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Freezing damage of bovine erythrocytes: Simulation using glycerol concentration changes at subzero temperatures
CHYOBIOLOGY
13, 587-598
Freezing
(1976)
Damage of Bovine Erythrocytes: Simulation Using Glycerol Concentration Changes at Subzero Temperatures1 S. P. LEIBO
Biology
Division,
Oak Ridge National
Laboratory,
When a cell is frozen and thawed, it must be exposed to the following changes in the properties of the suspending solution: (i) lowered temperature, (ii) increased concentration of the extracellular solution as water is removed in the form of ice during freezing, and (iii) decreased concentration during thawing. Without actually freezing the cells, I have attempted to simulate some of the physical-chemical changes to which bovine erythrocytes are exposed when frozen and thawed in glycerol solutions. These experiments followed from a mathematical and experimental analysis ‘of glycerol permeation in the bovine erythrocyte (5) and a correlative study ‘of that cell’s freezing sensitivity (6). The permeation study demonstrated that the permeability constant of the bovine erythrocyte for glycerol is relatively low with ‘a high activation energy. This means that it is possible to control the intracellular glycerol concentration of that cell by varying the time and/or temperature of exposure to glycerol. The freezing study demonstrated that protection against freezing damage does not require permeation by glycerol. This conclusion followed from the fact that the same percentages of cells survived freezing even when there was a 300-fold difference in the effective intracellular concentration of glycerol. But that
study also identified a transient increased sensitivity to freezing that depended on the length ‘of exposure to glycerol prior to freezing. This increased sensitivity, or survival “dip,” is illustrated by the results shown in Fig. 1 that are reproduced from Mazur et al. (6, their Fig. 7). Those results show that the time of the dip is characteristic for the glycerol concentration to which the cells are exposed prior to freezing. The objective of the presenf study was to use that time-dependent dip as a marker to attempt to separate the effects on cell survival of various dilution treatments. The fundamental variable in these experiments was the time of exposure to glycerol prior to the treatment. The test of the relevance of these simulation experiments to freezing damge depended not just on the extent to which an equivalent percentage survival could be produced. Rather, a successful test would require that the simulation experiments yield a survival dip at the same time as that produced by freezing and thawing. Using the dip as a marker, therefore, I have attempted to distinguish between the effects of the following changes on cell survival: (i) change in temperature versus change in solute concentration, (ii) change in glycerol concentration versus change in electrolyte concentration, and (iii) changes that occur during freezing versus changes that occur during thawing. The results show that the response of bovine erythrocytes when expo,sed to large changes in the concentration of extracellular glycerol at
Received July 28, 1975. 1 Research sponsored by the Energy Research and Development Administration under contract with the Union Carbide Corporation. 587 1976 by Academic Press, Inc. Copyright All rights o6 reproduction in any form reserved.
Oak Ridge, Tennessee 37830
588
S. P. LEIBO 100
times. Then, the cell suspensions were subjected to the f’ollowing sequences of temperature and/or concentration changes:
1
(i) a temperature change with no concentration change; (ii) a concentration change with no temperature change; (iii) an increased concentration change followed by a decreased concentration change with no temperature change; (iv) a temperature change followed by an increased concentration change; and (v) a temperature change followed by an increased concentration change followed, in turn, by a decreased concentration change.
3M
0’ 0
10 TIME (min) IN GLYCEROL
20 AT 20 *C
30
FIG. 1. Survival, as percentage of unhemolyzed cells, of bovine erythrocytes as a function of time of exposure at 20°C to 1, 2, or 3 M glycerol prior to freezing to -196°C at 43”C/min. The data are taken directly from Mazur et al. (6) who describe the experimental details.
subzero temperatures parallels their response when frozen and thawed in glycerol solutions. MATERIALS
AND
METHODS
The rationale of the simulation experiments was to examine the individual effect of some of the physical-chemical changes that occur concomitantly as a cell suspension is frozen. The concatenation of changes that ‘occurs ,during freezing is first that the temperature is reduced. Then, as ice forms, there is an increase in the concentration of the suspending solution. During thawing, the sequence is reversed so that the concentration of the soIution is decreased. It is important to note that these concentration changes occur at subzero temperatures. The experimental approach, then, was to examine the reIative effects on ceH survival of each of those steps. Operationally, the experiments consisted of suspending bovine erythrocytes in concentrated glycerol solutions at room temperature for varying
The temperature change consisted of transferring cells suspended in 1, 2, or 3 M glycerol from room temperature to -5°C. A single concentration change consisted of transferring cells from the initia1 concentrations of 1, 2, or 3 M glycerol into a 6 M glycerol solution. A double concentration change consisted of transferring cells from the initial glycerol solutions into a 6 M glycerol solution and then back into the initial glycerol solution. Portions of the procedure were also repeated for erythrocytes suspended initially in I.4 M sucrose, since bovine erythrocytes are impermeable to it and display no survival dip when frozen in sucrose solutions (5, 6). Two aspects of this procedure require special discussion, namely, the choice of -5°C as the subzero temperature of exposure and the choice of 6~ glycerol as the “shocking” concentration. The temperature of -5°C was selected for several reasons: (i) The temperature had to be low enough to examine phenomena at subzero temperatures. (ii) The temperature had to be high enough so that erythrocytes in 1 M glycerol would not freeze but rather would only supercool. (iii) Since the respective freezing points of 1 M, 2 M, and 3 M glycerol prepared with isotonic 0.165 M NaCl are about -1.9”, -4.6”, and -8.l”C, a temperature of -5°C seemed a reasonable
SIMULATION
OF FREEZING
compromise. The concentration of 6 M glycerol was selected to mimic the element of s’olute concentration resulting from freezing, because 6 M glycerol prepared with 0.165 M NaCl has a freezing point of about -23°C which is in the range at which erythrocytes frozen in glycerol solutions begin to exhibit substantial hemolysis. The experiments were performed using bovine erythrocytes drawn by venipuncture into a heparinized flask and stored at 4°C for no more than 7 days until used. Just before use, the erythrocytes were washed three times by centrifugation and resuspension at the original cell concentration in 0.165 M phosphate-buffered saline (pH 7.0). All glycerol and sucrose solutions were prepared with 0.165 M saline buffer and, for convenience, will be designated only in terms ‘of the glycerol molarity. Each experiment consisted of the following steps: The washed erythrocytes were diluted with an equal volume of a glycerol solution at twice the desired final glycerol concentration at 260°C. Precise temperature control at this point was important because the activation energy for glycerol permeation in the bovine erythrocyte is about 21 kcal/mole (5). The cells, now suspended in 1, 2, or 3 M glycerol at 2O”C, were held for various periods of time; then 0.05-ml volumes were pipetted with Hamilton syringes into tubes previously equilibrated at the appropriate temperature (usually 20” or -5°C). After a 30-set pause to allow for temperature equilibration, 0.45ml volumes of glycerol solutions ,at the appropriate concentrations and temperatures were rapidly pipetted into the tubes to mix the cell suspensions. These suspensions were held for 1.0 min longer, although the results of holding the suspensions for 5.0 min at -5°C at this step were indistinguishable from the results with only 1.0 min holding. Then 4.5 ml of the third glycerol solution at the appropriate concentration and temperature was pipetted into the suspension. The diluted suspensions were held for 5 min longer at the test temperature
DAMAGE
589
and then placed into a 20°C bath for an additional 5 min before being centrifuged at 10,060g for 10 min at room temperature. The supernatants were removed, and the amount of hemoglobin present was read at 540 nm as cyanmethemoglobin. This value was then calculated as a percentage of the amount of hemoglobin released from totally hemolyzed control samples, which consisted of cells suspended in the first glycerol solution, handled with the experimental samples, but diluted in two steps with 0.01 M phosphate buffer at pH 7.0. In other words, the percent hemolysis was calculated as [ OD ( experimental) /OD ( total hemolysis control)] X 100, ‘and the results were expressed as “percent survival,” i.e., ( 100% minus percent hemolysis ) . Each treatment was repeated with different batches of blood three to five times, and the results are shown as the mean percent survival + 1 standard error. Although some treatments consisted of diluting a suspension with the same concentration of glycerol, all samples (with the exception of those whose response is shown in Fig. 5) were subjected to the same number of dilution steps because erythrocytes in hypertonic solutions can be sensitive to handling alone (5). The results are #labeled according to the glycerol concentration and the temperatures for each of the dilution steps. For example, in figure legends the label 2 M at 20°C’ 6 M at -5°C * 6 M at -5°C indicates that these samples were suspended in 2 M glycerol at 20°C for various times, cooled to -5°C diluted IO-fold at -5°C with 6 M glycerol, and diluted lo-fold again at -5°C with 6 M glycerol. All samples were warmed to room temperature before hemolysis was measured. The final figure for glycerol concentration for each curve indicates the concentration in which hemolysis was determined. Measurements showed that Beer’s law for cyanmethemoglobin was unaffected by glycerol concentrations up to 6 M. Measurements of the effect of temperature (Fig. 5) were obtained in a slightly different manner. In that experiment the
S. P. LEIBO
590
TIME hd
IN,
M GLYCEROL
AT 20”
C
FIG. 2. Survival of RBC suspended in I M glycerol at 20°C for varying times and then subjected either to freezing and thawing [dotted curve, data of Mazur et al. (6)] or to various dilution treatments at 20°C (A) or at -5°C (B).
