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Survival of higher animal cells after the formation and dissolution of intracellular ice.

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Survival of Higher Animal Cells after the Formation
and Dissolution of Intracellular Ice'
J. K. SHERMAN
Department of Anatomy, University of Arkansas School of Medicine,
Little Rock, Arkansas
Presence of ice within cells usually is assumed to assure death, while
larger ice formations are assumed to induce greater injury. Evidence is presented
which questions these assumptions.
MCSA and no. 440 parakeet tumors and CsH mouse skin were studied. Cell structure before freezing (control), while frozen, and after rewarming was correlated with
survival on transplantation. Control and rewarmed tissue was fixed in Bouin's fluid
and processed routinely. Microscopic sites and appearance of ice artifacts relative to
frozen cells were preserved by the author's modified freeze-drying technique. Tissues
were cooled to -75°C and -150°C at rates of from l"C/min to 40"C/sec and rewarmed at 180°C/min. Tumor transplants were inoculated in and around the pectoralis major muscle of parakeets and followed as to appearance, size and microscopic
structure. Using hair color as a marker, ventral to dorsal autografts of 8 mm full
thickness circles of skin were made in 4-5 week old female mice.
Data showed that (1) nucleus and cytoplasm were sites of ice formation in cooled
cells, ( 2 ) little or no structural damage occurred in frozen-thawed tumors while skin
often was altered noticeably, (3) % survival of tumors frozen slowly (l"C/min) was
equal to that of controls, (4) 17% of frozen-thawed skin grafts survived, ( 5 ) freezethaw survival was greatest following the formation of the largest ice artifacts which
were induced during slow freezing, ( 6 ) intracellular ice and structural alteration,
therefore, need not be incompatible with survival.
ABSTRACT
A consideration of intracellular ice formation has figured prominently in evaluations of the lethal consequences of cooling
protoplasm below its freezing temperature.
The theory of Stiles ('30), which assumes
that the formation of ice within cells
causes cell death and should be prevented
through vitrification by ultra-rapid cooling, was actively championed by Luyet
and collaborators ('52) for about two decades. During the past 10 to 15 years, the
need for avoiding intracellular ice formation through the use of special cooling
rates or treatment with protective waterbinding or dehydrating agents has dominated the rationale behind attempts at
preservation by freezing as well as explanations for success in preservation
(Smith, '61). Freezing of intracellular
water has been and still is generally considered to be fatal to living cells of both
plants and animals (Goetz and Goetz, '38;
Luyet and Gehenio, '40; Scarth, '44;
Heilbrunn, '43; Smith, '54; Levitt, '56;
Asahina, '56; Mazur, '61). At a recent
symposium, the assumed contention that
intracellular ice always results in cell
death was reaffirmed (Meryman, '62).
Even if it were to be shown that some
cells do survive intracellular ice crystallization, the logical extension of a theory
of freeze-thaw survival which is based
upon avoidance or minimization of ice,
would be a prediction of less chance for
survival as the size of ice formed increases
(Stiles, '30).
The purpose of this communication is
to present details of our own findings
(Sherman, '61a, '61b) to show that the
formation and dissolution of intracellular
ice not only is compatible with the functional survival of mouse skin and two
parakeet tumors (MCSA and no. 440)
cooled to - 75 and - 196"C, but also that
survival is greatest when the size of intracellular ice formed is largest.
MATERIALS AND METHODS
Tissue used as biological test material
in the evaluation of functional survival
1This work was supported by research grant RG-
6418 from the National Institutes of Health.
171
172
J. K. SHERMAN
following environmental insult must not
only survive the treatment but also exhibit
valid, easily detectable, criteria of such
survival. Skin of the C,H mouse has been
shown to withstand exposure to freezing
conditions (-- 78°C) in the absence of a
protective substance, as evidenced by successful grafts in auto-transplantation
(Briggs and Jung, '44). We have found
(Sherman, '61b) that cells of the MCSA
rhabdomyosarcoma (Schlumberger and
Zack, '59) and no. 440 kidney tumor
(Schlumburger, '60) of the parakeet
(MeZapsettacus udulatus) survive freezing
to - 75 and - 196°C. Homo-transplantation of the tumors provide a reliable means
€or evaluation of survival.
