The relevance of cryoprotectant “toxicity” to cryobiology

CRYOBIOLOGY

23, I-13

(1986)

The Relevance

of Cryoprotectant

“Toxicity”

GREGORY American

Red

Cross

Trunsplantafion

Laborrrtoty,

to Cryobiology’s2

M. FAHY 9312

Old

Georgetown

Road,

Bethesda,

Maryland

20814

Cryoprotective agents are essential for the cryopreservation of almost all biological systems. These additives, however, do not usually permit 100% survival after freezing and thawing, though from a theoretical point of view they should be able to fully suppress all known types of freezing injury. In view of the known biological and physicochemical effects of cryoprotectants, it is suggested that the toxicity of these agents is a key limiting factor in cryobiology. Not only does this toxicity prevent the use of fully protective levels of additive, but it may also be manifested in the form of cryoinjury over and beyond the cryoinjury due to classical causes. Evidence for this extra injury (“cryoprotectantassociated freezing injury”) is reviewed. It is suggested that better suppression of toxicity is possible and will lead to advances in cryopreservation. C 1986 Academic Press. Inc.

Most cryobiologists date the beginning of the modern era of cryobiology from the discovery of the cryoprotective properties of glycerol by Polge, Smith, and Parkes in 1949 (35). Since this landmark discovery, cryoprotective agents have been used to secm-c the survival of innumerable living systems after storage at low, stabilizing temperatures. But despite this impressive degree of success, few cryopreservation techniques today permit 100% survival after freezing and thawing, and the preservation of many important systems has proven unsatisfactory or unsuccessful. In other words, cryoprotectants are generally not panaceas, even when used at optimized cooling and warming rates, As cryobiology matures as a discipline, we will become increasingly concerned with discovering why this is the case. In this paper, we raise the possibility that at least part of the problem is due to the cryoprotectants themselves. Our subse-

quent speakers will describe the significant biological effects of cryoprotective agents, effects which may explain the toxicity of these chemical species. In this introductory presentation, I will more globally review the evidence that cryoprotectant toxicity is of considerable significance in cryobiology despite the general neglect of this subject by cryobiologists to date. There are two fairly obvious reasons why cryoprotectant toxicity should be of concern to cryobiologists. First, the toxicity of cryoprotectants limits the concentrations of additive that can be used before freezing and therefore limits the cryoprotective efficacy of these agents. Second, there is now a significant amount of evidence that cryoprotective agents, despite their benefits, can actually play a direct role in producing cryoinjury. FULLY

CRYOPROTECTIVE

CONCENTRATIONS

VS TOXIC

OF CRYOPROTECTANTS

The first point can be illustrated diagrammatically as shown in Fig. 1. In this diagram, WC see the influence of initial cryoprotectant concentration on the temperature at which freezing injury is theoretically expected to first begin to occur. In this diagram, the factor associated with injury is assumed to be Mazur’s “U value” or un-

Received September 9, 1985; accepted September 16, 1985. ’ Presented at the symposium Biological Effects of Cryoprotective Agents at the Cryobiology meeting, June 1985, Madison, Wis. ? Contribution No. 665 from the American Red Cross. Supported in part by NIH Grants BSRG No. 2 507 RR05737 and GM 17959. I

001 l-2240/86 Copyright All rights

$3.00

0 1986 by Academc Prear, Inc. of reproduction in any form rcervcd.

