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Effect of hypertonic stress on mammalian cell lines and its relevance to freeze-thaw injury
CRYOBIOLOGY
15, 517-529
Effect
( 1978
)
of Hypertonic Stress on Mammalion and Its Relevance to Freeze-Thaw
Cell Injury
Lines
J. B. GRIFFITHS
Microbiological
Research
Establishmmt,
The cooling rate giving the best cell survival from a freeze-thaw cycle has been described as a compromise between the injurious ‘effects of physical and chemical changes to the medium brought about by slow cooling and the effect of intracellular ice at faster than optimum cooling rates (17). The nature of these solution effects and in particular hypertonic stress injury have been extensively investigated (14, 15, 19, 21, 23, 25). ZCells dehydrate and shrink in response to hypertonic conditions (4, 6, 7, 19) but whether cell damage is a consequence of high salt concentration and denaturation (15), the loss of ce1I water and< reduction in ceII volume ( 14) or to the mechanical effects of resisting further cell shrinkage (19) is uncertain. The effect of hypertonic@ on erythrocytes has been exhaustively studied both with and without the additional stress of low temperatures (thermal shock) (4, 6, 7, 21, 22, 25). However, much less is known about the effect of hypertonic conditions on mammalian cell Iines and how far the thermal shock data for erythrocytes can be extrapolated to freeze-thaw damage in this type of cell, In order to try and define freeze-thaw damage in mammalian cells the effects of cooling (9, 10) and thawing rates (11 j have been studied at intermediate temReceived 3,
September
29,
1977;
accepted
March
1978.
Potion,
Salisbu y, W&shire,
U.K.
peratures between 0” and -196°C. The surviva1 of cells has been measured using a ceI1 counting procedure (recovery index) together with a radiochromate assay to measure cell injury. These studies have led to the recognition of lethal and nonIethal damage to cells at various stages of the freeze-thaw cycle (II ). To gain further insight into the cellular processes affected by cooling the radiochromate method was extended to the use of other radiochemicals, many of which have far more specific attachment sites on the cell than radiochromate (8). This study raised the question of exactIy what role did dehydration and osmotic stress play in cel1 injury at both slow and fast cooling rates. This present investigation describes the effect of hypertonic stress alone on two mammalian celI lines in order to clarify the roles of thermal and dilution shock (5, 16) during freeze-thaw cycles and to extend the pubIished data on erythrocytes to more complex nucIeated fibrobIast and epithelial cells. MATERIALS
AND
METHODS
Cell Lines and Culture Procedure The human diploid cell line (MRC-5) and the Chinese hamster ovary cell line, clone A (CHO) were used. Culture procedures have been described previously (9, 10).
517
All
Copyright @ 1978 rights of reproduction
by Academic Press, Inc. in zny form reserved.
518
J, B. GRIFFITHS
Hypertonic Experiments in Sodium Chloride
tion was identica1 whether additions were made to the buffer or not.
Conffuent cultures of cells were harvested using 0.1% pronase (MRC-5 cells) or 0.25% trypsin (CHO cells) and suspended in Eagle’s basal medium (BME) with 10% calf serum at 6 X lO*/ml. The cell suspensions were gently agitated for 30 min at 37°C to allow cell repair to occur foIIowing possible trypsin or pronase damage. Some cell loss occurred during this period but this stabilized and there was found to be no advantage in extending this period beyond 30 min. The cells were then centrifuged (2OOg for 2 min) and resuspended in Earle’s Basal Salt SoIution (EBSS) at 6 x 10B/ml. One miIIiIiter aliquots were added to 16 centrifuge tubes and the cell suspensionswere then diluted to a final concentration of 2 m1 (3 X 108/ ml) with a range of sodium chloride solutions (in a buffered salt solution: KCl, 0.2 g/I; Na2HP04, 1,15g/I; KHZP04, 0.2g/l; MgC126H20, O,lg/l). The cells were diluted slowly (2 min/concentration step) and stepwise from 0.18 to 0.24, 0.40, 0.64, 0.80, 1.20, 1.60, to 2.40 M NaCl. After dilution to 2 ml there were duplicate samples at each of these eight NaCI concentrations, A slight drop in pH occurred with this procedure from an initial value of 7.3 at 0.16 M to pH 7 at 2.4 M NaCl. These samples were then divided into two, one part for hypertonic measurements, the other was sIowly diluted back to isotonic (0.16 M NaCl) concentration with various mixtures of EBSS and diIute NaCl solutions. Sampies were taken for the various parameters under study by centrifugation of the cells out of the saline solutions (15Og/3 min). This occurred after the cells had been exposed to hypertonic conditions for 20 min at a constant temperature throughout of 20°C. Dimethylsulphoxide (final concentration 10% ) was present at all times unless stated otherwise in the text. The addition of DMSO was made when diluting buffer concentrates so that salt concentra-
