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To be published in
Proceedings Symposium on Cryosurgery
UCLA Medical School
March 11-12, 1967
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CESTI BUICUS
no 13.00 mx .65
Peter Mazur
Biology Division, Oak Ridge National Laboratory,
Oak Ridge, Tennessee
LEGAL NOTICE
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with the Commission, or bio employment mul much contractor.
Research sponsored by the U. S. Atomic Energy Commission under contract with
the Union Carbide Corporation.
DISTRIBUTION OF THIS DOCUMENU IS UNLIMITED
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Running head: Celi Injury in Cryosurgical freezing
Send proof to: Dr. Peter Mazur
Biology Division
Oak Ridge National Laboratory
P. O. Box Y
Oak Ridge, Tennessee 37830
I. INTRODUCTION
The central aim of cryosurgery 16 to kill all cells in a diseased
target area while producing minimal injury in the surrounding healthy tissue.
Achieving this aim involves two decisions. The first is to estimate the
boundary of the diseased portion of the tissue or organ. The second 18 to
determine the conditions of freezing that should be employed to maximize
Injury within that boundary. It is sometimes assumed that all cells within
a visibly frozen aroa will be killed, but this assumption 18 unlikely to be
factors that underlle cellular injury at subzero temperatures, and it is
the purpose of this paper to review some of what 18 known about these factors.
The basic causes of freezing injury are understood best in isolated cells,
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and, hence, the discussion will to consider the cause of ingurgitsingte
bits. Although the resulting conclusions may not apply quantitatively to
tissues and organs, they should apply qualitatively.
II. PHYSICAL-CHEMICAL FACTORS UNDERLYING INJURY IN SINGLE CELLS
When cells are subjected to subzero temperatures, they are subjected to
lowered temperatures as well as to the consequences of ice formation. If
injury occurs, a first question t ok 18 to what extent is it due to cooline
and to what extent to ice formation?
A. The Effects of Lowered Temperature:
Lowering the temperature of biological systems obviously produces
profound biochemical and physiological changes: Respiration and growth,
muscular contraction, nerve conduction -- all slow or cease. But in most
cases the alterations are reversible; returning the temperature to normal
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restores normal function. This 18 generally true at subzero temperatures
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as well. Yeast and bacterial cells, fungus spores, and bacterial
viruses can be cooled without damage to -25° to -40° C, provided that the
suspending medium does not freeze (Mazur, 1966; Leibo and Mazur, 1967).
Survival might be possible at even lower temperatures if it were possible
to keep the extracellular medium unfrozen.
There are, however, some striking exceptions to the generalization Just
stated. Nonhibernating mammals usually cannot be cooled more than a few
degrees below their body temperature without being killed. Moreover, even
certain single cells are killed under special conditio:18 by sudden chilling
to temperatures near O° C. Rapid chilling, for example, is highly injurious
to bull spermatozoa and to cells of Escherichia coli in the logarithmic
growth phase. On the other hand, it is completely innocuous to human
spermatozoa and to cells to E. coli in the stationary phase. The cause of
injury, when it occurs, is obscure; however, several hypotheses have been
Buggested (Smith, 1961; Strange and Dark, 1962).
B. Events in Single Cells During Ice Formation:
A number of investigators have observed single cells under the microscope
during freezing. They invariably note that ice appears in the extracellular
medium before it appears inside the cells; and they generally report that
the cells themselves remain unfrozen at temperatures as low as -5° to -15° C,
even in the presence of extracellular ice.
Since the freezing point of most cells is above -1° C, any cell that is
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unfrozen below -1° C is by definition, supé-cooled. The supercooled state is
thermodynamically unstable, herunettthewert r oppodofibelentity
Toursuperiorettore will be eliminated in two possible ways: by dehydration or
by intracellular freezing.