GREGORY M. FAHY

innocuous. All of the postulated problems in cryobiology -cell packing (33), channel size constraints (27), optimal cooling rate differences for mixed cell populations (29, osmotically mediated injury (30, 41), and the rest-can be solved in principle by the selection of a sufficiently high concentration of cryoprotectant prior to freezing. In the extreme case, all ice formation could be suppressed completely by using a concen, J ‘ O%V/V IO%V/V ZO%V/V 3O%v/v 40% v/v tration of agent sufficient to ensure vitrifiINITIAL GLYCEROL CONCENTRATION cation of the biological system in question FIG. 1. Hypothetical relationship between the (16). Hence, if there were no constraints on threshold temperature for cryoinjury and the initial the choice of cryoprotectant concentration, concentration of glycerol, based on U = 0.25 or U = 100% survival of all or most cryopreserved 0.50 at the point of onset of injury. U = the unfrozen biological systems should be possible to obweight fraction of water (27). Calculated from T = tain. -(0.7541 - (0.56865 - SX: - IX,)“z)/(6.405 x IO-)), Ref. (8), X, = (1 + (MW,(R/MW, + 2/MW,)(lOO/W~ But the second point, of course, is that - I)l(UR + II))-‘)-‘, and U = (Wq/W,)(W,JWt.). the concentration of cryoprotectant reRef. (27). where X, = solute mole fraction. S and I are quired for the complete suppression of parameters defined in Ref. (8). MW,. MW,, and MW, injury on these bases can be are the molecular weights of water. glycerol, and freezing enough, in fact, to injure rather NaCI, respectively: R = the weight ratio of glycerol to high-high NaCl: W$= the initial weight percent of water, WY= than protect the system at hand. Figure 2 the initial weight percent of solute, and W,,,and W.r are shows how. in the case of fetal pancreas, the weight percentages of water and solute. respecthe use of higher prefreeze concentrations tively, at temperature T. Initial concentration of salt leads not to a complete was assumed to be 0.16 M at all initial glycerol con- of cryoprotectant centrations. U at 0.0 M glycerol was derived from data suppression of freezing injury but, instead, in Ref. (45). to increased injury (26). h\

100%

SURVIVAL

I

frozen fraction of water (27), but similar diagrams could be constructed for any number of other “endpoints”, depending upon the factor or factors assumed to be responsible for injury. For red cells, the U value associated with the onset of hemolysis is about 0.25 (27), and injury is expetted to begin to occur at the temperatures indicated by the lower curve. For granulocytes, however, the U value is probably closer to 0.5 (43), and injury should be expected to begin at the higher temperatures associated with this U value. Two conclusions can be drawn from this diagram. First, it is clear that with sufficiently high cryoprotectant concentrations, damaging

sufficiently

factors

should

be suppressible to as to become

low temperatures

1

0 Me2S0

2

CONCENTRATION

3 CM)

FIG. 2. Relationship between initial concentration of dimethyl sulfoxide and freeze-thaw recovery of fetal pancreas. Reproduced, by permission of the publisher, from Ref. (26).

CRYOPROTECTANT

TOXICITY

CRYOPROTECTANT-ASSOCIATED FREEZING INJURY

Let us now consider evidence for a contribution of cryoprotectant toxicity to freeze-thaw injury. Most of the available evidence has been obtained in systems cryoprotected with dimethyl sulfoxide, and we will consider these systems first. Dimethyl sulfoxide. Perhaps the earliest evidence for a damaging role of dimethyl sulfoxide during freezing was presented by Meryman in 1966 (28) (Fig. 3). Meryman froze and thawed human red blood cells at 0.3”Cimin in the presence of varying initial concentrations of this cryoprotectant and obtained a most peculiar and interesting result. For higher concentrations of additive, survival was seemingly independent of the initial dimethyl sulfoxide (MeSO) concentration, and depended instead solely on temperature. When these data are analyzed (Fig. 4), it emerges that hemolysis occurred

60

quite independently of the U value and other similar endpoints (Fig. 4A). Instead (Fig. 4B), these cells were killed in direct proportion to the concentration of dimethyl sulfoxide plus salt produced by freezing, providing striking evidence, though not recognized at the time, of cryoprotectant-associated freezing injury. Eleven years later, Fahy and Karow (15) noticed that dimethyl sulfoxide toxicity rather than other factors might explain the observed responses of rat hearts to freezing. As shown in Fig. 5A, rat hearts frozen to - 10°C after perfusion with 5% vi v Me,SO recovered fully, whereas hearts frozen to lower temperatures using higher initial concentrations of Me,SO recovered poorly, despite the fact that the latter hearts presumably contained less ice than the former and were exposed to lower salt concentrations. When the data are examined in terms of exposure to high Me,SO concentrations, however (Fig. 5B), it is obvious

HUMAN ERYTHROCYTES WITH DIMETHYSULFOXIDE IN 6 S S

50

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3

AND CRYOBIOLOGY

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2.OM

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2.5M

30

0 -5

-IO

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Degrees

-20

-25

-30

-3s

Centigrode

FIG. 3. Hemolysis of human red cells frozen to the indicated temperatures at 0.3”Cimin in different concentrations of dimethyl sulfoxide and thawed at 0.3”Umin. B.S.S. = 20 mM KH?PO,, 20 mM K2HP04, and 60 mM potassium citrate (approx. 250 mOsm). Reproduced. by permission of the publisher, from Ref. (28).

