Factors influencing survival of mammalian cells exposed to hypothermia

Factors influencing survival of mammalian cells exposed to hypothermia

CRYOBIOLOGY 22, 484-489 (1985) Factors Influencing Survival of Mammalian to Hypothermia II. Effects of Various J. KRUUV, Guelph-Waterloo Hyperto...

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CRYOBIOLOGY

22, 484-489 (1985)

Factors

Influencing

Survival of Mammalian to Hypothermia

II. Effects of Various J. KRUUV, Guelph-Waterloo

Hypertonic

D. J. GLOFCHESKI,

AND

Cells Exposed

Media J. R. LEPOCK of Biology, University of

Program for Graduate Work in Physics and Department Waterloo, Waterloo, Oniario, N2L 3G1, Canada

Survival of Chinese hamster lung (V79) cells, exposed as a function of time to hypothermia in tissue culture, in isosmotic and various hypertonic media was measured using a colony assay. The mechanism of hypothermic cell killing is different above and below 7°C in this cell line. Addition of NaCl or mannitol to increase the tonicity to 400 mOsm greatly decreased the survival at 10°C while addition of KC1 had no significant effect. When these experiments were repeated at 5”C, addition of either NaCl, KCI, or mannitol was detrimental to long-term cell survival. Furthermore, addition of mannitol to the medium did not improve survival when cells were stored at 7°C. Addition of KC1 at 5 or 10°C or NaCl at 5°C only affected the cells’ ability to accumulate sublethal damage, while addition of mannitol at 5 or 10°C affected both of the above and the cold sensitivity of the cells. Addition of NaCl at 10°C only affected the latter. These experiments suggest that prevention of cell swelling by these conditions, while possibly necessary during clinical hypothermic organ storage, is detrimental 0 1985 Academic Press, Inc. to single cell survival at these temperatures.

cell volume in isosmotic medium between 5 and 10°C (8), i.e., cells swell at 5°C and shrink at 10°C. These phenomena may be related to the fact that there is a lipid phase change, centered around 8”C, in mitochondrial membranes and plasmalemma in these cells (1, 7). In this paper, we report on the effects of media made hypertonic (400 mOsm) by addition of NaCl, KCl, or mannitol on survival of V79 cells stored at 5, 7, and 10°C.

Hypertonic flush and perfusion solutions are commonly used when organs are stored at hypothermic temperatures prior to transplantation (2), mainly to counteract the generalized tissue edema (3) which would otherwise cause blockage of the microcirculation. We decided to investigate if hypertonic solutions of approximately the same osmolality as those used clinically could decrease survival in single cells exposed to hypothermia in tissue culture. Arrhenius plots of the rate of cell death in the mammalian cell line chosen, Chinese hamster V79 cells, show a “break” at around 7 to 8°C (7). This implies that there are two different mechanisms of cell killing in the temperature range above and below about 7°C. Survival is optimal at 10°C and decreases if cells are stored at temperatures above or below this in isosmotic medium. Also, there is a reversal in direction of change of

MATERIALS

484 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

METHODS

The cells used were originally derived from the V79 S-171 line of Chinese hamster fibroblasts; the subline was S-171-Wl (5). The plating efficiency of this line is about 80-95%. The cells were maintained at 37°C in an atmosphere of 95% air + 5% carbon dioxide in exponential growth phase using Eagle’s basal medium (BME powder formula with Hanks’ salts) supplemented with antibiotics [penicillin G (78 IU/ml) and streptomycin sulfate (78 pg/ml], sodium bi-

Received July 26, 1984; accepted March 26, 1985.