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Mguro 1 depicts a supercooled cell that has just become surrounded by
extracellular ice. Equations (1) and (2) show the effect of decreasing
temperature on the vapor pressure of water outside and inside the cell,
respectively. The equations show that, 11 no water were to leave the
cell (1.6., dinx,/01 - 0), the vapor pressure inside the cell would become
increasingly greator than that outside, a statement that is equivalent to
saying that the coll would become increasingly supercooled as cooling
progresses. But equation (3) shows that this very incsseuse in the vapor
pressure difference will force water to leave the cell; and it shows that
the greater the vapor pressure difference, the greater will be the rate of
water loss (1.e., the bigger the absolute value of - av/at).
Aɛ water leaves the cell and freezes externally, the resulting dehydration
increasus the concentration of intracellular solutes and decreases the
concentration of water (x, decreases). This in turn reduces the internal
vapor pressure, De, and reduces the difference between P, and Pe. But the
closer De drops to pay the lower becomes the rate of water 1088.
Qualitatively, we can summarize this "leedback" situation as follows:
If a cell is cooled sufficiently slowly, it will never supercool, but will
maintain nearly continuous equilibrium with the ice outside by contimuous
dehydration. On the other hand, 17 the cell is cooled rapidly, water will not
be able to leave it rapidly enough to maintain equilibrium, and the cell will
become increasingly supercooled. Since the supercooled water 18 unstable, it
will eventually freeze inside the cell. In other words, slow cooling will
cause debydration and extracellular ice formation; rapid cooling will produce
both intracellular and extracellular freezing.
"The terms "slow" and "rapia" can be assigned numerical values if one has
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values for the variables in equations (1), (2), and (3). The values are
Imnown for yeast cells, and figure 2 gives several numerical solutions applinable (F-2)
to yoast. The curves show the relation between cooling velocity and cell
water content at various temperatures, assuming that intracellular freezing
does not occur. The curve "Eq" describes the coll water content to: a cell
that remains in continuous equilibrium with the outside ice, and thus avoids
becoming supercooled. At -4° C, for example, an equilibrated cell will have
lost about 75% of its normal water content.
It can be seen that the water content of cells cooled at 1• c/min remains
close to the equilibrium values, whereas that of cells cooled at 100° c/mia or
faster 18 much higher than equilibrium. For instance, 1r a yeast cell 18 cooled
at 100° C/min, its water content at -20° C will be about 55% of normal, instead
of the equilibrium value of 4%. Accordingly, the cell would be supercooled
about 18 degrees (horizontal distar.ce from the 100°/min curve to curve "Eq" for
a water content of 55%). Since cells in contact with extracellular ice are
unlikely to supercool this extensively (Mazur, 1965), the residual intracellular
water would be almost certain to freeze within the cell.
This prediction has been supported experimentally. Figure 3 gives the (F-3)
survival of frozen and thawed yeast as a function of cooling velocity. Survival ...
drops abruptly when the cooling velocity is increased from 10° to 200°C/min;
and, associated with this drop, 18 the appearance of supercooled water in the .
cell, as shown by the dotted curve derived from the curves in Mgure 2.. This
correspondence between the loss in viability and the appearance of supercooling
suggests that the 1088 18 indeed the result of intracellular freezing.
The curves in Figure 2 led to the prediction that cells cooled at 1° c/min
would become almost completely dehydrated, whereas cells cooled to say 300° C/min
would only be partially dehydrated by the time they froze at say -15° C. The
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photomicrographs in Figure 3 support this prediction. The cell.c abuenas in
these photographs were cooled at 1 or 280º c/min to -79° C and then fixed at
that temperature by the addition of prechilled ethanol. The difference in their
. . with cooling rates
tot size the two beds 18 apparent. Other evidence favoring the idea
that the abrupt drop in survival 18 associated with intracellular ice has
been published previously (Mazur, 1966, 1967).
