Use of Two-Step Cooling Procedures to Examine Factors Cell Survival Following Freezing and Thawing J. FARRANT, Clinical
C. A. WALTER, Research
Ccntrc,
HEATHER Watfod
Despite a general view to the contrary some reports indicate that the formation of intracellular ice is not always lethal to living cells (2, 3, 5, 24, 25). The relationship between the amount of intracellular ice within each cell and cell survival has never been determined. Knowledge of this relationship may help to explaiu how iutracellular ice results in injury and also how cooling rate or two-step procedures protect the cells. Intracellnlar ice has been associated with injury on rewarming, particularly with increased injury at slower rates of thawing ( 10, 1.5, 16). Comparisons of survival after slow or rapid thawing procedures may thus provide a presmnptive test of intracellular ice iuvolvement in cell injury. The present paper attempts to determine the association between survival and intracellular ice using a two-step freezing technique. The ice within Chinese hamster cells was detected indirectly by susceptibility to injury following slow thawing and directly by electron microscopy of freeze-substituted cells. The results are compatible with the hypothesis that the critical factor for damage is the total amount of ice per cell. METHODS Cells
The Chinese hamster lung ( V79-379A) and the conditions
fibroblasts of culture
Received January 28, 1977. 1 Present address: Division of Biomedical Engineering and Applied Sciences, University of Alberta, Edmonton, Alberta, Canada.
Copyright 0 1977 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
Road,
LEE, Harrow,
Influencing
AND L. E. McGANN United
1
Kingdom
aud preparation of the log phase cells for freezing were as described previously (8, 12, 13). In order to provide sufficient material for the ultrastructural examination 0.5 to 1.0 x 10” cells were frozen in each O.l-ml sample both for the survival assay alld the parallel ultrastructural examination. This is in contrast to the 120 cells present in each 0.2-ml sample when only survival estimates were required. The cells were frozen in Eagle’s minimal essential medium supplemented by lO?L foetal calf serum ( Gibco ) and containing dimethylsulphoxide (DMSO, 575 v/v). The tubes used for freezing were sterile glass test tubes (km length, 7-mm outer diameter, S-mm inner diameter). All survival estimates were done from triplicate freezing tubes within each experimental treatment.
Freezing und Thawing Constant temperature alcohol baths were used to provide the holding temperature as previously described (12, 13). The tubes were thawed rapidly by immersion in a water bath at +37”C (640”C/min). Slow thawing was done by suspending the tubes in air in an expanded polystyrene box ( 29 “C/min ) . To provide more uniform conditions of “slower” thawing, sample tubes were transferred from liquid nitrogen into constant temperature alcohol baths (e.g., at -15°C for 5 min) so that at the end of that time some samples could be thawed for survival estimations while others were plunged into liquid nitrogen
ISSN 0011.2240
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81%
-196
I 10
0 Time (minutes)
FIG. 1. Illustration of two-step freezing of Chinese hamster tissue culture cells frozen in dimethyl sulphoxide (DMSO, 5%, v/v). The percentage figures give survival after the different cooling procedures. All samples were thawed rapidly. A sample cooled rapidly to -196°C did not recover, but protection was acquired with time at -25°C. A sample thawed directly from -25°C showed the damage caused after exposure to the holding temperature itself.
