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MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS -1963
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To be published in
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JUN 2 2 1969
Proceedings Conference on
Cryobiology and Cryosurgery,
Bulletin of the Millard Fillmore
Hospital, Burfalo, New York ·
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FACTORS AFFECTING CELL INJURY IN CRYOSURGICAL FREEZING+, 2
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Peter Mazur
Biology Division, Oak Ridge National Laboratory,
Oak Ridge, Ternessee
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Research sponsored by the U. S. Atomic Energy Commission under contract
with the Union Carbide Corporation.
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A fuller version will appear in Proceedings Symposium on Cryosurgery,
UCLA Medical School, March 11-12, 1967, Thomas, Publ.
LEGAL NOTICE
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This report was prepared as an account of Government sponsorsd work. Neither the United
States, nor the Commission, nor any person acting on behalf of the Commissions
A. Makes any warranty or representation, expressed or implied, with respect to the accu-
racy, completeness, or ugafulness of the information contained in this report, or that the use
of any information, apparatus, method, or proceso disclosed in this report may not infringe
privately owned righto; or
B. Assumos any liabilities with roopact to the use of, or for damagos resulting from the
use of any information, apparatus, metbod, or process disclosed in this report,
As used in the above, "person acting on behalf of the Commission" Includes may one
ploys or contractor of the Commission, or omployee of epob contractor, to the extent that
such employee or contractor of the Commission, or employee of such contractor prepares,
disseminatos, or provides accoss to, any information pursuant to his employment or contract
with the Commission, or his employment with such contractor,
DISTRIBUTION OF THIS DOCUMENT IS UNLIMITED
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INTRODUCTION
The central aim of cryosurgery is 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 is to
determine the conditions of freezing that should be employed to maximize
injury within that boundary. It is sometimes assumed that all cells within the
a visibly frozen area will be killed, but this assumption is unlikely to be
valid. To understand why it may not be valid, one must be aware of the
factors that underlie cellular injury at subzero temperatures, and it is
the purpose of this paper to review some of what is known about these factors.
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. It
injury occurs, a first question is to what extent is it due to cooling and to
What extent to ice formation?
A. The Effects of Lowered Temperature:
Lowering the temperature of biological systeins 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
restores normal function. There are, however, some striking exceptions - to
this generalization. Nonhibernating mammals usually cannot be cooled more than
a few degrees below their body temperature without being killed, and certein
single cells are killed under special conditions by sudden chilling to
temperatures near 0° C. The cause of injury, when it occurs, is obscure.
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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
unfrozen below -1 ° C 1s, by definition, supercooled. The supercooled state
is thermodynamically unstable, and will be eliminated in two possible ways:
by dehydration or by intracellular freezing.
Since the vapor pressure of supercooled water is higher than that of ice,
water will tend to flow out of a cell and freeze externally. Hence, if a cell
is cooled sufficiently slowly, it will never supercool, but will maintain
nearly continuous equilibrium with the ice outside by continuous dehydration.
On the other hand, if 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 supercooled water is unstable, it will eventually freeze
Inside the cell. In other words, slow cooling will cause dehydration and
extracellular ice formation; rapid cooling will produce both intracellular and
extracellular freezing.
The terms "slow" and "rapid" can be assigned numerical values if one
knows the surface to volume ratio of a cell or tissue and its permeability to
water. Calculations for yeast indicate that the cells will dehydrate if cooled
slower than 10° C/min, but will freeze intracellularly when cooled more rapidly.
Calculations for mammalian red blood cells indicate that intracellular freezing
will occur only when the cooling rate exceeds 2,000° C/min. Both these
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calculations have been confirmed by experiment. The cooling rate required
for intracellular freezing in large masses of tissue, in contrast, may be
as low as 0.1° c/min because of the low surface to volume ratio and
permeability to water.
Intracellular freezing is almost invariably lethal in isolated single
cells, and appears to be equally deleterious to cells in tissues and organs,
although there is some disagreement on this letter point.
Cells can also succumb to freezing even when intracellular freezing does
not occur. Injury in this case is probably due to exposure to the concentrated
solutes or dehydration produced during slow freezing or slow thawing.
A priori, one would predict that maximum lethality would be produced by
the sequence of rapid freezing and slow thawing, and this has generally been
found to be the case. The rapid freezing tends to cause intracellular ice
formation, and the slow thawing exposes the cells that have not undergone
intracellular freezing to the deleterious consequences of concentrated solutes.
III. MAXIMIZING THE EFFECTIVENESS OF CRYOSURGICAL FREEZING
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There is comparatively little difficulty in determining the cooling 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 lajury profoundly, it is
important to know the distribution of temperatures and rates in that portion
of the animal that does freeze.
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
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tissues and cells are subjected to freezing, for they are completely surxcunded
by the heat sink (cool.ing bath), and eveatually reach a temperature close to
that of the coolant. But in cryosurgical freezing, the heat sivik (the probe)
is usually inside the target volume to be frozen and the target volume is
completely surrounded by a source of heat; namely, air and the patient's body.
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.
.: Consider the situation diagrammed in Figure 1 where a probe has been
embedded in a target tissue and subjected to the following sequence of events:
The probe is cooled to -100° C for 100 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 warn, and 200 seconds later the ball of ice has
completely melted.
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 the distribution of temperatures in the various
Intermediate regions are those indicated in the figure.
3. Region A. The cells in region A will cool from 37° to about -80° C in
100 seconds, or at 1.2° /sec. But the cooling rate between oº and -30° C
will be about 300°. C/min, a rate that is almost certain to produce intracellular
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When warning 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.
However, it will only take about 20 seconds for the cells in region 9 to
warm from -80° to 0° C.
In summary then, the colls in region A will cool and warm at about
300° c/min. Intracellular freezing will undoubtedly occur and, as a result,
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most of the cells will probably be killed. However, some may not undergu
interna]. freezing and may, therefore, survive.
Region C. The cells in this region will cool to a minimum temperature
between oº and -20° C. The cells in the center of the region will cool from
37° to about -10° C in 100 secords, a rate of about 30° c/min. The warming
rate of cells in region C will be even higher than the 300° c/min assumed
for the cells in region A. Accordingly, the cells in region C will only
spend about 30-60 seconds in the frozen state.
On the basis of the known respons of single celis, 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 -10° 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 temperature 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 traction could
survive. In region C, in contrast, the probability of survival would be
appreciable, especially near the outer edge of the ice sphere.
The possibility that some cells in the target area will survive freezing
can be greatly lessened by modifying the above sequence of freezing and thaving
in two ways.
... 1. Provide sufficient cooling so that the sphere of ice expands well
beyond the estimated outer limits of the target area. To be more precise, the
ice sphere should be allowed to expand until all of the estimated target has
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memer.commme consommation cominciarum.com imagebamcom imagens
been cocled 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' warning 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 sell is likely to
remaia 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 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 about i Moler).
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
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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 mm beyond the outer limits of the target area,
The likelihood of killing all cells in the target area can probably be
increased by subjacting the target to two or more cycles of freezing to below
-20° C and slow thawing. However, the effectiveness of miltiple freezing is
difficult to predict a priori.
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Fig. 1. Schematic representation of tissue subjected to cryosuryical
freezing. The probe surface is assumed to be at -100® C. The
dotted line represents the boundary of the tissue to be.
destroyed. The solid l.ine is the boundary of the sphere of
ice. (See text for details.)
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