J. Phys. Chem. 1903, 87,4273-4276
4273
Glass-Forming Tendency, Stability of the Amorphous State, and Cryoprotection of Red Blood Cells P. Boutron Laboratolre d'%"aologle, D6partement de Recherche Fondamentale, Centre d'Etudes Nuc&lres, 85 X-3804 I Qenoble C E E X , France, and Laboratohe Louls N h l , CNRS, 166 X, 38042 Qenoble CEDEX, France (Received August 23, 1982; I n Flnal Form: February 23, 1083)
The glass-forming tendency and the so-called stability of the wholly amorphous state have been studied for aqueous solutions containing one or two poly- or monoalcohols: glycerol, ethylene glycol, 1,2-propanediol,etc. These solutes are cryoprotectors. They protect cells against freezing damage by impeding ice crystallization. The cryoprotection of red blood cells has been assessed at various cooling rates in aqueous solutions of glycerol and 1,2-propanediol.The erythrocytes are better protected by the 1,2-propanediolsolutions at high cryoprotector concentration and high cooling rates, because of the higher stability of the wholly amorphous state, and also at low cooling rates, where less ice crystallizes outside the cells.
I. Introduction Many cells are killed at any cooling and warming rates if they are cooled to boiling liquid nitrogen temperature in suspension in a buffered medium. They may survive at optimal cooling and warming rates if they are protected by substances of very low toxicity 'called cryoprotectors. When cells are cooled, pure ice first crystallizes outside the cells. If the cells are cooled too slowly, due to the osmotic pressure they loose their water and are killed by the too high salts concentration.' If they are cooled too quickly, the water does not have enough time to flow out of the cells and they are killed by intracellular ice crystallization.' They may survive at intermediate cooling rates, where the damaging effects of salta do not have enough time to occur. When the cells are cooled and rewarmed very rapidly in the presence of high cryoprotector concentrations they may also survive if the inside and outside solutions remain wholly amorphous or if the ice crystals remain very small. The cryoprotectors protect the cells by decreasing the quantity of ice crystallized on cooling and rewarming by two mechanisms. As any solute, they lower the temperatures of ice crystallization a t equilibrium. Furthermore they favor supercooling and not only does the eutectic not crystallize even at very low cooling rates but the ice (or eventually the hydrate) itself may crystallize incompletely, leaving the solution in a partially or wholly amorphous state. The glass-forming tendency on cooling and the stability of the wholly amorphous state on rewarming aqueous solutions of cryoprote~tors~-~ and their influence on the cryoprotection of red blood cells have been studied.8 These properties increase very rapidly with concentration, but so also does the toxicity. The aim of this study was to determine among some solutions of cryoprotectors of very low toxicity what gives the highest glass-forming tendency and stability of the wholly amorphous state for (1)J. Farrant, "General Observations on Cell Preservation" in "Low Temperature Preservation in Medicine and Biology", M. J. AshwoodSmith and J. Farrant, Ed., Pitman Medical, 1980,pp 1-18. (2)P. Boutron and A. Kaufmann, Cryobiology, 15, 93-108 (1978). (3)P.Boutron and A. Kaufmann, Cryobiology, 16, 83-9 (1979). (4) P. Boutron, A. Kaufmann, and N. Van Dang, Cryobiology, 16, 372-89 (1979). (5)P. Boutron and A. Kaufmann, Cryobiology, 16, 557-68 (1979). (6)P. Boutron, D. Delage, and B. Roustit, J.China. Phys., 77,567-70 (1980). (7)P. Boutron, D. Delage, B. Roustit, and C. Karber, Cryobiology, 19, 550-54(1982). (8)P. Boutron and F. Arnaud, Cryobiology, in press. 0022-3654/83/2O87-4273$0 1.5010
the same water content (i.e., for approximately comparable toxicities). Red blood cells were chosen because their structure is relatively simple, the measurement of their survival rates is easy, and their behavior in the most commonly used cryoprotective solutions is already known.g Aqueous solutions of dimethyl sulfoxide (Me2SO)and glycerol, the most widely used cryoprotectors, ethylene glycol, 1,2-propanediol (or propylene glycol), and 1,3propanediol, and aqueous ternary systems with two of these solutes were studied. In these solutions no hydrates were observed in our experiments (except with Me2S0).2~3~5~6 In systems where glycerol or 1,2-propanediol were partially replaced by ethanol or 1-propanol the hydrates of the monoalcohols were 11. Measurements on the Aqueous Solutions of Cryoprotectors The temperatures of the various phase transitions and the heats of solidification and of fusion were obtained with a Perkin-Elmer DSC-2 differential scanning calorimeter. The solutions were cooled and rewarmed between -153 "C and a temperature above that at which melting was complete at programmed rates varying from 2.5 to about 300 OC/min. The states between the transitions were observed by X-ray diffraction.
