CRYOBIOLOGY
16, 424-429
(1979)
The Effect of Cooling and Warming Rates on the Survival Cryopreserved L-Cells T. AKHTAR, MRC Cryobiology
Group,
D. E. PEGG,
It has been known for many years that cooling rate and thawing rate affect cell survival after freezing (see Smith (10) for many examples). The influence of cooling rate has received the greater attention: In 1963, Mazur (3) showed that the probability of intracellular freezing is largely determined by cooling rate, and in subsequent studies (6) he elaborated the now wellestablished two-factor hypothesis of freezing injury. According to this theory, cell damage at slow cooling rates is caused by solute concentration or cellular dehydration, whereas injury at fast cooling rates is. due to intracellular freezing and recrystallization during rewarming. Survival is therefore maximal at an intermediate cooling rate dependent on the water permeability of the cell. Warming velocity is also important, and until recently it was generally held that rapid thawing was required for maximal cell recovery (6, 10); this was attributed to a reduction in the extent of recrystallization during thawing. However, the discovery that viable mouse embryos could be recovered after freezing only when slow cooling was combined with slow thawing (12) has led to a closer examination of the influence of thawing rate. It is now known that fetal rat pancreases can be preserved by a similar technique (5) and that erythrocytes cooled slowly in the presence of 2 M glycerol can be recovered only if they are rewarmed slowly (8). These observations are of great theoretical interest, and have been discussed in Received
March
22, 1979; accepted
May
29, 1979. 424
001 l-2240/79/0X)424-06$02.00/O Copyright All tights
AND
University Depariment of Surgery, Cambridge CE2
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
of
J. FOREMAN Douglas
House,
Trumpington
Road,
detail by Mazur elsewhere (4): We were particularly impressed by their potential importance for organ preservation. Whole organs cannot be cooled or satisfactorily rewarmed rapidly by existing techniques (9) and the possibility that adequate cell survival might be obtained by combining slow cooling with slow warming has obvious attractions. However, data supporting this approach are available for relatively few cell types, and the present study was undertaken to determine, in a comprehensive manner, the way in which cooling and warming rate determine the survival of a reasonably typical nucleated mammalian cell in the presence of the most commonly used cryoprotective compounds, glycerol and dimethyl sulfoxide (Me2SO). MATERIALS
AND
METHODS
Cells. Murine lymphoma cells, adapted to grow on glass in tissue culture (L-cells, strain L132.C3H), were used. On the morning of the experiment, the L-cells were detached from glass vessels by incubation with 0.25% trypsin in phosphate-buffered saline (PBS, containing (per liter) 137 rniV NaCI, 2.7 mM KCI, and 9.6 mM phosphate at pH 7.4) for 5 min and were then deposited by centrifugation for 5 min at 1000 rpm in an MSE minor centrifuge. After washing once in L15 (Leibovitz) medium (Gibco Biocult) supplemented with 10% fetal calf serum (Gibco Biocult), glutamine (2.0 mmohliter), penicillin ( lo5 D/liter), and streptomycin (100 mg/liter), the cells were resuspended in the same medium at a concentration of 2 x lo6 per milliliter, then kept at 0°C until they were used.
EFFECT
OF COOLING
AND
WARMING
RATES ON L-CELLS
425
al. (11); it measures the proportion of cells that will adhere to a glass surface. No single assay can be equated with “viability” in its broadest sense, but the ability to attach to a glass surface is one property of healthy L-cells which dead L-cells lack. The assay is therefore appropriate for measuring the overall effect of cryobiological variables, and it is simple, convenient, and reproducible, but it should not be assumed to equate with any other “viability” test. In all probability it underestimates the survival that would be recorded by a cell division assay. The procedure used was as follows: The contents of each ampoule were transferred to a 200-ml glass culture flask, together with 2.5 ml of L15 medium that was used to wash out each ampoule, and a further 17 ml of supplemented L15 medium was added slowly over approximately 10 min at 0°C. 0.3”Clmin Each ampoule was placed The flasks were incubated at 37°C for 18 hr, inside a loo-ml glass after which time the supernatant fluid was measuring cylinder which poured off, and the adherent cells were was in turn placed within a washed with 10 ml of L15 medium and then Dewar flask, the whole havdetached by incubation with 10 ml of 0.25% ing been precooled to trypsin in PBS followed by 10 ml of EDTA - 100°C. solution (0.6 mmol sodium ethylenedi1.O”C/min Each ampoule was placed aminetetracetate and 146 mmol sodium inside a Dewar flask prechloride per liter). The