature at which it will be stored. In freeze-thaw procedures, but not in vitrification protocols, ice formation is induced in the extracellular solution before cooling commences. Upon

247

removal from storage, the sample is thawed and cryoprotective chemicals are removed from the system by dilution. Below we discuss briefly the methodology and motivation of each step in the cryopreservation procedure.

Cryoprotective additives Ever since the discovery in 1949 that glycerol afforded protection to sperm during cryopreservation5’, it has become common practice to add one or several cryoprotective agents to freezing media. Cryoprotective chemicals can be divided into two categories: (i) permeating cryoprotectants, e.g. dimethyl sulphoxide (DMSO), glycerol, 1,2-propanediol, which can pass through cell membranes; and (ii) nonpermeating cryoprotectants, e.g. polymers such as polyvinyl pyrrolidone, hydroxyethyl starch and various sugars, which cannot enter cells. The mechanism by which these agents protect cells against stresses encountered during cryopreservation is not known, but it is thought that permeating cryoprotectants reduce cell injury due to solution effects by reducing potentially harmful concentrations of electrolytes in the cell. Cryoprotectants have also been thought to stabilize cell proteins entropically if they are preferentially excluded from the hydration shell of the protein5rs5’, while there is evidence that polar solvents such as DMSO stabilize the plasma membrane by electrostatic interactions5”. Furthermore, protection against intracellular ice formation has been attributed to the colligative effects of cryoprotectant chemicals54. In high concentrations, cryoprotective additives result in an increased viscosity of the extra- and intracellular solutions, and thus dramatically reduce the rates of ice nucleation and crystal growth. In fact, this is the basis for vitrification protocols, which employ very large concentrations of additives (6-9 M)“” in order to suppress ice formation altogether during cryopreservation. Despite the protection they afford to cells during cryoprotective chemicals can freezing and thawing, themselves be damaging to cells, especially when used Toxicity can be reduced by in high concentrationss6. decreasing the time or temperature of cell exposure to the cryoprotectant, or by using lower concentrations of the additive. Paradoxically, there is also evidence that the presence of cryoprotectants can cause increased retention of cytoplasmic water and enhance the probability of intracellular ice formation, thus causing cell injury’gZ57. Furthermore, the addition and removal of cryoprotectants before and after cryopreservation can cause damage to cells due to excessive osmotic forces. Because cryoprotectants typically enter and leave the cell at a rate slower than water, the initial response of a cell exposed to a cryoprotective solution is to lose water by exosmosis. Likewise, a cell containing cryoprotective solutes will initially swell when placed in an isotonic environment, as water enters the cell by osmosis. Even though the cell volume will eventually return to its isotonic value as the cryoprotectant permeates the cell membrane and equilibrates, excessive volumetric excursions and the attendant high osmotic water fluxes can be deleterious. Biomaterials

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Cryopreservation

Thus, cryoprotectants are usually added and removed gradually, changing the concentration of the extracellular solution in a stepwise fashion. However, the cryoprotectant addition benefits of slow, stepwise must be balanced against the deleterious effects of increased exposure times to the cryoprotectant. A mathematical model of water and cryoprotectant transport was used by Levin and Miller4 to theoretically predict optimal protocols for loading and diluting cryoprotectants.