Recovery Index (RI) A fulI description and evaIuation of this method has been pubIished (9) Recovery Index is a measure of the cells surviving a certain treatment and retaining the abiIity to survive 18 to 24 hr in culture and to divide, Identical aliquots of hypertonically stressed cells and contro1 ceIIs from the same initial population were incubated in growth medium (BME for CHO cells, MEM for MRC-5 ceIIs plus 10% calf serum) for 18 hr (CHO) or 24 hr (MRC-5), The cells were then harvested (by pronase or trypsin) and counted in a Coulter Counter. The number of cells grown from the experimental sampIes were expressed as a proportion of the numbers grown from the control cell suspension. Typan
Blue Staining
Cell counts were made in a haemocytometer after dilution in 0.1% trypan blue (in EBSS ) . This method is wideIy used for assessmentof viability on the basis that it is only taken up by highly damaged and leaky cells (2). However, in this investigation where celIs are subjected to high osmotic pressures it is used to indicate increased cell permeability rather than viability. Release of Radiochemicals Cell cultures were incubated with various radiochemicaIs, for up to 18 hr at 37”C, and then harvested and prepared for experiments as described above. During the experiment the release of radiochemicals from the cells into the various saline buffers was measured by adding 10 ml Brays Scintillation fluid to 1 ml of cell-free saline buffer and counting in a Nuclear Enterprise Counter. Gamma counting (chromate and rubidium) of the 1 ml samples was done directIy in a Nuclear Enterprise counter with the gamma head attach-
EFFECT
OF HYPERTONIC
STRESS ON MAMMALIAN
merit, The radioactivity remaining in the cells was also measured after dissolving the cells in 0.2 N NaOH. The values for release of radiochemicals from ceIIs were calculated as a percentage of the tota cpm (cells plus saline buffer). Results in this paper refer to the difference in percentage reIease between control samples (in isotonic buffer ) and the samples from cells subjected to hypertonic concentrations of NaCl. Radiochemicals were obtained from the Radiochemical Centre ( Amersham) and were used as follows: amino acid “C colIection (CFB 103; C7, 8, 9, 10, 15), 0.1 &i/ml for 3 hr; sodium chromate ( Wr ) (CJS. lP), 0.5 &i/ml for 2 hr (9, 10, 11); L-[1-3H]fucose (TRA 366), 0.25 ,&i/ml growth medium for 18 hr; rubidium chloride (eGRb) (RGS IP), 150 pCi/ml of growth medium with 3 x lo8 cells/ml in suspension for 1 hr; [5-H3]uridine (TRA 178), 0.25 pCi/ml growth med,ium 5 hr. The use of these radiochemicaIs to indicate freeze-thaw injury has been described (8). To summarize; amino acid release marks cell destruction with large losses of structural protein; chromate release is from denatured cell protein and can be from both lethal and non-lethal damage (11); fucose labels the carbohydrate component of membrane gIycoproteins; rubidium release is an indicator of cation permeabihty and marks the completion of cehular shrinkage; uridine is an RNA precursor and is thus mainIy cytoplasmic and was found to be released immediately after cell death. Cell Water Volume (CWV) The [‘“Cl thiourea : [3H]mannitol ratio method was used to measure the volume of intracehular fluid (18). The cell suspensions were puIsed with 4 ,&i/m1 of thiourea and 2 &i/m1 of mannito1 for 20 min at 37°C. The cells and suspending saline were separated by centrifugation (15Og/2 min) and the radioactivity of the cells and medium measured in Brays solu-
CELLS
519
tion using a dual label technique in a Liquid Scintillation Counter (Intertechnique SL-30). The medium vohrme was measured accurately by pipette so that the radioactive counts in the medium couId be used to convert cell counts (cpm) into volume. The intracellular voIume was taken as the difference between the apparent thiourea space and the apparent mannitol space ( 18). Cell Diameter Memurements Cells were photographed using a Zeiss photomicroscope ( x500) and cell diameter measurements made from the photographs. The longest diameter, and the diameter at right angles to this was measured in each ceI1 and averaged. Sample size was a minimum of 30 cells. This photographic method enabIed a11measurements to be made within a very short time of each other whilst the cell was under stress in the various hypertonic buffers or immediately after return to isotonicity. Lethal and Sublethal Chromate Release The relationship between RI and tota chromate reIease (PCR) allows the relative amounts of chromate released due to IethaI and sublethal (repairable) damage to be assessed using probabihty theory mathematics in the following equation: In (RI) = K, - Kz (PCR) K1 is a measure of sublethal damage and Kz a measure of the probability of death and PCR-K1 represents the chromate released as a direct consequence of cell death. The derivation and application of this equation has been published (11). RmULTS