111. Crystallization on Cooling and Glass-Forming Tendency The wholly or partially amorphous state can be obtained much more easily in the polyalcohol solutions than with pure water. Pure water would have to be cooled at rates estimated to be 2oooO OC/s'O or even lo7 OC/s" to obtain amorphous ice, which is impossible. Amorphous ice can be obtained only from the vapor.'O As the polyalcohol or M e a 0 concentration increases, ice crystallization becomes very difficult. Very different substances, such as salts, also give easily an amorphous state in their solutions.12 These latter cannot be used as cryoprotectors, since cells are very sensitive to salts concentration. (9)G.J. Morris and J. Farrant, Cryobiology, 9, 173-81 (1972). (10)L. G. Dowel1 and A. P. Rinfret, Nature (London), 188, 1144-8 (1960). (11)D.R. Uhlmann, J.Non-cryst. Solids, 7,337-48 (1972). (12)C.A. Angel1 and E. J. Sare, J. Chem. Phys., 52, 1058-68 (1970).
0 1983 American Chemical Society
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The Journal of Physical Chemistry, Vol. 87,No. 21, 7983
Boutron
TABLE I: Heats of Solidification on Cooling Aqueous Solutions of Alcohols in the Perkin-Elmer DSC-Pa cooling rate, "C/min solution 3 20 160 80 40 20 10 65% water-35% 1,2-propanediol 55% water-45% 1,2-propanediol 55% water-45% ethylene glycol 55% water-45% glycerol 65%water-l,2-propanediol-l5% 1-propanol/ (1,2-propanediol + 1-propanol) iceb clathrateb total 55% water-45% 1,3-propanediol
0 0 0
3.6 0 5.7 5.7 6.2
0.7 0
0.5 3.2 0 5.6 5.6
5.0 0 6.1 11.9
16.6 0 14.8 19.1
1.5 5.2 6.7 18.4
11.3 3.4 14.7
21.3
2.5
21.9
0
0
16.7 20.6
16.4 20.2
20
21.2
0
21.8 6.2 13.4 20.8 0 20.8
0
20
21.2 19.3
a The heats of solidification are represented by the number of milligrams of ice whose solidification at 0 "C would liberate the same amount of heat as that from 100 mg of solution. When it is not mentioned only ice has crystallized. To know the exact quantity of ice crystallized, one must take into account the variation of the latent heat of solidification of ice with temperature, etc. The values of the heats in calories per 100 g of solution can be obtained by multiplying the above values See ref 7 for explanations. by 79.78.