Seeding

of extracellular

ice

When cryopreserving by a freeze-thaw method, ice formation in the extracellular medium should be deliberately initiated by seeding at low degrees of supercooling. If ice formation is not induced by seeding, ice will form spontaneously when the solution is cooled sufficiently far below its equilibrium melting point. Because this process is stochastic in nature, ice formation will occur at random, unpredictable temperatures. Consequently, survival rates will be highly variable between repeated trials with the same freezing protocol. Furthermore, the extremely rapid crystallization which results when ice forms in a highly supercooled solution can cause damage to cells and tissues. Moreover, it has been shown that if extracellular ice formation is initiated at high degrees of supercooling, the probability of intracellular ice formation is drastically increased”“.““. This phenomenon results from the increased cooling rates experienced by the sample as a consequence of thermal fluctuations caused by the release of latent heat during extracellular ice formation”‘, or from the delayed onset of freeze-induced cell dehydration, which results in increased retention of intracellular water, and thus higher probabilities of ice formation in the cell”“. To avoid the problems of uncontrolled ice formation in the extracellular solution, it is advisable to deliberately induce extracellular ice formation at temperatures slightly below the solution melting point. Extracellular ice formation is typically initiated by cooling the solution to a few degrees below its equilibrium melting temperature and then touching the sample with a chilled needle or similar implement. Ice will form at the point of contact and spread through the biological sample. To allow this crystallization process to complete, and to thermally and chemically equilibrate the sample after the release of solutes and heat of fusion resulting from ice formation, it is common to hold the specimen at the seeding temperature for some time before starting the When cooling protocol. cryopreserving larger specimens, an alternative method for induction of extracellular ice formation can be used. The sample is cooled to the desired ice seeding temperature, then initiated rapid cooling is until ice forms spontaneously at the sample surface, at which time the sample is immediately rewarmed to the seeding large temperature”. In samples, the interior temperature of the specimen remains approximately constant at the seeding temperature during rapid cooling, and only the surface temperature decreases. 13iolll;ltc,rials19%.

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Consequently, ice formation is induced at the sample crystallization progresses into the surface and supercooled interior. This method of ice seeding has the advantage that manipulation of individual specimens is not required, and this approach may therefore be favourable when processing large quantities of tissue.

Cooling The rate of cooling to the final storage temperature can profoundly affect the fate of the frozen biological sample (see Figure 1). The effect of cooling rate on cell water content and on the characteristic intracellular ice formation temperature is illustrated in Figure z for cultured hepatocytes frozen in the presence of 1.33 M DMSO. At rapid cooling rates, the intracellular water volume remains almost constant during cooling to -8O”C, because there is insufficient time for the water to leave the cell by exosmosis. At slow cooling rates, cell water can leave to equilibrate with the extracellular ice, and high levels of dehydration ensue with concomitant increases in solute concentration. Increased concentrations of intracellular solutes increase the viscosity of the cytoplasm and depress the equilibrium melting point of the solution, thus decreasing the cytoplasmic supercooling. As a result, the characteristic intracellular ice formation temperature (at which 50%) of the cells have developed intracellular ice) is significantly depressed at slow cooling rates. Conversely, at fast cooling rates, cytoplasmic supercooling increases rapidly and intracellular ice formation occurs at relatively high temperatures. However. at extremely rapid cooling rates (e.g. >2OO C min ’ for cultured hepatocytes; see Figure 21, the intracellular ice formation temperature is depressed due to kinetic effects”‘. The dependence of cell dehydration on cooling rate is of practical use, inasmuch as slow or intermediate cooling rates can he used to control the concentration and intracellular volume of the

----------_________

--\

0

Cooling

.\ , ________---------.

tlmal g ale

Supraoptlmal

Rates

Rate (“Cimin)

Figure 1 Survival signature of a hypothetical frozenthawed cell, showing the competing modes of damage at slow and rapid rates of cooling, and the resulting optimal cooling rate. Freezing injury at suboptimal cooling rates is attributed to ‘solution effects’ and mechanical interactions with the extracellular ice, while damage at supraoptimal rates of cooling is thought to be due to intracellular ice formation or osmotic rupture (see text for details).

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249 Storage Although temperatures below -80 ‘C are generally required for successful preservation of cells and tissues for extended periods of time, shelf-life increases dramatically as the storage temperature is reduced. At -196°C (the boiling point of liquid nitrogen) there is insufficient thermal energy for chemical reactions”‘, and the only deterioration that can occur in a biological sample is DNA damage by background radiation and cosmic ray?. The shelf-life of cells stored at liquid nitrogen temperatures has been estimated to be of the order of 10” years’.