washings were cooled to - 100°C. pooled, the flask was examined microsS.O”C/min Each ampoule was allowed copically to ensure that the cells had been to thaw in still room air. removed, and the total number of cells reEach ampoule was placed trieved was determined. All cell counts 60”C/min inside a 146 x 13-mm Pyrex were performed with a Coulter Model B glass test tube which was counter, and the recovered cells were exthen placed in a shaking pressed as a percentage of the cells origiwater bath at 37°C. nally frozen. Each observation was the 2OOYYmin Each ampoule was thawed in mean of 18 replicate experiments. a 37°C shaking water bath. Controls for cryoprotectant toxicity. Thawing curves were obtained using am- Samples (0.5 ml) in each cryoprotectant poules containing 0.5 ml of L15 medium were incubated for 10 min and then treated and 30 SWG copper-constantan ther- in precisely the same way as the thawed mocouples; the warming rates listed above samples. Presentation of results. Graphs of surwere obtained when the mean temperature gradient between -60 and 0°C was deter- vival against the logarithm of cooling rate with thawing rate as parameter, and of surmined. Survival assay. The assay is an adapta- vival against the logarithm of thawing rate tion of the method described by Taylor et with cooling rate as parameter, were not
Freezing and thawing techniques. Aliquots of cell suspension were diluted with an equal volume of 4 M glycerol or 3.2 M Me&SO in the same supplemented L15 medium and held at 0°C for 10 min. Final cryoprotectant concentrations were therefore 2 M glycerol or 1.6 M Me.$O. Onehalf-milliliter aliquots of cell suspension were dispensed into 2-ml glass ampoules which were heat sealed and then placed in a seeding bath at -12°C for 2 min. Each ampoule was seeded by flicking and then transferred to a gas-phase liquid nitrogen cooling machine set at a temperature of -7S”C (2). The fully loaded apparatus was then cooled to - 100°C at 0.1, 0.3, 1.0, or lO.O”C/min. After reaching -lOO”C, the ampoules were rewarmed at 0.3, 1.0, 8.0, 60, or 2OO”Umin in the following manner:
426
AKHTAR,
PEGG, AND FOREMAN
TABLE 1 The Effect of Cooling and Warming Rate on the Survival of L-Cells Cooled to - lM)“C in the Presence of 2 M Glycerol” Survival (%)” at indicated cooling rates (“Urnin)
Warming rate (“C/n-k) 200 60 8 1 0.3
0.3
1.0
11.3 5 0.3 13.0 -e 2.6 9.6 k 4.8 15.6 i 2.3 15.7 t 2.2
18.7 + 2.0 13.0 5 1.2 8.7 _’ 0.3 6.3 2 0.3 9.3 f 0.3
0.1 13.0 2 8.0 k 8.6 2 10.5 ? 12.3 k
2.5 2.5 0.9 1.9 2.0
10 12.6 2 9.0 2 7.3 2 4.0 t 3.0 t
1.4 1.0 0.8 1.0 2.3
fl Each observation is the mean 2 SEM of 18 observations
especially helpful in interpreting the data. Survival contours, mapped on a grid of log cooling rate against log warming rate made visualization much easier. Contour diagrams were constructed on an ICL 1903 computer using the program of McGann and Farrant (7). This method involves the construction of a 50 X 50 matrix of “smoothed” data by fitting four-term polynomial equations to the experimental observations alternately in the direction of each axis until a stable response surface is obtained. RESULTS
The control survival rates obtained without freezing and thawing were SO + 5% in the case of 2 M glycerol and 37.5 -+ 0.5% with 1.6 M MezSO (mean + SEM; n = 18 in each case). The survival rates after freezing
are shown in Tables 1 and 2 and Figs. 1 and 2. It can be seen, particularly from the figures, that at moderate cooling rates, say 3 to S”C/min, survival increased quite markedly as the warming rate was increased, and that a maximum occurred at the commonly used combination of cooling at about l”C/min and thawing at 2OO”Clmin. At slower cooling rates of 0.2 to 0.3Wmin however, there was a much flatter response to changes in warming rate: As the warming rate was increased to lO”C/min, survival decreased somewhat with both cryoprotectants but further increase in warming rate had no effect in the glycerol experiments, and caused a small increase in sure viva1 with Me$O. The most striking feature of both experiments was the demonstration of two distinct peaks of maximal survival, one at the conventional combina-
TABLE 2 The Effect of Cooling and Warming Rate on the Survival of L-Cells Cooled to - 100°C in the Presence of 1.6 M Dimethyl Sulfoxide Warming rate (“Clmin)
Survival (%)R at indicated cooling rates (“Urnin) 0.1
0.3
1.0
10
200
36.6 t 4.4
41.6 +- 2.7
45.6 f 3.4
33.0 + 7.0
60
30.6 2 1.8
38.0 t- 3.1
38.3 + 3.2
31.0 2 7.0
8
34.6 ? 3.5
32.3 5 0.7
30.3 2 1.8
27.0 k 4.3
1
36.6 + 2.8
34.7 5 2.3
29.0 -c 3.5
20.3 k 4.1
0.3
35.0 ? 4.9
36.0 + 4.0
21.3 L 3.5
15.3 k 6.7
n Each observation is the mean k SEM of 18 observations
EFFECT
d3
OF COOLING
AND
WARMING
Id0
I WARMING
RATE
(‘%/mm
)
FIG. 1. Diagram showing isosurvival contours on a grid of log cooling rate against log warming rate for L-cells frozen and thawed in the presence of 2 M glycerol.