washed cells were diluted with an equal volume of 4.0 M glycerol and held for 5.0 min at 2O.O”C; then O.l-ml samples were pipetted into tubes previously equilibrated at the test temperatures. Thirty seconds later, 9.9-ml volumes of either 2 M or 6 M glycerol were rapidly pipetted into each sample. The samples were held for 5 min at the test temperature before being warmed to 20°C for 5 min; then the amount of hemolysis was measured as described above. The response of erythrocytes suspended in 1.4 M sucrose and diluted at -5°C was measured in a fashion similar to that used with glycerol. The cells were diluted with an equal volume of 2.8 M sucrose in isotonic saline buffer, held at 20°C for varying times, cooled to -5°C diluted IO-fold with 2.8 M sucrose at that temperature, and then diluted lo-fold again with 1.4 M sucrose at -5°C before the sample was warmed to 20°C and its amount of hemolysis was measured. RESULTS
Cells Suspended in 1 M Glycerol When bovine erythrocytes suspended in 1 l\f glycerol at 20°C for varying times were
frozen at about 40”C/min to -196°C and then thawed rapidly, they exhibited a distinctive response (Figs. 1 and 2, dotted curve, and Ref. 6). Survival was low after short exposure times, but within 5 min the survival Ievel was equal to that displayed after 30 min exposure (Fig. 2A, dotted curve). In the present experiments, when bovine red cells suspended in 1 M glycerol at 20°C for varying times are subjected to a variety of dilution procedure,s without freezing, they exhibit an analogous response (Figs. 2A, B). The cells are most sensitive when diluted after only brief exposures to 1 M glycerol at 2O”C, but become less sensitive after about 5 to 10 min of exposure to 1 M glycerol. The first series of experiments was designed to simulate the concentration changes, without a temperature change, to which cells are exposed when frozen and thawed (Fig. 2A). The results show that subjecting cells to a single ( 1 M * 6 M) or to a double concentration change (1 M + 6 M + 1 M) produces significant hemolysis during the first few min of exposure to 1 M glycerol, but relativeIy little after 5 min or more of exposure. The former treatment is analogous to the concentration of solute
SIMULATION
OF FREEZING
that occurs during freezing, and the latter to the concentration and dilution of solute that occurs during freezing and thawing. The second series of experiments was designed to examine the role of such concentration changes when they occur at subzero temperatures (Fig. 2B). The results show that subjecting cells to a temperature change alone (20’ + -5°C) in the absence of a concentration change produces little hemolysis. On the other hand, subjecting cells to a single concentration change a temperature (1 M + 6 M) following -5°C) produces substanchange (20’ -+ tial hemolysis during the first few minutes of exposure to 1 M glycerol, but very much less hemolysis after 5 min or more of exposure. When the cells are exposed to a double concentration change (1 M -+ 6 M + 1 M) following a temperature change their response closely re(ZOO-+ -S’C), sembles that produced by freezing. Cells Suspended in 2 M Glycerol The behavior of red cells suspended in 2 M glycerol at 20°C for varying times is analogous (Figs. 3A, B). When the cells were frozen to -196°C and thawed rapidly,
DAMAGE
591
Mazur et al. (6) found that survival after 20 set in 2 M glycerol ,at 20°C was as high as after 30 min of exposure before freezing but that the cells displayed a dramatic dip in survival when frozen after 5 min in 2 M glycerol (Figs. 1 and 3, dotted curve). Again, the first series of experiments was designed to simulate the concentration changes alone that occur during freezing and thawing (Fig. 3A). The results show that cells exposed to a single concentration change (2 M + 6 M) exhibit a survival dip after about a 5 min exposure to 2 M glycerol. Cells exposed to a double concentration change (2 M + 6~+2h1) alsoexhibit a survival dip, though less distinct than that following a single concentration change. When cells suspended in 2 M glycerol are subjected to a single concentration change (2 M + 6 M) at -5°C they also exhibit a clear survival dip after a 5 min exposure to 2 M glycerol (Fig. 3B ) , And when cells are subjected to a double concentration change (2 M + 6 M + 2 M) following a temperature change (20’ + -5”C), they not only display a survival dip after 2 to 5 min exposure, but also exhibit virtually the
FIG. 3. Survival of RBC suspended in 2 M glycerol at 20°C for varying times and then subjected either to freezing and thawing [dotted curve, data of Mazur et al. (6)] or to various dilution treatments at 20°C (A) or at -5°C (B).
592
S. P. LEIBO
100,
A
20
i
Oo.0
0. TIME
lmln)
FIG. 4. Survival of RBC suspended in 3 M glycerol jected either to freezing and thawing [dotted curve, dilution treatments at 20°C (A) or at -5°C (B).
same survival following 10 to 30 min exposure as that shown when the erythrocytes are frozen in 2 M glycerol. Cells Suspended in 3 M Glycerol When erythrocytes suspended in 3 M glycerol at 20°C f,or varying times were frozen and thawed, they, like the cells in 2 M glycerol, exhibited a survival dip, but after 10 min rather than after 5 min (Figs. 1 ‘and 4, ,dotted curve, and Ref. 6). However, the response to the dilution treatments of the cells suspended in 3 M glycerol is not nearly as neat as in the cases described previously. When the cells are subjected to concentration changes with no temperature change ( Fig. 4A), they exhibit an unusual response: Survival seems to increase with time of exposure, then ,decreases, and then increases again. Despite this unusual response, the two solid curves in that figure are strikingly similar, the response is reproducible, and both curves bear a clear similarity to the response produced by freezing. When cells suspended in 3 M glycerol are subjected to concentration changes following a temperature change (Fig. 4B),
30
IN 3 M GLYCEROL
IT
20”
C
at 20°C for varying times and then subdata of Mazur et al. (S)] or to various
they are even more sensitive than cells frozen in 3 M glycerol, at least for the first 10 min of exposure. But even in these cases, the cells display a clear survival dip after 5 to 10 min of exposure to 3 M glycerol that roughly corresponds to that shown by the cells frozen in 3 M glycerol; their survival after 15 min of exposure closely parallels that of the frozen-thawed cells. Role of Temperature Since it was clear that the sensitivity of the erythrocytes subjected to these various concentration changes had ,a temperature component, it was of interest to examine this temperature effect. To do this, erythrocytes were suspended in 2 M glycerol at 20” C for 5 min, the time at which they exhibited maximum sensitivity in that concentration (see Fig. 3). They were then cooled to various temperatures and rapidly diluted with either 2 M or 6 M glycerol at those temperatures (Fig. 5). The transfer of cells from 2 M glycerol sat 20°C into 2 M at another temperature is equivalent to “thermal shock” (1, 2). The results show that bovine erythrocytes are extremely resistant to that effect [see also Fig. 7 of
SIMULATION
OF FREEZING
Mazur et al. (6) 1. But when they are subjected to a single concentration change (2 M + 6 M) at ‘lower temperatures, the cells exhibit a clear cooperative effect of temperature. That is, they are relatively, insensitive to this concentration change down to 10°C but become increasingly sensitive to it below that temperature.