5000 Wmin

500

Warming

0

-20

-40 Temperature

-60

-80

[“Cl

Figure 2 Predicted kinetics of water transport and intracellular ice formation in cultured hepatocytes during cooling to -80°C at various cooling rates, in the presence of 1.33 M DMSO. Solid lines show the intracellular water content, normalized to isotonic conditions. Filled circles indicate the temperature at which 50% of the cell population has undergone intracellular ice formation, for each cooling rate. Predictions were obtained using methods described in detail elsewhere4’. Note that the apparent second intersection of the water transport curve and the intracellular ice formation temperature curve for cooling at 1OO’C mini’ IS an artefact due to the finite number of cooling rates represented in the plot.

solution. Note, however, that for all cooling rates, cell dehydration stops at some characteristic temperature (- -40°C for cultured hepatocytes; Figure 2). This is due to the Arrhenius temperature dependence of the membrane permeability (Equation 2); at water sufficiently low temperatures, the cell membrane becomes impermeablelg. effectively Thus, the thermodynamic state of the intracellular solution can be manipulated by dehydration only within a finite temperature range. Even though the use of constant-rate cooling protocols is practically ubiquitous, there is no intrinsic reason why a constant rate of change of temperature should be optimal. Indeed, it has been shown that multistep protocols (piecewise linear) can yield superior results to simple linear cooling method8’. Pitt16 has demonstrated the potential benefit of nonlinear protocols, in which the rate of cooling changes continuously with time. The main drawback of using multistep or non-linear cooling protocols is the of parameters that must be increased number optimized to achieve acceptable survival rates postfreezing. For all but the most simple cooling methods, the number of experiments required for protocol optimization becomes prohibitive. However, recent advances in the ability to predict the effects of candidate cryopreservation protocols using mathematical models have permitted theoretical optimization of non-linear protocols’6*5g.

In addition to the cooling rate, the rate at which frozen samples are rewarmed to normothermic temperatures can be important. During warming, the sample devitrifies, and in the finite time necessary to attain the equilibrium melting point, further nucleation and crystal growth can occur. Furthermore, small, innocuous intracellular ice particles that may have formed during freezing can coalesce into larger, damaging crystals during warming, by a process known as recrystallization”. Devitrification and recrystallization are more likely to occur in rapidly cooled cells, because these will have a high degree of cytoplasmic supercooling and are more likely to contain intracellular ice nuclei. To minimize damage due to devitrification and recrystallization, rapid warming rates are used. However, in some cases rapid warming rates can yield lower postthaw survivals than slow rates, especially for cells that have been frozen at a slow rate6. It has been proposed that rapid warming damage is due to stresses that may occur during rapid osmotic rehydration of the cell, and is less likely to occur in rapidly cooled cells because these do not dehydrate significantly during coolingz8. Thus, even though the exact mechanisms by which the warming process influences rapid cell survival are not known, warming rates appear to be beneficial for rapidly cooled samples, while slow warming rates may be preferable for slowly cooled samples.

CRYOPRESERVATION

OF TISSUES

Demonstrably, reversible cryogenic storage of complex tissues is a difficult problem, and success generally requires careful control of each step of the cryopreservation Although the process. accumulated understanding of many important biophysical processes relevant to the cryopreservation of cells will be an invaluable aid for gaining insight into tissue freezing, there is evidence that the intrinsic response of cells to the cryopreservation process is different if the cells are part of a tissue or if they are isolated. In addition, the scale-up of cryopreservation procedures from a microscopic cellular level to a macroscopic tissue scale will introduce new problems related to heat and mass transfer phenomena in larger systems, as well as modes of injury specific to tissue freezing. These problems are discussed below. Biomaterials