Efect of Increasing Hyptertonicity on MRC-5 Cells (Fig. I ) CelI size and release of various radiochemicals were measured as MRC-5 cells were subjected to increasing concentrations of sodium chloride in EarIe’s Buffered Salts
520
J. B. GRIFFITHS
Solution (EBSS) ranging from isotonic (0.18 M NaCl) to 2.40 M NaCI. The results, summarised in Fig. 1, show that MRC-5 cells rapidly shrink and Iose water down to a minimum value at 0.64 M NaCl. During this period there was virtualIy no release of radiochemicals or loss of cells. Increasing the NaCl molarity above 0.64 M caused a gradual increase in celI dmiameter and cell water vohnne, some release of rubidium, chromate and amino acids but very little release of fucose or uridine. There was approximately 15% loss of ce1I.s and this occurred between 0.8 and 1.6 M NaCl and was accompanied by the loss of radiochemicals. Cell viabiIity, which fell from 95% at 0.8 to 14% at 1.6 M NaCI, was mea-
NoCt
sured by trypan bIue staining and was, therefore, a better measure of cell permeability changes than actual ability of the cell to recover and resume growth (2, 9, 12), The morphological appearance of the cells was normal to 0.84 M NaCl but: at higher concentrations the cells lost their regular spherical shape and some apparently dead celIs appeared. There was very little evidence of complete cell destruction. The cells that were assumed to be dead were enIarged and “ghost-like” with a definite form, with and without very large vesicles, and often with denatured cell material adhering to one part of the cell. This morphoIogica1 appearance perhaps expIains the low release of fucose (eel1
CONCENTRATION
IMI
FIG. 1. Effect of subjecting MRC-5 cells to hypertonic concentrations of NaCl. V-V, cell water volume; T-T, cell diameter (V, viable cells; D, dead cells); X-X, total celI count; I--I, percentage viability. Percentage release of chromate (O-O), rubidium ( l -e ), uridine ( O--O), amino acids ( A-A ) and fucose ( A--A ).
EFFECT
OF HYPERTONIC
STRESS QN MAMMALIAN
CELLS
521
0.6 NaCt CONCENTRATON
ItAl
FIG. 2. Effect of returning MRC-5 cells to isotonic NaCl from various hypertonic concentrations of NaCl. V-V, cell water volume; V-7, cell diameter; X-X, total cell count; W-W, percentage viability; @- -8 recovery index. Percentage release of chromate (O-O), rubidium ( l -e ), uridine ( O-C! ), amino acids ( A-A ), and fucose ( A-A ).
shape maintained) and uridine (stiI1 within the vesicles) and relatively higher release of chromate and amino acids from the denaturation of the ceI1. Efiect of Returning MRC-5 Cells from Hypertonic to Isotonic Condith.s (Fig. 2) The cells were returned slowly and stepwise from their final hypertonic concentration to isotonic EBSS. This involved an S-fold increase in volume of all cell suspensions which caused some damage even to the cells which were kept in isotonic EBSS throughout the experiment. This damage was indicated by the initial release of amino acids, uridine and chromate
in Fig. 2 as well as a slight decrease in cell water voIume and diameter. AIthough ceI1 size increased on return to isotonic conditions it was stiI1 smaller than norma in the ceIIs subjected to NaCl concentrations up to 0.4 M. Above this salt concentration the cells returned to near their original volume and this was accompanied by additional release of chromate, amino acids and fucose. The morphological appearance of the cells remained good after returning from 0.4 M NaCl but at higher molarities they began to deteriorate so that after exposure to 0.8 M NaCl aII cells were adversely affected, showing heavy blebbing and loss of cell material. Cells exposed to 1.6 M N&l or above were extremeIy dam-
522
J, B. GRIFFITBS
aged with extensive granulation, vesicIes and protrusions but mainly intact. The cell loss from 2.4 M NaCl was less than 40% and this occurred mainly after returning from NaCl molarities of 0.8 to 1.2. Recovery index (RI) and cell viability (by trypan blue staining) declined at a rapid but steady rate to very low values after exposure to 2.4 M NaCI. The additional release of radiochemicals that did occur due to returning the cells to isotonic conditions occurred after exposure to concentrations above 0.8 M NaCl, the most significant being amino acids, chromate and uridine. There was very little release of
NoC4
FIG.
3. Effect
of subjecting
water volume; V-7, Percentage ( A-A
CHO
release of chromate ( O-O fucose ( A-A ).
) and
CONCENTRATION
cells
cell diameter;
fucose and this correlated with the relatively small cell loss. Analysis of the RI and chromate release data using the probability equation, In (RI) =K1 -I& (PCR), where K1 is a measure of the non-lethal damage to cells (11) showed that in this experiment there was virtually no non-lethal chromate release (K, = 1.0). The other instance where all the chromate reIease was lethal was during slow cooling of cells after exposure to temperatures between 0 and -4O”C, i.e. the period of thermal shock, dehydration and shrinkage in the cooling cycle.