001 I-2240185 $3.00

AND

EFFECTS

OF HYPOTHERMIA

carbonate (2.75 g/liter), and 15% fetal calf serum (all components from GIBCO Laboratories, Grand Island, N.Y.). The resulting mixture will, henceforth, be called regular growth medium. The experimental medium was prepared by adding either NaCl, KCl, or mannitol to regular growth medium until the total osmolality increased from 325 to 400 mOsm. No precipitate was observed at any of the experimental temperatures or 37°C. For asynchronous cell studies, exponential-phase cells were removed from plastic petri dishes with 5-min treatments of 0.25% trypsin at 37°C. Cell concentrations were adjusted at this time to suit the treatment by inoculating enough single cells into the experimental flasks so that 100-200 colonies would result per plastic flask at the end of the colony assay period (16). The cells were then allowed to attach and repair trypsin damage for 3 hr in complete growth medium, preequilibrated to 37°C and pH 7.4. At this point the medium was removed and replaced by either preequilibrated (37°C and pH 7.4) regular growth medium (controls) or preequilibrated experimental medium immediately after which the flasks were sealed and placed in the cold in a circulating water bath. The temperature in the medium in the flasks equilibrated to the desired temperature in less than 5 min in this system with the drop to room temperature being achieved in less than a minute. The average cellular multiplicity (i.e., total number of cells per plate divided by the number of clumps of cells per plate) at this time was approximately 1.02 so that the calculated single-cell survival fraction ( 17) would be essentially the same as the “colony surviving fraction,” which is the experimental value obtained when the number of colonies on a plate is divided by the number of cells inoculated. After the exposure to the cold, the flasks were incubated for 24 hr at 37°C so that any unattached, viable cells could reattach. The medium was then removed from both the

ON

CELL

SURVIVAL

485

experimental and control flasks and replaced by regular growth medium after which the flasks were incubated at 37°C for 5-7 days; the resulting colonies were stained with methylene blue. The fraction survival, shown in the figures, at each “cold dose” is obtained by multiplying the “colony surviving fraction” at that dose by 100 and dividing through by the plating efficiency (expressed as a percentage). Hence, assuming a plating efficiency of 90%, if one single cell (out of Ill,1 11 cells originally plated) survived the treatment to form a single colony, the fraction survival would be 0.00001. Standard errors (SE) were used to express the experimental variability and were represented as vertical bars in all figures unless smaller than the points as plotted. Three replicate flasks were used for each experimental point. Control experiments, where the three different hypertonic experimental solutions were left on the cells for 7 days at 37”C, showed that the plating efficiency of the cells was not affected by exposure to these solutions. Since we have previously shown that survival curves of mammalian cells exposed to cold (7, 13, 14) have the same shapes as survival curves of cells exposed to ionizing radiation, standard survival curve analysis procedures were used (4). Since the “tail” of a survival curve is expected to be a straight line on a semilogarithmic plot, the slope and elevation of a linear regression line may be calculated. Testing for significant differences between slopes and elevations of the simple linear regression lines (the cell survival curves) was then done by analysis of covariance (19). In accord with standard terminology (4), the survival curve can be represented by two numbers, n and D,. Extrapolation of the linear portion of the curve to the log axis will yield the extrapolation number, n. The D, is defined as the time (i.e., the “cold-dose”) required to reduce the survival by a factor of l/e on the linear portion

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KRUUV,

GLOFCHESKI.

of the semilog graph. The 0, is inversely related to the cold-sensitivity of the cells. If the curve has a “shoulder,” the size of the shoulder is a quantitative estimate of the amount of sublethal damage a cell can accumulate before this damage becomes lethal. This size is given by the parameter, D,, the quasithreshold “dose,” that can be found by extrapolation of the linear portion of this curve to the point where it meets the horizontal line drawn through 100% survival (i.e., fraction survival = 1). Alternatively, D, = D, x In(n).

AND LEPOCK

in the case of Na+ and a reduction in both

D, and D, in the case of mannitol.

High mannitol medium has a relatively small effect at 7°C entirely due to a 14% reduction of D,. Again it does not improve cell survival when compared to the control (Fig. 2). At SC, use of high Na+ or KS medium leads to significantly decreased cell survival compared to controls (Fig. 2) with the effect mainly being due to a decrease in D, and with high K+ again being less detrimental than high Na+ medium to the cells. Storage of ceils in high mannitol solutions at 5°C produced the only variable set of data from experiment to experiment RESULTS in all of our hypothermic survival results. Figure 1 shows survival curves of asynThe survival curves of the mannitol experchronous, attached cells as a function of iments that resulted in the highest and the time at 10°C in regular growth medium and lowest survivals are shown in Fig. 2. Two in medium made hypertonic by the addition other mannitol experiments resulted in surof either NaCl, KCl, or mannitol. It can be vivals between these two extremes (not seen that the high K+ medium has a relashown). In all cases except one (discussed tively small effect on cell survival, the main below), both D, and D, were adversely afeffect being a decrease in D,; however, it fected. Only one of these four experiments is important to note that it does not improve showed any significant increase in survival cell survival when compared to the 10°C in the mannitol-treated cells with respect to control. On the other hand, storage in high the control; however, this only occurred at Naf or mannitol medium at 10°C signifione point in time (the 200-hr point in the cantly decreases cell survival largely due to a decrease in D, to 48% of the control value 1 lr

0.1

.‘ .