The survival curve dhe Figure 3 applies to cells that were warmed and
thawed rapidly. It 18 replotted in Figure 4 along with curves for cells that (-ifj
were waimed slowly or ultra-rapidly. Three points emerge from the comparison:
1. When cells we cooled rapidly enough to undergo intracellular
freezing, survival varied over a 10-million fold range depending on the warming
velocity. The current explanation for this dependence is the following: The
intracellular ice crystals produced by rapid cooling are small. Although
small ice crystals are relatively innocuous, they are unstable; therefore,
when warming 18 slow, they tend to grow to a damaging size by processee
referred to as recrystallization or grain growth. However, when warming 18
ultra-rapid, melting occurs before there has been time for any appreciable
growth, and survival remains comparatively high.
2. When yeast cells in the cooled at less than 1° c/min, they underkant
little if any intracellular freezing. Even so, only some 10 to 40% survive?
I have
While the exact cause of this injury is not understood, it may be caused by
the dehydration that occurs during cooling. The conversion of water to ice
results in an increase in the concentration of electrolytes; and a decrease
in the spatial separation of macromolecules; and it may result in the removal
of water that is critical to the cell. Each of these consequences could
conceivably be lethal, and each has been suggested as a major cause of freezing
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injury (Lovelock, 1953; Levitt, 1962; Meryman, 1967).
3. Some cells survive even the harshest conditions of freozing and
tharing. Only the dilution late technique used for assaying microbial
survival is sensitive enough to detect the surviva
sough to detect the survival of one coll in a hundred
a point that
million, I shall return to the comportat later in discussing the applicability
of these findings to cryosurgery.
Two other factors that influence survival are the minimum temperature to
which cells are cooled, and the length of time they spend at that temperature.
Although colle differ considerably in their response to these two factors,
certain generalizations can be made. Temperatures between -5° and -45° C are
the most critical. Within that range, the immediate lethality of freezing
usually increases with decreasing temperature. Temperatures below about
-45° C usually exert no further lethality (assuming, of course, that the cooling
velocity remains constant).
Cells that survive the immediate consequences of freezing may later
succum! to being maintained at subzero temperatures, The rate of death 18
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usually greatest when cells are held above -30° C., Wittle water remains
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unfrozen, het onder and the decreased quantity of liquid water and the
lowness of the temperature combine to slow adverse chemical reactions. .
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III. FREEZING INJURY IN MAMALIAN TISSUES
A. Ice Formation in Tissues :
Theoretically, the same factors ought to operate during the freezing
of mammalian tissues as operate in yeast cells. In other words, slow cooling
should produce dehydration and extracellular freezing, while rapid cooling
should cause intracellular freezing. However, the numerical values to be
assigned to the term "slow" and "rapia" may be far different from those in
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In contrast
yout. This important quantitative aspect 18/11ustrated well/ with human
erythrocytes. The electromicrographs in Figure 5 show that slowly cooled
red blood cells do in fact dehydrate, whereas rapidly cooled cells undergo
intracellular froozing. But the "slow" and "rapid" cooling in this case
are about 1000 and 5000° c/min, respectively, versus 2.0 and 100° c/min in
the case of yeast. The reason why red colls freeze intracellularly only when it
cooling 18 nxtremely rapid 18 because their very high permeability to water
allows them to dehydrate much more readily during cooling. In fact, the
equations when in figure 1 correctly predict the cooling rate required to
yield intracellular ice in erythrocytes whicon the appropriate permeability
constant and surface area are interted in equation 3 tanning the gove (Mazur,
1966b). I
the cooling rates required for intracellular freezing in large masses
. are likely to be low...
of tissue magna contato corout. Suppose Figure 1 represented a sphere
of tissue 1 mm in diameter rather than a yeast cell 5 u in diameter. The
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calculated water contents as a function of cooling rate would then be those
shown in Figure 6 rather than those in Figure 2, and one would predict that
intracellular freezing would occur in the tissue even with cooling as slow as
1° c/min.