4

GREGORY M. FAHY 70

70.

1

I

I

4

5

B

60

60 -

50 L!? 03 2 40

50 -

z g

40 -

30

8 20 10 0 0.1

0.2

0.3

0.4

0.5-:,

2

3

U

6

MD+“NaCI

FIG. 4. Analysis of data from Fig. 3. (A) Lack of relationship between hemolysis and U. U = MW,(iOO/W$ - I)(R/MW, + 2/MW,)(X,/’ - l)/(R + 1),X, = (-1 + (P - 0.03864ST - 1.641 x 10-4ST2)l”-)/2S, and notation is as described in the legend to Fig. 1. (B) Relationship between hemolysis and the molarity of dimethyl sulfoxide plus the molarity of salt reached during freezing. M, + M, = (n, + n,)/(V, + v,,,~,), where no and n, = number of moles of dimethyl sulfoxide and salt, respectively. V, = volume of solute. V, = the partial molal volume of water, and n, = number of moles of water. n, = (nn + 2n,)(X,-’ - I), where X, is calculated from temperature as in (A). B.S.S. was assumed to be equivalent to 0.16 M NaCl for these calculations.

that the hearts which were the more damaged were those which were exposed to higher concentrations of Me,SO in the frozen state.

In 1978, Fishbein and Winkert (17) drew similar conclusions from their extensive studies of freezing injury to catalase. Figure 6 gives the results of one of their experi-

* @

-2 T(“C)

-6

-10

-14

COMPLETE SEVERE

RECOVERY INJURY

-18

T(W)

FIG. 5. (A) Lack of relationship between injury to rat hearts and the salt concentrations produced by freezing. Salt concentrations calculated as described in Ref. (8). (B) Relationship between injury to rat hearts and the calculated (8) Me,SO concentrations reached during freezing. 5, 10, and 15% = initial concentrations of Me$O (v/v). Reproduced with modifications from Ref. (7).

CRYOPROTECTANT

TOXICITY

5

AND CRYOBIOLOGY lOOr

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80

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2%

0 ;

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401

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5

6

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HOURS AT -400

FIG. 6. Percentage loss of activity of catalase with time at -40°C. Note marked augmentation of injury by Me+30 (DMSO). Reproduced by permission of the publisher, from Ref. (ii’). Copyright 1979. the American Chemical Society.

ments, in which the presence of Me,SO greatly increased the amount of damage done to the enzyme by freezing. These results may have more general significance because the damaging effects of the cryoprotectant were attributable to its effects on the pH prevailing at subzero temperatures, and similar effects on pH are likely to be found under normal freezing conditions for cells. Almost simultaneously, Richard Kahn (18) presented similar evidence for platelets (Fig. 7). The ability of platelets to accumulate serotonin after freezing and thawing in the absence of Me,SO is indicated here by the open triangles. When Me+30 was introduced before freezing, two effects emerged, one positive and one negative. When platelets were frozen in 2, 4, or 7.5% Me,SO, their ability to accumulate serotonin was maintained after freezing to higher salt concentrations then when they were frozen without Me,SO, suggesting that low concentrations of Me,SO could directly protect platelets from salt-induced injury. On the other hand, when 15% Me,!30 was employed, more injury was observed than could be accounted for on the

\

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30-

201 1

3

2

4

5

OSMOLAL

NoCf

7

6

0

FIG. 7. Recovery of human platelet serotonin uptake capacity as a function of the calculated osmolality of NaCl produced during freezing. 2, 4, 7.5, and 15% indicate the initial concentrations of Me2S0 (presumably v/v). Open triangles: platelets frozen without Me,SO. Reproduced. by permission of the publisher, from Ref. (18).

basis of exposure to NaCI, and Kahn concluded that the extra injury observed was attributable to toxic effects of dimethyl sulfoxide in the frozen state. In fact, Kahn found (Fig. 8) that when recovery was plotted against the concentration of di-

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FIG. 8. Recovery of human platelet serotonin uptake capacity as a function of the calculated osmotic concentration of MeSO (DMSO) encountered during freezing. Reproduced. by permission of the publisher, from Ref. (18).