A. Quantity of Ice Crystallized. When dilute solutions of polyalcohols are cooled (below 30% 1,2-propanediol,5 40% or less glycerol4),the quantity of ice crystallized is independent on the cooling rate used (up to 300 'C/min). For intermediate concentrations it is first stationary then decreases as the cooling rate increases and becomes zero at the highest cooling rates if the concentration is sufficient (Table I). If the concentration is even higher, the solution remains wholly amorphous at all the cooling rates used. When the wholly amorphous state of a solution was observed by X-ray diffraction, a diffuse ring similar in location and width to that of pure amorphous water2JoJ3 was observed, though the amorphous solution contains much solute. The smaller the cooling rate at which the amount of ice begins to decrease and the faster this quantity decreases, the higher is the glass-forming tendency of the solution. When replacing one polyalcohol solution by another, the quantity of ice formed almost always changed in the same sense over the whole range of cooling rates used. One can thus classify without ambiguity these solutions according to their glass-forming tendency. When the solutions also contain ethanol or 1-propanol, the ice crystallization may be replaced by clathrate crystallization at the highest cooling rates (Table I). In ref 7 is given an explanation of how the respective quantities of ice and clathrate formed were distinguished and measured. B. Comparison with Equilibrium and Deviation from Ideality. If the ice is in equilibrium with the residual solution when it has reached ita maximum value and becomes independent of the cooling rate, the amorphous residue would have a eutectic composition. This is generally assumed by cryobiologists. To make comparison,14the variation of the solidification heat of water with temperat~re,'~ the heat of mixing with the residual solution of water originating from the fusion of the ice etc., have been taken into account. It has been found that the amorphous residue of the aqueous glycerol solutions contained about 50% or less glycerol. This is much less than the composition of the eutectic which contains 66.7% glycerol.'6 Less ice crystallizes in the 1,2-propanediol solution^.^ The relative difference varies from about 7% for 15% solute to 30% for 30% solute. The amorphous residue is also much less concentrated than the presumed eutectic, with about 65% 1,2-propanedi01.~
\
I
-150
r
r
1
I
'
-1 00
I
I
r
I
I
I
I
i
I
- r
I
- 50
I
c
0
Flgure 1. Typical warming thermogram of a system of intermediate concentration without hydrate (solutlon with 55 % water, 27 % 1,2propanediol, and 18% glycerol warmed at 5 'Clmin after cooling at about 300 'Clmln). dCldt is the derivative of the specific heat vs the time; its scale is not represented.
One notes also that in an ideal solution the quantity of ice crystallized at equilibrium above the eutectic temperature, as well as the freezing point depression, is determined by the mole fraction of the solute: it is a colligative property. Ross1' has shown that, in the present solutions, the freezing points are lowered by partial hydration of the molecules.
IV. Crystallization on Rewarming and Stability of the Wholly Amorphous State Ice crystallizes on rewarming a solution in a wholly (or almost wholly) amorphous state if the warming rate is not sufficiently rapid. On the warming thermogram of a solution where no hydrate crystallizes (Figure l) one sees the glass transition, an exothermic peak at Td corresponding to the crystallization on rewarming often called a devitrification peak, and a melting peak at T , (see ref 7 for the exact meaning of Td and T,). Theoretically2 as well as experimentally on all the systems in~estigated,~-' the temperature Td varies linearly with the logarithm of the warming rate to a good approximation, except when Td becomes too close to the melting peak. The critical warming rate above which no crystallization occurs could be observed directly at the warming rates available with the apparatus if the cryoprotector concentration was sufficient. It could be obtained by extrapoThe lation of the logarithmic variation of Td smaller the critical warming rate, the higher is the "stability" of the amorphous In systems where the hydrate crystallizes,supplementary peaks appear when the relative monoalcohol (ethanol or 1-propanol) concentration becomes suffi~ient.~.' The sta(17) H. K. Ross, Ind. Eng. Chenz., 46,601-10 (1954).
The Journal of Physical Chemistry, Vol. 87, No. 21, 1983 4275
Cryoprotection of Red Blood Cells
TABLE 11: Critical Warming Rates ("C/min)for Several Solutions
wt % 1,2-propanediol1,2solutes water 1-propanol,p = 1 5 b propanediol wt %
35 40 45 50 ~~
65 60
a x io4
55 50
2
loo=
7.5 x 7.6 260
50
Me,SO
solutes glycerolethanol, p = 20b
ethylene glycol
1,3-
glycerol
propanediol
lo*
x 104
1.7
x 104
20
1.2 x 200
1.4 x
1 0 7
io8
2 x ioi3 4 x 107
3x 1x
ioi6 lo8
This has not been measured, but its value is 140 and 50 "C/min respectively when 10% and 20% of the 1,2-propanediol Maximum stabi1ity;p is the ratio of second solute/both solutes in percentage (w/w). is replaced by 1-propanol. bility of the amorphous state can no longer be defined.