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This theoretical presentatietatene prediction may or may not be observed in
practice, depending on whether or not it 18 possible for islands o.. supercooled
cells to be as much as 0.5 mm from extracellular spaces. But the prediction
1s qualitatively valid, for several authors have observed the presence of
intracellular ice in rapidly cooled tissues and its absence in slowly cooled
tissue. One example 18 shown in Figure 7 which depicts electromicrographs
of frozen-dried muscle. Comparable results have also been obtained by Menz
Minz and Luurt
and Luyet (1961). There also observed that if a rapidly cooled muscle fiber
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was warmed to -15° C for just a few seconds and then recooled to be low -60° C
for fixation, the resulting intracellular ice crystals were much larger than
those in Figure 7b, an observation indicating that recrystallization occurred.
The prediction that cells in tissues may undergo intracellular freezing
even when cooling is as slow as lº c/min 18 in agreement with the experimental
observations of Heard (1955) and Sherman (1962) on the freezing of tumors and
skin.
B. Effect of cooling Rate, Warming Rate, and Intracellular Ice Formation
on the survival of Mammalian Tissue Cells:
.
Smith (1961) thoroughly reviewed the available information on the
freezing of mammalian tissues. Several points in her review are pertinent to
the present discussionsgenduterorganegary):
1. Although most mammalian tissues are badly injured by freezing in
the absence of a protective additive, there is wide variation in susceptibility.
In some cases, dessert tenders of survival is obtained regardless of the
cooling and warning velocities; in other cases, no survival is obtained.
2. Tissue cells are more likely to be killed when cooling is rapid
(> 100c/min) than when it is slow (1-5° C/min).
3. Tissue cells are more likely to be killed when warming is slow
(1-10° c/min) than when it 18 rapid (> 100° c/min).
4. The sequence of rapid cooling and slow thawing is generally
nadmally deleterious.
(+Particularly pertinent are items on pp. 150, 156, 158, 160, 161, 171, 174,
178, 215, 217, 221, 223, 238, 237, and 441.
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and Medawar's
Now Medewer Billingham data (1952) on the survival of frozen and
thawed skin are good Illustrations of these points (Table 1). Their
criterion of viability was the ability of the frozen and thawed skin to
grow upon transplantation. Sherman (1962) noted similar effects of cooling
velocity on mouse skin, and also reported the numerical values of the cooling
velocity.
The question of the viability or survival of tissue cells is a vital
but difficult one, the meaning of which depends on whether one is interested
in the preservation of tissue, or in its destruction. For example, does the
fact that rapidly frozen and thawed skin did not show any evidence of
pigmentation after transplantation mean that each and every melanoblast had
been killed? Unfortunately, few if any tissue techniques have the sensitivity
of the microbiological dilution-plate assay in uni mbiguously detecting very
low levels of survival.
The causes of injury in frozen and thawed tissues are not certain. By
extrapolation from studies on single cells, one can hypothesize that the
fact that damage is greatest following rapid freezing and slow thawing means
that intracellular freezing is a major cause of injury. Sherman (1962),
however, disagrees with this hypothesis. He finds microscopic evidence for of
intracellular ice in both slowly and rapidly frozen mouse skin, even though
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been questioned on the basis that it is difficult to know f ortal whether
the growth of a graft would have occurred from those cells that were seen to
which
in fact
number to be in the freeze-dried sections.
It seems quite certain, however, that intracellular freezing is not the
14
only cause of cell injury. The fact that most tissues and most isolated
12
mammalian cells such as blood cells are damaged or destroyed by freezing,
even with very slow cooling, would suggest that the concentration of solutes
and the dehydration produced by slow cooling are important contributors to
the observed injury.
IV. MAXIMIZING THE EFFECTIVENESS OF CRYOSURGICAL FREEZING
There is comparatively little difficulty in determining the couling and
warming rates and final temperatures of small volumes of frozen cell suspensions
or tissues. But this is decidedly not the case in cryosurgical freezing where
only a portion of the body is frozen. Nevertheless, since cooling and warming
velocities and final temperatures influence cell injury profoundly, it 18
important to know the distribution of temperatures and rates in that portion
of the animal that does freeze.