6

GREGORYM. FAHY Na’ 200

r,.,. NaCl CONCENTRATION ELEVATED BY FREEZING I

I

2

2

3

4

5

6

MOLAR FACTOR

N&l

CONCENTRATION

4

3 CONCENTRATION (1=0.95%

W/V NaGI)

(X ISOTONIC)

FIG. 9. Relationship between injury to rabbit renal cortex and the concentration of NaCl the tissue was exposed to either as a result of freezing (filled symbols) or in the absence of freezing (open symbols). The sodium, water, and hippuran (Na+, HzO, and HIP) contents of the tissue were measured 60 min after restoration of active metabolism at 25°C and are expressed relative to the contents of these substances in paired control tissue. Loss of ability to extrude Na+, prevent water gain, and actively accumulate hippuran is apparent for the frozen samples but not for the samples exposed to NaCl without freezing. Reproduced with modifications by permission of the publishers, from Ref. (9).

methyl sulfoxide reached during freezing, an inverse correlation between recovery and Me+30 concentration could be demonstrated. In 1980 I published the first of a detailed series of studies on rabbit kidney cortex which led to similar conclusions (9). In this first experiment, the tissue was frozen to various temperatures after pretreatment with 15% w/v (1.92 M) Me,SO, and the concentrations of sodium chloride and Me+30 encountered by the tissue during freezing were calculated. This made it possible to determine the relationship between the recovery of the tissue and the concentrations of these solutes during freezing. Other tissue samples were exposed to similar concentrations of either NaCl or Me,SO without freezing, and the damage produced by these solutes was compared to the damage produced by freezing.

FIG. 10. Recovery of rabbit renal cortex after freezing in 5 or 15% w/v Me,SO so as to produce similar concentrations of NaCl in the frozen state. Recovery measured as in Fig. 9. Note the uncoupling of freezing injury from salt concentration. Reproduced with modifications by permission of the publisher, from Ref. (9).

The first result is shown in Fig. 9. Here injury is measured by the ability to extrude sodium and water and to actively accumulate Hippuran, damage being indicated by either upward or downward departures from the control values, which are indicated as 100%. It is clear that the concentrations of NaCl tolerated by unfrozen kidney tissue greatly exceed the concentrations present when slices were damaged by freezing. This discrepancy is not due to some directly damaging effect of ice because, as seen in Fig. 10, when the same amount of ice is frozen out after pretreating the slices with 5% rather than 15% dimethyl sulfoxide, so that the same concentrations of sodium chloride are reached as in the previous freezing experiment, no injury is observed. The failure of salt concentration or ice formation to simulate freezing injury contrasts sharply with the results of treating tissue with dimethyl sulfoxide at subzero temperatures (Fig. 11). Here we see that exposure to Me,SO in the absence of freezing (open points) resulted in injury which closely paralleled the injury associ-

CRYOPROTECTANT

TOXICITY

7

AND CRYOBIOLOGY

3 +a F ‘y 2.5

2 1.0

1.2 mNPS

0 30%

40%

50%

DIMETHYL SULFOXIDE CONCENTRATION (%w/v)

FIG. 11. Comparison between freezing injury and injury produced by treating rabbit renal cortex with Me,SO at temperatures close to the MeSO-waterNaCl liquidus. Recovery measured as in Figs. 9 and 10. Note similarity between injury observed when Me,SO is elevated either with or without freezing. Reproduced with modifications by permission of the publisher, from Ref. (9).

ated with similar concentrations of Me,SO in the frozen state (filled points). The natural conclusion to be drawn from these studies is that when kidney slices are frozen in the presence of 1.9 M Me,SO, it is exposure to MezSO during freezing which is likely to be the primary determinant of injury. If this conclusion is correct, then using concentrations of Me,SO higher than I .9 M before freezing should not succeed in improving freeze-thaw recovery because the basic problem of Me,SO toxicity will not be affected by this maneuver even though exposure to other factors such as ice formation might be strongly reduced. A test of this prediction was first reported in 1982 (12). The data and their interpretation are shown in Fig. 12. Kidney slices were frozen to -40°C in 20, 25, or 30% Me,SO. As can be seen, the recovery of the slices, which is indicated in this tigure by the post-treatment steady state K+/Na+ ratio (12), was similar regardless of the prefreeze Me,SO concentration. In addition, some slices