593
DAMAGE
100
1
1.4 KAT 20’ --2.8XAT~5’--I.4XAI-5Y
I
T
Cells Suspended in 1.4 M Sucrose One final test was performed to test the relevance of these dilution procedures to freezing ,damage of bovine erythrocytes. It had been found previously (6) that bovine erythrocytes frozen in I.4 M sucrose, isosmotic with 2 M glycerol, did not exhibit evidence of a survival dip as a function of time in sucrose prior to freezing (Fig. 6, dotted curve). When cells suspended in a sucrose solution are subjected to a double concentration change (I.4 M + 2.8 M + 1.4 M) following a temperature change (20” + -5”C), they ‘also display no evi-
20-
0’ 0
1
TIME
(mini
10 20 IN 14 M SUCROSE
30 AT 20°
C
FIG. 6. Survival of RBC suspended in 1.4 M sucrose at 20°C for varying times and then subjected to freezing and thawing [dotted curve, data of Mazur et al. (6)] or to dilution into 2.8 M sucrose and then back into 1.4 M sucrose at -5°C.
dence of a dip, although they are somewhat more resistant to the dilution treatment than to freezing (Fig. 6). DISCUSSION
1 01 20
10 TEMPERATURE
0 (“Cl
-10
FIG. 5. Survival of RBC suspended in 2 M glycerol for 5.0 min and then diluted with either 2 M or 6 M glycerol at various temperatures.
It is now generally accepted that the “two-factor hypothesis of freezing injury” is valid for many cell types (3, 4, 7). According to this hypothesis, cells killed by being cooled at rates greater than optimum are damaged by the formation of intracellular ice ,during freezing and its recrystallization during thawing. The two-factor hypothesis also states that cells killed by being cooled at rates less than optimum are killed by “solution effects.” This intentionally ambiguous phrase refers to the changes produced in the physical-chemical properties of solutions as they freeze. These changes include (i) lowered temperature,
S. P. LEIBO
594
TABLE Comparison
of Response of Bovine
Treatment
Transfer Initial
2OT
1
X
2 3 4
X x X X X X
; 7
Erythrocytes
to Various
sequence Initial
-5%
2OT
Treatmentsa of survival
At dip
-5°C
l&I
X x X
Dilution Percentage
6M
2oT!
1
At recovery
2M
3M
1M
2M
3M
-
97
-
-
-
-
X
81 53 22 r
90 70 56
67 42
95 91 71
92 66
81 80
X
i3 9
45 31 18
35 12 37
23 83 35
70 52 56
67 77 71
X X X freezing-thawing
.(LThe cells were suspended initially in 1, 2, or 3 M glycerol at treatments or to freezing and thawing (Treatment 7). The sequence of each dilution treatment. For example, Treatment concentration of 1, 2, or 3 M 20°C into 6 M glycerol at -.5’C, of 1, 2, or 3 M at -5’C. The survival percentages are from fined in the text.
(ii) ice crystal formation and liquid water removal as the ice grows, (iii) increase in solute concentration as water is removed,
FIG. 7. Calculated response of RBC suspended in 1, 2, and 3 M glycerol at 20°C as a function of time. The data are redrawn from Mazur et al. (5), who describe the details of the analytical as well as the experimental methods used to calculate these values. (A) Calculated water volume of the cell as a percentage of that contained by the cell in isotonic solution. ( B ) Calculated intracellular glycerol concentration as a percentage of that contained in the cell after exposure to each solution for 300 min at 20°C.
20°C, and then subjected to one of six dilution crosses, read from left to right, indicate the 6 consisted of transferring cells from an initial and then back into the initial concentration Figs. 2-5, with the dip and the recovery de-
(iv) solute crystallization and/or solidification, and (v) changes in pH. It has frequently been asserted that any one or combination of these changes may be responsible for the damage to cells frozen at rates less than optimum. One striking characteristic of the response of bovine erythrocytes frozen in glycerol is that they display a maximum in sensitivity (the dip) and then “recover” from that sensitivity. The simulation experiments were an attempt to separate the effects of some physical-chemical changes on cell survival, using the dip and the recovery as time-dependent markers ‘of the cell’s response. That is, the simulation experiments did not merely consist of searching for a treatment that would yield the same percentage survival as that produced by freezing. Rather, they were an attempt to identify a series of stresses to which the cells would respond with the same time-dependency as the cells display in response to freezing. On this basis, then, it is possible to rank the dilution treatments as to their damaging consequences (Table 1) . The relative effect of each dilution sequence is the same regardless of the initial glycerol concentration. However, the relative effect of dilution does seem to differ somewhat
SIMULATION
OF FREEZING
at the time of the dip from that at the time of recovery. That is, at the dip, the relative effectiveness of each sequence increases in the following order: (i) transfer of cells with no concentration change at 20°C (ii) transfer with no concentration change at -5°C (iii) transfer into increased concentration at 20°C (iv) transfer into increased followed by decreased concentration at 20°C (v) transfer into increased concentration at -5°C and (vi) transfer into increased followed by decreased concentration at -5°C. At recovery, the relative effectiveness differs only in that sequences (iv) and (v) appear to be reversed. As noted previously, the response of the cells to freezing and thawing seems most closely approximated by their response to dilution sequence six. This approach to examining some aspects of “solution effect” damage was suggested by the fact that bovine erythrocytes exhibit a dip when frozen in glycerol but not when frozen in sucrose. This seemed significant for the following reasons: When cells are suspended in a hyperosmotic solution of a permeating solute, the cells shrink initially in response to the osmotic pressure gradient across the membrane. Then, as the solute TABLE Comparison
of Values of Bovine
Glycerol concentration h)
Dip 1 2 3 Recovery 1 2 3
a Data from Mazur
enters the cells, they increase in volume by water uptake so ‘as to remain in osmotic equilibrium. When the cells are finally also in equilibrium with respect to the extracellular concentration of the permeating solute, they will have returned to their volume in isotonic solution. On the other hand, when cells are suspended in a hyperosmotic solution of a nonpermeating solute, the cells shrink and remain shrunken. A detailed analysis of permeation of bovine erythrocytes in concentrated glycerol solutions and their response to freezing in these solutions has been performed (5, 6). Among other things, that analysis clearly showed the fmollowing: The higher the glycerol concentration of the suspending solution, the greater the extent to which the cells shrink initially and the longer they require to return to their original volume. But the higher the glycerol concentration, the faster the rate at which glycerol enters the cells. These relationships, redrawn from Mazur et al. (5), are shown in Figs. 7A and 7B. The simulation experiments suggest the following explanation of the response of a cell suspended in a permeating solute and then frozen and thawed. (i) The cell shrinks and then gradually expands as the solute 2
Erythrocytes Time bin)
595
DAMAGE
Frozen in Glycerol water vohne (70 of isotonic)
Solutions”
Intracellular glyCWO1
(osmoles)
20 15 20 15 20 15
1 1.8 5 15 10 20
23.7 2.5.5 25.2 29.9 28.9 29.5
0.19 0.18 1.42 1.60 3.08 3.09
9 7 18 25 37 29
20 15 20 15 20 15
5 10 10 30 20 30
35.8 36.5 32.8 39.4 38.7 35.0
0.52 0.54 1.70 1.85 3.34 3.25
35 32 56 56 71 60
et al. (5, 6) ; see text for details.