tion of cooling at about l”C/min and warming at 200”Cimin, and the other at much slower cooling and thawing rates, 0.2 to 0.3”Clmin in each case. These two peaks can be identified by direct inspection of the data in Tables 1 and 2, but are more easily appreciated in the contour diagrams. DISCUSSION
The absolute survival rates obtained in this study were rather low, and this may
0:3
I WARMING
IO RATE
(‘C/mm
100 )
FIG. 2. Diagram showing isosurvival contours on a grid of log cooling rate against log warming rate for L-cells frozen and thawed in the presence of 1.6 M dimethyl sulfoxide.
RATES
ON
L-CELLS
427
well be due to osmotic damage during removal of the cryoprotectants, since the unfrozen controls also showed rather low recoveries, and glycerol gave worse results than Me.SO: Dilution at room temperature would probably have given higher survivals, but no attempt was made to improve this aspect of the technique. Hamster lung tibroblasts have also given lower survival rates when frozen slowly with glycerol than with MezSO (6). L-Cells exhibited the expected single peak of survival when cooling rate was varied and a single rapid warming rate was used. Freezing injury under these conditions is convincingly explained by Mazur’s two-factor hypothesis, and in the following discussion, the cooling rate giving maximal survival with rapid thawing will be designated “optimal.” When the warming rate was reduced after cooling at “optimal” or “supraoptimal” cooling rates, survival decreased: This again was the expected result, attributed by Mazur to recrystallization during the thawing process (6). When the warming rate was reduced after cooling at “suboptimal” rates there was much less effect on survival, a result which is qualitatively similar to that reported by Mazur and his collaborators for hamster lung fibroblasts frozen with MezSO (6). However, in our glycerol experiments there was a tendency for the survival of slowly cooled cells to increase as the warming rate was reduced (see the column showing recoveries after cooling at 0.3”C/min in Table 1). A similar phenomenon was observed by Miller and Mazur (8) in their study of erythrocytes frozen with glycerol, and is probably due to solute loading during slow cooling which produces osmotic lysis if thawing is too rapid. Our results show some marked similarities to those reported by Miller and Mazur for erythrocytes (8) but there are also considerable differences: It is therefore interesting to compare the two studies in detail. To facilitate the comparison, the erythrocyte data are shown in Fig. 3 as a
AKHTAR,
428
WARMING
RATE
(‘C/m
PEGG, AND FOREMAN
)
FIG. 3. Diagram showing isosurvival contours on a grid of log cooling rate against log warming rate for human erythrocytes frozen in 2 M glycerol. The data are those of Miller and Mazur (8).