0.1

0.00001~ 0 0.000011

0

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300 400 TIME (HOURS)

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FIG. 1. Survival curves of asynchronous attached cells exposed to high mannitol- (circles; broken line), Na+- (diamonds; solid line), or K+- (triangles; broken line) supplemented (400 mOsm) medium or to isosmotic medium (squares; solid line) as a function of time at 10°C.

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400 300 TIME (HOURS)

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600

FIG. 2. Survival curves of asynchronous, attached cells exposed to high mannitol- (circles; broken line), Na+- (diamonds; solid line), or K+- (triangles; broken line) supplemented (400 mOsm) medium or to isosmotic medium (squares; solid line) as a function of time at 5°C and to high mannitol-supplemented (400 mOsm) medium (stars; broken line) or to isosmotic medium (“X”; solid line) as a function of time at 7°C.

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OF HYPOTHERMIA

ON CELL

SURVIVAL

487

curve shown in Fig. 2) leading to an in- with other cells, tissues, or organs, it is not crease in D, but decrease in D, in this curve known what actual temperatures Ranges I only. Hence, it must be concluded that ad- and II encompass in other cells. It is sugdition of mannitol in these 5°C experiments gested that future investigations be interconfers no long-term benefits to the cells as preted in terms of Range I and II rather than in absolute temperatures. Furtherfar as survival is concerned. more, researchers should become aware of DISCUSSION which Range they may be experimenting in It has been shown that at least some of with their system, since procedures which the mechanisms and possibly targets for may be successful in one Range may be incold damage are different for exposures to effective or even detrimental in another (7). temperatures above and below 7°C in this The results of this work suggest that, in cell line (7, 14). For the purposes of this general, use of hypertonic solutions during discussion, these temperature ranges will hypothermic storage is less harmful in be referred to as Range I (+ 28 to 7°C) and Range I than 11. If loss of cell viability is partially related II ( + 7 to O’C), respectively. Cell death due to “cold” exposures in the 10 to 25°C range to the inability to reaccumulate K+ (18), (Range I) is proportional to the tempera- then hypothermic preservation in K+-rich ture-time integral of exposure (i.e., total medium may have some benefit as advoamount of metabolism) suggesting either cated by many groups over the years. Our the accumulation to a critical level of a results show that high K+ medium is the toxic product or the depletion of a vital and least harmful of the hypertonic solutions necessary metabolite; cell death below 7°C tested for hypothermic preservation. While there is no doubt that storage in does not fit this integral (14). Furthermore, the membrane lipid perturber, butylated hy- hypertonic medium at 5°C (Range II) is detdroxytoluene (BHT), is effective in im- rimental to cell survival (Fig. 2), the inferproving survival below but not above 7°C ence of possible mechanisms from these (7). The results in the present communica- cell survival curves is not clear cut. Since tion do not contradict the above. In the extrapolation of these curves gives a negacase of high Nat medium, D, was affected tive D,, we are probably dealing with mulat 10°C but D, at 5°C. High mannitol me- ticomponent (i.e., “resistant tail”) curves. dium affected both the cold sensitivity (D,) This situation could arise from an additive and D, at both temperatures, while high K + cell cycle killing effect where the tail is due medium only interfered with the cells’ to the population most resistant to the two ability to accumulate sublethal damage different stresses, i.e., hypertonicity and (i.e., a decrease in DJ at both 5 and 10°C. 5°C hypothermia. In fact, the survival reThe fact that high K+ (or mannitol) medium sponse curve for the cell cycle for these two affected the survival curve parameters in a stresses is similar (12, 13); the “resistant qualitatively similar fashion above and tail” is probably due to an early G, and/or below 7°C may simply mean that the dif- late S-phase population. One of the ferent cold-injury mechanisms are both common features of these two stresses is sensitive to high Kf effects. Alternatively, an increase in cytoplasmic viscosity (1 I), there are several injury mechanisms in- the additive effects of which would cervolved in each of these two temperature tainly influence the rate of metabolism ranges with some mechanisms common to within the cytoplasm. It has previously been shown that 10°C both ranges. Since detailed survival studies as a func- is the optimum storage temperature in isostion of temperature have not been done motic media for this cell line (7). Also,

