(SANITIZED)UNCLASSIFIED SOVIET PAPER ENTITLED, "SURVIVAL OF SOME ORGANISMS AND CELLS OF INTRACELLULAR ICE FORMATION"(SANITIZED)

• Document Type: CREST
• Collection: General CIA Records
• Document Number (FOIA) /ESDN (CREST): CIA-RDP80-00247A003800040001-4
• Release Decision: RIPPUB
• Original Classification: C
• Document Page Count: 18
• Document Creation Date: December 27, 2016
• Document Release Date: February 20, 2014
• Sequence Number: 1
• Publication Date: October 8, 1964
• Content Type: REPORT

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SURVIVAL OF SOME ORGANISMS AND CELLS OF INTRACRLIJULAR ICE
FORMATION
L.K. Lozina-Lozinsky
Institute of Cytology, Academy of Sciences of the USSR,
Leningrad.
. A durable maintenance of biological systems in living but not
active state (anabiosis) is available in the case of almost com-
plete cessation of biochemical processes. The state' of real ana-
biosis can be attained only on condition of more intensive cooling
or drying. However only those cells, microopganisms and inver-
tebrate animals which are specially adapted to drying in nature
can endure a complete loss of water.
Practically the conservation of living cells, organs and
whole organisms is brought about during freezing. In most cases
the latter may be performed at low temperatures in the presence
of protective agents (glycerin, ethylenglycol, sugars). The
formation of ice in the organism and its tissues does not give
evidence to the fact that all cellular functions have stopped,
At -100 in frozen caterpillars Mamestra sp. about 50/0 of water
becomes ice butt at the same time respiration is easily detected
(Lozina-Lozinsky, 1942). Under Similar condition as well as at
supercooling biochemical processes proceed and living systems
become destroyed as a result of prolonged preservation.
Three types of freezing are known to occur at the action
of extremely low temperatures: 1) extracellular formation of ice
in tissues; 2) intracellular crystallization; 3) freezing
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of cells accomDanied by the formation of amorphous ice
(vitrification).
the
A gradual and prolonged cooling tetemperature below
freezing point at minimal supercooling is necessary for
extracellular ice formation. This way Of freezing is dangerous
for those biological systems (mostly for animals) which are
sensitive to the disturbances in the coordination of func-
tions occuring in the process of slow cooling.
Vitrification is the least dangerous type of freezing, but
it almost never occur in pure state as even at overrapid
cooling a part of water in protoplasm is crystallized,
(Luyet, 1962) Moreover it is next to impossible to induce
vitrification in non microscopically small objects as MI ?
ns.oeeeeT7-Aa ultra rapid decrease in temperature, Lrme4a.
The formation of crystallized ice inside the cell most
frequently occur on Condition of relatively rapid cooling,
e.g. When the temperature is decreased at the rate of 20 grter
min and after supercooling, The intracellular freezing as a
rule results in the death of cells and the organism and there-
fore it is generally considered that if the organism can endure
freezing that means that crystallization took place outside
the cells. This point of view seems rather questionable. We
know cases when at rapid cooling after supercooling and tem-
perature jump cells and organisms containing the normal
amount of water in their tissues survive even upon the de-
crease of temperature to -196 and -2G90.
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MOM
Some insects (Losina7Losinsky, 1937, 1942, 1963a, 1963b,
1963c, Asahina, 1959; Asahina, Aoki, Shinczaki, 1954; Asahi-
na, Aoki, 1958; Salt, 1957), littoral animals (Poljansky,
1953, Kanwisher, 1955), epidermal cells (Keeley and 0th.,
1952; Mider, Morton, 1939; Billinham, Medawar, 1952, 1954;
Lapchinsky and Lebedeva, 1962) and cornea of mammals
(Henaff, 1960) can tolerate rapid freezing (but not vitri-
fication). For yeast cells quick cooling and slow thawing
othe, more dangerous than slow cooling and rapid thawing (Mazur,
1960; Rumyantseva, 1963). It indicates intirectly that at
rapid freezing crystallization occur inside the cells some
of which survive:
The cells ofsome cancerous tumors can tolerate freezing
at the temperatures of liquid gases. Contradictory data of
various authors show that in one caseythe effect of slow
cooling is favourable while in the other rapid cooling
is more preferable. This can be due to the fact that some
biological objects can stand only extracellular freezing,
whereas the other' remain alive during intracellular cry-
stallization. In many cases a favourable effect of rapid
thawing speaks for the fact that the time of warming is not
enough for the cell to be damaged by ice crystalls growing .
inside it.
