CRYOBIOLOGY
27,479-482 (1990)
Fracture Faces and Other Interfaces as Ice Nucleation Sites’ ROBERT J. WILLIAMS2 AND DAVID L. CARNAHAN American Red Cross Transplantation Laboratory, Jerome Holland Laboratory for the Biomedical Sciences, 15601 Crabbs Branch Way, Rockville, Maryland 20855 It has been recognized for many years that much of the ice formation injuring vitrified biological samples occurs during warming rather than cooling. Since the solutions in which this phenomenon occurs are quite viscous, it has frequently been assumed that heterogeneous or homogeneous nuclei do form during cooling, but that their growth is inhibited until the solution is again warm and fluid enough. In the aqueous sucrose solutions that we have been examining, the temperature of homogeneous nucleation as a function of concentration was investigated in Professor Luyet’s laboratory by Rasmussen and MacKenzie [cf. (Z)]. More recently, heterogeneous nucleation was systematically studied by Charoenrein and Reid (1). These data are summarized in Fig. 1. At low concentrations, the freezing (nucleation) points can be studied during cooling, and devitrification is observed only in rapidly cooled samples. Above the concentrations at which the nucleation and devitrification curves intersect, about 60% (w/w) for samples without and about 70% for samples with heterogeneous nuclei, ice formation is only seen at the devitrification temperature after warming from the nucleation temperature. If these observations are correct, no nuclei should form above the concentrations at which the nucleation temperatures interReceived July 18, 1989; accepted December 26, 1989. ’ Presented at the Symposium on Vitrification at the 26th Annual Meeting of the Society for Cryobiology, Charleston, South Carolina, June 1989. 2 To whom correspondence should be addressed.
sect the glass transition temperature. Freezeable water is still present in samples less concentrated than 80%, the concentration at which the melting point intersects the glass transition temperature, the point called Tg’, but it will not be seen to freeze because of lack of nucleation, Our experiments challenge these interpretations. Figure 2 contains a series of thermograms of a 70% sucrose solution from which heterogeneous nuclei have been carefully excluded (see below) and which has been cooled to -40, -60, -8o”C, etc., and rewarmed. After the startup artifact, the thermograms are eventless until the cooling temperature has reached - 8O”C, in which case a small endothermic event is seen at about -2S’C, above the -70°C glass transition. This is a melting of ice which was nucleated at or below the glass transition temperature. The amount is small, less than 1% of the freezeable water in this sample, and further cooling does not produce more ice until the sample has been cooled to - 17O”C,which causes a large increase in the amount of ice formed. This last event is easy to explain: the glassy sample has fractured between - 140 and - 170°C and ice has nucleated and grown within the cracks and into the glassy melt between the cracks. Cracking can of course only occur below the glass transition temperature when the sample has become sufficiently brittle. This aggressive type of ice formation, which in this example permits the freezing of half the freezeable water in a very viscous solution, has been described in detail elsewhere (4). The smaller events seen at more moder-
479 001L-2240190$3.00 Copyright 0 1990 by Academic Ress. Inc. All rights of reproduction in any form rcscwcd.
