Manifestations of cell damage after freezing and thawing

CRYOBIOLOGY

178-185 (1988)

25,

Manifestations LOCKSLEY

of Cell Damage after Freezing an.d Thawing’

E. McGANN,”

HONGYOU YANG,t AND MICHELE

WALTERSONS

*Department of Pathology, University of Alberta, Edmonton, Alberta, Canada; fLaboratory of Low Temperature Medicine, Jiamusi Medical College, Jiamusi, China; and fSkin Bank, University of Alberta Hospital, Edmonton, Alberta, Canada The nature of the primary lesions suffered by cells during freezing and thawing is unclear, although the plasma membrane is often considered the primary site for freezing injury. This study was designed to investigate the nature of damage immediately after thawing, by monitoring several functional tests of the cell and the plasma membrane. Hamster fibroblasts, human lymphocytes, and human granulocytes were subjected to a graded freeze-thaw stress in the absence of cryoprotective compound by cooling at - l”C/min to a temperature between - 10 and - 4o”C, and then were either warmed directly in water at 37°C or cooled rapidly to - 196°Cbefore rapid warming. Mitochondrial function in the cells was then assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetr~olium bromide (MTT), fluorescein diacetate (FDA), colony growth, and osmometric response in a hypertonic solution. Cells behaved as osmometers after cooling at - l”C/min to low temperatures at which there were no responses measured by other assays, indicating that the plasma membrane is not a primary site for injury sustained during slow cooling. These results also indicate that the FDA test does not measure membrane integrity, but reflects the permeability of the channels through which fluorescein leaves the cells. Fewer cells could respond osmotically after cooling under conditions where intracellular freezing was likely, implying that the plasma membrane is directly damaged by the conditions leading to intracellular freezing. A general model of freezing injury to nucleated mammalian cells is proposed in which disruption of the lysosomes constitutes the primary lesion in cells cooled under conditions where the cells are dehydrated at low temperatures. 8 1988 Academic press. IX.

It has long been known that freezing and thawing can damage living cells, and there have been many investigations into the nature of this injury. A general conclusion to date is that the plasma membrane is a primary site for freezing injury, although the actual mechanisms are unclear (10). Other investigators have proposed the mitochondria (15, 17), lysosomes (13), and other organelles (18, 14) as sites of freezing injury. Many of the studies on the mechanisms of freezing injury have used assays that test the integrity or function of membranes to assess recovery in cells or membrane model systems. Lethal injury to cells is eventually expressed as a breakdown in the Received September 10, 1987; accepted December 22, 1987. r Supported by the Medical Research Council and the Firefighters Bums Trust Fund.

semipermeable properties of the plasma membrane, independent of the cause and site of the initial lesion. Conditions such as the stresses encountered during freezing and thawing will result in changes to the more sensitive components or structures within the cell. If these changes are lethal, there will be a progressive development of damage to other regions within the cell, eventually leading to a loss of all functions associated with a living cell. Measurement of the integrity of the plasma membrane after a freeze-thaw stress will therefore inevitably lead to the conclusion that the plasma membrane is a site of freeze-thaw injury, whether or not damage to the membrane is a primary lesion. It is of interest to investigate the primary sites of freezing injury in order to understand the nature of this injury and to develop strategies for avoiding damage during 178

OOll-2240/88 $3.00 Copyright 0 1988 by Academic Press, Inc. AlI rights of reproduction in any form reserved.

MANIFESTATIONS

preservation of cellular, tissue, and organ systems. The purpose of this work was to investigate the sites of injury by subjecting living cells to a graded freeze-thaw stress and to assess the interrelationships between, and the sequence of changes in, membrane integrity, semipermeable properties, mitochondrial function, and reproductive integrity. A comparison of responses of cells immediately after thawing with their responses after an incubation period allows demonstration of the types of damage evident immediately after thawing, and of the secondary damage that develops subsequently. MATERIALS

Cell Preparation

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top in the platelet band. The granulocyte and lymphocyte bands were harvested from the gradient, washed twice in PBS with BSA, and resuspended in PBS with BSA. The resulting cell suspensions usually contained about 95% granulocytes with erythrocytes as the contaminant or 95% lymphocytes with monocytes as the main contaminant. Hamster fibroblasts (V79) were grown in tissue culture as monolayers attached to the bottom of tissue culture flasks (Corning, NY), in Eagle’s minimal essential medium (MEM;GIBCO) supplemented with 10% fetal calf serum (FCS;GIBCO). Cells in the exponential growth phase were detached from the flasks by a lo-min incubation at 37°C with 0.25% trypsin, and resuspended in MEM with FCS.