596
S. P. LEIBO
enters. (ii) When frozen, the cell is exposed to a second hyperosmotic solution as water is removed from the extracellular solution in the form of ice. (iii) To remain in osmotic equilibrium, the cell shrinks a second time by losing water, this time at a subzero temperature. (iv) The cell membrane undergoes a phase transition at subzero temperatures, rendering it less able to tolerate shape changes required by the volume change. (v) The extent of freezing damage reflects the interplay of several variables of the cell itself, of the suspending solution, and of the freezing-thawing treatment. These include: intracellular glycerol concentration and its corollary, cell volume; the rate ‘and extent to which the cell must shrink (during freezing) or swell (during thawing) at subzero temperatures to achieve osmotic equilibrium; the cell’s permeability constant for water, and the temperature coefficient of that constant. (The latter variables dictate the rate at which the cell can lose water at a subzero temperature. ) The dip and recovery of bovine erythrocytes frozen in glycerol solutions seem significant. Table 2 summarizes data from Mazur et al. (5, 6) listing, for three glycerol concentrations at two exposure temperatures, the times at which the cells were maximally sensitive to freezing (the dip) and the times by which the cells ,had recovered from that maximal sensitivity. The table also lists the calculated volumes of water that the cells contained under those conditions, as well as the osmoles of intracellular glycerol and the corresponding survival values. The data in the table illustrate several pertinent points: (i) For a given concentration of glycerol, the times of the dip and the recovery increase with decreasing temperature of exposure. (ii) For a given temperature of exposure to glycerol, the times of the dip and the recovery increase with increasing glycerol concentration. (iii) However, regardless of the glycerol concentration or the temperature of exposure, the calculated water vol-
ume at the dip is about 27% (range 25.2 to 29.9% ) of the cell water volume in isotonic solution, and at recovery is about 36% (range 32.8 to 39.4% ) of the isotonic cell volume. .To attempt to explain such striking behavior, the following relevant facts must also be considered: (i) As a solution begins to freeze at temperatures above its eutectic point, the concentration in the unfrozen portion is fixed by temperature alone regardless of the initial concentration of the solution. (ii) Therefore, cells frozen in 1, 2, or 3 M glycerol must be exposed to approximately the same increased glycerol concentration at the same subzero temperature, e.g., 6 M glycerol at -23°C. (iii) However, the intracellular osmolality of glycerol at a given time prior to freezing is a function of the glycerol concentration of the initial suspending solution [Mazur et al. (6); Table 2, Fig. 7B]. (iv) The corollary of this is that the cell volume at a given time prior to freezing is also a function of the glycerol concentration of the initial suspending solution. (v) Therefore, at a given time, to achieve osmotic equilibrium with a second increased concentration of solution (e.g., 6 M glycerol), the lower the initial glycerol concentration the more the cell will be required to dehydrate. The demonstrated response of bovine erythrocytes exposed to large changes in glycerol concentration of the suspending medium suggests that some facets of freezing damage can be mimicked without actually freezing the cells. Moreover, the data also suggest that it may be possible to “dissect out” the contributions of some other physical-chemical changes to freezing damage of cells. For example the increased electrolyte concentrations to which cells are exposed when frozen have frequently been proposed as a fundamental cause of freezing damage (1, 2). It is clear from the present experiments that damage to bovine erythrocytes is not a result of the concentration of extracellular electrolyte, since it
SIMULATION
OF FREEZING
was possible to mimic a time-dependent aspect of freezing damage using glycerol solutions prepared in isotonic NaCl alone. Furthermore, comparison of the data in Figs. 2-4, together with the corresponding ratios of glycerol:salt (6), suggests that the intracellular electrolyte concentration is not the primary cause of freezing damage either. For example, after 5 min in glycerol at 20°C 83% of the cells survive transfer from 1 M into 6 M glycerol at -5”C, whereas only 33% survive transfer from 3 M in,to 6 M mat-5°C. Although the intracellular electrolyte concentration produced in the former case must be much higher than in the latter, the former is less damaging. The data in Figs. 2B and 5 demonstrate that temperature change alone (thermal shock) can play only a minor role in freezing damage of bovine erythrocytes [see also Mazur et al. ( 6 ) 1. But comparison of the data in Fig. 6 with those in Figs. 2-4 suggests that the time-dependent volume changes which these cells undergo when suspended in glycerol solutions at room temperature are critically important to the overall survival of these cells when they are exposed to large concentration changes. There is also a difference in the response of cells suspended in a given glycerol solution exposed to a single concentration change (e.g., 1 M at 20°C + 6 M at -5°C) compared with that of cells exposed to a double concentration change (e.g., 1 M at 2O"C+ 6 M at -5°C --) 1 M at -5°C). This suggests that freezing damage may result from certain deleterious events that occur during cooling and ‘others that occur deduring warming. If the simulation scribed in this paper does in fact mimic freezing damage, then the results suggest that a cell’s sensitivity to events during cooling is separable from its sensitivity during warming. In other words, during freezing the cell is required to shrink at subzero temperatures. The extent to which it will shrink depends ‘on its intracellular glycerol concentration ‘and water volume at the time of freezing. During thawing the cell is re-
DAMAGE
597
quired to swell at subzero temperatures. The extent to which it will swell depends not ,only on the conditions during freezing but also on the initial concentration of the suspending medium. That concentration will determine the volume to which the cell must swell as the solution melts. The aspect of these observations that is most intriguing is the transient maximum in sensitivity-the survival dip. I can offer no quantitative explanation of it at present. It must be related to the time-dependent changes in cell volume that the bovine erythrocyte undergoes in glycerol solutions, since the dip is not observed with sucrose solutions. Furthermore, the time of the dip in a given concentration of glycerol does seem to be related to some critical cell volume. Finally, it must have a temperature component, since the dip is most obvious when the cell is exposed to concentration changes at subzero temperatures. Despite this missing element, I suggest that a major factor in freezing damage is the relative volume of a cell at the time of freezing and the extent to which it must shrink or swell to achieve osmotic equilibrium at subzero temperatures in partially frozen or thawed solutions. SUMMARY
When a cell is frozen and thawed, it is exposed to (i) lowered temperature, (ii) increased solute concentration during freezing, and (iii) decreased solute concentration during thawing. Without actually freezing the cells, an attempt has been made to simulate physical-chemical changes to which bovine erythrocytes are exposed when frozen and thawed in glycerol solutions. Experimentally, the study consisted of suspending erythrocytes in 1, 2, or 3 M glycerol mat20°C for various times and then exposing them to each of several dilution sequences. The dilution sequences were: (i) transfer from the initial glycerol concentration at 20°C into the same concentration at -5°C (ii) transfer into an in-
598
S. P. LEIBO
creased glycerol concentration at 20” C, (iii) transfer into an increased followed by a decreased glycerol concentration at 20°C (iv) transfer into an increased glycerol concentration at -5°C and (v) transfer into an increased followed by a decreased glycerol concentration at -5°C. This last sequence is analogous to the exposure that cells undergo at subzero temperatures to increased solute concentration during freezing and decreased solute concentration during thawing. This dilution sequence yielded a survival pattern very similar to that obtained when bovine erythrocytes are frozen and thawed, and thus does appear to mimic freezing damage. It is concluded that a major factor in freezing damage is the extent to which a cell must shrink or swell t’o achieve osmotic equilibrium at subzero temperatures in partially frozen or thawed solutions. ACKNOWLEDGMENTS Many followed perature Division,
of the ideas described in this paper have from continual discussions of low-tembiology with colleagues in the Biology ORNL. I especially thank Peter Mazur
for his valuable contributions to this work, and also thank William D. Fisher and John S. Cook for theirs. Portions of this work were first presented at the Ninth Annual Meeting of the Society for Cryobiology [Cryobiology 9, 320 ( 1972)]. REFERENCES 1. Farrant, J., and Morris, G. J. Thermal shock and dilution shock as the causes of freezing injury. Cryobiology 10, 134-140 ( 1973). 2. Lovelock, J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim. Biophys. Acta 11, 28-36 (1953). 3. Mazur, P. Cryobiology: The freezing of biological systems. Science 168, 939-949 (1970). 4. Mazur, P., Leibo, S. P., and Chu, E. H. Y. A two-factor hypothesis of freezing injury. Exp. Cell Res. 71, 345-355 (1972). 5. Mazur, P., Leibo, S. P., and Miller, R. H. Permeability of the bovine red cell to glycerol in hyperosmotic solutions at various temperatures. .I. Membr. Biol. 15, 107-136 ( 1974). 6. Mazur, P., Miller, R. H., and Leibo, S. P. Survival of frozen-thawed bovine red cells as a function of the permeation of glycerol and sucrose. .I. Me&r. Biol. 15, 137-158 ( 1974). 7. Meryman, H. T. Freezing injury and its prevention in living cells. Ann. Rev. Biophys. Bioeng. 3, 341-363 (1974).