Collection of shin. One hundred and
thirty eight four to five week old female
C,H mice were used. Hair on mid-ventral
and mid-dorsal surfaces of anaesthetized
mouse was cut close to skin with an electric clipper. Exposed areas were prepared
for graft removal and deposition by first
being wiped with 70% alcohol, then
marked with circles of neutral red described by stained cork borers of suitable
diameter. A circle ( 8 mm) of skin from
venter was excised from each mouse with
a pair of scissors and placed in sterile
Ringer's solution in a small Petri dish for
one hour prior to either transplantation,
fixation or freezing.
To permit a study of cell structure before freezing, while frozen and after rewarming, relative to functional survival,
each ventral circle of skin was then treated
in one of the following ways: (1) placed
in Bouin's solution for fixation as a control, ( 2 ) frozen and thawed, then placed
in Bouin's fluid for fixation, ( 3 ) frozen
and then dried in the frozen state by
vacuum sublimation (freeze-drying) to
preserve ice artifacts, or ( 4 ) frozen and
thawed, then transplanted to dorsum €or
evaluation of functional survival. Fixed
tissues were prepared on slides by usual
paraffin method.
Transplantation of skin. The method
employed for ventral to dorsal auto-transplantation of skin grafts was modified
from that described by Briggs and Jung
('44). Main changes instituted were:
( 1 ) use of ether instead of nembutal as
the anaesthetic - for minimal loss of ani-
mals and their rapid recovery of normal
activity, ( 2 ) use of no sutures but rather
tight fitting grafts - for speed in the
operation and clean field for observation
of healing at edges, ( 3 ) use of no bandages or covesings over graft -for daily
observations on progress of graft as to
gross changes in tissue and hair at or near
surface, ( 4 ) smaller ventral grafts and
still smaller dorsal wounds - for increased
chances of complete rather than incomplete "takes," and (5) use of fluid medium
and long holding time in medium prior to
transplantation - for correlation with
other experiments involving one hour pretreatment with glycerol.
The circular wound on the dorsum of
the mouse, which was the site of graft
deposition, was prepared by removing the
6 mm circle of full-thickness skin outlined
by a no. 3 cork borer. It was excised just
prior to transplantation of the ventral
graft. The 8 mm circle of skin from the
venter was transferred to this prepared
wound from its Ringer's solution bath
after blotting on gauze. It was usually
rotated 90" to 180" to change hair growth
direction, and carefully fitted so as to
approximate edges and flatten graft. Since
the circular wound tended to widen and
the graft to shrink, the cut edges were
approximated easily by gentle probing with
forceps.
Freezing. To accomplish slow freezing,
the excised circle of ventral skin was
placed dermis side down at the bottom of
a small glass stendor dish. This dish was
placed within a larger one, covered, then
transferred to the surface of a slab of dryice in an insulated box (- 75°C) for 65
to 75 minutes. Skin was frozen rapidly by
immersing it for one to five minutes in
a bath of isopentane cooled either to
- 150°C with liquid nitrogen or to - 75°C
with dry ice. The cooling curves were
traced through a thermorecorder (Minneapolis Honeywell model no. Y153 X 18P-11-111-30) through a copper Constantine
thermocouple inserted into the skin. Average rate of cooling to - 75°C was l . l ° C /
min. in dry-ice, 4O"C/sec. and 10"C/sec.
in isopentane baths.
Thawing. Skin was thawed quickly
( 180"C/min.) by removing inner stendor
dish, placing it in a room temperature
SURVIVAL WITH INTRACELLULAR I C E
bath and twice pouring room temperature
Ringer’s solution directly on and over the
frozen tissue. After two minutes in the
fluid, the tissue was removed, blotted on
gauze and transplanted or fixed.