Human lymphocytes and granulocytes were isolated from the peripheral blood of Freeze-Thaw Protocol normal human volunteers. Peripheral blood A freeze-thaw protocol was selected to was collected in heparinized tubes and provide a graded stress resulting in a promixed with a solution of phosphategressive increase in damage (9). Aliquots of buffered saline (PBS) (GIBCO, Grand Is- cell suspension (0.2 ml) in PBS with BSA, land, NY) and 6% (w/v) dextran (seven without cryoprotectant, were dispensed parts blood to five parts dextran solution). into glass tubes (5 x 50 mm) and immersed The majority of erythrocytes were removed in an alcohol bath (FTS, Stoneridge, NY) at from the suspension by sedimentation at -5°C. After 1 min, the samples were unit gravity and room temperature for 30 seeded to induce ice formation, and another min. The resulting supernatant was washed minute was allowed for removal of the latwice in PBS with 0.15% bovine serum al- tent heat of fusion. The bath was then bumin (BSA) (Miles Laboratory, Naper- cooled at a programmed rate of - l”C/min, ville, IL). A stock Percoll (Pharmacia, Swe- and at predetermined temperatures samples den) solution was prepared by mixing 90 ml were either thawed rapidly (300Wmin) of Percoll with 10 ml of PBS at 10 times from various subzero temperatures by imisotonic concentration (GIBCO). A work- mersion in water at 37°C or cooled rapidly ing Percoll solution, consisting of 22.5 ml of (60OWmin) by immersion in liquid nitrostock Percoll solution and 13.5 ml of PBS gen, stored for 30 min, and then thawed with 0.15% BSA, was centrifuged at rapidly at 37°C. 11,SOOgfor 2 hr to create a continuous density gradient (density, 1.02-1.13 g/ml). The Assays washed cell suspension was layered on conColony growth. Hamster fibroblasts were tinuous density gradients of Percoll and diluted to 750 cells/ml so that experimental centrifuged at 500g for 30 min to separate aliquots of 0.2 ml could be directly seeded the cell suspension on the basis of density into tissue culture flasks to give 150 cells/ into bands of erythrocytes, granulocytes, flask. After an incubation period of 7 days, lymphocytes, monocytes, and debris at the the tissue culture medium was removed,

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and the cells were fixed with 70% isopropanol. The colonies were then stained with methylene blue, rinsed in distilled water, and counted. Triplicate samples were used for each experimental condition, and cell recovery was calculated as the mean of the experimental colony counts expressed as a percentage of the mean control colony counts. The uncertainties expressed in all graphs were the standard errors of the mean. MTT. Tetrazolium salts can be used to assess the activity of various dehydrogenase enzymes (16). Tetrazolium is cleaved in active mitochondria, so the reaction occurs only in living, metabolically active cells. The experimental procedure used for the MTT assay was similar to that described by Mosmann (11). After experimental treatment, 5 x lo4 cells in 100 and 10 t.J of an MTT (Sigma, St. Louis, MI) solution (5 mg/ml) were added to each well of a 96well flat-bottom plate (Flow Labs, VA). The plate was incubated at 37°C for 2 hr and then centrifuged at 800g for 5 min. The media was removed and 100 u.1of isopropanol was added to each well. The plate was then shaken for 60 set, and the absorbance in each well was recorded at a wavelength of 570 nm relative to the reference reading at 630 nm (Dynatech, VI). The mean absorbance of eight blank wells was used as the background reading and subtracted from the measured absorbencies. Triplicate samples were used for each experimental condition, and each sample was assessed in eight wells of the microplate. Cell recovery was expressed as the mean absorbance of the experimental samples as a percentage of the mean absorbance of the control samples. FDA. The FDA assay was similar to that originally described by Rotman and Papermaster (12). Fluorescein diacetate (FDA; Sigma), at a concentration of 10 pM, was added to each cell suspension for a 15-min period after thawing before assessment by fluorescent microscopy. A constant volume