13, 587-598
Freezing
(1976)
Damage of Bovine Erythrocytes: Simulation Using Glycerol Concentration Changes at Subzero Temperatures1 S. P. LEIBO
Biology
Division,
Oak Ridge National
Laboratory,
When a cell is frozen and thawed, it must be exposed to the following changes in the properties of the suspending solution: (i) lowered temperature, (ii) increased concentration of the extracellular solution as water is removed in the form of ice during freezing, and (iii) decreased concentration during thawing. Without actually freezing the cells, I have attempted to simulate some of the physical-chemical changes to which bovine erythrocytes are exposed when frozen and thawed in glycerol solutions. These experiments followed from a mathematical and experimental analysis ‘of glycerol permeation in the bovine erythrocyte (5) and a correlative study ‘of that cell’s freezing sensitivity (6). The permeation study demonstrated that the permeability constant of the bovine erythrocyte for glycerol is relatively low with ‘a high activation energy. This means that it is possible to control the intracellular glycerol concentration of that cell by varying the time and/or temperature of exposure to glycerol. The freezing study demonstrated that protection against freezing damage does not require permeation by glycerol. This conclusion followed from the fact that the same percentages of cells survived freezing even when there was a 300-fold difference in the effective intracellular concentration of glycerol. But that
study also identified a transient increased sensitivity to freezing that depended on the length ‘of exposure to glycerol prior to freezing. This increased sensitivity, or survival “dip,” is illustrated by the results shown in Fig. 1 that are reproduced from Mazur et al. (6, their Fig. 7). Those results show that the time of the dip is characteristic for the glycerol concentration to which the cells are exposed prior to freezing. The objective of the presenf study was to use that time-dependent dip as a marker to attempt to separate the effects on cell survival of various dilution treatments. The fundamental variable in these experiments was the time of exposure to glycerol prior to the treatment. The test of the relevance of these simulation experiments to freezing damge depended not just on the extent to which an equivalent percentage survival could be produced. Rather, a successful test would require that the simulation experiments yield a survival dip at the same time as that produced by freezing and thawing. Using the dip as a marker, therefore, I have attempted to distinguish between the effects of the following changes on cell survival: (i) change in temperature versus change in solute concentration, (ii) change in glycerol concentration versus change in electrolyte concentration, and (iii) changes that occur during freezing versus changes that occur during thawing. The results show that the response of bovine erythrocytes when expo,sed to large changes in the concentration of extracellular glycerol at
Received July 28, 1975. 1 Research sponsored by the Energy Research and Development Administration under contract with the Union Carbide Corporation. 587 1976 by Academic Press, Inc. Copyright All rights o6 reproduction in any form reserved.
Oak Ridge, Tennessee 37830
588
S. P. LEIBO 100
times. Then, the cell suspensions were subjected to the f’ollowing sequences of temperature and/or concentration changes:
1
(i) a temperature change with no concentration change; (ii) a concentration change with no temperature change; (iii) an increased concentration change followed by a decreased concentration change with no temperature change; (iv) a temperature change followed by an increased concentration change; and (v) a temperature change followed by an increased concentration change followed, in turn, by a decreased concentration change.
3M
0’ 0
10 TIME (min) IN GLYCEROL
20 AT 20 *C
30
FIG. 1. Survival, as percentage of unhemolyzed cells, of bovine erythrocytes as a function of time of exposure at 20°C to 1, 2, or 3 M glycerol prior to freezing to -196°C at 43”C/min. The data are taken directly from Mazur et al. (6) who describe the experimental details.
subzero temperatures parallels their response when frozen and thawed in glycerol solutions. MATERIALS
AND
METHODS
The rationale of the simulation experiments was to examine the individual effect of some of the physical-chemical changes that occur concomitantly as a cell suspension is frozen. The concatenation of changes that ‘occurs ,during freezing is first that the temperature is reduced. Then, as ice forms, there is an increase in the concentration of the suspending solution. During thawing, the sequence is reversed so that the concentration of the soIution is decreased. It is important to note that these concentration changes occur at subzero temperatures. The experimental approach, then, was to examine the reIative effects on ceH survival of each of those steps. Operationally, the experiments consisted of suspending bovine erythrocytes in concentrated glycerol solutions at room temperature for varying
The temperature change consisted of transferring cells suspended in 1, 2, or 3 M glycerol from room temperature to -5°C. A single concentration change consisted of transferring cells from the initia1 concentrations of 1, 2, or 3 M glycerol into a 6 M glycerol solution. A double concentration change consisted of transferring cells from the initial glycerol solutions into a 6 M glycerol solution and then back into the initial glycerol solution. Portions of the procedure were also repeated for erythrocytes suspended initially in I.4 M sucrose, since bovine erythrocytes are impermeable to it and display no survival dip when frozen in sucrose solutions (5, 6). Two aspects of this procedure require special discussion, namely, the choice of -5°C as the subzero temperature of exposure and the choice of 6~ glycerol as the “shocking” concentration. The temperature of -5°C was selected for several reasons: (i) The temperature had to be low enough to examine phenomena at subzero temperatures. (ii) The temperature had to be high enough so that erythrocytes in 1 M glycerol would not freeze but rather would only supercool. (iii) Since the respective freezing points of 1 M, 2 M, and 3 M glycerol prepared with isotonic 0.165 M NaCl are about -1.9”, -4.6”, and -8.l”C, a temperature of -5°C seemed a reasonable
SIMULATION
OF FREEZING
compromise. The concentration of 6 M glycerol was selected to mimic the element of s’olute concentration resulting from freezing, because 6 M glycerol prepared with 0.165 M NaCl has a freezing point of about -23°C which is in the range at which erythrocytes frozen in glycerol solutions begin to exhibit substantial hemolysis. The experiments were performed using bovine erythrocytes drawn by venipuncture into a heparinized flask and stored at 4°C for no more than 7 days until used. Just before use, the erythrocytes were washed three times by centrifugation and resuspension at the original cell concentration in 0.165 M phosphate-buffered saline (pH 7.0). All glycerol and sucrose solutions were prepared with 0.165 M saline buffer and, for convenience, will be designated only in terms ‘of the glycerol molarity. Each experiment consisted of the following steps: The washed erythrocytes were diluted with an equal volume of a glycerol solution at twice the desired final glycerol concentration at 260°C. Precise temperature control at this point was important because the activation energy for glycerol permeation in the bovine erythrocyte is about 21 kcal/mole (5). The cells, now suspended in 1, 2, or 3 M glycerol at 2O”C, were held for various periods of time; then 0.05-ml volumes were pipetted with Hamilton syringes into tubes previously equilibrated at the appropriate temperature (usually 20” or -5°C). After a 30-set pause to allow for temperature equilibration, 0.45ml volumes of glycerol solutions ,at the appropriate concentrations and temperatures were rapidly pipetted into the tubes to mix the cell suspensions. These suspensions were held for 1.0 min longer, although the results of holding the suspensions for 5.0 min at -5°C at this step were indistinguishable from the results with only 1.0 min holding. Then 4.5 ml of the third glycerol solution at the appropriate concentration and temperature was pipetted into the suspension. The diluted suspensions were held for 5 min longer at the test temperature
DAMAGE
589
and then placed into a 20°C bath for an additional 5 min before being centrifuged at 10,060g for 10 min at room temperature. The supernatants were removed, and the amount of hemoglobin present was read at 540 nm as cyanmethemoglobin. This value was then calculated as a percentage of the amount of hemoglobin released from totally hemolyzed control samples, which consisted of cells suspended in the first glycerol solution, handled with the experimental samples, but diluted in two steps with 0.01 M phosphate buffer at pH 7.0. In other words, the percent hemolysis was calculated as [ OD ( experimental) /OD ( total hemolysis control)] X 100, ‘and the results were expressed as “percent survival,” i.e., ( 100% minus percent hemolysis ) . Each treatment was repeated with different batches of blood three to five times, and the results are shown as the mean percent survival + 1 standard error. Although some treatments consisted of diluting a suspension with the same concentration of glycerol, all samples (with the exception of those whose response is shown in Fig. 5) were subjected to the same number of dilution steps because erythrocytes in hypertonic solutions can be sensitive to handling alone (5). The results are #labeled according to the glycerol concentration and the temperatures for each of the dilution steps. For example, in figure legends the label 2 M at 20°C’ 6 M at -5°C * 6 M at -5°C indicates that these samples were suspended in 2 M glycerol at 20°C for various times, cooled to -5°C diluted IO-fold at -5°C with 6 M glycerol, and diluted lo-fold again at -5°C with 6 M glycerol. All samples were warmed to room temperature before hemolysis was measured. The final figure for glycerol concentration for each curve indicates the concentration in which hemolysis was determined. Measurements showed that Beer’s law for cyanmethemoglobin was unaffected by glycerol concentrations up to 6 M. Measurements of the effect of temperature (Fig. 5) were obtained in a slightly different manner. In that experiment the
S. P. LEIBO
590
TIME hd
IN,
M GLYCEROL
AT 20”
C
FIG. 2. Survival of RBC suspended in I M glycerol at 20°C for varying times and then subjected either to freezing and thawing [dotted curve, data of Mazur et al. (6)] or to various dilution treatments at 20°C (A) or at -5°C (B).