Freeze-drying. The technique of freezedrying was employed in the procedure for
preserving the site and character of ice
formed in and around cells during freezing. Its first application in evaluating the
relationships between ice formation and
functional cellular survival was with human spermatozoa (Sherman, ’54a and b).
The writer’s method has been improved so
that not only a suspension of cells but
also an organ or its parts can be studied.
Its basic steps have been described elsewhere (Sherman, ’62).
Parakeet tumors. Tissues were obtained from birds carrying tumors in their
breasts. Sterile handling procedures were
employed were practicable. Feathers over
tumor area were plucked, skin swabbed
with 70% alcohol and tumor was carefully removed. Tissue was placed in Petri
dish with Ringer’s solution and either
minced (MCSA) or cut into 2 mm cubes
(no. 440). The tissues then were treated
essentially as described for skin in order
to permit study of structure before freezing, in the frozen state and after thawing,
each relative to survival on homo-transplantation. One hundred and sixteen
three to five month old parakeets of both
sexes were recipients of the tumor tissue
in our study.
Variation from skin procedures included: ( 1 ) less than 15 minutes during
initial stay in small amount of fluid, ( 2 )
0.2 cm3 MCSA tumor was inoculated in
and around the pectoralis major muscle
on right side of animal; 2 mm cube of no.
440 tumor was inserted onto the surface
of the muscle through a subcutaneous
pocket whose opening was later sealed
with celloidin. Two to six birds were inoculated with tissue from one tumor, ( 3 )
progress of tumor was traced after transplantation at one, two and three week intervals with MCSA and four, five and six
weeks for no. 440. Biopsies were taken
for microscopic examination when palpation showed a successful growth after
transplantation (“take”).
173
OBSERVATIONS
Shin transplants. Although progress of
successful grafts in terms of time often
vaned from animal to animal, the general sequence of events described below
was consistent (figs. 1-4). The interesting aspect of this variability and consistency was that no differences were noted
between the behavior of those control and
frozen-thawed grafts which became functional parts of the integument.
Day 1 t o day 7. Healing of adjacent
edges with maintenance of distinct fusion
line and occasional scabs at this point.
Graft is flat and flexible (fig. 2).
Day 8 to 14. Superficial part of stratum corneum (disjunctum) began to
slough, breaking off and carrying hair with
it, leaving skin smooth (fig. 3 ) . Grafts
with normal color and consistency of
vascularized skin usually persisted, as reported by Briggs and Jung (’44).
Day 21 on - N e w hair growth apparent. This hair was usually less pigmented than original, making color difference between ventral graft and new
dorsal location even more pronounced
(fig. 4).
Color, length, texture and direction of
hairs served as the combined criterion for
a successful graft. Also, several series of
experiments were run in which the graft,
with some adjacent host tissue, was removed, fixed and processed for microscopic examination after 1, 3, 9 and 14
days to further substantiate the validity of
the classification as a take. The successful graft clearly remained distinct as to
its epidermis, dermis and appendages with
the fusion area between host and graft
quite apparent (fig. 5 ) .
Tumor transplants. No difference in
progressive growth of a successful tumor
transplant was noted between unfrozen
control tissues and tissues which had been
frozen slowly. The average time for a
take detected by palpation usually was in
the lower end of both the range of two to
three weeks for MCSA and that of three
to six weeks for no. 440. However, on
the basis of rate of increase in size, rapidly
frozen tumors usually took the maximal
time to develop as a take. Biopsies of control and experimental tumors were obtained for microanatomical study at the
174
J. K. SHERMAN
time a take was declared. Comparisons
revealed no features in microstructure
with which one could distinguish one
take from another relative to treatment of
original transplant (fig. 6).