AND

WALTERSON

of cell suspension was assessed using a hemocytometer that yielded approximately 300 cells in the control samples. This procedure accounts for cells which may lyse during the freeze-thaw process. Triplicate samples were used for each experimental condition. Cell recovery was expressed as the percentage of cells with fluorescence compared with the control sample. Osmotic response. Osmometric behavior after experimental treatment was assessed using a method similar to that described by Law et al. (4). Volume distributions of cell suspensions were measured using a Coulter ZBI electronic particle counter (Coulter Electronics, Hialeah, FL) and the data were stored on an IBM PC microcomputer using a custom interface (8). After treatment, two measurements of volume distributions were performed for each of the triplicate samples. One was in isotonic PBS (osmotic pressure, 0.282 Osmkg) and the other in a hypertonic solution of PBS (osmotic pressure of 1.35 Osm/kg). The hypertonic solution was prepared by mixing equal volumes of 10x PBS (GIBCO) and distilled water to yield a nominal 5X isotonic concentration. Cells were exposed to the appropriate solution for 5 min at room temperature before measurements. Cells with an intact, semipermeable plasma membrane shrank and remained shrunken in the hypertonic solution. No change in viability, as measured using any of the techniques described, resulted from the hypertonic exposure. RESULTS

Cells cooled at l”C/min to subzero temperatures without cryoprotectant suffer increasing damage as shown in Fig. 1. Recovery of hamster fibroblasts measured by reproductive integrity shows that cell damage begins below - 10°C and increases at lower temperatures (Fig. la). Recovery was low for samples cooled to - 196°C from the higher subzero temperatures, increased at intermediate temperatures, and subse-

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OF CELL DAMAGE

Fibroblast Lymphocyte

* X

q

Granulocyte

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q

-20

TEMPERATURE

(‘C)

TEMPERATURE

-30

-40

(‘Cl

FIG. 1. Responses of cells cooled at l”C/min to various subzero temperatures and then either thawed by warming at 300Wmin or cooled at 600YYmin to - 196°Cbefore warming. Cell recovery was assessed after thawing using colony growth of hamster fibroblasts (a) or MTT (b).

quently decreased at the lower temperatures, as reported elsewhere (9). Measurements of colony growth allow time for the repair of sublethal freeze-thaw damage (7). When assayed using MTT, the recovery of hamster fibroblasts was lower than that obtained using colony growth, and the response of human lymphocytes to the same freeze-thaw stress (Fig. lb) was similar to that of hamster tibroblasts. Human granulocytes, however, were significantly more sensitive to injury, with virtually no recovery after the suspension was seeded at -5°C and thawed directly. This granulocyte response is similar to results using phagocytosis as the assay for recovery (2, 3, 6). Figure 2 shows cell recovery measured

Lymphocyte anulocyte

using FDA. Immediately after thawing, hamster fibroblasts show a pattern of recovery with FDA similar to that shown by colony formation, but with a higher level of recovery at all sub-zero temperatues. Human lymphocytes and granulocytes also showed higher recoveries with FDA than with MTT, but even with FDA, granulocytes showed no recovery when thawed from - 196°C. After a 2-hr incubation period, cell recoveries for lymphocytes and granulocytes measured using FDA were similar to the results using MTT. The ability of cells to respond osmotically in a hypertonic solution was used to test the semipermeable properties of the plasma membrane. Figure 3 shows the results of these tests after the cells were exposed to

x

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+

A

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fl

-20 TEMPERATURE

(‘C)

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-30 (‘Cl

FIG. 2. Responses of cells cooled at l”C/min to various subzero temperatures and then either thawed by warming at 3OOWmin or cooled at 600Wmin to - 196°Cbefore warming. Cell recovery was assessed using FDA either immediately after thawing (a) or after incubation for 2 hr at 37°C (b).

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the same freeze-thaw stresses as in the previous experiments. The left column shows that hamster fibroblasts behaved as osmometers immediately after freeze-thaw conditions that caused significant damage measured by colony growth, MTT, and FDA, indicating that the plasma membrane 282 mOsm/kg

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maintains its semipermeable properties immediately after lethal injury during slow cooling. For cells cooled from 0°C to - 196°C at 600”C/min, conditions under which intracellular freezing is likely, a significant proportion was osmotically inactive immediately after thawing, indicating -

from

CONTROL

-10%

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to liquid

nitrogen

thaw

1r

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-20%

from -3O’C

‘row -4O’C

mOsm/kg

-40%

to liquid

lo liquid

nitrogen

nitrogen

lo Itqutd nttrogen

thaw

FIG. 3. Volume distributions of hamster fibroblasts in isotonic (282 mOsm/kg) or hypertonic mOsm/kg) PBS solutions after cooling 1Wmin to various subzero temperatures.