washed cells were diluted with an equal volume of 4.0 M glycerol and held for 5.0 min at 2O.O”C; then O.l-ml samples were pipetted into tubes previously equilibrated at the test temperatures. Thirty seconds later, 9.9-ml volumes of either 2 M or 6 M glycerol were rapidly pipetted into each sample. The samples were held for 5 min at the test temperature before being warmed to 20°C for 5 min; then the amount of hemolysis was measured as described above. The response of erythrocytes suspended in 1.4 M sucrose and diluted at -5°C was measured in a fashion similar to that used with glycerol. The cells were diluted with an equal volume of 2.8 M sucrose in isotonic saline buffer, held at 20°C for varying times, cooled to -5°C diluted IO-fold with 2.8 M sucrose at that temperature, and then diluted lo-fold again with 1.4 M sucrose at -5°C before the sample was warmed to 20°C and its amount of hemolysis was measured. RESULTS
Cells Suspended in 1 M Glycerol When bovine erythrocytes suspended in 1 l\f glycerol at 20°C for varying times were
frozen at about 40”C/min to -196°C and then thawed rapidly, they exhibited a distinctive response (Figs. 1 and 2, dotted curve, and Ref. 6). Survival was low after short exposure times, but within 5 min the survival Ievel was equal to that displayed after 30 min exposure (Fig. 2A, dotted curve). In the present experiments, when bovine red cells suspended in 1 M glycerol at 20°C for varying times are subjected to a variety of dilution procedure,s without freezing, they exhibit an analogous response (Figs. 2A, B). The cells are most sensitive when diluted after only brief exposures to 1 M glycerol at 2O”C, but become less sensitive after about 5 to 10 min of exposure to 1 M glycerol. The first series of experiments was designed to simulate the concentration changes, without a temperature change, to which cells are exposed when frozen and thawed (Fig. 2A). The results show that subjecting cells to a single ( 1 M * 6 M) or to a double concentration change (1 M + 6 M + 1 M) produces significant hemolysis during the first few min of exposure to 1 M glycerol, but relativeIy little after 5 min or more of exposure. The former treatment is analogous to the concentration of solute
SIMULATION
OF FREEZING
that occurs during freezing, and the latter to the concentration and dilution of solute that occurs during freezing and thawing. The second series of experiments was designed to examine the role of such concentration changes when they occur at subzero temperatures (Fig. 2B). The results show that subjecting cells to a temperature change alone (20’ + -5°C) in the absence of a concentration change produces little hemolysis. On the other hand, subjecting cells to a single concentration change a temperature (1 M + 6 M) following -5°C) produces substanchange (20’ -+ tial hemolysis during the first few minutes of exposure to 1 M glycerol, but very much less hemolysis after 5 min or more of exposure. When the cells are exposed to a double concentration change (1 M -+ 6 M + 1 M) following a temperature change their response closely re(ZOO-+ -S’C), sembles that produced by freezing. Cells Suspended in 2 M Glycerol The behavior of red cells suspended in 2 M glycerol at 20°C for varying times is analogous (Figs. 3A, B). When the cells were frozen to -196°C and thawed rapidly,
DAMAGE
591
Mazur et al. (6) found that survival after 20 set in 2 M glycerol ,at 20°C was as high as after 30 min of exposure before freezing but that the cells displayed a dramatic dip in survival when frozen after 5 min in 2 M glycerol (Figs. 1 and 3, dotted curve). Again, the first series of experiments was designed to simulate the concentration changes alone that occur during freezing and thawing (Fig. 3A). The results show that cells exposed to a single concentration change (2 M + 6 M) exhibit a survival dip after about a 5 min exposure to 2 M glycerol. Cells exposed to a double concentration change (2 M + 6~+2h1) alsoexhibit a survival dip, though less distinct than that following a single concentration change. When cells suspended in 2 M glycerol are subjected to a single concentration change (2 M + 6 M) at -5°C they also exhibit a clear survival dip after a 5 min exposure to 2 M glycerol (Fig. 3B ) , And when cells are subjected to a double concentration change (2 M + 6 M + 2 M) following a temperature change (20’ + -5”C), they not only display a survival dip after 2 to 5 min exposure, but also exhibit virtually the
FIG. 3. Survival of RBC suspended in 2 M glycerol at 20°C for varying times and then subjected either to freezing and thawing [dotted curve, data of Mazur et al. (6)] or to various dilution treatments at 20°C (A) or at -5°C (B).
592
S. P. LEIBO
100,
A
20
i
Oo.0
0. TIME
lmln)
FIG. 4. Survival of RBC suspended in 3 M glycerol jected either to freezing and thawing [dotted curve, dilution treatments at 20°C (A) or at -5°C (B).
same survival following 10 to 30 min exposure as that shown when the erythrocytes are frozen in 2 M glycerol. Cells Suspended in 3 M Glycerol When erythrocytes suspended in 3 M glycerol at 20°C f,or varying times were frozen and thawed, they, like the cells in 2 M glycerol, exhibited a survival dip, but after 10 min rather than after 5 min (Figs. 1 ‘and 4, ,dotted curve, and Ref. 6). However, the response to the dilution treatments of the cells suspended in 3 M glycerol is not nearly as neat as in the cases described previously. When the cells are subjected to concentration changes with no temperature change ( Fig. 4A), they exhibit an unusual response: Survival seems to increase with time of exposure, then ,decreases, and then increases again. Despite this unusual response, the two solid curves in that figure are strikingly similar, the response is reproducible, and both curves bear a clear similarity to the response produced by freezing. When cells suspended in 3 M glycerol are subjected to concentration changes following a temperature change (Fig. 4B),
30
IN 3 M GLYCEROL
IT
20”
C
at 20°C for varying times and then subdata of Mazur et al. (S)] or to various
they are even more sensitive than cells frozen in 3 M glycerol, at least for the first 10 min of exposure. But even in these cases, the cells display a clear survival dip after 5 to 10 min of exposure to 3 M glycerol that roughly corresponds to that shown by the cells frozen in 3 M glycerol; their survival after 15 min of exposure closely parallels that of the frozen-thawed cells. Role of Temperature Since it was clear that the sensitivity of the erythrocytes subjected to these various concentration changes had ,a temperature component, it was of interest to examine this temperature effect. To do this, erythrocytes were suspended in 2 M glycerol at 20” C for 5 min, the time at which they exhibited maximum sensitivity in that concentration (see Fig. 3). They were then cooled to various temperatures and rapidly diluted with either 2 M or 6 M glycerol at those temperatures (Fig. 5). The transfer of cells from 2 M glycerol sat 20°C into 2 M at another temperature is equivalent to “thermal shock” (1, 2). The results show that bovine erythrocytes are extremely resistant to that effect [see also Fig. 7 of
SIMULATION
OF FREEZING
Mazur et al. (6) 1. But when they are subjected to a single concentration change (2 M + 6 M) at ‘lower temperatures, the cells exhibit a clear cooperative effect of temperature. That is, they are relatively, insensitive to this concentration change down to 10°C but become increasingly sensitive to it below that temperature.