contour diagram. The most striking difference is, of course, that the optimal cooling rate is some two orders of magnitude higher for erythrocytes, and this is certainly due, as Mazur demonstrated in 1963 (3), to the high water permeability of these cells. It is also clear that erythrocytes are insensitive to warming rate when cooled at their optimal cooling rate but become remarkably sensitive to slow thawing when cooled more rapidly. Sensitivity to slow thawing is very probably due to the presence of small, intracellular ice crystals (1, 6) and if so, our data would indicate that intracellular ice is present in L-cells frozen with glycerol or Me$O under conventional “optimal” conditions, that is, at the cooling rate found to be optimal for rapid thawing, whereas this is not the case with red cells. The 90% survival contour in Miller and Mazur’s experiments falls somewhat obliquely across the grid, but is a single peak: With the Lcells there was also a ridge of high survival running obliquely from the conventional optimal combination of cooling at - l”C/min and thawing at -2OO”C/min, to cooling and warming at 0.2-0.3YYmin. However, the center of the ridge exhibited a depression, quite distinct when glycerol was used as the
cryoprotectant but also discernible with Me2S0. We suggest that this difference is also due to the fact that small intracellular ice crystals are present in cells cooled at l”C/min: As both cooling and warming rates are reduced there will be an increased tendency to recrystallization but a simultaneous reduction in the amount of intracellular ice available to recrystallize. This would cause an initial decrease in survival, which, when superimposed on the anticipated increase due to improved tolerance to solute loading, would produce the observed ridge with two peaks. Our data do not extend very far into the region where solute effects would be seen in the complete absence of intracellular freezing, and consequently we did not find a zone comparable to that found in red cells cooled at 0.1 to 1.O”C/min. These results seem to us to be entirely explicable on the basis of the known mechanisms of intracellular freezing and solution effects, with particular emphasis on recrystallization and osmotic damage caused by solute loading. The detail of the pattern differs from that observed with erythrocytes for two reasons: Red cells have a much higher permeability to water and they do not contain intracellular ice when cooled at a rate giving maximal survival with rapid thawing. From the practical viewpoint that was the principal reason for this study, the results are encouraging: Survival of nucleated cells was obtained by a combination of slow cooling and slow warming using rates that are practicable for whole organs. SUMMARY
A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to lO.O”C/min) and warming rate (0.3 to 200”Umin) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow
EFFECT OF COOLING AND WARMING
cooling and rapid warming (-1 and 2OOW min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3Wmin. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent. ACKNOWLEDGMENT We are grateful to Dr. P. Mazur for his review of this manuscript, to Dr. J. Farrant for access to the contour computer program, and to Mr. A. R. Hayes for preparing the contour diagrams. REFERENCES 1. Farrant, J., Lee, H., and Walter, C. A. Effects of interactions between cooling and rewarming conditions on survival of cells. In “The Freezing of Mammalian Embryos,” Ciba Foundation Symposium 52, New Series (K. Elliot and J. Whelan, Eds.). Elsevier, Amsterdam, 1977. 2. Hayes, A. R., Pegg, D. E., and Kingston, R. E. A multirate small-volume cooling machine. Cryobiology 11, 371-377 (1974). 3. Mazur, P. Kinetics of water loss from cells at subzero temperatures and the likelihood of in47, tracellular freezing. J. Gen. Physiol. 347-369 (1963).
RATES ON L-CELLS
429
4. Mazur, P. Slow-freezing injury in mammalian cells. In “The Freezing of Mammalian Embryos,” Ciba Foundation Symposium 52, New Series (K. Elliot and J. Whelan, Eds.), pp. 19-42. Elsevier, Amsterdam, 1977. 5. Mazur, P., Kemp, J. A., and Miller, R. H. Survival of fetal rat pancreases frozen to -78 and -196°C. Proc. Nut. Acad. Sci. 73, 4105-4109 (1976). 6. Mazur, P., Leibo, S. P., Farrant, J., Chu, E. H. Y., Hanna, M. G. Jr., and Smith, L. H. Interactions of cooling rate, warming rate, and protective additive on the survival of frozen mammalian cells. In “The Frozen Cell,” Ciba Symposium, (G. E. W. Wolstenholme and M. O’Connor, Eds.). Churchill, London, 1970. 7. McGann, L. E., and Farrant, J. Survival of tissue culture cells frozen by a two-step procedure to - 196°C 1. Holding temperature and time. Cryobiology 13, 261-268 (1976). 8. Miller, R. H., and Mazur, P. Survival of frozen- thawed human red cells as a function of cooling and warming velocities. Cryobiology 13, 404-414 (1976). 9. Pegg, D. E., Jacobsen, I. A., Arm&age W. J., and Taylor, M. J. Mechanisms of cryoinjury in organs. In “Organ Preservation II” (D. E. Pegg and I. A. Jacobsen, Eds.). Churchill Livingstone, Edinburgh, 1978. 10. Smith, A. U. “Biological Effects of Freezing and Supercooling.” Edward Arnold, London, 1961. Il. Taylor, R., Adams, G. D. J., Boardman, C. F. B., and Wallis, R. G. Cryoprotection-permeant vs nonpermeant additives. Cryobiology 11, 430-438 (1974). 12. Whittingham, D. G., Leibo, S. P., and Mazur, P. Survival of mouse embryos frozen to -l%“C and -296°C. Science 178, 411-414 (1972).