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Intrinsic differences between cultured and isolated cells for investigating the A common starting point cryobiological behaviour of a tissue is to characterize the biophysical properties of individual cells isolated from it. For example, McGann et nl.“” have studied isolated chondrocytes in an effort to gain insight into the freezing response of articular cartilageCi4. In addition, there are no practical techniques for directly measuring the water transport parameters (L,,, and EL,,) for cells which are anchored to an extracellular matrix, since conventional methods for determining the membrane permeability require direct measurement of volumetric changes of cells in response to osmotic (although an indirect measurement variations technique has been used by Yarmush et al.““). However, several recent reports suggest that cells vary with cryobiological behaviour regards to their depending on whether they are freshly isolated or maintained in culture. Rat hepatocytes cultured in a double collagen gel show a higher tendency for intracellular ice formation than do isolated hepatocytes, i.e. intracellular ice forms at higher temperatures and lower cooling rates’“. Similar observations have been reported for bovine cornea1 endothelial cells grown in culture flasks”’ and hamster fibroblasts cultured on glass”“. These differences in intracellular ice formation kinetics between cultured and isolated cells indicate that the biophysical parameters which govern the water transport and intracellular nucleation dynamics during freezing are different for isolated cells versus cells in tissue. Biophysical parameters for isolated and cultured rat hepatocytes were compared directly by Yarmush et nl.““-, who found that the water permeability parameters L,,, and EL,’ (see Equation I) were an order of magnitude larger for isolated cells; i.e. although isolated hepatocytes dehydrate faster than cultured cells at high temperatures, the permeability of isolated cells decreases more rapidly as a function of decreased temperature. The nucleation parameters !I and I\’(see Equation 2) were also elevated in isolated hepatocptes when compared with hepatocytes in culture”“, indicating that intracellular ice formation requires higher degrees of cytoplasmic supercooling, but that nucleation kinetics are more rapid. The reason for the observed differences between isolated and cultured cells are not known, but cell-cell and cell-matrix interactions have been suggested as possible factors in this phenomenon”“.““. Another possibility is that the process of isolating cells from tissue (e.g. by enzymatic digestion of the extracellular matrix) changes the biophysical properties of the cells. From a practical standpoint, the intrinsic difference between the cryobiology of cultured and isolated cells not only means that caution must be used when extrapolating cryobiological data from isolated cell suspensions to the analysis of tissue cryopreservation, but also that freezing of cells in culture may require significantly different strategies than the cryopreservation of isolated cells. For example, the activation energy for water transport (EL,,) determines the range of temperatures in which the cell dehydration process is Biomaterials

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active for a given cell type, and can thus have a major effect on the intracellular ice formation characteristics of the cell’“, and hence also on the requirements to avoid cell damage. Figure 3A and B shows, in the phase diagram of the intracellular solution of cultured and freshly isolated hepatocytes, respectively, the liquidus (T,,,), as well as the predicted intracellular ice formation temperature, as a function of intracellular DMSO concentration. As can be seen from the intracellular ice formation temperature curve ( TIlr:), a concentration of at least 9.1 M DMSO is required to avoid intracellular ice formation when cooling the cell to liquid nitrogen temperature. Although this concentration is in principle attainable by loading cells with a low, non-toxic concentration of DMSO, and taking advantage of the freeze-induced dehydration process to increase the intracellular concentration of cryoprotectant, the time required to reach the necessary level of dehydration may be excessive if the membrane permeability is too low. Indeed. using Equation I, it can be shown that the characteristic time constant for cell dehydration (T) increases exponentially with decreased temperature, and thus that there exists a characteristic temperature below which the dehydration process will require protocols of extreme duration and concomitant overexposure of the biological sample to concentrated solutions. Thus, there is a finite range of temperatures within which it is practical to use the cell dehydration process to control the composition of the cytosol. In Figure 3, this region is demarcated by the temperature at which r=lh. Furthermore, because freeze-induced dehydration requires the presence of extracellular ice, the liquidus curve (T,,,) marks a second constraint on the locus of thermodynamic states in which water

~140 0

2

4

6

DMSO Concentration

8

[M]

10

0

2 DMSO

4

6

Concentration

8

10

[M]