The question concerning the resistance of cells to
freezing is not quite clean especially. due to the fact that
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WI&
it is rather embarrassing to obsurve the formation of cry-
stals inside the cell and the nucleus. Our attention was
Centered on the solution of the Question whether extra- or
intra cellular ice formation takes olace in the organisms
and their cells capable uf enduring extremely low tempera-
tures and freezing.
Material and Methods
The caterpillars of the Pyrausta nubilalis Hub. tole-
racing in fro2;en state the temperatures of -30 -79, -19G
and -269 (Losina-Losinsky, 1937, 1962, 1963a, b) during dia-
pause or after hardening were used for the experiments.
Whole caterpillars and isolated pieces of organs (salivary
gland, tracheae, nerve chain, pericardium, etc.) were sub-
jected to freezing. The viability of caterpillars and their
cells was judged about by the ability of contractile uissues
to respond to electric current and the ability of cells of
other organs and tissues to form granules and stain orange-
inr3h, a
-red when dyed vvital dye, neutral red.
The cells of Ehrlich's ascitic carcinoma were chosen for
experiments out. of malignant 'tumors known for their tolerance
to extremely low temperature.
The viability of carcinoma cells was determined by the
growth of tumors in rots after the injection of 0.2 ml
suspension of carcinoma cells frozen to -79 and -196? during mal
hours and days.
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MID
OMB
? To reveal the processes going inside the cells and their
nuclei during freezing and thawing we used luminiscence micro-
scope at upper falling light. The objects were located in a
special cooling chamber on the table of the luminiscence micro-
scope (Losina-Losinsky, 1964; Losina-Losinsky and Moroz, in
press). The preparations were stained acridine orange (AO)
before freezing. Microphotographs were taken during cooling
and thawing. -
The 'rate of cooling of the objects in the chamber was
altered due to gradual decrease in.temperature of isopentane
wherein the preparation was submerged, or by location of the
latter'/Preliminarily cooled isopentane or directly to liauid
nitrogen in place of isopentane. The moment of crystallization
and thawing was establishedr5TidebTaiTriimicroscopic_a_l_lndj
the temperature of surrounding media in the chamber.
The time of freezing can be easily fined due to the apacity
and whitening of the media or a tissue. In another second the
luminescent structure of cells becomes visible.
Experimental data
ativ
Cellular freezingeaterpillars Pyrausta nubilalis.
- ? ---... -
The degree of supercooling of cells and pieces of organs
in vitro as well as at freezing of whole caterpillar varies
and asa rule the beginning of crystallization occurs at the
temperatures from -150 to -240 and more seldom at lower or
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higher temperatures. In all the experiments with the excep-
objects were immersed in lioUid nitrogen,
0.
tion of those wh77ENTOTTing proceeded at the rate of 4-60
per min and pieces of organs were considerably supercooled.
The procedure of freezing is demonstrable in the salivaY -
gland where large cells contain giant nuclei, whereas the
cells in his organ make one layer. The net or honeycomb
line structure in the nuclei appears just after freezing
(Fig.1). In case the temperature of the object is rapidly
decreasing no alterations are possible. However if cooling is
slow or or in the case the temperature becoms constant or begins
to increase the "septa" of the honeycomb structure disappear;
meshes grow in size folming so called darg "Cavities" of
irregular shape (Fig.2). Such a change in the structure of
the nuclei and cytoplasm is in accord with the processes
described by Meryman (Meryman, 1957) as "recrystallization".
Recrystalliztion or the growth of crystals observed upon
stabilization or increase of temperature was more than once
noted during freezing of various solutions (Rey, 1959) Luyet,
1962), but was not described for cell nuclei. No doubt, the
honeycomb structure appearing in our -creparations since the
moment of freezing corresponds to the phenomena of crystalli-
zation and recrystallization detected by the above mentioned
authors by meansof "freezing-drying" method or in some
other way. In the case of .our method the Meshes or dark
"cavities" correspond to ice crystals whereas the anplication
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411111,
of the freezing-drying method makes it possible to observe
the structure which appears as a result of ice sublimation
under vacuum and thus fix only the final stage of the whole
procedure of freezing
However the correspondence in the appearence of similar
structures in different objects confirms the conclusion
saying that the development of honeycomb structure in the
nuclei and cytoplasm visible in luminescence light results
from the growth of crystals. The formation of the honeycomb
structure in the nuclei upon freezing is due to the fact
that since the moment of freezing nucleoplasm of the
? nucleus green fluorescing is moved to the periphery of the
growing ice crystal. The fact that in cytoplasm meshes are
? less visible can be explained by lesser intensity of fluo-
rescence and probably by lesser density of the latter. Any-
how the honeycomb structure of endoplasm upon freezing is
perfectly visible in Paramecium candatum whose Cytoplasm is
well fluorescing
The size and hence, the number of "cavities" (crystals)
in the cell varies considerably depending on temperature.