A. Cooling Rates, Warming Rates, and Temperatures in Cryosurgical Freezing:
The chief distinction between cryosurgical freezing and the types of
freezing discussed so far is that in the former the distance from the heat
sink becomes a critical factor. This is not the case when isolated samples of
tissues and cells are subjected to freezing, for they are completely surrounded
by the heat sink (cooling bath), and eventually reach a temperature close to
that of the coolant. But in cryosurgical freezing, the heat sink (the probe)
is usually inside the target volume to be frozen and the target volume. 18
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completely surrounded by a source of heat; namely, air and the patient's body.
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As a result, only those cells in contact with the probe cool to its temperature.
All others cool to some temperature between that of the probe and -37° C.
Meryman's (1960) measurements on model systems of starch in water indicate
that sorts of kinetics of freezing that occur in cryosurgery. Figure 8 shows
the results he obtained when a cylinder containing a starch solution was applied
to a metal cold plate at -72° C. The ensuing changes in temperature at various
distances
(F-8)
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1. Id

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13
the graph
from the cold plate are shown in Figure SB. Several features of the curve
are of interest:
1. The temperature near the cold plate drops rapidly in the first
five minutes, whereas that in the distal portions remains at 35° C.
2. The thermal gradient (slope of the curves) is higher near the
cold plate than away from it.
3. The cooling rate is higher near the cold plate than it is away
from it.
4. As steady state conditions are approached (the steady state being
one in which the temperature in any portion remains constant), the thermal
gradient becomes approximately linear.
5. After 30 minutes of cooling, the portion 1 cm from the cold plate
has cooled to -50° C, but the portions 5-6 cm from the cold plate are still
above 0° C.
The differences in the cooling rate in different portions of the starch
solution are shown more clearly in Figure 9 where temperature is plotted as a
function of time. The region 1 cm from the cold plate cools from 0° to -30° C
at about 10° C/min whereas the region 4 cm from the cold plate cools at about
1° C/min over the same temperature range.
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B. Cell Survival in Cryosurgical Freezing:
To relate these findings to cryosurgical freezing, consider the
diagram in Figure 10 of a probe embedded in a target tissue fotoddenster, and
of virits
assume the following sequence constantmed: The probe 18 cooled to -100° C for 100 (F-11:
seconds at the end of which time the ball of ice has expanded to just beyond
the boundary of the target tissue. The probe is then allowed to warm, and
2
200 seconds later the ball of ice has completely melted.
14
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· The likelihood of killing the cells in different regions of the tissue
will depend on (a) the rate of cooling in that region, (b) the minimum
temperature attained, (c) the time spent at or near the minimum temperature,
and (a) the time required for warming and thawing. Without actually making
measurements, all that can be said with certainty is that after 100 seconds
of cooling, the temperature of the cells lying against the probe will be close
to -100° C, and that of the cells at the edge of the ice ball will be 0° C;
but let us assume that stemme second the distribution of temperatures in
the various intermediate regions are those indicated in the figure.
Region A. The cells in region A will have cooled from 37° to about -80° C
in 100 seconds, a rate of about 1.2° / sec. But the curves in Figure 9 show
that the cooling rate 18 about four times higher between oº and -30° C than
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between 30º and -50°. So we were going estimate that the cooling rate between 0°
and -30° C in our cryosurgical model will have been about 5° /sec or 300° C/min,
a rate that is almost certain to have produced intracellular freezing.
When warming is initiated by stopping the flow of coolant to the probe,
heat flows into the frozen mass of ice from the surrounding tissue and blood
supply, and the frozen mass is assumed to thaw completely in 200 seconds.