V. Comparison of the Various Cryoprotective Solutions No significant maxima in the stability of the amorphous state were obtained by replacing partially, for the same water contents, one polyalcohol by another or by MezS0.2p3p5*6 Maxima were obtained only in the systems water-glycerol-ethanol4 and water-1,2-propanediol-lpropan01.~The maxima seem to appear only in systems where one of the two solutes gives hydrates. Critical warming rates of the binary systems and of the compositions of the two ternary systems corresponding to the maxima are given Table 11. The wholly amorphous state could be obtained below 45% solutes only in the binary and ternary systems with 1,2-propanediol. For this reason, the critical warming rates could not be determined with 35 or 40% of the other solutes. The 1,2-propanediolsolutions have a far higher stability of the amorphous state than the others, except where 15% of the 1,2-propanediol is replaced by 1-propanol. In this case a reduction of lo4 in the critical warming rate is obtained in the presence of 65% water. In Table I are given the heats of solidification observed at various cooling rates in some of the solutions studied. The 1,2-propanediol solutions have also a far better glass-forming tendency than the other binary systems. The system where 15% of the 1,2-propanediol is replaced by 1-propanol is slightly less good, due to the crystallization of a small amount of clathrate at the highest cooling rates.7 VI. Survival Rates of Red Blood Cells When Frozen in If-Propanediol and Glycerols 1,2-Propanediolhas been chosen because it has the most interesting physical properties and glycerol is commonly used to protect erythrocytes as well as many other cells against freezing damage. A. Experiments. The erythrocytes were in a phosphate buffer solution with the same composition as that used by Miller and Mazur18 and containing the cryoprotector. They were cooled to -196 " C in straws of 0.5 cm3, stored in liquid nitrogen, and generally thawed by immersion in a water bath at 37 O C . The cooling rates ranged from 1 to 3500 oC/min.s The survival rates were measured by the rate of haemolysis.ls B. Results. The survival of the erythrocytes is given Figures 2-4. It increases at the low cooling rates (below that corresponding to the maximum) as the glycerol or 1,2-propanediol concentration is increased, since less ice crystallizes outside the cells: they lose less water and are less damaged by the concentrations of the salts.' Just above the maximum, the survival is only a little sensitive to the increase of cryoprotector concentration. Adding cryoprotector has two opposite effects. Since less ice crystalizes outside, more water remains in the cells, which (18) R. H. Miller and P. Mazur, Cryobiology, 13, 404-14 (1976).
70
40
30 20
A
5
10
500 lo00 4
50 100
Cool~ng rate ( " C k " ) Figure 2. Survival (%) of red blood cells after cooling at dlfferent rates to -196 OC in A l o % , 0 15%, X 20% and 0 30% (w/w) glycerol and rewarming the straws in water at 37 OC.
t
S u r v i v a l (7.)
100
50c
/
4ol 30
I
A
/
A'A/
/d
5 A
Figure 3. Survival (%) of red blood cells after cooling at different rates to -196 OC in A l o % , 0 15%, X 20% and 0 30% (w/w) 1,2propanediol and rewarming the straws in water at 37 OC and in 30% (w/w) l,P-propanediol, straw rewarmed in a kleanex in water at 37 OC (for 2000 'Clmin cooling rate) and in the air (for 3500 OC/min cooling rate).
+
favors ice crystallization; but they contain also more cryoprotector, which impedes the crystallization. Between about 100 and 1000 "C/min, glycerol is more effective than 1,2-propanediol,maybe because more water remains in the cells in the presence of 1,Zpropanediol than in the presence of glycerol. The most interesting feature is that at the highest cooling speed (at 3500 OC/min), the survival rate drops to a very low value in the presence of 30% glycerol, while,
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The Journal of Physical Chemistty, Voi. 87, No. 21, 1983
80
/+
6ol / d$
50
4- /
2ol
t!