(1350

MANIFESTATIONS

that conditions leading to intracellular freezing are also involved in the loss of semipermeability of the plasma membrane. DISCUSSION

These experimental results point to several important conclusions regarding the nature of freeze-thaw injury, and to the mechanisms of the assay systems used. After slow cooling, cells behave as osmometers, indicating a semipermeable plasma membrane, when mitochondria are nonfunctional as measured by the MTT assay and the cells are unable to retain fluorescein. This indicates that the plasma membrane is intact, and therefore not a primary site of injury sustained during slow cooling. The lipid portion of the plasma membrane may sustain damage during freezing and thawing, but could reseal during or after thawing. Whether the cells that suffer transient leakiness during the freezethaw stress are the same cells that are osmotically active yet nonfunctional after thawing remains to be determined. After rapid freezing, conditions where intracellular freezing is likely, loss of the semipermeable property of the plasma membrane was evident in a portion of the cell population immediately after thawing, implying that the plasma membrane may be directly damaged by the conditions leading to intracellular freezing. This supports the concept that the type of damage suffered by cells is dependent on the cooling conditions (5). A post-thaw incubation period allows time for the development of secondary damage, as demonstrated by the experiments with FDA. Impairment of mitochondrial function is evident immediately after thawing under some conditions where the FDA assay shows no change in intracellular fluorescence. However, on incubation, significant changes are evident using FDA, indicating the damage develops during the incubation period, which is then manifested as changes detectable using the FDA assay.

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This type of damage is therefore secondary, resulting from a previous lesion. A post-thaw incubation period also allows time for the repair of sublethal damage (7) as demonstrated by the difference between the results from the colony growth and MTT assays. In fibroblasts, the colony growth assay for reproductive integrity is a functional test for virtually all active systems within the cell. Cell recovery after freezing to subzero temperatures is higher when measured using colony growth than when measured using MTT. Therefore some of the damage measured by MTT must be repaired during the period of incubation for colony growth. Repair of sublethal freeze-thaw damage has been previously reported for these cells (7). A comparison of the MTT assay with the other tests for cell recovery reported here (colony growth and FDA) shows that the MTT assay is a very sensitive test to detect freeze-thaw damage, a conclusion supported by the experiments with granulocytes, where our results with the MTT assay compared well with those using phagocytosis (2, 3, 6) and chemotaxis (2). Results for the different cell types illustrate the similarity of responses of human lymphocytes and hamster fibroblasts and the different responses of human granulocytes. These observations are consistent with the interpretation (13) that the lysosomes are sensitive organelles in granulocytes that are easily activated by perturbations such as the osmotic stresses encountered during freezing and thawing. The similar responses of two very different cell types-human lymphocytes and hamster libroblasts-would indicate that these two have similar sites of freezing injury. Rotman and Papermaster (12) reported that after freezing, thawing, and incubation, no fluorescence was observed in intact cells, whereas esterase activity could be demonstrated in cell-free extracts, and concluded that intracellular fluorescence due to FDA is a measure of the integrity of

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the plasma membrane. Our results show that the plasma membrane maintained its semipermeable properties immediately after thawing under conditions where significant cell damage was evident when measured using MTT and FDA. It is unlikely that a general loss of membrane integrity, sufficient to allow leakiness to fluorescein (MW 416.39), would allow the cells to remain effectively semipermeable. It therefore appears that the loss of fluorescence in damaged cells is more specific to the movement of fluorescein across the plasma membrane or to the hydrolysis of FDA in the cytoplasm than to a general loss of membrane integrity. Rotman and Papermaster (12) proposed that fluorescein accumulates inside the cell after hydrolysis of FDA because the nonpolar FDA can penetrate the plasma membrane more rapidly than the polar fluorescein, and they measured the kinetics of release of intracellular fluorescein. The loss of fluorescein occurs in intact cells and is not necessarily an expression of cell damage. We propose that damage to the cell, such as freeze-thaw damage, causes changes in the channels by which fluorescein moves across the plasma membrane, resulting in an accelerated loss of fluorescein. The FDA assay is therefore another specific test rather than a general measure of membrane integrity. We propose a general model of freezing injury to nucleated mammalian cells consistent with our experimental observations. Disruption of the lysosomes constitutes the primary lesion in cells cooled under conditions where the cells are dehydrated during the cooling process. This leads to an early impairment of mitochondrial function and then to changes in other specific structures such as channels for fluorescein in the plasma membrane. There is evidence that the time course of damage to mitochondria by lysosomal enzymes (14) is much shorter than that of damage to the plasma membrane (1). The plasma membrane is a primary site of freezing injury during rapid