X-X,
to hypertonic
total
), rubidium
IMI
concentrations of cell count; H-m, ( e-0 ), uridine
NaCI. V-V, cell percentage viability. ( 0-U ), amino acids
EFFIXT
OF HYPERTONIC
STRESS ON MAMMALIAN
CELLS
523
FIG. 4. Effect of returning CHO cells to isotonic NaCl from various hypertonic concentrations of NaCl. V-T/, cell water volume; V-7, cell diameter; X-X, total celI count; m-m, percentage viability; m--m, recovery index. Percentagerelease of chromate (O-O), rubidium ( 0 -@ ), uridine (O--O), amino acids ( A-A ) and fucose ( A-A ).
Effect of Increasing Hypertonicity on CHO Cells (Fig. 3) This experiment was carried out in an identical manner to that described for MRC-5 celIs (in Fig. 1) for comparative purposes as CHO and MRC-5 cells have been shown to have different behavioural patterns during freeze-thaw cycIes (S-11). However, there were many similarities in their behavior in hypertonic NaCI. The degree of shrinkage to a minimum value at 0.64 M NaCI was identical but CHO cells remained at this reduced level whilst the NaCI moIarity was increased to 1.2 or 1.6 before abruptly rising to normal (contro1) values. The only other difference between the two cell lines was that CHO cells re-
leased a significantly higher amount of uridine and a much higher quantity of rubidium than MRC-5 cells, The morphological appearance of the celIs rapidly changed from their normal smooth spherical shape to become smaller and crinkly (accompanied by a small amount of fucose reIease) at 0.4 M NaCl. The cells then became very “angular” (at 0.8 M NaCl) and this was followed by dense granulation and denaturation (at 1.2 M NaCl) and finally swollen “ghost-like” cells and signs of cell disruption (at 1.2 to 2.4 M NaCI). Effect of Returning CHO Cells from Hypertonic to Isotonic Conditions (Fig. 4) CHO cells remained smaller than control cells to a similar degree to MRC-5
J. B. GRIFFlTHS
524
index and percentage chromium release of concentrations of NaCl before and after returning to isotonic NaCl. Recovery index; W--II DMSO-free; O-0, 10% DMSO. Percentage chromium release: at hypertonic NaCl concentrations, l -a (DMSO-free) and O-O (10% DMSO); after returning to isotonic NaCl from various hypertonic concentrations -A--A (DMSO-free) and A--A (10% DMSO). FIG.
5. Effect of 10% DMSO
on recovery
MX-5 and CHO cells subjectedto hypertonic
cells except that they did not return to norma size until after being subjected to 1.2 M NaCI (compared to 0.64 M for MRC-5 cells), Recovery Index (RI) began to decline significantIy after exposure to molarities greater than 0.8 M NaCl. Return to isotonicity caused large additional release of amino acids, fucose and chromate, but not of rubidium or uridine. A comparison between Figs. 2 and 4 show that the release of rubidium and uridine was very much higher in CHO ceIIs but fucose and amino acids were only slightly higher and chromate reIease was less than for MRC-5 ceIIs. Chromate release (PCR = 17) was mainly lethal, although there was some
non-lethal release (Ki = 2.5). Morphological changes occurred after exposure to molarities above 0.64 when the 1~11sbecame larger and more dense with extensive vesicle formation and some cell Ioss (about 30%). Effect of DMSU on Cells Subjected to Hypertonic NaCl Concentrations (Fig. 5) Recovery index and percentage chromate reIease of MRC-5 and CHO cells subjected to increasing concentrations of NaCl, and after dilution back to isotonic EBSS in the presence and absence of 10% DMSO are shown in Fig. 5. Recovery index was always lower at each hypertonic NaCl con-
EFFECT
OF HYPERTONIC
STRESS ON MAMMALIAN
centration in the absence of DMSO but only became really significantly different after exposure to molarities of 1.2 and above. The addition of DMSO had the effect of delaying chromate release from 0.4 to 0.64 M NaCl at hypertonic salt concentrations but above 16 M NaCl there was very Iittle difference in chromate release with and without DMSO. After the cells had been brought back to isotonic concentration DMSO only had its main protective effect on MRC-5 cells exposed to above 1.6 M NaCl. CHO cells were very significantly protected by DMSO during their return from all hypertonic concentrations up to 2.4 M NaCl. The results in Fig. 5 definitely show DMSO to have a protective effect on cells subjected to hypertonic stress. An analysis of chromate release in the absence of DMSO (using the equation In RI = Kr - KZ PCR) showed a change in the relationship between RI and PCR as the hypertonicity increased, MRC-5 cells subjected to 0.16 to 0.40 M NaCl showed a similar K, to that in the presence of DMSO (K, increased from 1.0 to 1.7) but after exposure to between 0.64 and 2.4 M NaCl the non-IethaI chromate release became much greater (Kr = 9.0). SimilarIy CHO celIs after exposure to 0.16 to 0.8 M NaCI had a K1 of 7.5 (compared to 2.5 with DMSO) but this increased to 17.5 after exposure to 1.2 to 2.4 M NaCl. DISCUSSION