14

2

3 .4 U

5

9

10

11 12 13 mD

FIG. 12. Interpretation of the effects of freezing rabbit renal cortex in varying (20, 25, or 305% w/v) concentrations of MeSO. All tissue was frozen to - 40°C unless otherwise noted. Recovery is indicated by the steady-state K’/Na+ ratio of the tissue after Me,SO washout and 90 min incubation at 25°C. (Al Lack of correlation between recovery and the nonpenetrating solute (NPS) molality. NPS assumed to be equivalent to 0.16 M NaCl in terms of phase diagram behavior. (B) Lack of correlation between recovery and U value. C/ was calculated from T as described in the legend to Fig. 4. (C) Correlation between freezing injury and the molality of MeSO (rnn). /no = no&v, p,,n,). where /in. II,. v,,, and the calculation of II,% are as described in the legend to Fig. 4 and p,$ = I kg/ liter. Original data from Ref. (12).

were frozen to -50°C in 25% Me$O to allow discrimination between nonpenetrating solute molality (A), U value (B), and Me,SO molality (C) as causes of injury. It is apparent that nonpenetrating solute molality and I/ value either have no effect on viability or have an extremely erratic and unlikely effect. By contrast, all of the data can be correlated quite nicely with the molality of Me,SO. Confirmatory, unpublished results have recently been obtained in my laboratory. A second test of the toxicity hypothesis was recently made possible by the observation that mixtures of various amides and Me,SO are less toxic to nonfrozen kidney slices than is Me,SO alone (11). These mixtures should, therefore, also permit higher freeze-thaw recoveries than Me,SO alone according to the hypothesis. In fact, as shown in Fig. 13, when kidney slices are frozen to -40°C in the presence of mixtures of Me$O and amides, which are indicated here as DA, DF, and DU, the re-

8

GREGORY M. FAHY

covery after thawing is indeed higher than when Me,SO, indicated here as D, is used as the sole cryoprotectant (13). This is true despite the fact that when mixtures are used more ice actually forms (U values are lower) than when Me,SO is used alone (14). The enhanced freeze-thaw recovery in the presence of amides is hard to explain in terms other than prevention of Me,SO toxicity at -40°C in the frozen state. The fact that freezing injury is not entirely abolished by the use of amides is also explicable because even Me,SO-amide mixtures are toxic when sufficiently concentrated. Ethylene glycol. Some evidence for a role of ethylene glycol-associated freezing injury is available in the case of hearts. Armitage and Pegg (1) showed, in 1979, that the death of rabbit hearts which had been perfused with ethylene glycol and frozen could be simulated without freezing by exposing the hearts to ethylene glycol concentrations which were equivalent to the concentrations of ethylene glycol produced in their freezing experiments. A second piece of evidence comes from an attempt by Rapatz to preserve rat hearts (39) by slowly introducing and removing ethylene glycol while gradually lowering or raising temperature so as to “hug” the ethylene glycol-water freezing point or liquidus curve (38). His efforts with rat hearts were not successful, and he implied that this might be because this agent was toxic even at temperatures close to its aqueous liquidus curve. But if ethylene glycol is indeed toxic at its aqueous liquidus temperatures, the implication is that this agent must be toxic during the course of normal freezing. Methanol and ethanol. Rapatz also obtained similar results using methanol as the cryoprotective agent (38). In this case the failure of hearts to recover was clearly because of toxicity from methanol at temperatures near the freezing points of its aqueous solutions. Meryman, too, has

K+/Na+

03

0.4

u

05

FIG. 13. Lack of relationship between U value and recovery of rabbit renal cortex when the latter is frozen to -40°C in 30% w/v Me$O (D), 30% w/v “DA” (an equimolar mixture of Me$O and acetamide), 30% w/v “DF” (a mixture containing 2 mol of Me$O/mol of formamide), or 30% “DU” (a mixture containing 2.3 mol of Me$O per mol of urea). Viability data from Ref. (13). The enhanced K+/Na+ ratio in the DA, DF, and DU groups is attributable to reduced cryoprotectant toxicity rather than to any favorable alteration of phase diagram behavior. U was calculated directly from the known (14) solute concentrations of the relevant solutions before and after freezing to -40°C assuming no alterations in partial molal volumes of any of the cryoprotectants during formation of solutions and assuming 2% of the original solution volume was occupied by RPS-2 solutes. U = (~r/W$$)(lOO/Wr - I). Before freezing, wr/W$ = 0.478, 0.474, 0.477. and 0.470 for D, DA, DF. and DU. respectively. At -4o”C, Wr = 48.5. 51.9, 51.3, and 55.2, respectively.