593
DAMAGE
100
1
1.4 KAT 20’ --2.8XAT~5’--I.4XAI-5Y
I
T
Cells Suspended in 1.4 M Sucrose One final test was performed to test the relevance of these dilution procedures to freezing ,damage of bovine erythrocytes. It had been found previously (6) that bovine erythrocytes frozen in I.4 M sucrose, isosmotic with 2 M glycerol, did not exhibit evidence of a survival dip as a function of time in sucrose prior to freezing (Fig. 6, dotted curve). When cells suspended in a sucrose solution are subjected to a double concentration change (I.4 M + 2.8 M + 1.4 M) following a temperature change (20” + -5”C), they ‘also display no evi-
20-
0’ 0
1
TIME
(mini
10 20 IN 14 M SUCROSE
30 AT 20°
C
FIG. 6. Survival of RBC suspended in 1.4 M sucrose at 20°C for varying times and then subjected to freezing and thawing [dotted curve, data of Mazur et al. (6)] or to dilution into 2.8 M sucrose and then back into 1.4 M sucrose at -5°C.
dence of a dip, although they are somewhat more resistant to the dilution treatment than to freezing (Fig. 6). DISCUSSION
1 01 20
10 TEMPERATURE
0 (“Cl
-10
FIG. 5. Survival of RBC suspended in 2 M glycerol for 5.0 min and then diluted with either 2 M or 6 M glycerol at various temperatures.
It is now generally accepted that the “two-factor hypothesis of freezing injury” is valid for many cell types (3, 4, 7). According to this hypothesis, cells killed by being cooled at rates greater than optimum are damaged by the formation of intracellular ice ,during freezing and its recrystallization during thawing. The two-factor hypothesis also states that cells killed by being cooled at rates less than optimum are killed by “solution effects.” This intentionally ambiguous phrase refers to the changes produced in the physical-chemical properties of solutions as they freeze. These changes include (i) lowered temperature,
S. P. LEIBO
594
TABLE Comparison
of Response of Bovine
Treatment
Transfer Initial
2OT
1
X
2 3 4
X x X X X X
; 7
Erythrocytes
to Various
sequence Initial
-5%
2OT
Treatmentsa of survival
At dip
-5°C
l&I
X x X
Dilution Percentage
6M
2oT!
1
At recovery
2M
3M
1M
2M
3M
-
97
-
-
-
-
X
81 53 22 r
90 70 56
67 42
95 91 71
92 66
81 80
X
i3 9
45 31 18
35 12 37
23 83 35
70 52 56
67 77 71
X X X freezing-thawing
.(LThe cells were suspended initially in 1, 2, or 3 M glycerol at treatments or to freezing and thawing (Treatment 7). The sequence of each dilution treatment. For example, Treatment concentration of 1, 2, or 3 M 20°C into 6 M glycerol at -.5’C, of 1, 2, or 3 M at -5’C. The survival percentages are from fined in the text.
(ii) ice crystal formation and liquid water removal as the ice grows, (iii) increase in solute concentration as water is removed,
FIG. 7. Calculated response of RBC suspended in 1, 2, and 3 M glycerol at 20°C as a function of time. The data are redrawn from Mazur et al. (5), who describe the details of the analytical as well as the experimental methods used to calculate these values. (A) Calculated water volume of the cell as a percentage of that contained by the cell in isotonic solution. ( B ) Calculated intracellular glycerol concentration as a percentage of that contained in the cell after exposure to each solution for 300 min at 20°C.
20°C, and then subjected to one of six dilution crosses, read from left to right, indicate the 6 consisted of transferring cells from an initial and then back into the initial concentration Figs. 2-5, with the dip and the recovery de-
(iv) solute crystallization and/or solidification, and (v) changes in pH. It has frequently been asserted that any one or combination of these changes may be responsible for the damage to cells frozen at rates less than optimum. One striking characteristic of the response of bovine erythrocytes frozen in glycerol is that they display a maximum in sensitivity (the dip) and then “recover” from that sensitivity. The simulation experiments were an attempt to separate the effects of some physical-chemical changes on cell survival, using the dip and the recovery as time-dependent markers ‘of the cell’s response. That is, the simulation experiments did not merely consist of searching for a treatment that would yield the same percentage survival as that produced by freezing. Rather, they were an attempt to identify a series of stresses to which the cells would respond with the same time-dependency as the cells display in response to freezing. On this basis, then, it is possible to rank the dilution treatments as to their damaging consequences (Table 1) . The relative effect of each dilution sequence is the same regardless of the initial glycerol concentration. However, the relative effect of dilution does seem to differ somewhat
SIMULATION
OF FREEZING
at the time of the dip from that at the time of recovery. That is, at the dip, the relative effectiveness of each sequence increases in the following order: (i) transfer of cells with no concentration change at 20°C (ii) transfer with no concentration change at -5°C (iii) transfer into increased concentration at 20°C (iv) transfer into increased followed by decreased concentration at 20°C (v) transfer into increased concentration at -5°C and (vi) transfer into increased followed by decreased concentration at -5°C. At recovery, the relative effectiveness differs only in that sequences (iv) and (v) appear to be reversed. As noted previously, the response of the cells to freezing and thawing seems most closely approximated by their response to dilution sequence six. This approach to examining some aspects of “solution effect” damage was suggested by the fact that bovine erythrocytes exhibit a dip when frozen in glycerol but not when frozen in sucrose. This seemed significant for the following reasons: When cells are suspended in a hyperosmotic solution of a permeating solute, the cells shrink initially in response to the osmotic pressure gradient across the membrane. Then, as the solute TABLE Comparison
of Values of Bovine
Glycerol concentration h)
Dip 1 2 3 Recovery 1 2 3
a Data from Mazur
enters the cells, they increase in volume by water uptake so ‘as to remain in osmotic equilibrium. When the cells are finally also in equilibrium with respect to the extracellular concentration of the permeating solute, they will have returned to their volume in isotonic solution. On the other hand, when cells are suspended in a hyperosmotic solution of a nonpermeating solute, the cells shrink and remain shrunken. A detailed analysis of permeation of bovine erythrocytes in concentrated glycerol solutions and their response to freezing in these solutions has been performed (5, 6). Among other things, that analysis clearly showed the fmollowing: The higher the glycerol concentration of the suspending solution, the greater the extent to which the cells shrink initially and the longer they require to return to their original volume. But the higher the glycerol concentration, the faster the rate at which glycerol enters the cells. These relationships, redrawn from Mazur et al. (5), are shown in Figs. 7A and 7B. The simulation experiments suggest the following explanation of the response of a cell suspended in a permeating solute and then frozen and thawed. (i) The cell shrinks and then gradually expands as the solute 2
Erythrocytes Time bin)
595
DAMAGE
Frozen in Glycerol water vohne (70 of isotonic)
Solutions”
Intracellular glyCWO1
(osmoles)
20 15 20 15 20 15
1 1.8 5 15 10 20
23.7 2.5.5 25.2 29.9 28.9 29.5
0.19 0.18 1.42 1.60 3.08 3.09
9 7 18 25 37 29
20 15 20 15 20 15
5 10 10 30 20 30
35.8 36.5 32.8 39.4 38.7 35.0
0.52 0.54 1.70 1.85 3.34 3.25
35 32 56 56 71 60
et al. (5, 6) ; see text for details.