Gn the immersion of a piece of salivary gland in liquid
nitrogen the number of crystallization loci which appear
immediately in the nucleus and cytoplasm. is very great and
one can clearly observe apacity and fine honeycomb structure:
When the freezing temperature ranges from -15. to -300 the
number of "cavities" is considerably less. It goes on de-
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creasing with the growth of crystals, and by the end of the
process it amounts to about a dozen. In the small nuclei
of trachea only one mesh remains, and the nucleus assumes
the appearence of a fluorescing ring (Fig.3).
Now let us refer to the changes which occur upon freezing
Of cells depending of cold resistance of caterpillars. At
attiw,
the oeginning andVend of diapause and libernating period
e.i. when cold resistance is decreased more small crystals
form in the cell nuclei upon freezing. These crystals grow
and form. large "cavities" when thawed which, especlly in
the case when caterpillars cannot stand extremely low tempe-
ratures, are preserved after thawing (Fig.4). Otherwise
structural changes caused by crystallization inside the
nucleus appear to be irreversible, this fact being in accord-
ance with some other phenomena of the injury ?and death of
tho cells and the organism. In all cytoplasm of diapausing
caterpillars such irreversible changes were not noted even
as a result of profound and rapid freezing. IL can be
suggested that cytoplasm of caterpillars of the corn borer
can tolerate freezing and is more resistant than the nucleus.
In the period of maximal cold resistance of caterpillars
the honeycomb structure in the nucleus disappear upon thawing,
and the appearance of the nucleus becomes the same as .before
freezing, i.e. the changes proved to be reversible (Fig.5).
At the same time the cells of organs subjected to freezing
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to -196? when dyed with neutral red, formed granules after
thawing and stained orange-red which was Indicative of
their living state.
It can be assumed that in caterpillars which tolerate
extremely low temperatures the proteins, nucleoproteins and
other substances ;u-e able to resist the denaturating effect
of crystallization and increased concentration of electro-
lytes.
The intensity of cooling influences the degree of re-
versibility of the nuclear structure upon freezing, although
the character of structure does not give' anypossibility
to determine the temperature at intracellular crystallization.
At any rate irreversible changes in the cell nuclei of
salivary gland occur more often if they are frozen at -196?
than at -79?.
In caterpillars of the corn borer which lost their cold-
resistance after the completion of. diapause the freezing.
at _1960 causes not only irreversible changes, but also
results in the break of aells and sometimes of the nuclei
themselves and desintegration of tissues. These phenomena
were observed during freezing in vitro and in vivo.
The rate of thawing influences the, reversibility of
changes in the nuclear str,,.cture. In Lhe experiments when
thawing of caterpillar's organs was carried out. in physio-
.logical solution heated to Li5-;52? a honeycomb structure was
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either absent or found not in all the nuclei of salivary
gland and tracheae. This phenomenon was observed in Feb-
usu.
rupLry: When under 1.-4Ral condition of thawing (at 18, 22?) tile,
honeycomb structure did not disappear. It is noteworthy
that thawing of whole cater.oillars at high temperatures
significantly increased their cupacity of restoring after
the effect of extremely low temperatures as compared to
slow thawing at room temperature or at 00
.
The protective effect of glycerol.
As the protective effect of glycerol upon freezing is
well known we made the following series of experiments.afta
A piece was cut from the paired salivary gland of the corn
borer caterpillar and placed for 20-0 min in 20 glycerol
prepared on Bidle-Efrussi solution while the second extract
was located in the same solution without glycerol. The
experiments were performed on caterpillars with weak cold
resistance. After freezing to -'54. and -4C? the objects were
-thawed at room temperatureApon freezing in both preparation
we observed intracellular ice formation, small "cavities" in
the nuclei which grew in size at thawing. Upon the complete
thawing the "cavities" of the nuclei remained in the gland
which had not been soaked in glycerol, whereas in the gland
treated with glycerol they completely disappeared and the
nuclei assumed the same appearence as before cooling.