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these cells shrink at 10°C in isosmotic medium, whereas they swell at 5°C (8). If cell shrinking at the single cell level, as in our experiments, is important for survival, then storage at 10°C in hypertonic medium would not be expected to increase survival as cells are already shrunken in the 10°C control. Figure 1 confirms these predictions. On the other hand, storage of cells in hypertonic medium at 5°C should prevent cell swelling. Unfortunately, this procedure is detrimental to cell survival. Hence, cell swelling is probably unrelated to the mechanism of cell death due to hypothermia, at least at the single cell level. This is in agreement with data which shows that BHT greatly improves survival of cells exposed to 5°C (7) while not decreasing the rate or amount of cell swelling at this temperature (8). Even though the 5°C results with mannitol were relatively unpredictable, in view of the 7 and 10°C experiments and the four 5°C experiments, it seems safe to say that high mannitol medium at 5°C does not improve cell survival above the isosmotic control for long-term preservation. The variability may be related to the fact that mannitol slowly leaks into cells (15) and that leakage into and out of cells at low temperatures is rather inconsistent especially if some of the plasmalemma lipids have undergone a phase transition, which is the case for these cells at temperatures below 8°C (1, 7). Since the hypertonic solutions tested did not improve cell survival at 5 or 10°C and since no cell swelling is observed at 10°C (8), which is the optimum storage temperature in this cell line (7), it is suggested that organ storage at 10°C (or the low temperature end of Range I) in isosmotic solutions be considered more widely in order to increase the length of time an organ remains viable for clinical transplantation purposes. Furthermore, since post-hypothermic cell lysing at 37°C is greater after 5 than after 10°C storage (9), there may be fewer com-

AND

LEPOCK

plications in transplant patients after organ storage at the latter temperature. These experiments do not entirely mimic the “clinical” situation. First, the hypertonic medium was added at 37°C rather than at lower temperatures. However, experiments in our laboratory (Kruuv, unpublished) have shown that with the cooling rates used in our system, there was no significant difference in cell survival whether the hypertonic medium is added at 37, 22, or 5°C. Second, the cells were allowed to remain in the hypertonic medium for 24 hr at 37°C after the hypothermic exposure to ensure reattachment of all cells before the colony assay. It is well known that cold inhibits readhesion of cells in culture (6) and, hence, removal of the medium immediately following the hypothermia might lead to the loss of any viable cells which may have detached during the cold exposure. It is possible that the sublethal damage accumulated during the experiment in the cells that survived exposure to the hypertonic medium in the cold could be additive with sublethal damage accumulated due to exposure to 400 mOsm medium at 37”C, resulting in lethal damage. While we cannot rule out this possibility, it is not probable since the majority of sublethal cold-induced (SC) injury is repaired in 1.5 hr at 37°C (10). Furthermore, the control experiments showed that no decrease in survival was observed in cells exposed to 400 mOsm medium at 37°C for 168 hr. This implies that little, if any, sublethal damage, of the type that accumulates to result in lethal damage, is piling up in the cells under these conditions. The model used is, however, not completely incompatible with the clinical situation. Successful kidney preservation is limited to 72 hr of continuous hypothermic (5 to 10°C) perfusion while 120 hr usually results in nonviable kidneys. While not obvious in the figures presented in this manuscript, the 5, 7, and 10°C control curves generally all have “shoulders.” The average D, values for this cell line are 53, 51,