There is extensive information about the effect of osmotic stress on erythrocytes (4, 6, 7, 15, 16, 19, 21, 22, 25) but very few data pertaining to this aspect of the cooling cycIe for mammalian cell lines. This present study was carried out to investigate the responses of such cell lines during hypertonic stress at a constant temperature and to compare this with similar data obtained during freeze-thaw experiments (8). The first conclusion to be made from the data in this nauer is that cell shrinkage
CELLS
525
(dehydration) occurs extensiveIy as the molarity of NaCI increases from isotonic (0.16 M) to 0.64 M. The minimum cell size is reached at 0.64 M and both MRC-5 and CHO cells show an identical degree of size change (46-45s ) . This was not predictable as the two cell lines are quite dissimiIar both in morphology and in growth potential. The second conclusion is that shrinkage is not damaging to either cell line as there was no radiochemical reIease or loss of ceIIs during this period. This confirms various conclusions drawn from studies with erythrocytes on shrinkage at constant temperatures (3, 5, 23). Radiochemical release occurs after cells reach their minimum size and are subjected to further increases in hypertonicity, InitialIy rubidium is released followed by chromate, amino acids and, in CHO cells only, uridine and this is accompanied by a gradual increase in ceI1 size from the minimum value at 0.64 M NaCl. Rubidium is a marker of the intracellular space occupied by potassium (1) and its release marks the Ioss of membrane integrity and permeability to cations (3). This increase in membrane permeabibty is also indicated by the increase in trypan blue staining and subsequent sweIling of cells. Thus damage occurs when the cells can shrink no further resulting in some Ioss of viability and ceII destruction; perhaps due to salt denaturation of protein (13) or disruptive sweIIing of cells, Some discrimination between obviousIy dead cells and probable viabIe celIs could be made by microscopical examination and this showed that the dead cells were significantly larger (Fig. 1). CHO cells retain their minimum voIume at greater NaCl molarities than MRC-5 cells and lose more rubidium and uridine demonstrating that they have a higher degree of membrane leakiness and are able to withstand a greater Ioss of cytopIasmic constituents before crossing the threshold for recovery. When this happens cell swelling occurs, vesicles on the cell surface are
526
J, B. GRIFFITHS
FIG. 6. A comparison of radiochemical release by CHO and MRC-5 cells subjected to various hypertonic concentrations of KaC1 and after their return to isotonic NaCI. A, amino acids; C, chromate; F, fucose; R, rubidium; U, uridine. ZZ release at stated NaCl concentrations; 0, extra release on return to 0.16 M NaCI.
formed but there is no widespread lysis at hypertonic NaCl concentrations. After subjecting the cells to hypertonic NaCl they were then brought back into isotonic EBSS, a process analogous to the warming phase of freeze-thaw cycles, Cells did not return to their control (normal) size until after exposure to 0.64 (MRC-5) or 1.2 M NaCl (CHO). The recovery index became significantly reduced only after the cells had’ begun to swell back to control size, and this was followed by considerabIe radiochemical release. This is summarized in Fig. 8 so that the extra reIease caused by the return to isotonicity can be more easily seen. There was no further release of rubidium as could be predicted if this is a marker of shrinkage, but uridine release occurred in MRC-5 celIs at this stage rather than during increasing hypertonicity as was the case with CHO cells. The results are indicative of MRC-5 celIs succumbing to hypertonic stress at a low NaCl molarity (0.64 or below) and becoming denatured (presumably by saIt damage). Thus dilution back to isotonic conditions
causes litle extra damage, as shown by the Iow release of chromate, amino acids and fucose (Fig. 6). CHO ceIIs, on the other hand, are able to withstand molarities of at least 1.2 M NaCI, possbiIy due to the protective leakage of cytoplasmic constituents (e.g., rubidium). However, they are prone to dilution damage resulting in n higher amino acid, chromate and fucose release than MRC-5 cells. Microscopical examination reveals more vesicle formation, disruptive swelling and loss of cell material from CHO than from MRC-5 ~11s. It should be noted that CHO cells with an RI of 0.11 after exposure to 2.4 M NaCl are far more stressed than during a freezethaw cycle with an RI of 0.53 after cooling at 200”C/min. This RI occurs after exposure to 1.2 M NaCl which, in the presence of DMSO, is probabIy greater than the NaCl concentration at -40°C during a freeze-thaw cycle (21). A stated aim of this investigation was to determine if freeze-thaw data on radiochemical release ( 8) could be interpreted in terms of hypertonic stress.A comparison
EFFECT
OF HYPERTONIC
STRESS ON MAMMALIAN TABLE
CELLS
527
1
Comparative Effects of Freeze-Thaw Cycles Using Various Cooling Rates to - 196°C Exposure to 2.4 M NaCl and Return to Isotonic NaCl (0.16 M) After Exposure to 2.4 M NaCl on the Recovery Index and Percentage Release of Radiochemicals from CHO and MRC-5 Cells MRC-5 NaCl
RI Radiochromate Rubidium Uridine Fucose Amino acids
eonc.