10
Cooling rate CC/min) Flgure 4. Comparison of the survival (%) of red blood cells after
cooling at different rates to -196 and 0 15% (w/w) glycerol.
O C
in
+ 15% (w/w) 1,Ppropanediol
with 30% 1,2-propanediol, after a minimum around 5o(t-1000 OC/min, it increases again to high values at 2000 OC/min or more if the cells are rewarmed rapidly. This can be explained as follows. Around 500-1000 OC/min, ice crystallizes inside the cells which contain much water. However, high 1,2-propanediol concentration is sufficient to impede ice crystallization inside the cells on cooling if they are cooled a t 2000 OC/min or more (high glass formation tendency). On the contrary, at the highest speed, glycerol is unable to impede ice crystallization inside the cells. A higher glycerol concentration would be necessary to observe a minimum and an increase of survival at 3500 "C/min.Ig High survivals above 2000 OC/min in the presence of 30% 1,2-propanediol are observed only if the cells are rewarmed rapidly. If they are rewarmed slowly (Figure 3), the survival rate is low. However, even when the straws are rewanned in water at 37 "C, the warming rate, of about 5000 to 7000 OC/min, is much smaller than the critical warming rate above which no crystallization would occur with 30% 1,Zpropanediol. May be, even after a previous cooling of 2000 OC/min or more some water has left the cells, or the constituants of the erythrocytes increase the critical warming rate. More probably they survive because intracellular ice crystallization is not in itself injurious. They are injured only if the ice crystals are too large.l9 The critical warming rate above which erythrocytes cooled rapidly can survive certainly increases with that above (19)Tokio Nei, Cryobiology, 13, 278-94 (1976).
Boutron
which no crystallization occurs, since the later the crystals appear, the later they reach their critical size for cell survival. More complete results a t high cooling rates and a quantitative comparison with the physical properties of the solutions will be published elsewherea8 At the lowest cooling rates (Figure 4 and Figures 2 and 3) 1,2-propanediol protects the cells better than glycerol. At these rates even for 30% cryoprotector the quantity of ice outside the cells has reached its maximum value; but in this case also less ice crystallizes in the presence of 1,2-propanediol than of glycerol (section IIIB). The cells are therefore less damaged by the concentrations of the salts. There is no simple relation between this difference and the difference of the molecular weights of glycerol and 1,2-propanediol since the equilibrium is not reached outside the cells which are not in ideal solutions and the cells are cooled well below what would be the eutectic temperature.
VII. Conclusion By inveatigating aqueous solutions of polyalcohols, much better glass formation tendencies and stabilities of the amorphous State have been found than with the most usual cryoprotectors. These properties seem related to the cryoprotection of cells cooled and rewarmed rapidly. If this zone of cooling and warming rates, where the cells survive because all is vitrified inside and outside the cells, could be extended to smaller rates and higher water contents and join the peak of maximum survival for many cells, it could be very useful. Indead this peak is dependent on the permeability of the membranes and corresponds to different cooling rates for different kinds of cells, while the zone of complete vitrification does not depend on this permeability. By extending this zone, one can hope to freeze without damage whole organs or living beings. For this, the glass formation tendency and stability of the amorphous state would have to be investigated in many systems. Probably very asymmetric and ramified molecules of polyalcohols would be the most interesting, because the polyalcohols are of particularly low toxicity, and asymmetric molecules favor the amorphous state more than the symmetric ones. For instance 1,Zpropanediol is much better than l,&propanediol or glycerol. In addition the relation between the stability of the amorphous state, the glass-forming tendency, and the nucleation and growth of the ice (or clathrate) crystals would have to be investigated for better comprehension of the freezing damage. Registry No. Me2S0, 67-68-5; ethylene glycol, 107-21-1;glycerol, 56-81-5; 1,2-propanediol, 57-55-6; 1-propanol, 71-23-8; 1,3-propanediol, 504-63-2; water, 7732-18-5; ethanol, 64-17-5.