EFFECTS OF HYPOTHERMIA

and 80 hr at 5, 7, and 10°C respectively (7). Examination of survival curves, where the shoulder region has been carefully investigated (14, lo), reveals that cell survival is very high (85-100%) at these temperatures for at least 72 hr. On the other hand, the fact that 72 hr of hypothermic perfusion yields a functioning kidney does not necessarily mean that 100% of the “kidney cells” will turn out to be survivors when the kidney is transplanted. Hence, despite the differences from the clinical situation, the study of the effects of hypothermia on mammalian cells in tissue culture cannot only provide some insight into mechanisms of “cold damage” at the cellular level but may also prove to be of value to those using hypothermia in the clinical setting. ACKNOWLEDGMENTS This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada. REFERENCES 1. Al-Qysi, H. M. A. “Physical Properties of Mammalian Cell Membranes and Alterations after Hyperthermia.” Ph.D. thesis, University of Waterloo, Waterloo, Ontario, Canada (1984). 2. Collins, G. M., Green, R. D., and Halasz, N. A. Importance of anion content and osmolarity in flush solutions for 48 to 72 hr hypothermic kidney storage. Cryobiology 16, 217-220 (1979). 3. Downes, G., Hoffman, R., Huang, J., and Belzer, F. 0. Mechanism of action of wash-out solutions for kidney preservation. Transplantation 16, 46-53 (1973). 4. Elkind, M. M., and Whitmore, G. F. “The Radiobiology of Cultured Mammalian Cells,” pp. 7115. Gordon & Breach, New York, 1967. 5. Frim, J., Kruuv, J., Frey, H. E., and Raaphorst, G. P. Survival of unprotected, mammalian plateau-phase cells following freezing in liquid nitrogen. Cryobiology 13, 475-483 (1976). 6. Kolodny, G. M. Effect of various inhibitors on readhesion of trypsinized cells in culture. Exp. Cell Res. 70, 196-202 (1972). 7. Kruuv, J., Glofcheski, D. J., Cheng, K.-H.,

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Campbell, S. D., Al-Qysi, H. M. A., Nolan, W. T., and Lepock, J. R. Factors influencing survival and growth of mammalian cells exposed to hypothermia. I. Effects of temperature and membrane lipid perturbers. J. Cell Physiol. 115, 179-185 (1983). 8. Kruuv, J., Glofcheski, D. J., and Lepock, J. R. Cell size distributions as a function of time in hypothermia. Cryo-Lett. 5, 327-332 (1984). 9. Kruuv, J., Glofcheski, D. J., and Lepock, J. R. Cell size distributions as indicators of lysing during hypothermic storage. Cryo-Lrtf. 5, 333342 (1984). IO. Kruuv, J., Nelson, R. J., Frey, H. E., and McCann, L. E. Recovery from sublethal coldinduced damage in mammalian cells. Exp. Cell Res. 74, 514-518 (1972). 11. Lepock, J. R., Cheng, K-H., Campbell, S. D., and Kruuv, J. Rotational diffusion of tempone in the cytoplasm of Chinese hamster lung cells. Biophys. J. 44, 405-412 (1983). 12. McGann, L. E., Kruuv, J., and Frey, H. E. Effect of hypertonicity and freezing on survival of unprotected synchronized mammalian cells. Cryobiology 9, 107-l 11 (1972). 13. Nelson, R. J., and Kruuv, J. Survival of synchronized mammalian cells following exposure to cold. Exp. Cell Res. 70, 417-422 (1972). 14. Nelson, R. J., Kruuv, J., Koch, C. J., and Frey, H. E. Effect of sub-optimal temperatures on survival of mammalian cells. Exp. Cell Rex. 68, 247-252 (1971). 15. Pegg, D. E. Perfusion technology. In “Organ Preservation for Transplantation” (A. M. Karow and D. E. Pegg, Eds.), 2nd ed., p. 480. Marcel Dekker, New York, 1981. 16. Puck, T. T., and Marcus, P. 1. A rapid method for viable cell titration and clone production with HeLa cells in tissue culture: The use of Xirradiated cells to supply conditioning factors. Proc.

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(1955). 17. Sinclair, W. K., and Morton, R. A. X-ray and ultraviolet sensitivity of synchronized Chinese hamster cells at various stages of the cell cycle. Biophys. J. 5, 1-25 (1965). 18. Southard, J. H., Kuniyoshi, M., Lutz, M. F., Ametani, M.. and Belzer, F. 0. Comparison of the effect of 3- and 5-day hypothermic perfusion of dog kidneys on metabolism of tissue slices. Cryobiology 21, 285-295 (1984). 19. Zar, J. H. “Biostatistical Analysis,” pp. 228-235. Prentice-Hall, Englewood Cliffs, N.J., 1974.