Survival. Seventeen per cent of the
ventral skin transplants survived as functional grafts after slow freezing but none
met this criterion of survival when frozen
rapidly. It was appreciated that the final
temperature reached was not the same for
rates of cooling compared (- 75°C and
- 150°C) and that this might be a factor
in freeze-thaw survival. To eliminate this
variable, rapid freezing was accomplished
in a bath of isopentane cooled to - 75°C
by dry-ice. The rate of cooling realized
(lO"C/sec.) proved also to be fast enough
to prevent survival (table 1).
Results of freeze-thaw survival of skin
as a function of rate of cooling (table l ) ,
show that some transplants of skin do
survive slow freezing (l.l"C/min.) but
none persist as functional grafts following
rapid freezing (10 or $O"/sec.). This
trend toward a n inverse relationship between speed of freezing and survival was
evident also with MCSA and no. 440 parakeet tumors (table 2). It is of interest to
note that essentially no loss accompanied
slow freezing of these tumors while rapid
freezing resulted in a substantial (29%
and 39% ) drop in per cent survival. Slow
freezing of no. 440 tumors undoubtedly
does not favor greater survival than controls (62 to 72% ) but merely is without
adverse effect.
Histology. Observations on sectioned
and stained frozen-dried preparations of
skin and tumor tissues revealed that ice
always formed within cells during both
rapid and slow freezing. Slow freezing resulted in the formation of large ice artifacts which were visible within every cell
observed. Intracellular ice formations in
tissues frozen at a rate of 1-1.3"C/min.
were 5-15 times larger than in tissues
frozen at 38-4O0C/sec. The larger artifacts of ice formation which accompanied
slow cooling, often distorted cells markedly, sometimes squeezing nuclear and
cytoplasmic contents peripherally, against
their distorted membranes (figs. 7, 8, 14,
18). On the other hand, intra-nuclear and
TABLE 1
Functional survival o f frozen-thawed' mouse skin as tested by auto-transplantation
Treatment
Rate of
cooling
to - 75°C
Number
of grafts
Number
of takes2
-
19
23
19
19
17
4
0
0
%
Unfrozen control
Slow freeze (to - 75°C)
Rapid freeze (to - 150°C)
Rapid freeze (to - 75°C)
l.l"C/min
40"C/sec
10"C/sec
89
17
0
0
'Rate of thaw was constant at lSO°C/min.
ZA take is considered here as a transplant which is retained as a permanent functional part of
the integument, different only in attributes of structure and color which distinguish it as to its origin.
TABLE 2
Functional survival of frozen-thawed' parakeet tumors as tested by homo-transplantation
Tumor
Treatment
Cooling rate
(to -75°C)
No. of
transplants
No. of
takes2
Survival
24
18
15
15
13
5
62
72
33
19
21
18
18
19
10
95
90
56
%
-
440
Unfrozen control
Slow freeze
Rapid freeze
l.O"C/min.
38"Wsec.
MCSA
Unfrozen control
Slow freeze
Rapid freeze
1.3"C/min.
40"C/sec.
-
1Rate of thawing was constant at 170"C/min.
% Atake is defined here as the retention of a transplant which continues to grow and function
characteristic of its nature as a tumor.
SURVIVAL WITH INTRACELLULAR ICE
175
intra-cytoplasmic alterations were slight healthy, dividing cells underlying them.
and difficult to detect in rapidly frozen In the absence of controls, there was no
cells whose contours were unchanged by report of a comparison of fat body deminute, not readily detectable ice forma- velopment between the frozen-thawed extions in the frozen state (figs. 9, 15, 19). perimentals and unfrozen controls on
Alterations induced during freezing and either a gross or microscopic level. This
thawing included nuclear hyperchromatic- would have helped in evaluating this point.
ity, shrinkage and pycnosis, as well as It would appear, however, even in the
granulization and vacuolization of the absence of clarifying details, that Salt’s
cytoplasm. Skin thawed after having been work questions the universal assumption
cooled slowly consistently showed more of the lethal nature of intracellular ice.