(M)
cells Cooling
CHO rate
(DC.hin)
2.4
2.4 to 0.164
1.7
25
20.5 16 6.5 4.5 14.5
0.08 30.5 16 17.5 7.5 21
0.91 10.5 40.5 15.5 5.5 9
0.29 23 42.5 27 15.5 14.5
b 200
0.03 57 31 41.5 41 35
NsCl
mm.
(M)
cells Cooling
rate
(T/minP
2.4
2.4 to 0.m
10
25
13.5 36.5 30 4 7.5
0.11 24 36.5 33 13.5 25.5
0.95 11.5 43.5 5 4 5
0.91 9.5 42 16 10 9
200
0.53 31 40 24 21.5 20.5
a Exposed to 2.4 M NaCl and then returned to 0.16 lf NaCl. 1 Cooling rate data hasbeenpreviouslyprablished (8).
of radiochemical release in hypertonic and shock than CHO cells, The data in this freeze-thaw experiments (Table I) can paper suggest that they are able to shrink only provide circumstantial evidence, par- to the same degree as CHO cells but are ticularly for fast-frozen cells where, if unable to withstand increases in osmohypertonic damage occurs, this must be Iarity at their minimum volume and theremanifested during thawing ( 5). Rubidium fore lose membrane integrity at relatively release from MRC-5 ceIIs is considerably low NaCl molarities (0.4-0.64 M NaCl). lower in the hypertonic experiments and is During freezing MRC-5 cells show a far not fohowed by uridine release as in freez- greater release of membrane labels than ing experiments (S), CHO ceils show a far CHO cells which would allow early inhigher rubidium release, almost equivaIent tracelluIar ice formation and explain the to that released in freeze-thaw experiments extremely low RI of 0.03. CHO cells would and this is followed by uridine, as in freez- on the evidence of this hypertonic investiing experiments. The low level of fucose gation, be able to withstand molarities release indicates that only minimal mem- 1ikeIy to be met during a freezing experibrane destruction occurs, which also cor- ment and therefore be ,better able to rerelates with the freeze-thaw data in that duce intracellular ice formation to a minifucose is only released after exposure to mum. In fact the data indicate that they temperatures below -40°C (i.e. after are more likely to be damaged during reshrinkage is complete) except during con- warming (dilution). The fact that all ditions in which intracellular ice formation chromate release is lethal during both hyoccurs (8). A limited amount of ceIlular pertonic stress (with DMSO present) and materia1 is lost, as indicated by chromate after exposure to temperatures between 0 and amino acid reIease, but this was about and -40°C indicate the stress may be the level that occurs during freeze-thaw similar for both conditions, experiments with SIOW cooling rates DMSO was included in all these ex(TabIe 1). periments for comparative purposes with Thus freeze-thaw stress causes a higher the freeze-thaw data (Table 1). The rerelease of radiochemicals than hypertonic stress from MRC-5 cells indicating a much sults sum’marized in Fig. 5 include some hypertonic experiments carried out in the greater susceptibility to cold ( thermal)
528
J. B. GRIFFITHS
absence of DMSO and cIearly demonstrate a protective effect by DMSO, partiruIarly for CHO cells which show a very high chromate reIease in the absence of DMSO during their return to isotonic EBSS. As there was not such a difference in RI values with and without DMSO after the cells had been subjected to high NaCl concentrations the data can be extrapoIated with some confidence to conclude that DMSO prevents disruptive swelling and disintegration of cells, and therefore aids the cells to maintain their integrity whilst they are being forced to swell under high osmotic pressure and, more especiaIIy, when they are being returned to isotonic conditions. This conclusion is substantiated by the fact that much of the extra chromate release in the absence of DMSO was non-lethal, i.e. the extra release was not correlated to extra cell death but to the disruption of dead cells. The effect of DMSO (10% ) was to delay the release of chromate and loss of RI to slightly higher NaCl molarities. In the presence of 10% DMSO (data in Figs. I-4) considerabIe cell shrinkage occurs and cation leak (measured by rubidium loss) started at Iower NaCl concentrations than 0.75 M (25) or 0,8 M (16) or even higher concentrations (6, 7) quoted for erythrocytes.
used for erythrocytes but not for nucIeated mamamlian cell lines. The findings were that considerable ceII shrinkage occurred, with a minimum size at 0.6 M NaCl, but that this caused no cell injury or death. Injury, measured by cation leakage and release of membrane and cytoplasmic labels occurred whilst the cell was swelling after reaching its minimum volume. MRC-5 cells succumbed at reIatively Iow salt concentrations and became denatured, CHO cells withstood far high saIt concentrations but were then damaged during dihrtion back to isotonic conditions. Comparison of the data obtained from hypertonic stress experiments and freeze-thaw experiments showed many similarities for CHO cells and indicated that the cell membrane could withstand high salt concentrations both at constant and changing temperatures but were prone to injury on dilution back to isotonic conditions. MRC-5 ceIIs were shown to be very prone to cold shock and the results indicated that they probably succumb to damage and death during the hypertonic phase of cooling rather than thawing thus explaining their much lower survival from freeze-thaw experiments than CHO cells. The influence of DMSO in delaying cell damage to higher salt concentrations and lessening disruptive swelling during dilution were aIso demonstrated.