But Meryman (1960) has shown that it takes only about 1/10 as long for the
temperature throughout a sphere of ice to rise to the melting point (-0.56° C
for human tissues) as it does for complete melting. This is due (a) to the
high thermal diffusivity of ice and (b) to the fact that it only takes 0.5
calories to warm 1 gm of ice 1°C in contrast to the 80 calories required to
melt 1 g of ice. As a result, we may estimate that the cells in region A
- arate of
will warm From -80° to 0° c in about 20 seconds, or 250° C/min.
In sumary then, the cells in region A will have cooled and warmed at
about 300 c/min. Intracellular freezing will undoubtedly have occurred,
20
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and, as a result, most of the cells will probably have been killed.
However, some may have survived, for we have seen from Figure 4 and Table 1
that a small fraction of yeast and rabbit skin cells do survive the sequence
of rapid cooling and rapid warming.
Region C. The cells in this region will have cooled to a minimum
tenerature between oº and -20° C. The cells in the center of the region
will have cooled from 37° to about -10° C in 100 seconds, a rate of about
30° c/min. Critical to survival, however, is the rate of cooling from 0°
to -10° C, and that rate may have been lower than 30º c/min 1f the steady
state has been approached during the 100 seconds of cooling. The warming
rate of cells in region C will have been even higher than the 300° C/min
assumed for the cells in region A. Accordingly, the cells in region C will
only have spent about 30-60 seconds in the frozen state.
On the basis of the known response of single cells, a considerable
fraction of the cells in region C is likely to survive. The probability of
intracellular freezing will be low, both because of the rather slow cooling
and because many cells have the ability to supercool to -10° C or below.
Moreover, any supercooled cells, or islands of supercooled cells, will not
undergo much dehydration in the 30 seconds that their temperature remains
between -1° and -20° C; and, if they do not dehydrate extensively, their
internal solute concentration will not reach a lethal level. Furthermore,
the cells near the outer edge of the ice will have cooled only one or two
degrees below 0° C; hence, even if they were held at that 'cemperature long
enough to dehydrate to equilibrium, the resulting solute concentration or
dehydration would not be sufficient to be injurious.

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In summary, then, although the cells near the probe. in region A are
likely to be killed, there is a possibility that a small fraction could
survive. In region C, in contrast, the probability of survival would be
appreciable, especially near the outer edge of the ice sphere.
--
C. Maximizing Lethality in the Target:
The possibility that some cells in the target area will survive
freezing can be greatly lessened by modifying the above sequence of freezing
and thawing in two ways.
1. Provide sufficient cooling so that the sphere of ice expands well
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beyond the estimated outer limits of the target area.
To be more precise, the
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ice sphere should be allowed to expand until all of the estimated target has
been cooled below -20° C as measured by thermocouples.
2. Allow the frozen sphere to warm slowly. It would be desirable to
have at least 10 to 30 minutes elapse between the onset of warming and the
disappearance of all ice. This would result in the cells' warming at about
10° to 50° C/min from their minimum temperature to the melting point.
There are several reasons why cooling cells to below -20° C and thawing
them slowly should maximize lethality. First of all, no cell is likely to
remain supercooled below -20° C. Most of them will probably undergo
intracellular freezing, and either be killed immediately or during the.
subsequent slow thawing. Secondly, any cell that does not freeze
intracellularly, and consequently survives the initial freezing, is still
likely to succumb during slow thawing while it is exposed to concentrated
electrolytes during the minute or so it takes the temperature to rise from
-21.1° C (the eutectic point of a sodium chloride solution) to, let us say,
-4° C (at which temperature the concentration of unfrozen solution would be
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The penalty for allowing the sphere of ice to extend sufficiently
far beyond the target area to drop its temperature below -20° C will be
injury to some of the surrounding healthy tissue. However, since thermal
gradients of 10° c/mm or more are commonly obtained in cryosurgical freezing,
it will probably not be necessary for the outer boundary of the ice sphere
to extend more than 2-5 m beyond the outer limits of the target area.