16, 424-429
(1979)
The Effect of Cooling and Warming Rates on the Survival Cryopreserved L-Cells T. AKHTAR, MRC Cryobiology
Group,
D. E. PEGG,
It has been known for many years that cooling rate and thawing rate affect cell survival after freezing (see Smith (10) for many examples). The influence of cooling rate has received the greater attention: In 1963, Mazur (3) showed that the probability of intracellular freezing is largely determined by cooling rate, and in subsequent studies (6) he elaborated the now wellestablished two-factor hypothesis of freezing injury. According to this theory, cell damage at slow cooling rates is caused by solute concentration or cellular dehydration, whereas injury at fast cooling rates is. due to intracellular freezing and recrystallization during rewarming. Survival is therefore maximal at an intermediate cooling rate dependent on the water permeability of the cell. Warming velocity is also important, and until recently it was generally held that rapid thawing was required for maximal cell recovery (6, 10); this was attributed to a reduction in the extent of recrystallization during thawing. However, the discovery that viable mouse embryos could be recovered after freezing only when slow cooling was combined with slow thawing (12) has led to a closer examination of the influence of thawing rate. It is now known that fetal rat pancreases can be preserved by a similar technique (5) and that erythrocytes cooled slowly in the presence of 2 M glycerol can be recovered only if they are rewarmed slowly (8). These observations are of great theoretical interest, and have been discussed in Received
March
22, 1979; accepted
May
29, 1979. 424
001 l-2240/79/0X)424-06$02.00/O Copyright All tights
AND
University Depariment of Surgery, Cambridge CE2
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
of
J. FOREMAN Douglas
House,
Trumpington
Road,
detail by Mazur elsewhere (4): We were particularly impressed by their potential importance for organ preservation. Whole organs cannot be cooled or satisfactorily rewarmed rapidly by existing techniques (9) and the possibility that adequate cell survival might be obtained by combining slow cooling with slow warming has obvious attractions. However, data supporting this approach are available for relatively few cell types, and the present study was undertaken to determine, in a comprehensive manner, the way in which cooling and warming rate determine the survival of a reasonably typical nucleated mammalian cell in the presence of the most commonly used cryoprotective compounds, glycerol and dimethyl sulfoxide (Me2SO). MATERIALS
AND
METHODS
Cells. Murine lymphoma cells, adapted to grow on glass in tissue culture (L-cells, strain L132.C3H), were used. On the morning of the experiment, the L-cells were detached from glass vessels by incubation with 0.25% trypsin in phosphate-buffered saline (PBS, containing (per liter) 137 rniV NaCI, 2.7 mM KCI, and 9.6 mM phosphate at pH 7.4) for 5 min and were then deposited by centrifugation for 5 min at 1000 rpm in an MSE minor centrifuge. After washing once in L15 (Leibovitz) medium (Gibco Biocult) supplemented with 10% fetal calf serum (Gibco Biocult), glutamine (2.0 mmohliter), penicillin ( lo5 D/liter), and streptomycin (100 mg/liter), the cells were resuspended in the same medium at a concentration of 2 x lo6 per milliliter, then kept at 0°C until they were used.