SUMMARY
The effect of subjecting the mammalian cell Iines MRC-5 and CHO to hypertonic salt concentrations (0.16 to 2.4 M) and returning them to isotonic conditions was investigated. Parameters for measuring cell size, viability and release of radiochemical markers were used to determine the relative susceptibilities of the two cell lines to hypertonic stress and the relative effects of increasing and decreasing hypertonicity. The aim of this study was to determine how great a role hypertonic stress plays in freeze-thaw damage of mamalian cells. This type of study has been extensiveIy
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tonic conditions; a model system for investigating sulfoxide.
freezing Cqobiplogy
damage. 3. Ql 131-138
Dimethyl ( 1972 ).
EFFECT
OF HYPERTONIC
STRESS ON MAMMALIAN
5. Farrant, J., and Morris, G. J, Thermal shock and diIution shock as the causes of freezing injury. Cryobiology 10, 134-140 (1973). 6. Farrant, J., and Woolgar, A. E. Human red cells under hypertonic conditions, a model system for investigating freezing damage. I. Sodium Chloride. ClyobioEogy 9, 9-15 (1972). 7. Farrant, J., and Woolgar, A. E. Human red eelIs under hypertonic conditions: a model system for investigating freezing damage. 2. Sucrose. Cryobiology 9, 16-21 ( 1972 1. 8. Griffiths, J, B., and Beldon, I. Assessment of cellular damage during cooling to -196°C using radiochemical markers. Cryobiology 15, 391-402 ( 1978). 9. Harris, L. W., and Grifliths, J. B. An assessment of methods used for measuring the recovery of mammalian cells from freezing and thawing. Cryobiology 11,80-84 (1974). 10. Harris, L. W., and Griffiths, J. B. The effect of cooling on celluIar changes during the freeze-thaw cycle. Cqobiology 11, 85-91 (1974). Il. Harris, L. W., and Griffiths, J, B. Relative effects of cooling and warming rates on mammalian cells during the freeze-thaw cycle. Cyobblogy 14, 662469 ( 1977). 12. House, W. Viability of BHK 21 line of hamster Bbroblasts after storage at subzero temperatures. J. Path. Bact. 92, 325-329 (1966). 13. Karow, A. M., Jr., and Webb, W. R. Tissue Freezing. A theory for injury and survival. Cryobiology 2, 99-108 ( I965 ). 14. Litvan, G. G. Mechanisms of cryoinjury in biological systems. Cryobiology 9, 182-191 (1972). 15. Lovelock, J. E. The mechanism of the protective action of glycerol against haemol-
CELLS
529
ysis by freezing and thawing. Biochim. Biophys. Actu 11, 2%36 ( 1953). IS. Lovelock, J, E. Haemolysis by thermal shock. Mt. J. Haemutd. 1, 117-129 (1955). 17. Mazur, P. Cryobiology: The freezing of biological systems. Science 108, 939-949 (1970). 18. Meisler, A. I. Studies on contact inhibition of growth in the mouse fibroblast, 3T3. I. Changes in cell size and composition during ‘unrestricted’ growth. J. Ceil Sci. 12, 847-859
(1973).
19. Meryman, H. T. Modified model for the mechanism of freezing injury in erythrocytes. Nature (London) 218, 333-336 (1968). 20. Meryman, H. T. The exceeding of a minimum tolerable celI volume in hypertonic suspension as a cause of freezing injury. In ‘The Frozen Cell” (G. E. W. Wolstenholme and M. O’Connor, Eds.), pp. 51-64. Churchill, London, 1970. 21. Meryman, H. T. Osmotic stress as a mechanism of freezing injury. Cyobiology 8, 489-500
( 1971).
22. Morris, G. J. Lipid loss and haemolysis by therma shock: lack of correlation. Cryobiology 12, 192-201 ( 1975). 23. Morris, G. J., and Farrant, J. Effects of cooling rate or thermal shock haemolysis. CryobioZogy 10, 119-125 (1973). 24. Walter, C. A., Knight, S. C., and Farrant, J. Ultrastructural appearances of freeze-substituted lymphocytes frozen by interrupting rapid cooling with a period of -26°C. Cryobidogy 12, 103-109 (1975). 25. Woolgar, A. E., and Morris, G. J. Some combined effects of hypertonic solutions and changes in temperature on post hypertonic hemolysis of human red blood cells. Cfy~biobgy 10, 82-86 (1973).