found apparent toxicity from methanol and ethanol during freezing of red cells protected by these alcohols (29, 31). Glycerol. Even glycerol may play a damaging role during freezing and thawing. Most of the data relevant to this possibility of which I am aware have been obtained using human erythrocytes as the experimental material. The first evidence again comes from the work of Meryman in 1966 (28). Figure 14 is a reproduction of his figure showing that progressively higher glycerol concentrations caused freezing injury of red cells to occur at progressively higher unfrozen fractions of liquid (- “L” values). For example, injury took place at an L value of

CRYOPROTECTANT

TOXICITY

AND CRYOBIOLOGY

60-

50 -

NaCl

S-40% :” a .F 30.-s -1 0 0= 20e n IO -

01

-5

-10

-15

-20 Degrees

-25 Centqrode

-30

-35

-40

FIG. 14. Lack of consistent correlation between the volume of the unfrozen portion of the medium and the occurrence of 10% hemolysis following the freezing and thawing of human erythrocytes. The double curves were assumed to enclose the actual liquid volume vs temperature curves for the various glycerol solutions studied (initial glycerol concentrations for each solution indicated between the appropriate double curves). Dashed lines indicate results of three separate experiments. Reproduced, by permission of the publisher. from Ref. (28).

IO-20% when the cells were frozen in 0.5 M glycerol, but the injurious L value was 30% when cells were frozen in 2 M glyc-

who observed that glycerol seemed to render red cells more susceptible to U as well as to many other putative “solution effects” not shown here. They suggested erol, suggesting a contribution of glycerol that glycerol was most likely damaging due to the stress of freezing and thawing. to its osmotic effects at subzero temperaIn 1977, Fahy and Karow presented a reexamination of Lovelock’s original data tures (36). (Fig. 15) and found that glycerol appeared These three sets of data were obtained under three very different conditions of to reduce the mole fraction of electrolyte associated with a given level of hemolysis freezing and thawing and clearly suggest a after freezing and thawing (15). For ex- role for glycerol in producing cryoinjury. ample, in the absence of glycerol, 40% sur- These results seem not to be explicable in vival occurred at a sodium chloride mole terms of Mazur’s U value concept because fraction of about 0.040, whereas 40% sur- as the critical U value increases under the vival occurred at a mole fraction of only influence of glycerol, any confounding from about 0.024 after freezing in the presence factors such as electrolyte concentration of just 1 M glycerol. Again, extra damage will be minimized, not maximized; it is only has taken place which is correlated with the concentration of glycerol which glycerol concentration. changes in the appropriate direction for a The same effect was again reported in correlation with freezing injury. 1978 by Rail, Mazur, and Souzu (Fig. 16), The apparent damaging effect of glycerol

GREGORY M. FAHY 100

60

2

60

2 2 h 3

40

20

0 0

IO

20 -T

3’0

0.06

0.16

FRACTION

WATER

0.24 UNFROZEN

0.32 (U)

FIG. 16. Lack of relationship between freeze-thaw survival and U value for human erythrocytes frozen in various concentrations of glycerol (concentrations noted for each survival-ll value curve). Reproduced with modifications by permission of the publisher, from Ref. (36).

1°C)

FIG. 15. Lack of correlation between NaCl mole fraction and the hemolysis of frozen-thawed human red cells. Levels of hemolysis (ranging from 0 to 100%) are indicated in relationship to the mole fraction vs temperature curves for various initial concentrations of glycerol (0.0 to 2.00 M). For discussion see text. Reproduced, by permission of the publisher, from Ref. (15).

protection, again hinting at a possible damaging role for glycerol during freezing. The same conclusion has been drawn from recent detailed studies of the same system by Clark et al. (4).

requires a detailed explanation but it may very well be osmotic in nature, as suggested by Rall et al. (36), since very high concentrations of glycerol are not directly hemolytic (i.e., toxic) to red cells even at room temperature. Leibo has shown that abruptly changing the glycerol concentration in an unfrozen red cell suspension at - 5°C can be damaging (2 l), presumably because the cells are less tolerant of sudden volume changes at that temperature. In 1982, I reported further evidence using rabbit kidney cortical slices as the experimental material (12). Slices were frozen in 0, 0.9, and 3 M glycerol and the temperature at which injury first became statistically significant was determined. As can be seen (Fig. 17), the slices frozen in 3 M glycerol were more damaged than they should have been based on simple colligative cryo-