EFFECT
OF COOLING
AND
WARMING
RATES ON L-CELLS
425
al. (11); it measures the proportion of cells that will adhere to a glass surface. No single assay can be equated with “viability” in its broadest sense, but the ability to attach to a glass surface is one property of healthy L-cells which dead L-cells lack. The assay is therefore appropriate for measuring the overall effect of cryobiological variables, and it is simple, convenient, and reproducible, but it should not be assumed to equate with any other “viability” test. In all probability it underestimates the survival that would be recorded by a cell division assay. The procedure used was as follows: The contents of each ampoule were transferred to a 200-ml glass culture flask, together with 2.5 ml of L15 medium that was used to wash out each ampoule, and a further 17 ml of supplemented L15 medium was added slowly over approximately 10 min at 0°C. 0.3”Clmin Each ampoule was placed The flasks were incubated at 37°C for 18 hr, inside a loo-ml glass after which time the supernatant fluid was measuring cylinder which poured off, and the adherent cells were was in turn placed within a washed with 10 ml of L15 medium and then Dewar flask, the whole havdetached by incubation with 10 ml of 0.25% ing been precooled to trypsin in PBS followed by 10 ml of EDTA - 100°C. solution (0.6 mmol sodium ethylenedi1.O”C/min Each ampoule was placed aminetetracetate and 146 mmol sodium inside a Dewar flask prechloride per liter). The washings were cooled to - 100°C. pooled, the flask was examined microsS.O”C/min Each ampoule was allowed copically to ensure that the cells had been to thaw in still room air. removed, and the total number of cells reEach ampoule was placed trieved was determined. All cell counts 60”C/min inside a 146 x 13-mm Pyrex were performed with a Coulter Model B glass test tube which was counter, and the recovered cells were exthen placed in a shaking pressed as a percentage of the cells origiwater bath at 37°C. nally frozen. Each observation was the 2OOYYmin Each ampoule was thawed in mean of 18 replicate experiments. a 37°C shaking water bath. Controls for cryoprotectant toxicity. Thawing curves were obtained using am- Samples (0.5 ml) in each cryoprotectant poules containing 0.5 ml of L15 medium were incubated for 10 min and then treated and 30 SWG copper-constantan ther- in precisely the same way as the thawed mocouples; the warming rates listed above samples. Presentation of results. Graphs of surwere obtained when the mean temperature gradient between -60 and 0°C was deter- vival against the logarithm of cooling rate with thawing rate as parameter, and of surmined. Survival assay. The assay is an adapta- vival against the logarithm of thawing rate tion of the method described by Taylor et with cooling rate as parameter, were not
Freezing and thawing techniques. Aliquots of cell suspension were diluted with an equal volume of 4 M glycerol or 3.2 M Me&SO in the same supplemented L15 medium and held at 0°C for 10 min. Final cryoprotectant concentrations were therefore 2 M glycerol or 1.6 M Me.$O. Onehalf-milliliter aliquots of cell suspension were dispensed into 2-ml glass ampoules which were heat sealed and then placed in a seeding bath at -12°C for 2 min. Each ampoule was seeded by flicking and then transferred to a gas-phase liquid nitrogen cooling machine set at a temperature of -7S”C (2). The fully loaded apparatus was then cooled to - 100°C at 0.1, 0.3, 1.0, or lO.O”C/min. After reaching -lOO”C, the ampoules were rewarmed at 0.3, 1.0, 8.0, 60, or 2OO”Umin in the following manner:
426
AKHTAR,
PEGG, AND FOREMAN
TABLE 1 The Effect of Cooling and Warming Rate on the Survival of L-Cells Cooled to - lM)“C in the Presence of 2 M Glycerol” Survival (%)” at indicated cooling rates (“Urnin)
Warming rate (“C/n-k) 200 60 8 1 0.3
0.3
1.0
11.3 5 0.3 13.0 -e 2.6 9.6 k 4.8 15.6 i 2.3 15.7 t 2.2
18.7 + 2.0 13.0 5 1.2 8.7 _’ 0.3 6.3 2 0.3 9.3 f 0.3
0.1 13.0 2 8.0 k 8.6 2 10.5 ? 12.3 k
2.5 2.5 0.9 1.9 2.0
10 12.6 2 9.0 2 7.3 2 4.0 t 3.0 t
1.4 1.0 0.8 1.0 2.3
fl Each observation is the mean 2 SEM of 18 observations
especially helpful in interpreting the data. Survival contours, mapped on a grid of log cooling rate against log warming rate made visualization much easier. Contour diagrams were constructed on an ICL 1903 computer using the program of McGann and Farrant (7). This method involves the construction of a 50 X 50 matrix of “smoothed” data by fitting four-term polynomial equations to the experimental observations alternately in the direction of each axis until a stable response surface is obtained. RESULTS
The control survival rates obtained without freezing and thawing were SO + 5% in the case of 2 M glycerol and 37.5 -+ 0.5% with 1.6 M MezSO (mean + SEM; n = 18 in each case). The survival rates after freezing
are shown in Tables 1 and 2 and Figs. 1 and 2. It can be seen, particularly from the figures, that at moderate cooling rates, say 3 to S”C/min, survival increased quite markedly as the warming rate was increased, and that a maximum occurred at the commonly used combination of cooling at about l”C/min and thawing at 2OO”Clmin. At slower cooling rates of 0.2 to 0.3Wmin however, there was a much flatter response to changes in warming rate: As the warming rate was increased to lO”C/min, survival decreased somewhat with both cryoprotectants but further increase in warming rate had no effect in the glycerol experiments, and caused a small increase in sure viva1 with Me$O. The most striking feature of both experiments was the demonstration of two distinct peaks of maximal survival, one at the conventional combina-
TABLE 2 The Effect of Cooling and Warming Rate on the Survival of L-Cells Cooled to - 100°C in the Presence of 1.6 M Dimethyl Sulfoxide Warming rate (“Clmin)
Survival (%)R at indicated cooling rates (“Urnin) 0.1
0.3
1.0
10
200
36.6 t 4.4
41.6 +- 2.7
45.6 f 3.4
33.0 + 7.0
60
30.6 2 1.8
38.0 t- 3.1
38.3 + 3.2
31.0 2 7.0
8
34.6 ? 3.5
32.3 5 0.7
30.3 2 1.8
27.0 k 4.3
1
36.6 + 2.8
34.7 5 2.3
29.0 -c 3.5
20.3 k 4.1
0.3
35.0 ? 4.9
36.0 + 4.0
21.3 L 3.5
15.3 k 6.7
n Each observation is the mean k SEM of 18 observations
EFFECT
d3
OF COOLING
AND
WARMING
Id0
I WARMING
RATE
(‘%/mm
)
FIG. 1. Diagram showing isosurvival contours on a grid of log cooling rate against log warming rate for L-cells frozen and thawed in the presence of 2 M glycerol.