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mniP
Cooling and freezing of the cells of. Ehrlich
ascitic carcinoma
The cells of ascitic carcinoma stained orange acridine
(p_efore cooling or in the state of supercoolitare charac-
terizedby evenly green fluorescins nuclei. The nucleolL
fluoresce more brightly, cytoplasm observed in the falling
light is either. dark and transparent or has a slight green
fluorescence. A 2-3 hour soaking in A.O. (0.06%) increases
the fluorescence of cytoplasm, but at the same time one
4
can observe sighs of iia-cUoliz'atiOn, AaMage;formation of
vesicle-like protrusions. aaa?1-141a.
Intracellular freezing occurred at the temperature
about -200, sometimes at -350 and once even at -510. The.
moment of freezing was tazy to fix, but in this case it
was more difficult to observe the process of crystalli-
zation inside the nucleus than in the cells of caterpillar's
salivary gland. It can be due to 'lesser size of ascitic
cells despite the fact that relatively large cells were
found among them. Moreover it can be also explained by
,the fact that. the freezing. of the media inkreater degree
disturbed the fluorescence of nuclei.
However on some preparations we menaged to see clearly
the procedure of formation and growth of ice crystals in-
side the nuclei if jUdged by the formation of similar
meshes as in the cells of caterpillars of the corn borer'
(Fig. 6 . After the thawing of pareillOMOUS cells frozen
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at the temperatures to -196? the cells assumed the same or
almost the same appearence as before freezing:meshes were
either absent or the nuclei contained darker and more trans-
parent cavities or vacuoles sometimes as eight -(4-31-tt-:4+. In
a number of cases the density of the nucleus fluorescence
was uneven (Fig.?).
The nuclei of irregular shape were noted even in uncooled
cells, but the presence of devirticuli in some nuclei were
found only after freezing and thawing (after the action of
-43? and -190). It is of interest that first minutes after
thawing the nuclei appear/to be slightly altered: not seldom
one can observe meshes and vacuoles. and uneven fluorescence
of karioplasm; 1-2 hours after thawing these phenomena dis-
ai_Tear and it is impossible to tell these cells from the
control ones.
? The inoculation of carcinoma cells to rats after freezing
weeks
to -79 and -196 3-4 mri-Ek& later led to the death of all the
investigated animals due to the development of malignant
tumor. The results obtained from the experiments differred
from those of the control tests only by the fact that the
development of tumors and death of the rats under investi-
0-
0,Ition occurred 1-2 weeks later.
All the above makes us to conclude that the cells of
ascitic carcinoma are not injured irreversibly at the action
of extremely low temT)eratures and during intracellular
freezing.
Aloilyside with intracellular freezing we Observed extra-
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cellular freezing of ascitic cells upon slaw cooling to -180.
At this temperature crystallization started in the surround-
ing media (in the capillary space between cover slips). Then
the nuclei became shrunk and deformed, meshes were invisible.
This experiment show that upon slow cooling ascitic cells can
avoid intracellular cooling.
Conclusion
Perhaps intracellular freezing of tissues in the cater-
pillars of Pyrausta nubilalis can be observed only in vitro
.while in vivo freezing occurs outside the cells and, there-
fore, caterpillars are able to stand extremely law tempera-
tures? There are data which contradict thia.suggestiona 691e
caterpillars can be cooled at about the same rate 4s the
pieces of organs, so#etimes a little bit quicker or slower.
However, at all the rates tested in vitro freezing, occurred
inside. the cell.
-There-is no any reason to suggest that under Similar con-
ditions whole caterpillars freeze in some other. way. Supe
cooling was almost identical down to .712, 720? in vivo and
its. vitro. Crystallization started just after the jump of.
temperature: Therefore it is difficult to, suggest that upon
,rapixtcooling in solid carbonic acid or liquid. nitrogen cry,-
.stallization should not occur inside the cell. Moreover, the
state of cells investigated after freezing in vitro and after
freezing of whole caterpilla,was identical, particularly
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when neutral red was used. At the time of maximal stability
to extremely low temperatures irrevessible changes were
not defected in the nuclei. The reversibility of the struc-
ture upon freezing also speaks for the fact that cold re-
sistant cells can endure crystal ice.