596
S. P. LEIBO
enters. (ii) When frozen, the cell is exposed to a second hyperosmotic solution as water is removed from the extracellular solution in the form of ice. (iii) To remain in osmotic equilibrium, the cell shrinks a second time by losing water, this time at a subzero temperature. (iv) The cell membrane undergoes a phase transition at subzero temperatures, rendering it less able to tolerate shape changes required by the volume change. (v) The extent of freezing damage reflects the interplay of several variables of the cell itself, of the suspending solution, and of the freezing-thawing treatment. These include: intracellular glycerol concentration and its corollary, cell volume; the rate ‘and extent to which the cell must shrink (during freezing) or swell (during thawing) at subzero temperatures to achieve osmotic equilibrium; the cell’s permeability constant for water, and the temperature coefficient of that constant. (The latter variables dictate the rate at which the cell can lose water at a subzero temperature. ) The dip and recovery of bovine erythrocytes frozen in glycerol solutions seem significant. Table 2 summarizes data from Mazur et al. (5, 6) listing, for three glycerol concentrations at two exposure temperatures, the times at which the cells were maximally sensitive to freezing (the dip) and the times by which the cells ,had recovered from that maximal sensitivity. The table also lists the calculated volumes of water that the cells contained under those conditions, as well as the osmoles of intracellular glycerol and the corresponding survival values. The data in the table illustrate several pertinent points: (i) For a given concentration of glycerol, the times of the dip and the recovery increase with decreasing temperature of exposure. (ii) For a given temperature of exposure to glycerol, the times of the dip and the recovery increase with increasing glycerol concentration. (iii) However, regardless of the glycerol concentration or the temperature of exposure, the calculated water vol-
ume at the dip is about 27% (range 25.2 to 29.9% ) of the cell water volume in isotonic solution, and at recovery is about 36% (range 32.8 to 39.4% ) of the isotonic cell volume. .To attempt to explain such striking behavior, the following relevant facts must also be considered: (i) As a solution begins to freeze at temperatures above its eutectic point, the concentration in the unfrozen portion is fixed by temperature alone regardless of the initial concentration of the solution. (ii) Therefore, cells frozen in 1, 2, or 3 M glycerol must be exposed to approximately the same increased glycerol concentration at the same subzero temperature, e.g., 6 M glycerol at -23°C. (iii) However, the intracellular osmolality of glycerol at a given time prior to freezing is a function of the glycerol concentration of the initial suspending solution [Mazur et al. (6); Table 2, Fig. 7B]. (iv) The corollary of this is that the cell volume at a given time prior to freezing is also a function of the glycerol concentration of the initial suspending solution. (v) Therefore, at a given time, to achieve osmotic equilibrium with a second increased concentration of solution (e.g., 6 M glycerol), the lower the initial glycerol concentration the more the cell will be required to dehydrate. The demonstrated response of bovine erythrocytes exposed to large changes in glycerol concentration of the suspending medium suggests that some facets of freezing damage can be mimicked without actually freezing the cells. Moreover, the data also suggest that it may be possible to “dissect out” the contributions of some other physical-chemical changes to freezing damage of cells. For example the increased electrolyte concentrations to which cells are exposed when frozen have frequently been proposed as a fundamental cause of freezing damage (1, 2). It is clear from the present experiments that damage to bovine erythrocytes is not a result of the concentration of extracellular electrolyte, since it
SIMULATION
OF FREEZING
was possible to mimic a time-dependent aspect of freezing damage using glycerol solutions prepared in isotonic NaCl alone. Furthermore, comparison of the data in Figs. 2-4, together with the corresponding ratios of glycerol:salt (6), suggests that the intracellular electrolyte concentration is not the primary cause of freezing damage either. For example, after 5 min in glycerol at 20°C 83% of the cells survive transfer from 1 M into 6 M glycerol at -5”C, whereas only 33% survive transfer from 3 M in,to 6 M mat-5°C. Although the intracellular electrolyte concentration produced in the former case must be much higher than in the latter, the former is less damaging. The data in Figs. 2B and 5 demonstrate that temperature change alone (thermal shock) can play only a minor role in freezing damage of bovine erythrocytes [see also Mazur et al. ( 6 ) 1. But comparison of the data in Fig. 6 with those in Figs. 2-4 suggests that the time-dependent volume changes which these cells undergo when suspended in glycerol solutions at room temperature are critically important to the overall survival of these cells when they are exposed to large concentration changes. There is also a difference in the response of cells suspended in a given glycerol solution exposed to a single concentration change (e.g., 1 M at 20°C + 6 M at -5°C) compared with that of cells exposed to a double concentration change (e.g., 1 M at 2O"C+ 6 M at -5°C --) 1 M at -5°C). This suggests that freezing damage may result from certain deleterious events that occur during cooling and ‘others that occur deduring warming. If the simulation scribed in this paper does in fact mimic freezing damage, then the results suggest that a cell’s sensitivity to events during cooling is separable from its sensitivity during warming. In other words, during freezing the cell is required to shrink at subzero temperatures. The extent to which it will shrink depends ‘on its intracellular glycerol concentration ‘and water volume at the time of freezing. During thawing the cell is re-
DAMAGE
597
quired to swell at subzero temperatures. The extent to which it will swell depends not ,only on the conditions during freezing but also on the initial concentration of the suspending medium. That concentration will determine the volume to which the cell must swell as the solution melts. The aspect of these observations that is most intriguing is the transient maximum in sensitivity-the survival dip. I can offer no quantitative explanation of it at present. It must be related to the time-dependent changes in cell volume that the bovine erythrocyte undergoes in glycerol solutions, since the dip is not observed with sucrose solutions. Furthermore, the time of the dip in a given concentration of glycerol does seem to be related to some critical cell volume. Finally, it must have a temperature component, since the dip is most obvious when the cell is exposed to concentration changes at subzero temperatures. Despite this missing element, I suggest that a major factor in freezing damage is the relative volume of a cell at the time of freezing and the extent to which it must shrink or swell to achieve osmotic equilibrium at subzero temperatures in partially frozen or thawed solutions. SUMMARY
When a cell is frozen and thawed, it is exposed to (i) lowered temperature, (ii) increased solute concentration during freezing, and (iii) decreased solute concentration during thawing. Without actually freezing the cells, an attempt has been made to simulate physical-chemical changes to which bovine erythrocytes are exposed when frozen and thawed in glycerol solutions. Experimentally, the study consisted of suspending erythrocytes in 1, 2, or 3 M glycerol mat20°C for various times and then exposing them to each of several dilution sequences. The dilution sequences were: (i) transfer from the initial glycerol concentration at 20°C into the same concentration at -5°C (ii) transfer into an in-
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creased glycerol concentration at 20” C, (iii) transfer into an increased followed by a decreased glycerol concentration at 20°C (iv) transfer into an increased glycerol concentration at -5°C and (v) transfer into an increased followed by a decreased glycerol concentration at -5°C. This last sequence is analogous to the exposure that cells undergo at subzero temperatures to increased solute concentration during freezing and decreased solute concentration during thawing. This dilution sequence yielded a survival pattern very similar to that obtained when bovine erythrocytes are frozen and thawed, and thus does appear to mimic freezing damage. It is concluded that a major factor in freezing damage is the extent to which a cell must shrink or swell t’o achieve osmotic equilibrium at subzero temperatures in partially frozen or thawed solutions. ACKNOWLEDGMENTS Many followed perature Division,
of the ideas described in this paper have from continual discussions of low-tembiology with colleagues in the Biology ORNL. I especially thank Peter Mazur
for his valuable contributions to this work, and also thank William D. Fisher and John S. Cook for theirs. Portions of this work were first presented at the Ninth Annual Meeting of the Society for Cryobiology [Cryobiology 9, 320 ( 1972)]. REFERENCES 1. Farrant, J., and Morris, G. J. Thermal shock and dilution shock as the causes of freezing injury. Cryobiology 10, 134-140 ( 1973). 2. Lovelock, J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim. Biophys. Acta 11, 28-36 (1953). 3. Mazur, P. Cryobiology: The freezing of biological systems. Science 168, 939-949 (1970). 4. Mazur, P., Leibo, S. P., and Chu, E. H. Y. A two-factor hypothesis of freezing injury. Exp. Cell Res. 71, 345-355 (1972). 5. Mazur, P., Leibo, S. P., and Miller, R. H. Permeability of the bovine red cell to glycerol in hyperosmotic solutions at various temperatures. .I. Membr. Biol. 15, 107-136 ( 1974). 6. Mazur, P., Miller, R. H., and Leibo, S. P. Survival of frozen-thawed bovine red cells as a function of the permeation of glycerol and sucrose. .I. Me&r. Biol. 15, 137-158 ( 1974). 7. Meryman, H. T. Freezing injury and its prevention in living cells. Ann. Rev. Biophys. Bioeng. 3, 341-363 (1974).