before freeze-substitution at -80°C. Thus a system of interrupted rapid thawing was used as an alternative to slow thawing. Measurement
of Survival
This was as already described (12,13) except that a suitable dilution step was necessary after thawing in order that loo-120 cells only would be plated into each petri dish. The colonies were counted after 7 days of culture. The plating efficiency was 80-85 $3. Freeze-substitution After the various experimental proce dures the fibroblast culture cells in unstoppered tubes were transferred from liquid nitrogen and submerged in a 1: 1 mixture of absolute methanol and acetone at -80°C in universal glass bottles. Care was taken to ensure that the liquid nitrogen was first drained off from the sample tubes and that the tops of the universals were not screwed down until about 15 set after transfer in order to avoid possible explosions due to gaseous expansion of liquid
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nitrogen. The httlm wew stored in ~hvar HUkS containing al,solutc methallol ;llrtl crushed solid carbon dioxide and examined weekly. Complete substitution of visible ice occurred within 5 to 6 weeks, The substituted samples were placed in universal bottles containing a solution of osmic acid (l’i;, w/v) in acetone at -8O”C, and left for 24 hr before slowly warming to -40°C. After 24 hr they were put in crushed ice. The tubes were then removed from the bottles and centrifuged to pellet the cells which were then washed in two changes of acetone before preparation for electron microscopy. Electron
Microscop!/
The substituted fibroblast cells were infiltrated overnight in a 1: 1 mixture of acetone and Epon 812 on a rotating wheel to ensure adequate impregnation. Finally the cells were embedded in an Epon mixture composed of 5 ml of Epon 812, 4 ml of methyl nadic anhydride, 3 ml of dodecenylsuccinic anhydride, and 0.3 ml of benzyldimethylamine ( Taab Labs). The sample blocks were cured at +88”C for 5 days, and sections were cut on an LKB Ultrotome I using glass knives and picked up on 200-mesh copper grids. The sections in alcoholic were stained sequentially uranyl acetate followed by lead citrate, an d micrographs were taken on a Zeiss EM 9S2 electron microscope. RESULTS
Two-Step Freezing Figure 1 illustrates the protection given by two-step freezing. Spontaneous freezing of the bulk extracellular solution occurred during the initial cooling phase to -25°C. A sample cooled almost immediately from -25°C into liquid nitrogen showed negligible recovery of colony forming ability on rapid thawing. In contrast, an aliquot held at -25°C for 10 min acquired protection against injury on rapid cooling to -196°C
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FIG. 2. Electron micrograph of unfrozen log phase Chinese hamster fibroblast cells. It can be seen that in suspension these cells show a marked variation in cell size ( X4500). FIG. 3. Unfrozen culture cell showing the general structural features after conventional fixation. Note particularly the presence of vacuoles in the cytoplasm with and without an amorphous content (arrows) ( x4500). FIG. 4. Characteristic appearance of a fibroblast cell following rapid freezing to -196°C in DhlSO (5cj0, v/v) and freeze-substitution at -80°C. The structural morphology does not differ markedly from that of the unfrozen cells. Ice cavities are present throughout the vvhole cell but most are extremely small. There is an area of larger ice cavities at the top right of this cell, but the vacuoles can still be distinguished. Note the shrunken mitochondrion (arrow) adjacent to several others in which internal freezing has occurred (x4950). FIG. 5. Following rapid freezing to -196°C some cells contained large ice cavities after freeze-substitution. Note the large amount of ice that can form in a cell that has not had enough time to shrink during cooling (X.5400). and rapid thawing. A corresponding sampie thawed directly from the holding temperature recorded the extent of damage to the conditions of following exposure the holding temperature itself.
Unfrozen
Controls
Figures 2 and 3 show the appearance of unfrozen fibroblast culture cells following tryl>sinizatioii an d treatment with DMSO
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freczc-sul)stitution groups ~1s suflici~lrtly distinct to avoid confusion with vacuoles. Rapid Cooling to -196°C
-4
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-20
-40
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Temperature("C) FIG. 6. Survival ( %) of tissue culture cells frozen rapidly in DMSO (5%, v/v) to different final temperatures and held for 5 min before rapid thawing (line A). Between -35 and -45°C survival falls markedly. This injury is prevented if samples are held for 10 min at -25°C before cooling to the different final temperatures (line B). For convenience, in this and subsequent survival figures, the time-temperature relationships of the cooling and rewarming procedures are illustrated diagrammatically.