structural alteration than rapidly frozen
In a recent interesting paper, Salt (’61)
skin. In fact, the post-thaw microscopic studied intracellular and extracellular
structure of rapidly frozen skin usually was freezing in the larvae of the wheat stem
indistinguishable from the unfrozen con- saw fly, Cephus cinctus (Nort.). Data
trol (figs. 10, 11).
presented show that intracellular ice had
Less alterations in physical structure of formed within cellsaf the larvae. Howtumor cells than skin cells were observed ever, injury and premature death accomafter freeze-thawing, and these appeared panied ice formation of either type and
independent of cooling rate. Tumor no. no evidence was given for functional sur440 often showed slight shrinkage of nu- vival comparable with controls, either on a
clei and slight general hyperchromaticity tissue or cellular level.
(fig. 13), but MCSA cells were essentially
Rinfret and Dowell (’62) used x-ray
unchanged aside from a variable slight diffraction techniques to establish that ice
general hyperchromaticity (fig. 17).
forms within human red blood cells during cooling to - 196°C at the same rate
DISCUSSION
which results in their preservation. Ice
Salt (’59) reported that fat cells in the detected by x-rays had to be intracellular
larvae of the golden-rod gall fly Eurosta since cells immersed in liquid nitrogen
solidagines (Fitch) freeze internally at were without their extracellular aqueous
- 15 to - 25°C without harm to them- environment, being suspended in ethyl
selves or to the larvae, in spite of globula- and dibutyl phthalate. In the absence of
tion of cellular lipid. Cell hardness, the critical but difficult experiment in
changes in light transmission, and main- which hemolysis and in vivo survival is
tenance of cell size and shape served as tested for erythrocytes frozen in phthaevidence for intracellular ice formation. late, we cannot consider this noteworthy
However, sudden darkening, “flashing” or work as proof for compatability of intra“black-out” of cells which accompanies in- cellular ice with human red cell survival.
ternal freezing, as observed microscopi- However, their data strongly suggests that
cally with ordinary transmitted light, was this is the case.
not described, although he noted such
The author has used his freeze-drying
“flashing” as evidence for intracellular ice techniques since 1959 in studies on the
formation in fat cells of the wheat stem site and character of ice formed in many
saw fly (Salt, ’61). Also, it appears that mammalian tissues during cooling at
observations on cell structure during various rates to different final temperafreezing, thawing and subsequent develop- tures. It has been observed, without exment to puparium stage, were made ception, that ice forms within cells during
“through the transparent cuticle on fat cooling at the rates (1 to 25”C/min.) to
cells near the surface.” This makes it the temperatures (- 70 to - 196°C) orpossible that cells below the surface in dinarily employed in preservation by freezthe interior of the fat body remained un- ing. Such observations question the statefrozen during such cold exposure. If this ment that “in animal tissues and cell
were the case, surface cells could have suspensions ice formation is only seen
perished as a consequence of the freezing outside the cells at ordinary rates of freezand thawing, and then were replaced by ing” (Meryman, ’60). The assumption is
176
J. K. SHERMAN
that extracellular ice forms during slow
cooling and thus cells survive at these rates
because no intracellular ice is formed.
However, this is based upon observations
made at relatively high sub-freezing temperatures of about - 20°C (Chambers and
Hale, '32; Maximow, '29; Stiles, '30) and
not upon conditions existing during attempts at cellular preservation. Smith
and Smiles ('53) reported intracellular
ice formation during their direct observations on slowly cooled cells in tissue culture, but were unable to evaluate survival
in cells which remained intact. In anhydrous phthalate medium, Rinfret and
Dowel1 ('62) also have observed internal
freezing during slow cooling of human
erythrocytes. The cells appeared to remain intact on thawing but functional survival remains to be ascertained.