480
WILLIAMS
AND
CARNAHAN
covering of isopentane has also prevented cracking in a sample cooled to - 170°C (Fig. 4). Vitrification media differ widely in the conditions under which they form glasses, as well as in the stability of the glasses they form, but we propose that these studies with sucrose solutions may offer a representative model. Our results suggest that while it may not be possible to eliminate devitrification entirely in vitrified materials, there exists a rational procedure for reducing it to the minimum. Elimination of heterogeneous nuclei greatly reduces the concentration required to prevent devitrification, though the concentration required to prevent homogeneous nucleation in any given vitrification medium may still be impracticably high. Except in organisms which have evolved mechanisms for intentionally forming ice at high temperatures, biological materials seem to be generally 80 0 40 20 60 free of heterogeneous nuclei. This implies Sucrose % (w/w) FIG. 1. Supplemented phase diagram of sucrose: that their elimination from the vitrification media has the potential to alleviate many water. See text for sources of data. T,,,=,, = “liquidus”; TSOli, = “solidus”; TtipS = glass transition current problems. temperature; TheI = temperature at which heterogeThe most efficient sources of heterogeneous nucleation by INA bacteria is observed; Thorn= neous nuclei are probably the Pseudomotemperature for homogeneous nucleation of solution bacteria. These are now prevby fluctuations in the solution structure; Tdevit = tem- nas “INA” alent in our laboratory as a consequence of perature at which ice forms during warming after cooling below the glass transition temperature. their great utility in experiments where supercooling is not desirable. Thus, their presence cannot be avoided but their effect ate temperatures are missing from the anal- can be made negligible by standard treatogous thermograms in Fig. 3. These ther- ment in a steam autoclave and sterile transmograms differ from those in Fig. 2 in that fer of specimens and media in a laminar the sucrose in the sealed aluminum sample flow hood. The nuclei themselves, howpan was covered with a layer of isopentane, ever, are not intact bacteria but membrane rather than a layer of air. Thus, these small protein complexes which readily pass a events can be ascribed to the formation of 0,45+m filter and are not reliably removed ice nuclei at or below the glass transition by 0.22~pm filtration. As usual, cleaning up temperature on the air-solution interface. does not compensate for poor preparation. They are analogous to nuclei that have Nuclei of nonbiological origin, being particformed in cracks, but because the surface ulates, can be removed by appropriate filarea is more limited in a sample that has tration. not undergone extensive cracking, they Cracking as a surface phenomenon (3) are less aggressive in their growth. The should be entirely preventable, but it
481
FRACTURES AS ICE NUCLEI
70% sucrose WT:5mg Scan Rate: 20 deglmin
FIG. 2. Warming DSC thermograms of 70% sucrose solutions which have been cooled at 2WClmin to -40, -60, - 8O”C, etc. After the startup artifact, thermograms are eventless until the sample has been cooled to - 80°C or below, when a small endotherm appears at about - 20°C. This endotherm represents the nucleation of ice on the air-solution interface, at or below the glass transition temperature. The solid line at the top of the figure is a thermogram of a sample cooled to - 170°C before warming. Because the sucrose glasses are quite brittle, the sample has cracked, and the large endotherm represents the nucleation and aggressive formation of ice in the fracture interfaces.
SU45N viirT: 6.00 mg s‘CAN RATE:
20.00
deglmin
I / I
/
i
i -130
-110
-90
-70
j
1 -50
TEMPERATURE
i
I
I -30
-10
10
30
(C)
FIG. 3. Experiment as in Fig. 2, except that sample has been covered with isopentane. No surface ice formation is observed, indicating that the isopentane effectively covers the interface and prevents nucleation of ice.
482
WILLIAMS
AND
CARNAHAN
A
-40
-30
-10 -20 TEWERATWE (‘C,
0
10
FIG. 4. The effect of an isopentane covering on the resistance of 70% sucrose glasses to cracking. This sample had been cooled at 2O”Cimin to - 170°Cand thermograms made during warming. Covering the sample with isopentane (dotted line) apparently suppressed the fracturing induced by low temperatures
should be borne in mind that even in a highly stressed sample, the strain of fracturing propagates from a surface defect. In the 5- to 15-p,l samples used in the calorimeter, cracking was prevented by flooding the air-glass surface with an appropriate liquid. In samples large enough to be practicable for cryopreservation considerable stresses may build up as a result of uneven cooling. These can be ameliorated by proper annealing regimes or by the addition of appropriate plasticizers. But attention should also be paid to the container and its surface properties.
REFERENCES 1. Charoenrein, S., and Reid, D. S. The use of DSC to study the kinetics of heterogeneous and hw mogeneous nucleation of ice in aqueous systems. Thermochim. Actu 156, 373-381 (1989). 2. MacKenzie, A. P. Non-equilibrium freezing behaviour of aqueous systems. Phil. Trans. R. sm. London B 278, 167-189 (1977). 3. Westwood, A. R. C. Surface-sensitive mechanical properties. In “Chemistry and Physics of Interfaces I” (S. Ross, Ed.) pp. 159-171. ACS Publications, Washington, DC, 1965. 4. Williams, R. J., and Carnahan, D. L. Association between ice nuclei and fracture interfaces in sucrose: H,O glasses. Thermachim. Acta 155, 103-107 (1989).