Hence, there-is every reason to believe that some of the
biological systems, i.e. extremely cold resistant organisms
and cells of certain type can tolerate intracellular cry-
stallization without using protective agents from outside.
The reversible character of changes in some types of pro-
teins atd nucleoproteins, the capacity of the latter to
acquire resistance to freezing during the life cycle of an
animal and under the influence of environmental conditions
will give us an opportunity and perspective, in resuect of
durable conservation of certain living systems at the
action of extremely low temperatures and for the study of
cold resistance mechanisms.
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Literature
Asahina E. Nature, 184:1426, 1959.
Asahina K.Aoki. kiature, 182:327, 1958.
Asahina E., K.Aoki, Shinozaki. Bull. Ent.Res. 45:329, 1954.
Billingham R.E., P.B.Medawar. J.Exp.Biol. 29:454, 1952.
Henaff.L'o. Recent Research in Freezing and Drying.: e95 ,
1960. Oxford.
Konwisher J.W. Biol.Bull. 109:56, 1955.
Keeley R. L., A. S. Gomes, J. W.Brown. Plast. Re constr. Sur.
;
9:330, 1952. ICI-:2z2_ 1962.\
Lapchinsky A.G. and N. S.Lebedeva. Acta chi/Tt-z-i-ai-e-.-.M.
Losina-Losinsky L.K. Zool.Journ 16:614, 1937. (in kussian).
Losina-Losinsky L.K. "Priroda" 3-4:65, 1942. (in hussian)7
Losina-Losinsky L.K. Doc. Acad. Sc. U. S. S. 147:1247, 1962.
Losina-Losinsky L. K. Intern. Symp. on Cyt ?ecology. Leningrad:
39, 1963a.
Losina-Losinsky L.K. "Cell reaction on the extremal effects.
Collected Articles. (COOpHMH padoT
) N 4:34, 1963b.
Aced. of Sc. USSP. (In uss . and 8nt; 1 . Summary).
Losina-Losinsky L.K. Cytologia 5:220, 1963,c. (In liuscian).
Losina-Losinsky L.K. r,nd P.1!;.1ioroz. CyLologia (in press).
Luyet B. Proceed.Low Temper. hlicrobiol.Sympos. 1961. 1962.
Masur P. Recent
Oxford.
Meryman H.T.
Oxford.
Research in Freezing and Drying.:65, 1960.
Recent Research in Freezing
and Drying.
: 23, 1960. ?
STAT
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? Mider J.J.Morton. Amor.J.Cancer. 35:502, 1939.
Poljrmsky G.J. lia.716.Zool.Inst:. Acad. of Sc. USSP.
1953. (in kussian).
Rey L.R. Conservation de la Vie 'Jar le Froid. 1959. Pnris.
Rumyantseva V.M., "Cell reaction on the extremal effects.
Collected Articles. ( C6OpHIAK padoT
(In itussian, &IG1summary).
salt R.W. Nature, 184:1426, 1957.
Taylor AS Proceed. RoySoc.B.? 147;466, 1957.
N 4:54, 1963..
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Legends to Plates
Fig.1 Frozen salivary .gland of the caterpillar of Pyrausta'
nubilalis at -100, -1200. Luminescence microscopy. The
nuclei fluoresce more intensely and have honeycomb struc-
ture. Microphoto. Oc.10, ob.20:
Fig.2. Frozen salivary gland of the caterpillar of PyraUsta
'nubilalis at -25?: Recrystallization in the nuclei (the
same magnitude).
Fig:3. A piece of trachea of the caterpillar of Pyrausta
nubilalis after the freezing at -79? and thawing. Fluo-
rescence of nuclei at the. periphery appe9/as a thin rim:
:The cells perish. (The same magnitude):
Fig:4. Salivary Gland of the caterpillar after the repeated
action of extremely low temperatures and after thawing.
The nuclei show the honeycomb structure after thawing.
The cells perish:
Fig.. The same region of the salivary gland as in Fig.2
after thawing. The caterpillars at the time of maximal
cold resistance. The honeycomb nuclear structure after
crystallization and thawing has disappeared: (The same
magnitude).
-
Fig.6. Frozen cells of Ehrlich ascitic carcinoma at -25o . The
structure of the nuclei is perfectly visible. Luminescence
microscopy. Microphoto. 00.10, ob.40.
FiG.7. The sane cells as in fig.G just after thawing. In some
-cells the nuclei are reversibly structurated (below) while in
.others they 4.44-Ni4 40-an the same as before freezing.
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