tion of cooling at about l”C/min and warming at 200”Cimin, and the other at much slower cooling and thawing rates, 0.2 to 0.3”Clmin in each case. These two peaks can be identified by direct inspection of the data in Tables 1 and 2, but are more easily appreciated in the contour diagrams. DISCUSSION
The absolute survival rates obtained in this study were rather low, and this may
0:3
I WARMING
IO RATE
(‘C/mm
100 )
FIG. 2. Diagram showing isosurvival contours on a grid of log cooling rate against log warming rate for L-cells frozen and thawed in the presence of 1.6 M dimethyl sulfoxide.
RATES
ON
L-CELLS
427
well be due to osmotic damage during removal of the cryoprotectants, since the unfrozen controls also showed rather low recoveries, and glycerol gave worse results than Me.SO: Dilution at room temperature would probably have given higher survivals, but no attempt was made to improve this aspect of the technique. Hamster lung tibroblasts have also given lower survival rates when frozen slowly with glycerol than with MezSO (6). L-Cells exhibited the expected single peak of survival when cooling rate was varied and a single rapid warming rate was used. Freezing injury under these conditions is convincingly explained by Mazur’s two-factor hypothesis, and in the following discussion, the cooling rate giving maximal survival with rapid thawing will be designated “optimal.” When the warming rate was reduced after cooling at “optimal” or “supraoptimal” cooling rates, survival decreased: This again was the expected result, attributed by Mazur to recrystallization during the thawing process (6). When the warming rate was reduced after cooling at “suboptimal” rates there was much less effect on survival, a result which is qualitatively similar to that reported by Mazur and his collaborators for hamster lung fibroblasts frozen with MezSO (6). However, in our glycerol experiments there was a tendency for the survival of slowly cooled cells to increase as the warming rate was reduced (see the column showing recoveries after cooling at 0.3”C/min in Table 1). A similar phenomenon was observed by Miller and Mazur (8) in their study of erythrocytes frozen with glycerol, and is probably due to solute loading during slow cooling which produces osmotic lysis if thawing is too rapid. Our results show some marked similarities to those reported by Miller and Mazur for erythrocytes (8) but there are also considerable differences: It is therefore interesting to compare the two studies in detail. To facilitate the comparison, the erythrocyte data are shown in Fig. 3 as a
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WARMING
RATE
(‘C/m
PEGG, AND FOREMAN
)
FIG. 3. Diagram showing isosurvival contours on a grid of log cooling rate against log warming rate for human erythrocytes frozen in 2 M glycerol. The data are those of Miller and Mazur (8).