(5$7, v/v) before fixation (9), for comparison with subsequent electron micrographs of freeze-substituted Chinese hamster fibroblast cells. As can be seen in Fig. 2, the cells in suspension are not uniform in size. Figure 3 shows that the fibroblast cells are spherical in shape with numerous short projecting villi at the surface. The cells have large lobed nuclei and cytoplasm packed with free ribosomes. A very noticeable feature is the cytoplasmic vacuoles of varying size. However, as the later micrographs show, the overall distribution of ice cavity patterns in the different
Cells frozen rapidly to -196°C in DMSO (570, v/v) showed negligible recovery of colony forming ability after rapid thawing. Figures 4 and 5 illustrate the ultrastructure of rapidly frozen cells rewarmed to -80°C for freeze-substitution. The characteristic appearance of these cells is shown in Fig. 4. The cell has changed very little in shape and size compared with an unfrozen cell, but throughout the cytoplasm and nucleus there are extremely small ice crystal cavities. An interesting feature is that not all the mitochondria freeze internally. Some appear shrunken in response to the increased osmolality of the surrounding frozen cytoplasm, whereas others retain their shape and contain ice cavities. Figure 5 shows that some rapidly frozen fibroblast cells contain larger ice cavities of irregular shape situated mainly in the nucleus. Despite the presence of intracellular ice a degree of shrinkage has occurred. Temperature of Intracellular in Rapidhy Cooled Cells
Ice Formation
Figure 6 (line A) shows how the survival of cells rapidly cooled in DMSO (5c/o, v/v) falls drastically as the lowest temperature reached passes below about -30°C. For comparison, Fig. 6 (line B) shows that this injury can be prevented by 10 min at -25°C. To see whether or not the falloff in survival of these rapidly cooled cells correlated with any observed formation of intracellular ice, aliquots were taken after 10 min at -25, -35, or -45”C, plunged rapidly into liquid nitrogen, and rewarmed to -80°C for freezesubstitution. Figure 7 represents the most commonly observed appearance of cells cooled after holding at -25°C. These cells were se-
FIG. 7. The most comn~on appearance of cells held at -25°C before plunging to -196’C and freeze-sul,stitution at -8O’C. Virtually all of the cells \vere very shrunken and ice cavities could not he identified. The two cavities arro\vcd are considered to be vacuoles (x5850). In this figure and suhseqllcnt cxlectron nlicrographs a diagram of the cooling procedure before freeze substitution at -80°C is included. FIG. 8. Fibroblast cell after t\\,o-step cooling to -196°C with an initial holding temperature of -35”C, sho\ving the typical shrunken appearance with no clear indications of the presence of intracellular ice. The majority of cells in this group had this appearance ( x5850). FIG. 9. FiGroblast cell after two-step cooling to -196°C with an initial holding temperature of -35°C. This cell shows some degree of shrinkage but also contains rccognisalk ice cavities of medium size in the nuclrns and cytoplasm. Only x minority of cells in this group showed the obvious intracellular ice depicted here. The arrow indicates a group of vacuoles similar to those ohscrved in unfrozen cells ( X4050). PIG. IO. Fibroblast cell after two-step cooling to -196°C with an initial holding temperature of -45°C. Almost all of the freeze-substituted cells observed in this group contained obvious intracellular ice cavities. This micrograph shows a cell with numerous irregul;lrly shaped ice cavities throughout the cyt0phs111and NUCLEUS ( X3150).
verely shrmlken without any indications of ice crystals in either the nucleus, the cytoplasm, or the various organelles. Fig-
ures S and 9 show two examples of cells after holding at -35”C, rapid cooling to - 196”C, and freeze-substitution. In this
“is
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El’
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Diferences between -25 and -35°C Holding Temperatures on Injury Following Slow Thawing
FIG. 11. Comparison between slow thaw and rapid thaw injury of cells held at -25 or -35°C for 5 min before cooling in liquid nitrogen. The cells held at -25°C were destroyed by slow thawing; in contrast, the -35°C holding temperature conferred resistance to injury on slow thawing from -196°C.
group the majority of the cells were indistinguishable from those held at -25°C without obvious intracellular ice (Fig. 8). However, some cells were not grossly shrunken and had an uneven distribution of ice crystal cavities (Fig. 9). After exthe spectrum of cells posure to -45°C present was different; severely shrunken cells without intracellular ice were uncommon, and most of the cells contained varying amounts of intracellular ice (Fig. 10).
As we have described previously (12, 13), there is a marked difference in the slow thawing injury of hamster fibroblasts in DMSO (5%, v/v) cooled after holding temperatures of -25 and -35°C. This effect is again shown in Fig. 11. Cells held at -25°C during cooling tolerate rapid but not slow thawing from -196°C whereas cells held at -35°C withstand both. We have already shown that cells shrunken by holding at -25 (Fig. 7) and at -35°C (Fig. 8) and subsequently cooled to -196°C did not appear to contain ice cavities after freeze-substitution. In order to investigate whether or not ice nuclei were present that were too small to see, aliquots were rewarmed from -196°C to higher subzero temperatures for a period to allow possible recrystallization to occur.