We consistently find that the relationship between size of ice artifacts preserved
by freeze-drying and speed of freezing
agrees with physical predictions as well
as direct microscopic observations made
on various cells. Smears of egg albumin
on slides and slips, frozen at different
rates, showed distinctive ice patterns relative to cooling speed in direct observations
with a special cooling stage. These patterns were similar to those revealed by
means of preservation of ice artifacts by
freeze-drying. We found a similar "ice
picture" when freeze-drying was accomplished with frozen tissues kept at - 70
to - 75"C, instead of - 30 to -50°C.
Therefore, marked changes in the site and
character of ice formations were not induced when we compared survival of tissues frozen to --75°C with their ice picture as preserved by freeze-drying at
- 45°C. Further, a series of frozen tumor
tissues were kept at the temperature of
the drying chamber (- 45°C) during
freeze-drying of a paraIIel series and were
shown by transplantation to survive at the
end of this period. This served as a check
on the time of contact with ice at this
temperature during drying as a possible
confounding variable. It seems reasonable, therefore, to admit structural findings with this method as evidence for the
presence of intracellular ice under the
conditions evaluated. Of course, the ideal
biological material would be individual
cells, suited for direct observations during
freezing, and capable of being evaluated
as to functional survival after such observations. Tissues of higher animals do not
yet lend themselves readily to such studies with our present tools of research.
Mammalian eggs meet the requirements
for such an approach (Sherman and Lin,
'58) but preliminary studies in '59 showed
no survival following intracellular ice formation in these cells.
The inverse relationship between speed
of freezing and freeze-thaw survival suggested in this report has been demonstrated with human spermatozoa, in the
presence as well as the absence of glycerol
(Sherman, '54), and with glycerolated bull
spermatozoa (Sherman, '57b). Comparisons
were made with several rates of cooling,
including those specified in vitrification
procedures, in a test of the theory of survival based upon vitrification by rapid
cooling. The theory was found inapplicable. The trend of decreased survival with
increased rate of cooling with acc.7mpanying smaller ice formations, as reported
here, thus may apply also to cells treated
with the protective substance glycerol. Indeed, the author has observed this with
mouse skin.
The writer does not doubt from cited
experimental evidence and his own (Sherman, '57a, '58) that many cells do perish
because of direct or indirect effects of ice
crystals which form within them. However, he is equally convinced that some,
perhaps many, cells do survive such treatment in procedures used to preserve them
by freezing, and that evaluation by experimentation should replace assumption. This
appears to be true for the mouse skin and
parakeet tumors described.
ACKNOWLEDGMENT
The author acknowledges with appreciation the capable assistance of Mrs.
Marjory Mulkey.
LITERATURE CITED
Asahina, E. 1956 The freezing process of plant
cell. Contrib. Inst. Low Temp. Sci., 10: 81-126.
Briggs, R., and L. Jung 1944 Successful grafting of frozen and thawed mouse skin. Anat.
Rec., 89: 75-86.
Chambers, R., and H. 0. Hale 1932 The formation of ice in protoplasm. Proc. Roy. SOC.
B, 110: 33C352.
177
SURVIVAL WITH INTRACELLULAR ICE
Goetz, A., and S. S. Goetz 1938 Vitrification
and crystallization of organic cells at low temperature. J. Appl. Physics, 9: 718-729.
Levitt, J. 1956 Cold Hardiness in Plants. Academic Press Inc., New York.
Luyet, B. J. 1952 Survival of cells, tissues and
organisms after ultra-rapid freezing. In Freezing and Drying. Hafner, New York, 77-98.
Luyet, B. J., and P. M. Gehenio 1940 Life and
Death at Low Temperatures. Biodynamica, Normandy.
Mazur, P. 1961 Manifestations of injury in
yeast cells exposed to subzero temperatures.
I. Morphological changes i n freeze-substituted
and in “frozen-thawed” cells. J. Bact., 82:
662-672.