contour diagram. The most striking difference is, of course, that the optimal cooling rate is some two orders of magnitude higher for erythrocytes, and this is certainly due, as Mazur demonstrated in 1963 (3), to the high water permeability of these cells. It is also clear that erythrocytes are insensitive to warming rate when cooled at their optimal cooling rate but become remarkably sensitive to slow thawing when cooled more rapidly. Sensitivity to slow thawing is very probably due to the presence of small, intracellular ice crystals (1, 6) and if so, our data would indicate that intracellular ice is present in L-cells frozen with glycerol or Me$O under conventional “optimal” conditions, that is, at the cooling rate found to be optimal for rapid thawing, whereas this is not the case with red cells. The 90% survival contour in Miller and Mazur’s experiments falls somewhat obliquely across the grid, but is a single peak: With the Lcells there was also a ridge of high survival running obliquely from the conventional optimal combination of cooling at - l”C/min and thawing at -2OO”C/min, to cooling and warming at 0.2-0.3YYmin. However, the center of the ridge exhibited a depression, quite distinct when glycerol was used as the
cryoprotectant but also discernible with Me2S0. We suggest that this difference is also due to the fact that small intracellular ice crystals are present in cells cooled at l”C/min: As both cooling and warming rates are reduced there will be an increased tendency to recrystallization but a simultaneous reduction in the amount of intracellular ice available to recrystallize. This would cause an initial decrease in survival, which, when superimposed on the anticipated increase due to improved tolerance to solute loading, would produce the observed ridge with two peaks. Our data do not extend very far into the region where solute effects would be seen in the complete absence of intracellular freezing, and consequently we did not find a zone comparable to that found in red cells cooled at 0.1 to 1.O”C/min. These results seem to us to be entirely explicable on the basis of the known mechanisms of intracellular freezing and solution effects, with particular emphasis on recrystallization and osmotic damage caused by solute loading. The detail of the pattern differs from that observed with erythrocytes for two reasons: Red cells have a much higher permeability to water and they do not contain intracellular ice when cooled at a rate giving maximal survival with rapid thawing. From the practical viewpoint that was the principal reason for this study, the results are encouraging: Survival of nucleated cells was obtained by a combination of slow cooling and slow warming using rates that are practicable for whole organs. SUMMARY
A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to lO.O”C/min) and warming rate (0.3 to 200”Umin) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow
EFFECT OF COOLING AND WARMING
cooling and rapid warming (-1 and 2OOW min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3Wmin. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent. ACKNOWLEDGMENT We are grateful to Dr. P. Mazur for his review of this manuscript, to Dr. J. Farrant for access to the contour computer program, and to Mr. A. R. Hayes for preparing the contour diagrams. REFERENCES 1. Farrant, J., Lee, H., and Walter, C. A. Effects of interactions between cooling and rewarming conditions on survival of cells. In “The Freezing of Mammalian Embryos,” Ciba Foundation Symposium 52, New Series (K. Elliot and J. Whelan, Eds.). Elsevier, Amsterdam, 1977. 2. Hayes, A. R., Pegg, D. E., and Kingston, R. E. A multirate small-volume cooling machine. Cryobiology 11, 371-377 (1974). 3. Mazur, P. Kinetics of water loss from cells at subzero temperatures and the likelihood of in47, tracellular freezing. J. Gen. Physiol. 347-369 (1963).
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4. Mazur, P. Slow-freezing injury in mammalian cells. In “The Freezing of Mammalian Embryos,” Ciba Foundation Symposium 52, New Series (K. Elliot and J. Whelan, Eds.), pp. 19-42. Elsevier, Amsterdam, 1977. 5. Mazur, P., Kemp, J. A., and Miller, R. H. Survival of fetal rat pancreases frozen to -78 and -196°C. Proc. Nut. Acad. Sci. 73, 4105-4109 (1976). 6. Mazur, P., Leibo, S. P., Farrant, J., Chu, E. H. Y., Hanna, M. G. Jr., and Smith, L. H. Interactions of cooling rate, warming rate, and protective additive on the survival of frozen mammalian cells. In “The Frozen Cell,” Ciba Symposium, (G. E. W. Wolstenholme and M. O’Connor, Eds.). Churchill, London, 1970. 7. McGann, L. E., and Farrant, J. Survival of tissue culture cells frozen by a two-step procedure to - 196°C 1. Holding temperature and time. Cryobiology 13, 261-268 (1976). 8. Miller, R. H., and Mazur, P. Survival of frozen- thawed human red cells as a function of cooling and warming velocities. Cryobiology 13, 404-414 (1976). 9. Pegg, D. E., Jacobsen, I. A., Arm&age W. J., and Taylor, M. J. Mechanisms of cryoinjury in organs. In “Organ Preservation II” (D. E. Pegg and I. A. Jacobsen, Eds.). Churchill Livingstone, Edinburgh, 1978. 10. Smith, A. U. “Biological Effects of Freezing and Supercooling.” Edward Arnold, London, 1961. Il. Taylor, R., Adams, G. D. J., Boardman, C. F. B., and Wallis, R. G. Cryoprotection-permeant vs nonpermeant additives. Cryobiology 11, 430-438 (1974). 12. Whittingham, D. G., Leibo, S. P., and Mazur, P. Survival of mouse embryos frozen to -l%“C and -296°C. Science 178, 411-414 (1972).