Attempt to Detect Intracellular Ice in Cells Hekl at -25 or -35°C and Cooled to -196°C Figure 12 shows that cells held at -25°C for 10 min before cooling to -196°C are susceptible to injury if rapid
DhlSO 1541%I Fibrobiasts DAIS0 15S”h)
-15%
73% 714:
O’%
68F
Fibroblasts
12% 69% 709; 74% 79% -15-C -25°C
-35% 10'
-4oe 5'
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FIG. 12. Study on the survival ( % ) of cells held during cooling at -25°C for 10 min. Effect of interrupting the rapid thawing of cells with 5 min at different subzero temperatures. The cells were able to withstand rapid thawing from -196°C but interrupting the thawing particularly at temperatures of -25°C or above caused severe damage.
FIG. 13. Study on the survival (%) of cells held during cooling at -35°C for 10 min. Effect of interrupting the rapid thawing of cells with 5 mm at different subzero temperatures. These cells were able to withstand rapid thawing from -196°C and also interrupted thawing even at high subzero temperatures.
thawing is interrupted by 5 min at -2j°C or above. This interrupted thawing has the same deleterious effect as a slow continuous rate of thawing. In contrast, interrqlted thawing is tolerated well by cells hclcl for 10 min at -35°C before cooling to -196°C (Fig. 13). In an attempt to develop any ice nuclei that might be l>resent in these shrunken cells, samples were rewarmed to sulzero temperatures for 5 min before rapid return to -196°C. Figure 14 shows that for cells held initially at -25°C there is no recluction in survival following rapicl. thawing until the temperature of rewarming is above -45°C. In contrast, after a rewarmcell survival ing temperature of -25°C does not occ!ur. With an initial holding
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FIG:. 15. Study on tho survival (%) of cells held during cooling at -35°C for 10 min. Effect of re\varniing from -196°C to different subzero tenlperatrxes before rapid return to -19G”C md thawing either rapidly or slowly. Below a rc\vaming tempcraturc of -35°C there is little effect on the survival of cells thawed either rapidly or slowly. With a rewarming ternperaturc of -25°C the cells no longer tolerate slow tha\viu,q hut arc able to reamer if thauzd rapidly.
0
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Rewarming temperature
-60 f, CJ
FIG. 14. Study on the survival ( % ) of cells held during cooling at -25°C for 10 min. Elect of rewarming from -198°C to different s~~bzero
tcnlperatures before rapid return to -196°C and thawing either rapidly or slowly. Below a rewarlning telnperaturc of -45’C no effect is seen, but on rewarming to -25°C there is very little survival of functional cells even klftcr sulmquent mpid tha\ving from liquid nitrogen.
temperature of -35°C (Fig. 15) cells rewarmed from -196 to -25°C before rcturning to -196°C no longer tolcratc slow thawing, although they still retain the ability to recover if thawed rapiclly. A rewarming temperature of -25°C was therefore chosen for the studies in which cells were returned to -196°C before freeze-substitution at -80°C. Figure 16 shows that for cells held at -25°C initially, the loss of survival on rapid thawing from -196°C after rewarming to -25°C is accompnnicd by the appearance of obvious intracellular ice unities. Figure 17 is a corresponding micrograph of a cell from the -35°C group, rewarmed from -196 to -25°C. This cell also contained. ice cavities particularly in th(l nucl(~us.
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FIG. 16. Fibroblast cell showing foci of intracellular ice cavities. Although ice cavities were not evident in the shrunken cells using the -25°C two-step procedure (see Fig. 7), the presence of intracellular ice can be detected if the cells are rewarmed to -25°C and rapidly cooled to -196°C (x5850). Fro. 17. Micrograph of a fibroblast cell after the -35°C two-step procedure with ice cavities mainly in the nucleus detected by rewarming to -25°C and returning to -196°C (x4500). FIG. 20. Micrograph showing the freeze-substituted appearance of a fibroblast cell initially held at -25°C but then rewarmed to -15°C before cooling rapidly to -196°C. In these cells very small ice cavities were present in the nucleus. They were most prominent in localized areas just within the inner nuclear membrane (arrows). A large vacuole is indicated by an asterisk ( x5400). FIG. 21. Micrograph showing the freeze-substituted appearance of a fibroblast cell initially held at -35°C but then rewarmed to -15°C before cooling rapidly to -196°C. As in the cell held at -25°C (Fig. 20), ice cavities were mainly confined to just within the inner nuclear membrane ( arrows ) ( x7200).