27,479-482 (1990)
Fracture Faces and Other Interfaces as Ice Nucleation Sites’ ROBERT J. WILLIAMS2 AND DAVID L. CARNAHAN American Red Cross Transplantation Laboratory, Jerome Holland Laboratory for the Biomedical Sciences, 15601 Crabbs Branch Way, Rockville, Maryland 20855 It has been recognized for many years that much of the ice formation injuring vitrified biological samples occurs during warming rather than cooling. Since the solutions in which this phenomenon occurs are quite viscous, it has frequently been assumed that heterogeneous or homogeneous nuclei do form during cooling, but that their growth is inhibited until the solution is again warm and fluid enough. In the aqueous sucrose solutions that we have been examining, the temperature of homogeneous nucleation as a function of concentration was investigated in Professor Luyet’s laboratory by Rasmussen and MacKenzie [cf. (Z)]. More recently, heterogeneous nucleation was systematically studied by Charoenrein and Reid (1). These data are summarized in Fig. 1. At low concentrations, the freezing (nucleation) points can be studied during cooling, and devitrification is observed only in rapidly cooled samples. Above the concentrations at which the nucleation and devitrification curves intersect, about 60% (w/w) for samples without and about 70% for samples with heterogeneous nuclei, ice formation is only seen at the devitrification temperature after warming from the nucleation temperature. If these observations are correct, no nuclei should form above the concentrations at which the nucleation temperatures interReceived July 18, 1989; accepted December 26, 1989. ’ Presented at the Symposium on Vitrification at the 26th Annual Meeting of the Society for Cryobiology, Charleston, South Carolina, June 1989. 2 To whom correspondence should be addressed.
sect the glass transition temperature. Freezeable water is still present in samples less concentrated than 80%, the concentration at which the melting point intersects the glass transition temperature, the point called Tg’, but it will not be seen to freeze because of lack of nucleation, Our experiments challenge these interpretations. Figure 2 contains a series of thermograms of a 70% sucrose solution from which heterogeneous nuclei have been carefully excluded (see below) and which has been cooled to -40, -60, -8o”C, etc., and rewarmed. After the startup artifact, the thermograms are eventless until the cooling temperature has reached - 8O”C, in which case a small endothermic event is seen at about -2S’C, above the -70°C glass transition. This is a melting of ice which was nucleated at or below the glass transition temperature. The amount is small, less than 1% of the freezeable water in this sample, and further cooling does not produce more ice until the sample has been cooled to - 17O”C,which causes a large increase in the amount of ice formed. This last event is easy to explain: the glassy sample has fractured between - 140 and - 170°C and ice has nucleated and grown within the cracks and into the glassy melt between the cracks. Cracking can of course only occur below the glass transition temperature when the sample has become sufficiently brittle. This aggressive type of ice formation, which in this example permits the freezing of half the freezeable water in a very viscous solution, has been described in detail elsewhere (4). The smaller events seen at more moder-
479 001L-2240190$3.00 Copyright 0 1990 by Academic Ress. Inc. All rights of reproduction in any form rcscwcd.