Figure 3 Phase diagram of the intracellular solution of cultured (A) and isolated (6) hepatocytes, in the presence of DMSO. T, is the equilibrium melting temperature (the liquidus) and TllF is the predicted intracellular ice formation temperature. The line marked 'T = 1 h’ indicates the temperature at which the time necessary to achieve 95% dehydration of the cell is 1 h, as estimated using simulations of water transport. The dotted curve (in A) represents the trajectory of thermodynamic states of the intracellular solution during cooling at 1.C min-’ of cultured hepatoinitially containing 1.33~ DMSO. Mathematical cytes models used in the predictions of these curves are described in detail elsewhere4’; water transport parameters for isolated hepatocytes were taken from the literature65, while nucleation rate parameters for isolated hepatocytes were determined from published experimental data69; model parameters for cultured hepatocytes are given by Karlsson et a1.40.

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transport can be used to modify the intracellular solution. Therefore, the maximum intracellular cryoprotectant concentration that can be obtained in practice by freeze-induced dehydration can be estimated from the intersection of the liquidus curve and the r = 1 h constraint. As demonstrated in Figure 3A, it is possible to dehydrate cultured hepatocytes sufficiently to be able to avoid intracellular ice formation during cooling (by concentrating the intracellular DMSO to >9.1 M). In contrast, due to the higher water permeability activation energy for isolated hepatocytes, the maximum intracellular DMSO concentration that can practically be achieved using freeze-induced water transport is less than 4 M (Figure 3); at this concentration, intracellular ice formation will occur at - -25°C. Thus, cryopreservation of isolated hepatocytes will require a drastically different approach from that needed for hepatocytes in culture. For example, it may be necessary to cryopreserve isolated hepatocytes by vitrification (i.e. using high initial cryoprotectant concentrations and ultrarapid cooling rates). Although the curves in Figure 3 are theoretical estimates, they demonstrate that it may be inappropriate to design cryopreservation methods for tissues based on biophysical parameter measurements obtained on isolated cells. Hence, it will be important to develop improved experimental techniques for directly measuring the water transport and nucleation parameters for cells in tissues.

Mass transport limitations in tissue cryopreservation In the cryopreservation of biological tissue, as in cell cryopreservation, two mass transport processes are of critical importance: the addition and removal of cryoprotectant chemicals before and after freezing, and the redistribution of biological water during freezing and thawing. Whereas these processes were governed by membrane-limited water transport in the case of individual cells, one must take into consideration diffusive processes as well as cell-cell interactions to fully understand the behaviour of multicellular tissue during cryopreservation. Some insight into the nature of water transport in biological tissue during freezing has been gained from the development of mathematical models of this process. Levin et a1.70 considered the osmotic response of a one-dimensional array of cells during freezing, that water must diffuse with the constraint sequentially from one cell to its neighbour, with no parallel transport allowed through the interstitial space. Thus, while cells in the surface layer would respond to freeze-induced osmotic changes much like cells in suspension, interior cells would dehydrate only as a response to the increased intracellular in the dehydrated surface layers. tonicity Consequently, interior cells were predicted to lose water at a slower rate than surface cells during freezing, and to contain more water at the termination of freezing7’. Diller and Raymond” have recently developed a model of water transport in tissue, including multiple cell layers as well as an interstitial matrix through which water may flow. Although this