Rehyclration
Experiments
Experiments were next done in which a second holding temperature was added before the initial plunge into liquid nitro-
gen. Figure 18 illustrates survival data where the initial holding temperatures were -25 or -35°C for 10 min. After the change to different second holding tcm-
TWO-STEP
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t;r
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PROCISI)URE
“Sl 9 5 “‘d
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IL
FIG. 18. Studies on the survival of cells held during cooling at either -25 (circles) or -35°C (squares) for 10 min. Effect of exposure to a second holding temperature (T) for 5 min before cooling rapidly to liquid nitrogen and thawing either rapidly (RT) or slowly (ST). It can be seen that the susceptibility to injury following slow thawing from -196°C is solely dependent on the second holding temperature.
peratures cells were cooled to -196°C before thawing either rapidly or slowly. The figure shows that the susceptibility to injuly following both slow or rapid thawing from -196°C is solely dependent on the second holding temperature. For example, cells held for 10 min at -35°C (a regime conferring resistance to slow thawing injury from liquid nitrogen) become sensitive to slow thawing damage if the second holding temperature is raised to -25°C before plunging to - 196°C. Conversely, cells held at -25°C for 10 min are susceptible to slow thawing injury, but this susceptibility can be abolished by adding a second holding temperature at -35°C before cooling in liquid nitrogen (Fig. 18). Figure 19 shows the survival data and cooling procedures in an espcrimcnt in which freeze-substitution was also used.
FIG. 19. Studies on the survival of cells held for 10 min during cooling at different subzero temperatures with and without a second holding temperature of -15°C for 5 min before cooling to -196°C and thawing rapidly. Exposure to the second temperature of -15°C greatly reduced the final survival.
All samples were thawed rapidly from -196°C either after a single holding temperature of -25 or -35”C, or when a second temperature of -15°C preceded the cooling in liquid nitrogen. The exposure to the second temperature of -15°C greatly reduced the survival on rapid thawing. With initial holding temperatures of both -25 (Fig. 20) and -35°C (Fig. 21), cells rewarmed to -15°C before plunging into liquid nitrogen and freeze-substitution showed evidence of kc cavities. These cavities were clustered at the periphery of the nucleus within the imier nuclear memlxanc and also around the nucleolus. However, the cells still retained their shrunken appearance without definite indications of ice cavities in the cytoplasm. Figure 22 is a higher magnification of part of a nucleus of a ccl1 from the -23°C group showing in more detA the ice cavities formetl in this region. It appears that during the re\vnrming to -15°C water entered the nucleus from the cytoplasm; during the sub-
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FIG. 22. High magnification ice cavities (arrows) within
ET AL.
micrograph of part of a m&us showing the inner nuclear membrane as indicated
the clusters of small in Figs. 20 and 21
(x27,000). sequent rapid cooling to -196°C small ice crystals were localised in the area of rehydration. Development of Susceptibility of Cells Held at -25°C to Slow Thaw Injury Only if Cooled Below -80°C Experiments were done with cells held at -25°C to find at what temperature the intracellular ice forms during the subsequent cooling to -196°C. As before, susceptibility to injury on slow thawing was used as a test for the formation of ice nuclei. In Fig. 23 it can be seen that the
development of susceptibility to slow thawing injury only occurs between -80 and -196°C. Figure 24 examines the effect on the survival of cells shrunk at -25°C of storage time at - 80” C following slow and rapid thawing. It can be seen that after 35 days at -80°C slow thawing injury has developed, perhaps indicating the onset of intracellular ice nuclei at this temperature.