Maximow, N. A. 1929 Internal factors of frost
and drought resistance in plants. Protoplasma,
7: 259-291.
Meryman, H. T. 1960 Physical aspects of freezing. Ann. N. Y. Acad. Sci., 85: 503-509.
1962 Symposium: Preservation of Cells.
Fed. Proc. Program, 66.
Rinfret, A., and L. Dowell 1962 Personal Communication.
Salt, R. W. 1959 Survival of fat body cells i n
a n insect. Nature, 184: 1426-1427.
1961 A comparison of injury and survival of larvae of Cephus cincus Nort. after
intracellular and extracellular freezing. Can.
J. Zool., 39: 349-357.
Scarth, G. W. 1944 Cell physiological studies of
frost resistance: A review. New Physiologist,
43: 1-12.
Schlumberger, H. G., and G. Zack 1959 Neoplasia in the parakeet. IV. Transplantable
methylcholanthrene - induced rhabdomyosarcoma. Cancer Research, 19: 951-958.
Schlumberger, H. G. 1960 Unpublished data on
pathology of no. 440.
Sherman, J. K. 1954a Freezing and freeze-drying of human spermatozoa. Thesis, Univ. of
Iowa.
1954b Freezing and freeze-drying of human spermatozoa. Fertil. and Steril., 5: 357371.
1957a Freezing, thawing and freeze-drying of brine shrimp eggs. The Physiologist,
I : 77.
195713 Freezing and freeze-drying of bull
spermatozoa. Am. J. Physiol., 190: 281-286.
1961a Factors affecting survival of
mouse skin during freeze-thawing. Fed. Proc.,
20: 134.
1961b Functional survival of cells during the formation and dissolution of ice. Am.
Zool., 1: 388.
1962 Improved methods i n preservation
of human spermatozoa by freezing and freezedrying. Fertil. & Steril.
to be published.
Sherman, J. K., and T. P. Lin 1958 Survival of
unfertilized mouse eggs during freeze-thawing.
Proc. SOC.Exp. Biol. & Med., 98: 902-905.
Smith, A. U. 1954 Effects of low temperature
on living cells and tissues. In: Biological Applications of Freezing and drying. Academic
Press, Inc., New York, Chapter 1.
1961 Biological Effects of Freezing and
Supercooling. Williams & Wilkins Co., Baltimore.
Smith, A. U., and J. Smiles 1953 Microscopic
studies of mammalian tissues during cooling
and rewarming from -79°C. J. Roy. Micr.
SOC.,73: 134-139.
Stiles, W. 1930 On the cause of cold death.
Protoplasma, 9: 459-468.
-
-
PLATE 1
Graft from clipped mid-ventral area, in place on dorsum. Observe differences in hair.
x 10.
4
Successful ventral to dorsal transplantation macroscopically evidenced by variation in
characteristics of hair. x 10.
3 Venter to dorsum auto-transplantation after nine days. Note fusion line (arrow) at
healing surfaces between graft and graft site (see fig. 5 for microstructure), sloughing
of hair and healthy appearing skin. The graft and site are as one, flexible and freely
mobile on palpation. x 10.
2
1 Wound on clipped mid-dorsal area of mouse, prepared for reception of full thickness
skin graft from venter. Note blood vessels. x 10.
EXPLANATION OF FIGURES
SURVIVAL WITH INTRACELLULAR ICE
J. K. Sherman
PLATE 1
PLATE 2
EXPLANATION OF FIGURES
180
5
Section through full thickness of dorsal skin consisting of graft and
graft site tissue a t fusion (healing) site, nine days after transplantation. Following fusion line (arrow), with graft to its left, note differences between graft and graft site tissue in terms of thickness of epidermis, direction of hair follicles, density of stromal elements, and
presence of fat and muscle layers. X 125.