251

represents a more realistic model of tissue, incorporating both cell-cell and cell-interstitium transport, the results are qualitatively similar to those previously obtained by Levin et a1.70, with interior cells dehydrating more slowly than surface cells. It should be noted, however, that this effect is expected primarily in densely populated tissues. In tissues with sparse cell populations, e.g. cartilage, ice will intercalate the highly hydrated extracellular matrix, and thus interior cells will be in direct communication with extracellular ice, preventing delays in the osmotic response. Similarly, in highly vascularized tissue such as liver, interior cells can exchange water directly with ice in the vasculature7’. In contrast, during freezing of multilayer tissue such as skin, interior cell layers will tend to retain more than cells in the surface layer. This effect has also been observed in neoplastic liver tissue, which is largely avascular: compared with normal liver tissue, cells in the interior of tumours appeared to resist freeze-induced dehydration7’. In addition to water transport in tissues, one must consider the dynamics of cryoprotectant diffusion into and out of tissue during the addition and removal of cryoprotectants before and after freezing, in order to appreciate the effect of these procedures on tissue survival. Mathematical models of cryoprotectant transport in tissue have focussed mainly on the kinetics of organ perfusion: whereas early models considered the extravascular space to act as a single, homogeneous solution4’ 73, 74, Lachenbruch and Diller75 recently developed a model incorporating extravascular space and dynamic coupling between fluid exchange and elastic swelling of tissue during perfusion. More relevant to the cryopreservation of tissue samples is the diffusion of cryoprotective chemicals through the tissue. An analysis of DMSO diffusion through the collagen matrix of a hepatocyte double-gel culture has been presented by Bore1 Rinkes Schreuders et a1.77 have derived et a1.76, while equations describing the coupled diffusion of multiple chemical species through an interstitial matrix. By fitting their theoretical equations to experimental measurements of DMSO concentration, Bore1 Rinkes et a1.76 determined the time constant for DMSO diffusion into the collagen matrix containing a monolayer of hepatocytes, and found that at 22”C, a 15 min exposure to DMSO was required for 95% equilibrium of the tissue with cryoprotectant. Permeation of cryoprotectants into tissue and organs has also been measured directly, using nuclear magnetic or highresonance spectroscopy78-80 Several performance liquid chromatography81. researchers have observed a biphasic behaviour in the kinetics of cryoprotectant permeation, suggesting the existence of separate diffusion compartments in the tissue, with distinct time constants for diffusion78.81. Specific diffusion times depend both on the composition and geometry of the tissue, and may vary from the order of minutes to the order of hours. The long equilibration times necessary for cryoprotectant loading into tissues can have both beneficial and adverse effects on the tissue cell population. Whereas longer exposure times to cryoprotectant chemicals are associated with higher probabilities of toxicity, the Biomaterials

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252 gradual increase of extracellular solute concentration, due to the rate-limiting effect of diffusion, appears to reduce osmotic stress and obviates the need for incremental addition of cryoprotectants7’i. In addition to longer time constants for cryoprotectant equilibration, mass transport limitations in macroscopic tissue rise to spatial concentration also give samples measured the gradients. Carpenter and Dawson’l spatial distribution of DMSO in tissue during cryoprotectant loading, and found a significant lag (-1 h) for DMSO incorporation into the interior of a I cm” sample of porcine myocardium compared with DMSO uptake at the periphery of the sample, with corresponding spatial concentration gradients of the order of -1 M cm ‘. Thus, in large tissue samples, there may be significant differences in cryoprotectant concentration between the surface and interior of the tissue. Consequently, one may have a situation in which surface cells must be exposed to toxic concentrations of cryoprotectant in order to attain the minimal necessary cryoprotectant concentration in the tissue interior. Conversely, if care is taken not to harm outer cells during cryoprotectant addition. interior cells ma! have too low concentrations of oryoprotectant, and thus not be sufficiently protected against freezing. In order to fully understand the cases in which mass transport processes limit the effective use of cryoprotectants in tissue freezing, and to explore strategies for circumventing these problems. better models of cryoprotectant diffusion in tissue must be developed, and techniques to measure cryoprotectant distribution in tissues must be improved.