The two-step technique was first described by Luyet and Keane (11) and has since been applied to a wide variety of
TWO-STEP
FREEZING
biological systems (1, 8, 12, 13, 20, 22, 23, 26, 27). The present experiments used the two-step method to freeze Chinese hamster cells in DMSO (5c/c, v/v). They confirm that cellular shrinkage induced by holding at a subzero temperature correlates with protection against injury on thawing from liquid nitrogen. This agrees with our previous results with lymphocytes (27). It appears that intracellular ice forms on initial rapid cooling in some of the cells held at -35°C and in a high proportion of the cells held at -45°C. This formation of ice in unshrunken cells correlates with irrevocable damage even after rapid thawing. In cooling rate experiments, an increased sensitivity to injury on slow thawing is thought to be diagnostic of injury associated with intracellular ice. Slow
283
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RT ST
120
r. :**
?*
. .
RT
i
> .
20
ST r
o
u+k--kTF
40 Time (days)
FIG. 24. Study on the survival of cells held during cooling at -25°C for 10 min and then cooled rapidly to -80°C. Effect of storage time at -80°C on survival following rapid (RT,O ) or slow (ST,O) thawing. In this experiment survival estimations are given for individual samples. Up to 35 days at -80°C there is a slow development of susceptibility to injury on slow thawing.
RT Sl
-25 c
10' 4-v -T-C 5'
100
201 0
1, -50
\ I
I
-60
-70
Final telrlperature reached j Ci
FIG. 23. Study on the survival of cells held during cooling at -25°C for 10 min. Effect of final storage temperature on recovery after either rapid (RT) or slow (ST) thawing. It can be seen that the development of susceptibility to slow thaw injury only occurs with a final tcmperaturc of below -80°C.
thawing injury is more marked for rapidly cooled cells (those more likely to form intracellular ice) than for slowly cooled cells. There are several examples of cooling rate data illustrating this point (10, 15, 16). It is possible that the susceptibility to injury on slow thawing of cells rewarmed from liquid nitrogen after being held at -25°C is due to the presence of intracellular ice at -196°C. The resistance to slow thaw injuly in a high proportion of cells held at -35°C might then be explained by the absence of intracellular ice nuclei in the shrunken cells at the temperature of liquid nitrogen. This would be a reasonable assumption since the efFectivc force leading to cell shrinkage will be far greater at -35 than at -25°C. Thus cells
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held at -25”C, although grossly shrunken, may still contain enough water to freeze internally on subsequent cooling to -196”C, whereas insufficient water may be left within cells shrunken at -35°C for any ice nuclei to form. The optimal conditions for survival on rapid thawing from -196°C are obtained in cells held at -25°C for 10 min. Evidence that these shrunken cells contain intracellular ice nuclei at -196°C comes not only from the increased sensitivity to slow thawing injury but also from the appearance of visible intracellular ice cavities after recrystallization at -25°C. It is probable that the ice nuclei were present but not apparent after the initial cooling to -196”C, since the recrystallization temperature was -25”C, the same as the original holding temperature. This avoids rehydration and thus prevents the formation of new ice nuclei during the second cooling to -196°C. From other slow thawing data it appears that the ice nuclei form in the -25’C group below - 80°C or after a long time The viscosity and concenat -80°C. tration of the cellular contents of cells shrunken at -25°C probably requires such low temperatures for ice to nucleate. The presence of these intracellular ice nuclei in cells whose treatment leads to the optimal survival from rapid thawing is strong evidence that the presence of intracellular ice in itself is not necessarily lethal to cells. This agrees with other published reports (2, 3, 5, 24, 25). Although some cells form intracellular ice on reaching the holding temperature of -35”C, the majority become shrunken and resistant to injury on slow thawing from -196°C. However, the recrystallization experiments did not confirm the absence of intracellular ice nuclei at -196°C in these shrunken cells held initially at -35°C for 10 min. In these recrystallization studies it is possible that the ice nuclei observed may form during the second phase of cooling to -196°C since the re-
ET AL.