480
WILLIAMS
AND
CARNAHAN
covering of isopentane has also prevented cracking in a sample cooled to - 170°C (Fig. 4). Vitrification media differ widely in the conditions under which they form glasses, as well as in the stability of the glasses they form, but we propose that these studies with sucrose solutions may offer a representative model. Our results suggest that while it may not be possible to eliminate devitrification entirely in vitrified materials, there exists a rational procedure for reducing it to the minimum. Elimination of heterogeneous nuclei greatly reduces the concentration required to prevent devitrification, though the concentration required to prevent homogeneous nucleation in any given vitrification medium may still be impracticably high. Except in organisms which have evolved mechanisms for intentionally forming ice at high temperatures, biological materials seem to be generally 80 0 40 20 60 free of heterogeneous nuclei. This implies Sucrose % (w/w) FIG. 1. Supplemented phase diagram of sucrose: that their elimination from the vitrification media has the potential to alleviate many water. See text for sources of data. T,,,=,, = “liquidus”; TSOli, = “solidus”; TtipS = glass transition current problems. temperature; TheI = temperature at which heterogeThe most efficient sources of heterogeneous nucleation by INA bacteria is observed; Thorn= neous nuclei are probably the Pseudomotemperature for homogeneous nucleation of solution bacteria. These are now prevby fluctuations in the solution structure; Tdevit = tem- nas “INA” alent in our laboratory as a consequence of perature at which ice forms during warming after cooling below the glass transition temperature. their great utility in experiments where supercooling is not desirable. Thus, their presence cannot be avoided but their effect ate temperatures are missing from the anal- can be made negligible by standard treatogous thermograms in Fig. 3. These ther- ment in a steam autoclave and sterile transmograms differ from those in Fig. 2 in that fer of specimens and media in a laminar the sucrose in the sealed aluminum sample flow hood. The nuclei themselves, howpan was covered with a layer of isopentane, ever, are not intact bacteria but membrane rather than a layer of air. Thus, these small protein complexes which readily pass a events can be ascribed to the formation of 0,45+m filter and are not reliably removed ice nuclei at or below the glass transition by 0.22~pm filtration. As usual, cleaning up temperature on the air-solution interface. does not compensate for poor preparation. They are analogous to nuclei that have Nuclei of nonbiological origin, being particformed in cracks, but because the surface ulates, can be removed by appropriate filarea is more limited in a sample that has tration. not undergone extensive cracking, they Cracking as a surface phenomenon (3) are less aggressive in their growth. The should be entirely preventable, but it
481
FRACTURES AS ICE NUCLEI
70% sucrose WT:5mg Scan Rate: 20 deglmin
FIG. 2. Warming DSC thermograms of 70% sucrose solutions which have been cooled at 2WClmin to -40, -60, - 8O”C, etc. After the startup artifact, thermograms are eventless until the sample has been cooled to - 80°C or below, when a small endotherm appears at about - 20°C. This endotherm represents the nucleation of ice on the air-solution interface, at or below the glass transition temperature. The solid line at the top of the figure is a thermogram of a sample cooled to - 170°C before warming. Because the sucrose glasses are quite brittle, the sample has cracked, and the large endotherm represents the nucleation and aggressive formation of ice in the fracture interfaces.
SU45N viirT: 6.00 mg s‘CAN RATE:
20.00
deglmin
I / I
/
i
i -130
-110
-90
-70
j
1 -50
TEMPERATURE
i
I
I -30
-10
10
30
(C)
FIG. 3. Experiment as in Fig. 2, except that sample has been covered with isopentane. No surface ice formation is observed, indicating that the isopentane effectively covers the interface and prevents nucleation of ice.
482
WILLIAMS
AND
CARNAHAN
A
-40
-30
-10 -20 TEWERATWE (‘C,
0
10
FIG. 4. The effect of an isopentane covering on the resistance of 70% sucrose glasses to cracking. This sample had been cooled at 2O”Cimin to - 170°Cand thermograms made during warming. Covering the sample with isopentane (dotted line) apparently suppressed the fracturing induced by low temperatures
should be borne in mind that even in a highly stressed sample, the strain of fracturing propagates from a surface defect. In the 5- to 15-p,l samples used in the calorimeter, cracking was prevented by flooding the air-glass surface with an appropriate liquid. In samples large enough to be practicable for cryopreservation considerable stresses may build up as a result of uneven cooling. These can be ameliorated by proper annealing regimes or by the addition of appropriate plasticizers. But attention should also be paid to the container and its surface properties.
REFERENCES 1. Charoenrein, S., and Reid, D. S. The use of DSC to study the kinetics of heterogeneous and hw mogeneous nucleation of ice in aqueous systems. Thermochim. Actu 156, 373-381 (1989). 2. MacKenzie, A. P. Non-equilibrium freezing behaviour of aqueous systems. Phil. Trans. R. sm. London B 278, 167-189 (1977). 3. Westwood, A. R. C. Surface-sensitive mechanical properties. In “Chemistry and Physics of Interfaces I” (S. Ross, Ed.) pp. 159-171. ACS Publications, Washington, DC, 1965. 4. Williams, R. J., and Carnahan, D. L. Association between ice nuclei and fracture interfaces in sucrose: H,O glasses. Thermachim. Acta 155, 103-107 (1989).