Heat transport limitations cryopreservation

in tissue

Heat transport limitations associated with the scale-up of cryopreservation methods to tissue dimensions are largely analogous to the mass transport problems encountered in tissue cryopreservation. Thus, due to the macroscopic size of the specimen and its finite thermal conductivity, it is generally more difficult to achieve rapid cooling and warming rates in tissue than in cell suspensions. Similarly, there may be large thermal gradients from the surface to the interior of the systemH2. Although these temperature differentials may not be disadvantageous per se, they imply nonuniform rates of cooling through the tissue sample, with slower rates of temperature change in the interior of the sample compared with the surface. For vitrification methods of cryopreservation, the slow cooling rates in the tissue core necessitate the use of very high concentrations of cryoprotectant additives, which can be damaging”“. The non-uniform spatial distribution of cooling rates is also a problem in freeze-thaw techniques, because of the high sensitivity of survival to cooling rate (Figure 1). If the difference in cooling rates between the interior and exterior is larger than the width of the survival curve, then it may not be possible to recover full viability throughout the tissue. Diller and co-workers have modelled the temperature and cooling rate distributions in bulk tissue samples during freezing and combined these predictions with survival signature data for isolated cells to obtain I3iornatnrials

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estimates of total cell survival in the tissue and the spatial distribution of cell damage”“PH4. Although these models can provide useful insight into the effect of heat transfer phenomena in bulk systems on tissue cryopreservation, they are limited by the assumptions that the survival signature for isolated cells is valid for cells in tissue (see discussion above), and that the cell survival signature is identical for all cells in the sample. However, it should be noted that as a result of the slower water transport from cells in the core of densely populated tissue, these interior cells require slower rates of cooling than surface cells in order to achieve levels of dehydration sufficient to prevent intracellular ice formation; i.e. the survival curve shifts towards lower cooling rates’“. Thus, during cooling of a tissue sample, the shift in the optimal cooling rate may partiallv compensate for the reduced rates of cooling that -can be achieved in the interior of the specimen. The magnitude of this effect depends on the relative rates of mass and heat transfer in the tissue, and may be altogether absent in tissues such as cartilage or liver, where water flow in the tissue interior is not limited by mass transport constraints. may Likewise, heat transfer limitations cause problems during the warming of crvopreserved tissue. For example, if the interior of vitrified tissue samples is warmed too slowly, the sample mav undergo damaging devitrification and recrystallization processes~~. ($2.Hfi, Similar damage can also occur in tissues cryopreserved by freezing methods: cells which are not fully dehydrated can devitrify during thawing, and innocuous ice crystals that mav have formed inside cells during freezing may grow to damaging proportions if the warming rate is too slow. Indeed, Bore1 Rinkes et al.’ noted a strong dependence of the survival of frozen-thawed hepatocyte cultures on the warming rate (with rapid warming yielding higher survival) if the tissue had been cooled to below its intracellular ice formation temperature4”. In tissue with multiple cell layers, cells in the tissue interior, which tend to retain water during freezing, are more likely to undergo damaging recrystallization than dehydrated cells at the tissue surface, thus requiring faster rates of warming in the tissue core than at the sample surface. Whereas the warming rate in the tissue interior is slower than at the surface, one is faced with a situation opposite to that encountered during freezing: i.e. the optimal warming rate increases towards the interior, while the effective warming rate decreases towards the centre of the bulk sample. Thus, heat transfer limitations may be more critical during the warming phase of cryopreservation than during freezing.

Freezing

damage

in tissues

Although survival signatures of cryopreserved cultured cells appear qualitatively similar to the behaviour for isolated cell suspensions observed in Figure I, i.e. conforming to the two-factor hypothesis of cryoinjury”,H7, there is evidence of several modes of damage unique to the cryopreservation of tissues, during which do not arise cryopreservation of suspended cells. As discussed above, some of these