The likelihood of killing all cells in the target area can probably
be increased by subjecting the target to two or more cycles of freezing to
below -20° C and slow thawing. However, the effectiveness of multiple freezing
is difficult to predict à priori.
Finally, mention should be made of one peculiarity of the freezing
process that could make it difficult to achieve 100% kill on certain tissues.
If the tissue contains cells that are free to move in intercellular spaces,
the cells may be pushed ahead of the advancing ice boundary, and, therefore,
never be cooled to below 0° C. However, the faster the ice front advances,
the greater is the likelihood that the cells will be trapped within the ice
(Jackson and Uhlmann, 1966), and the rate of growth of ice through the target
zone will be greater if the outer edge of the target 18 cooled to below -20° C
than 1f it is cooled to just below 0° C. Another way to minimize the
possibility of free cells escaping contact with temperatures below 0° C would
be to freeze inwardly by having the heat sink surround the target rather than
be located within it. Although instruments to achieve this are certainly
feasible, there would be a number of self-evident complications.
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REFERENCES
Heard, Brian E. 1955. Nuclear crystals in slowly-frozen tissues at very
low temperatures. Comparison of normal and ascites tumour cells.
Brit. J. Surgery 42: 659-663.
Leibo, S. P. and P. Mazur 1967. Freezing of bacteriophage T4B. Biophysical
J. 7, PG 9 (Abstract).
Levitt, J. 1962. A sulfhydryl-disulfide hypothesis of frost injury and
resistance in plants. J. Theoret. Biol. 3: 355-391.
Love, R. M. 1966. The freezing of animal tissue. In "Cryobiology" (H. T.
Meryman, ed.), Academic Press, London, pp. 317-405.
Lovelock, J. E. 1953. The haemolysis of human red blood cells by freezing
and thawing. Biochim. Biophys. Acta 10: 414-426.
Mazur, P. 1965a. Causes of injury in frozen and thawed celle. Fed. Proc.
24, No. 2, Part III, S-175 - S-182.
Mazur, P. 1965). The role of cell membranes in the freezing of yeast and
other single cells. Annal. N. Y. Acad. Sci. 125: 658-676.
Mazur, P. 1966. Physical and chemical basis of injury in single-celled
md.croorganisms subjected to freezing and thawing. In "Cryobiology"
(H. 2. Meryman, ed.), Academic Press, London, pp. 213-315.
Mazur, P. 1966b. Theoretical and experimental effects of cooling and warming
velocity on the survival of frozen and thawed cells. Cryobiol. 2:
181-192.
Mazur, P. 1967a. Physical-chemical basis of injury from intracellular
freezing 10 yeast. Proc. Conference on Cryobiology, Sapporo, Japan
(in press).
Mazur, P. 19676. Physical and chemical changes during freezing and thawing
celle, with special reference to blood cells. Proc. XI Congress of
Billingham, R. E. and P.B. Midawar, 1952. The huzing
- dining and storage of brocammalian skin. J. Expir. Piel
20.apoi dit o .
2
1
.-
.
.
su
*
Int. Soc. Blood Transfusion, s. Karger, Publ., Basel, Switz...
Medewar, P. B. and Billingham, R. E. 1951. The viability of mammalian
skin after freezing, thawing, and freeze-drying. Ia "Freezing and
Drying." R. J. C. Harris, ed., pp. 55-62. London: Institute of
Biology
Menz, L. J. and B. J. Luyet. 1961. An electron microscope study of the
distribution of ice in single muscle fibers frozen rapidly. Biodynamica


8: 261-294.
.:
Meryman, H. T. 1960. The mechanisms of freezing in biological systems.
In "Recent Research in Freezing and Drying" (A. S. Parkes and A. v.
Smith, eds), Blackwell Sci. Pub., Oxford, pp. 23-39.
Meryman, H. T. 1967. In "Proc. Conf. on Cryobiology," Sapporo, Japan,
August 1966.