crystallization temperature ( -25°C) is higher than the initial holding temperature ( -35°C) introducing the possibility of cellular rehydration. Further experiments will therefore be needed to provide conclusive evidence for the absence of ice nuclei in shrunken cells cooled to -196°C after holding for 10 min at -35”C, but our present results support the idea that the slow thawing injury in the -25°C group is due to the recrystallization of ice already present in the cells at -196°C. If ice nuclei are present inside cells an increase in temperature may cause recrystallization. If ice is present on both sides of a cellular membrane changes in temperature will not impose osmotic gradients, since adjustments to cellular water contents will be made far more rapidly by the melting or formation of ice than by the bulk transport of water across membranes (6). This will hold unless the ice melts totally in one compartment. Cellular rehydration will thus only occur as the temperature is raised if no ice nuclei are present within the cells. The absence of intracellular ice will also allow shrinkage to take place as the temperature is lowered. The absence of ice nuclei during the period at the holding temperature itself is evident in both the -25°C and in the shrunken cells in the -35°C group. This is shown by the rehydration experiments. After the initial step of -35”C, it is possible that the second holding temperature of -25°C could allow the shrunken cells to rehydrate so that they contain sufficient water to allow ice nuclei to form during cooling to - 196”C, thus rendering them sensitive to slow thaw injury. Conversely, resistance of the -25°C cells to slow thaw injury when a second holding temperature of -35°C is added can be interpreted as providing extra shrinkage of the cells leading to little or no intracellular ice nuclei. The actual mechanism of intracell&r ice injury during rewarming remains undecided. Th e proposal most commonly made is that recrystallization during re-
TWO-STEP
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warming leads to the formation of large ice crystals whose size is the damaging agency (4, 14, 17-19, 21). The present data provide stronger support for the idea that it is the amount of intracellular ice per cell that is the critical factor for injury during thawing. With only a partially shrunken cell (e.g., -25°C for 10 min), the ice nuclei formed during cooling (below -80°C) may not grow to the total equilibrium amount of ice that is possible within the shrmiken cell. If thawing is slow, there is then time for this ice to grow and reach its equilibrium amount per cell and this is sufficient to cause injury. avoids this damaging Rapid thawing amount being reached. With a more severely shrunken cell (e.g., -35°C for 10 min) either no ice nuclei form or so few form that time for recrystallization during a slow or interrupted thawing procedure may still only produce within the cell an amount of ice per cell that is less than that needed to result in injury. It seems that the major problem for cell survival is in the disposal of these varying amounts of intracellular ice during rewarming. In other words, it may be the conditions imposed on the cells by the melting of different amounts of intracellular ice within each cell that leads to damage. As has already been suggested (6, 7)) variations in the total amount of ice per ccl1 (or cellular compartment) may affect the driving force for osmotic movements of water during thawing. A delay in the rate of rewarming in the locality of large amounts of ice due to absorption of latent heat may impose a transient temperature gradient across the cellular membranes. Different radii of ice crystals may alter the chemical potential of water inside and outside the cells. Finally, the temperature at which the cell is “unsealed” to osmotic water movements may he the temperature during rewarming at which the last crystal of intracellular ice melts; this is dependent on the amount of ice per cell.
PROCEDURE
2%
These fluctuations in the osmotic movement of water across cellular membranes during thawing due to different amounts of ice per cell may determine the extent of cellular injury. If this hypothesis is correct, injury will also depend on the ability of particular cellular membrane systems to withstand an osmotic stress. SUMMARY
The two-step cooling procedure has been used to investigate factors involved in cell injury. Chinese hamster fibroblasts frozen in dimethylsulphoxide (5Th, v/v) were studied. Survival was measured using a cell colony assay and simultaneous observations of cellular shrinkage and the localization of intracellular ice were done by an ultrastructural examination of freezesubstituted samples. Correlations were obtained between survival and shrinkage at the holding temperature. However, cells shrunken at -25°C for 10 min (the optimal conditions for survival on rapid thawing from - 196°C) contain intracellular ice nuclei at -196°C detectable by recrystallization. These ice nuclei only form below -80°C and prevent recovery on slow or interruptcd thawing but not on rapid thawing. Cells shrunken at -35°C for 10 min (just above the temperature at which intracellular ice forms in the majority of rapidly cooled cells) can tolerate even slow thawing from - 196”C, suggesting that they contain very few or no ice nuclei even in liquid nitrogen. Damage may correlate with the total amount of ice formed per cell rather than the size of individual crystals, and we suggest that injury occurs during rewarming and is osmotic in nature. ACKNOWLEDGMENTS We would like to thank Miss Fiona Barclay for her invaluable technical assistance, and Dr. Stella Knight for her helpful criticism of the manuscript. REFERENCES 1. Asahina, E. I’refrwzing as a mc+hod enabling animals to survive freezing at an extremely low temperature. Nature (London) 184, 1003-1004 ( 1959).
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