6
Biopsy of a MCSA tumor take, removed 16 days following transplantation of cells which were rewarmed after being frozen slowly
(1.3"C/min) to -77°C. Cells appear no different from those in unfrozen tissue (fig. 16). Note cell i n metaphase. x 1,125.
SURVIVAL WITH INTRACELLULAR ICE
J. K. Sherman
PLATE
a
181
PLATE 3
Note ice artifacts within cells of epidermis, hair follicle, dermis and sebaceous gland.
Intra-nuclear ice formation is particularly apparent. X 500.
Higher magnification of junction of epidermis with hair follicle shows intracellular ice
artifacts to better advantage. X 1,125.
Section through full thickness circle of ventral skin in frozen state after being frozen
rapidly (40"C/sec). Note the very small, difficult to detect ice artifacts in cells at this
junction of epidermis with hair follicle. Compare with figure 8. x 1,125.
7
8
9
Figs. 7-8 Sections through full thickness circles of ventral skin i n frozen-state after
being cooled slowly ( l.l"C/min) to -77°C. Frozen-dried.
EXPLANATION OF FIGURES
SURVIVAL WITH INTRACELLULAR ICE
J. K. Sherman
PLATE 3
PLATE 4
Section through full thickness circle of ventral skin thawed after being frozen slowly
(1.l0C/min) to -77°C. The most prominent change from unfrozen skin is seen i n the
nuclei. Pycnosis and general hyperchromaticity of epithelial cells is evident. Such
alterations were always found. Nevertheless, 17% of transplants survived. x 500.
Section through full thickness circle of ventral skin thawed after being frozen at a rate
of 40"C/sec. This tissue appears indistinguishable from the unfrozen control. Although
this was the usual situation, none of the tissues so treated survived after transplantation.
X 500.
10
11
EXPLANATION OF FIGURES
SURVIVAL WITH INTRACELLULAR ICE
J. K. Sherman
PLATE 4
PLATE 5
EXPLANATION OF FIGURES
12
Section of unfrozen no. 440 tumor. X 1,125.
13
Section of frozen and thawed no. 440 tumor. Nuclei usually were
smaller and more chromatic and cytoplasm more granular than unfrozen tissue, but difference was not dramatic. Cellular structure was
similar regardless of whether or not freezing was slow or rapid.
X 1,125.
186
14
Section of no. 440 tumor showing its appearance i n frozen-state after
being frozen slowly ( l.O"C/min). Intracellular ice artifacts distort
the cells, especially those artifacts in the nuclei. Frozen-dried. x 1,125.
15
Section of no. 440 tumor showing its appearance in frozen-state aftcr
having been frozen rapidly (38"C/sec). Ice formed within cells are
visible i n some nuclei but are not seen readily i n others. Cytoplasmic
ice often is not observed at this magnification. Frozen-dried. x 1,125.
SURVIVAL WITH INTRACELLULAR ICE
J. K. Sherman
PLATE 5
187
PLATE 6
EXPLANATION O F FIGURES
188
16
Section of unfrozen MCSA tumor. X 1,125.
17
Section of frozen and thawed MCSA tumor. Aside from a variable
slight general hyperchromaticity, cells are quite similar to unfrozen
controls. x 1,125.
18
Section of MCSA tumor showing its appearance i n frozen-state after
being frozen slowly (1.3'C/min). Intracellular ice artifacts distort
the cells, some pushing nuclear contents to periphery against their
membranes. Cytoplasmic ice artifacts also are prominent, although
much smaller. Frozen-dried. x 1,125.
19
Section of MCSA tumor showing its appearance i n frozen state after
having been frozen rapidly (40"C/sec). Intracellular ice artifacts are
seen as small clear areas in the nuclei as i n the other rapidly frozen
tissues, but cytoplasmic ice is not distinct. Frozen-dried. x 1,125.
SURVIVAL WITH INTRACELLULAR ICE
J. K. Sherman
PLATE 6
189
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survival, formation, ice, animals, intracellular, dissolution, higher, cells
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