Cryopreservation

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damage mechanisms are related to the heat and mass transport limitations, which are a consequence of the macroscopic phvsical dimensions of tissue. Thus, uneven distribution of cryoprotectant in the tissue may result in regions which are not sufficiently protected or regions which are damaged by overexposure to the cryoprotectant. Similarly, as a consequence of nonuniform rates of temperature change through the sample, optimal cooling and warming rates may not be achieved in large sections of the tissue. Moreover, thermal gradients can induce mechanical stress due to uneven expansion or contraction in the biomateria18z~R8. It has been shown that thermal stress components induced by freezing can become sufficiently large to cause fractures in the biological sample, with damage”“, 89. Similar concomitant problems may arise due to the heterogeneous composition of biological tissue: because different elements of the tissue vary in their thermophysical properties (e.g. thermal expansion coefficients), thermoeleastic stress may ensue even under uniform cooling or warming conditions”. This may be of engineered tissue, e.g. particular concern in closely which biomaterials are bioreactors, in integrated with non-biological materials. Another problem related to tissue heterogeneity arises in biomaterials in which several distinct cell types are co-cultured. It is known that the cooling rate required for optimal survival varies by several orders of magnitude between different cell types273g1. Thus, it may be impossible to simultaneously satisfy optimal cooling requirements for all cells in the tissue. it appears that cryoprotectants may Nonetheless, broaden the width of the survival curve (Figure 11, thus increasing the probability of simultaneously achieving high levels of survival in cells with distinct optimal cooling rates. A further cause of damage can occur in tissues with defined three-dimensional geometries, as a result of mechanical forces generated by the extracellular ice. demonstrated that an Rubinsky et ICI~.‘~.~~ have important mechanism of structural damage in tissue with a vascular system is due to the fact that extracellular ice forms preferentially in the intravascular Consequently, dehydration of surrounding space. tissue causes water to accumulate and freeze in the vascular system, ultimately resulting in overdistention and rupture of blood vessels and sinusoids. One may expect similar mechanisms of damage to be operative in engineered tissues which have vasculature or vascular-like enclosed spaces. For example, hollowfibre bioreactors, a common design for bioartificial to structural liver devices”z”3s”4, may be susceptible damage during cryopreservation as a result of The the intraluminal space. engorgement of approaches that rely on cryopreserving cells separately before introduction into the bioreactor may circumvent this problemg. Thus, the cryopreservation process is a potentially important design factor in tissue engineering. Lastly, it should be mentioned that there are many biological structures specific to tissue cultures, which may be sensitive to cryopreservation damage. For example, cell-cell and cell-matrix junctions, which

253

are important for the normal physiological function of multicellular tissue, have been suggested as potential targets of injury during tissue cryopreservation”“. Although there is evidence that intercellular junctions are affected by prolonged exposures to reduced temperatures”“, further research is needed to determine the effects of the cryopreservation process on junctional and complexes, the resulting consequences for post-thaw tissue function and differentiation.

SUMMARY Cryopreservation is a technology with potentially far reaching implications for the tissue engineering industry. The possibility of long-term banking of cells and tissues would provide a means for inventory control for both the manufacturer and end-user of the tissue product. This problem is especially acute when human materials are used, since the availability is often limited and unpredictable. Furthermore, the problem of transporting tissue products from the manufacturer to the consumer would be solved by cryopreservation. The practically unlimited shelf-life of cryopreserved biomaterials would allow products to be distributed to distant users at relatively low cost, thus facilitating penetration into overseas markets. However, for cryopreservation to become a practical tool in tissue engineering, cell and tissue damage caused by the cryopreservation process itself must be circumvented. This can only be achieved if the mechanisms of freezing damage are understood on a fundamental level. Cryobiology research has generated much information on the interactions of physicochemical, biophysical and biological processes during cryopreservation of cells. Although this body of knowledge has provided useful insights into the related problem of tissue cryopreservation, the increased complexity of biological tissues compared with isolated cells introduces many new problems to be solved before cryopreservation of tissue will become a reality. We have addressed some of the problems associated with tissue cryopreservation and reviewed the progress of research in these areas. It is clear that much remains to be learned about the effect of the cryopreservation process on tissues and the appropriate strategies for minimizing freezing injury to tissues. In summary, we hope to have demonstrated that tissue cryopreservation is a complex problem, but one that has potentially important implications for the field of tissue engineering.

ACKNOWLEDGEMENT This research was partially supported Institutes of Health (DK 46270).

by the National

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