Nei, T., Kojima, Y. and Hanofusa, N. 1964. Contrib. Inst. Low Temp. Sci.
Series B. No. 13.
Sherman, J. K. 1962. Survival of higher animal cells after the formation
and dissolution of intracellular ice. Anat. Rec. 144: 171-189.
Strange, R. E. and Dark. F. A. 1962. Effect of chilling on Aerobacter
aerogenes in aqueous suspension. J. Gen. Microbiol. 29: 719-730.
Smith, Audrey U. 1961. "Biological Effects of Freezing and Supercooling."
Monographs of the Physiological Society, No. 9. Williams and Wilkins
Co., Baltimore.
Table 1
Survival (+) or nonsurvival (o) of rabbit ear skin epithelium
and melanoblasts after slow or rapid freezing and thawing.
Freezing
Thawing
Rate
Rate
Epithelium
Melanoblasts
Rapid
Rapid
0
.
+
Rapid
Slow
0
0
Slow
Rapid
+
+
.
Tissues were suspended in Ringer's solution (from
Billingham and Medawar, 1952).
Figure Legends
Fig. 1 Schematic representation of events occurring in a supercooled cell
vapor pressure of the external ice and supercooled protoplasm with
temperature, and equation (3) describes the rate of water loss
from the cell. The parameters have the following meanings: DA
and P. are the internal and external vapor pressures, x, is the
mole fraction of solutes in the cell, T is temperature, and L
are the molar heats of vaporization and sublimation of water, R
18 the gas constant, v the volume of cell water, A the cell surface
area, k the permeability constant for water, and viº the molar
volume of water.
Fig. 2 Calculated percentages of intracellular water remaining in yeast
cells as a function of temperature and cooling velocity. The
curve Eq gives the equilibrium water content (from Mazur, 1965a).
Fig. 3 Survival, morphology, and extent of supercooling of yeast cells
as a function of cooling velocity. The survival curve 18
experimental; the supercooling curve 18 calculated from the
curves in Fig. 2. Survivals are those for cells suapended
in distilled water, cooled at the Indicated rates to -70°C,
cooled rapidly to -196°C, and warmed rapidly at 1400°c/min (from
Mazur 19670).
:
22
,
Fig. 4 Effect of cooling velocity on the survival of yeast cells warmed
at indicated rates. See Fig. 3 for details. (From Mazur, 1967a).
Fig. 5 Electron micrographs of thin sections of control and freeze-dried
rabbit erythrocytes. The slowly, moderately, and rapidly cooled
cells were cooled to -30°, -50°, and -150°C, respectively, and were
dried at -30°, -50°, and -70°C, respectively. (From Nei et al., .
1964).
Fig. 6 Calculated percentages of intracellular water remaining at various
temperatures in a cell i mm in diameter cooled at the indicated
rates. (From Mazur, 1966b).
Fig. 7 Cross sections of cod muscle: (a) unfrozen, (b) rapidly frozen,
showing intracellular ice formation, (c) slowly frozen, showing
extracellular ice only. (From Love, 1966).
Fig. 8 Thermal gradients during the freezing of cylinder of a solution
of 7% starch in water in contact with a metal plate at -72°C. The
numbers on the individual curves refer to the time elapsed since the
Initial contact with the cold plate. Redrawn from the data in Fig. 9.
Fig. 9 Temperature in various portions of a cylinder of 7%"starch solution
as a function of time in contact with a metal plate at -72°C. The
curves from left to right represent the temperatures 1, 2, 3, 4,
5, and 6 cm from the cold plate. (From Meryman, 1960).
918. 10 Schematic representation of tissue subjected to cryoburgical.
freezing. The prove surface is assumed to be at -200°c. The
dotted line represents the boundary of the tissue to be
destroyed. The solid line 18 the boundary of the sphere of
ico. (See text for details).
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DATE FILMED
9 / 11 /67








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