The apparent association between cryoprotectants and freezing injury as reviewed here suggests some interesting analogies. The presence of a cryoprotectant, similarly to the presence of ice, results in dehydration, and several cryoprotectant effects can be explained nonspecifically on this basis (2, 5, 11, 16). At least one cryoprotectant can dramatically inhibit catalase and peroxidase activity (37), and free radicals have been implicated in freezing injury (23, 24). Freezing has been shown to transiently increase membrane permeability, leading to and posthypertonic os“solute loading” motic shock (22, 32, 43); if cryoprotectants are present, they might also participate in these phenomena (9, 10, 26). Cryoprotectants alter the polarity of the cellular environment (2), and it is thought that this alteration in polarity, like freezing, can

ANALOGIES BETWEEN FREEZING INJURY AND CRYOPROTECTANT TOXICITY

CRYOPROTECTANT

-3ot INITIAL

,

,

1

2 MOLAAITY

TOXICITY

\ 3

OF GLYCEROL

FIG. 17. Relationship between Tk (the temperature at which freezing injury to rabbit renal cortex first becomes statistically significant) and the prefreezing concentration of glycerol. Size of plotted experimental points indicates uncertainty of Tk. A theoretical relationship similar to the curves drawn in Fig. I is provided for comparison. The theoretical curve lies substantially below the experimental curve at higher concentrations of glycerol, even though the Tk at 0.0 M glycerol has been assumed to be higher than it actually is. Thus, more injury is observed than can be accounted for on the basis of colligative cryoprotection. Redrawn and revised, by permission of the publisher. from Ref. (12). For one possible reconciliation between these data and those in (34), see (12). p. 415.

damage membranes (2). In fact, penetrating cryoprotectants under certain conditions can induce membrane fusion (6, 19, 42). Many specific effects of cryoprotectants are also known (20, 40, 44) which could easily become damaging when these agents are concentrated by freezing. More generally speaking, both freezing and cryoprotectants must alter the balance between the fundamental factors involved in maintaining the stability of biological structures. Hence, it ought to be true that a deeper understanding of cryoprotectant toxicity will improve our understanding of both cryoinjury and the biophysics of the molecular stability of the cell. CAVEATS

Having said this, it also needs to be clearly acknowledged that the hypothesis

AND CRYOBIOLOGY

11

of cryoprotectant-associated freezing injury is not as yet backed by an adequate amount of experimental evidence. The examples cited here are few, and although they are highly suggestive, their rigor can be challenged. Questions of interpretation can still be raised concerning such fundamental matters as the distinction between osmotic injury and true toxicity for, say, a cell frozen to -50°C. Some negative evidence, not reviewed here, also exists (27, 34). Finally, the phenomenon may not apply to particularly freeze-hardy systems such as mammalian embryos. CONCLUSION

Nevertheless, I submit that the detrimental effects of cryoprotectants are almost as relevant to cryobiology as are their cryoprotective effects, and that our understanding of cryobiology will remain incomplete until we have finished examining both sides of the cryoprotectant coin. We very much need the services of talented biochemists and biophysicists who can work out the mechanisms and the sites of injury so that we might be permitted to protect cells against the “side effects” of cryoprotectants. Of particular interest would be more studies which explore those effects of cryoprotectants which have been shown to be irreversible. We have substantial clues with regard to both where and how to look for answers. We also have evidence that deeper insight can lead to practical benefits (3, 13). REFERENCES 1. Armitage, W. J., and Pegg, D. E. Contribution of the cryoprotectant to total injury in rabbit hearts frozen with ethylene glycol. Cryobiology 16, 152-160 (1979). 2. Arnold, K., Pratsch, L., and Gawrisch, K. Effect of poly(ethylene glycol) on phospholipid hydration and polarity of the external phase. Biochim. Biophys. Acra 728, 121-128 (1983). 3. Baxter. S. J., and Lathe, G. H. Biochemical effects on kidney of exposure to high concentrations of dimethyl sulfoxide. Biochern. Phtrrmacol. 30, 1079- 1091 (1971).

12

GREGORY IV,. =. -rntl1 ’ T.X’

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