freezing when intracellular freezing is either a cause or a result of this damage. A practical implication of this hypothesis is that two modes of cryoprotection for slow cooling are desirable-one is the reduction of osmotic stresses by colligative action, and the other is the stabilization of the lysosomal membrane. REFERENCES 1. Desai, I. D., and Tappel, A. L. Erythrocyte hemolysis by isolated rat liver liver lysosomes. Proc. Sot. Exp. Biol. Med. 118,496-499 (1965). 2. Dooley, D. C., and Takahashi, T. The effect of osmotic stress on function of the human granulocyte. Exp. Hematol. 9, 731-741 (1981). 3. Lancaster, M. G., and Allison, F., Jr. Studies on the pathogenesis of acute inflammation. VII. The influence of osmolality upon the phagocytic and clumping activity by human neutrophils. Amer. J. Pathol. 49, 1185-1200 (1966). 4. Law, P., Rule, G. S., Sprott, D. A., Lepock, J. R., and Kruuv, J. Cell size in hypertonic medium for estimation of freeze-thaw survival in mammalian cells. Cryo-Let?. 1, 28-34 (1980). 5. Mazur, P. Cryobiology: The freezing of biological systems. Science 168, 939-949 (1970). 6. McGann, L. E., Grant, M., Turner, A. R., and Turc, J.-M. Osmotic limits of human granulocytes. Cryobiology 18, 622 (1981). 7. McGann, L. E., Kruuv, R. J., and Frey, H. E. Repair of freezing damage in mammalian cells. Cryobiology 9, 496-501 (1972). 8. McGann, L. E., Turner, A. R., and Turc, J-M. Microcomputer interface for rapid measurements of cell volume using an electronic counter. Med. Biol. Eng. Comput. 20, 117-120 (1982). 9. McGann, L. E., and Walterson, M. L. Cryoprotection by dimethyl sulfoxide and dimethyl sulfone. Cryobiology 24, 11-16 (1987). 10. Morris, G. J. Liposomes as a model system for investigating freezing injury. In “Effects of Low Temperatures on Biological Membranes” (G. J. Morris and A. Clarke, Eds.), pp. 241262. Academic Press, New York, 1981. 11. Mosmann, T. Rapid calorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. .I. Inmunol. Methods 65, 55-63 (1963). 12. Rotman, B., and Papermaster, B. W. Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. PNAS 55, 136141 (1966).

MANIFESTATIONS 13. Rowe, A. W., and Lenny, L. L. Cryopreservation of granulocytes for transfusion studies on human granulocyte lysosomes, kinetics of glycerol uptake, and cryopreservation with dimethyl sulfoxide and glycerol. Cryobiology 17, 198-212 (1980). 14. Sawant, P. L., Desai, I. D., and Tappel, A. L. Digestive capacity of purified lysosomes. Biochim. Biophys. Acta 85, 93-102 (1964). 15. Sherman, J. K. Comparison of in vitro and in sifu ultrastructural cryoinjury and cryoprotection of mitochondria. Cryobiology 9, 112-122 (1972). 16. Slater, T. F., Sawyer, B., and Strauli, U. D. Stud-

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ies on succinate-tetrazolium reductase systems. III. Points of coupling of four different tetrazolium salts. Biochim. Biophys. Acta 77, 383-393 (1963). 17. Tsvetkov, T., Tsonev, L., and Minkov, I. A quantitative evaluation of the extent of inner mitochondrial membrane destruction after freezing and thawing based on functional studies. Cryobiology 23, 433-439 (1986). 18. Volger, H., Heber, U., and Berzbom, R. J. Loss of function of biomembrane proteins during freezing. Biochim. Biophys. Acta 511, 455-469 (1978).