15, 517-529
Effect
( 1978
)
of Hypertonic Stress on Mammalion and Its Relevance to Freeze-Thaw
Cell Injury
Lines
J. B. GRIFFITHS
Microbiological
Research
Establishmmt,
The cooling rate giving the best cell survival from a freeze-thaw cycle has been described as a compromise between the injurious ‘effects of physical and chemical changes to the medium brought about by slow cooling and the effect of intracellular ice at faster than optimum cooling rates (17). The nature of these solution effects and in particular hypertonic stress injury have been extensively investigated (14, 15, 19, 21, 23, 25). ZCells dehydrate and shrink in response to hypertonic conditions (4, 6, 7, 19) but whether cell damage is a consequence of high salt concentration and denaturation (15), the loss of ce1I water and< reduction in ceII volume ( 14) or to the mechanical effects of resisting further cell shrinkage (19) is uncertain. The effect of hypertonic@ on erythrocytes has been exhaustively studied both with and without the additional stress of low temperatures (thermal shock) (4, 6, 7, 21, 22, 25). However, much less is known about the effect of hypertonic conditions on mammalian cell Iines and how far the thermal shock data for erythrocytes can be extrapolated to freeze-thaw damage in this type of cell, In order to try and define freeze-thaw damage in mammalian cells the effects of cooling (9, 10) and thawing rates (11 j have been studied at intermediate temReceived 3,
September
29,
1977;
accepted
March
1978.
Potion,
Salisbu y, W&shire,
U.K.
peratures between 0” and -196°C. The surviva1 of cells has been measured using a ceI1 counting procedure (recovery index) together with a radiochromate assay to measure cell injury. These studies have led to the recognition of lethal and nonIethal damage to cells at various stages of the freeze-thaw cycle (II ). To gain further insight into the cellular processes affected by cooling the radiochromate method was extended to the use of other radiochemicals, many of which have far more specific attachment sites on the cell than radiochromate (8). This study raised the question of exactIy what role did dehydration and osmotic stress play in cel1 injury at both slow and fast cooling rates. This present investigation describes the effect of hypertonic stress alone on two mammalian celI lines in order to clarify the roles of thermal and dilution shock (5, 16) during freeze-thaw cycles and to extend the pubIished data on erythrocytes to more complex nucIeated fibrobIast and epithelial cells. MATERIALS
AND
METHODS
Cell Lines and Culture Procedure The human diploid cell line (MRC-5) and the Chinese hamster ovary cell line, clone A (CHO) were used. Culture procedures have been described previously (9, 10).
517
All
Copyright @ 1978 rights of reproduction
by Academic Press, Inc. in zny form reserved.
518
J, B. GRIFFITHS
Hypertonic Experiments in Sodium Chloride
tion was identica1 whether additions were made to the buffer or not.
Conffuent cultures of cells were harvested using 0.1% pronase (MRC-5 cells) or 0.25% trypsin (CHO cells) and suspended in Eagle’s basal medium (BME) with 10% calf serum at 6 X lO*/ml. The cell suspensions were gently agitated for 30 min at 37°C to allow cell repair to occur foIIowing possible trypsin or pronase damage. Some cell loss occurred during this period but this stabilized and there was found to be no advantage in extending this period beyond 30 min. The cells were then centrifuged (2OOg for 2 min) and resuspended in Earle’s Basal Salt SoIution (EBSS) at 6 x 10B/ml. One miIIiIiter aliquots were added to 16 centrifuge tubes and the cell suspensionswere then diluted to a final concentration of 2 m1 (3 X 108/ ml) with a range of sodium chloride solutions (in a buffered salt solution: KCl, 0.2 g/I; Na2HP04, 1,15g/I; KHZP04, 0.2g/l; MgC126H20, O,lg/l). The cells were diluted slowly (2 min/concentration step) and stepwise from 0.18 to 0.24, 0.40, 0.64, 0.80, 1.20, 1.60, to 2.40 M NaCl. After dilution to 2 ml there were duplicate samples at each of these eight NaCI concentrations, A slight drop in pH occurred with this procedure from an initial value of 7.3 at 0.16 M to pH 7 at 2.4 M NaCl. These samples were then divided into two, one part for hypertonic measurements, the other was sIowly diluted back to isotonic (0.16 M NaCl) concentration with various mixtures of EBSS and diIute NaCl solutions. Sampies were taken for the various parameters under study by centrifugation of the cells out of the saline solutions (15Og/3 min). This occurred after the cells had been exposed to hypertonic conditions for 20 min at a constant temperature throughout of 20°C. Dimethylsulphoxide (final concentration 10% ) was present at all times unless stated otherwise in the text. The addition of DMSO was made when diluting buffer concentrates so that salt concentra-
Recovery Index (RI) A fulI description and evaIuation of this method has been pubIished (9) Recovery Index is a measure of the cells surviving a certain treatment and retaining the abiIity to survive 18 to 24 hr in culture and to divide, Identical aliquots of hypertonically stressed cells and contro1 ceIIs from the same initial population were incubated in growth medium (BME for CHO cells, MEM for MRC-5 ceIIs plus 10% calf serum) for 18 hr (CHO) or 24 hr (MRC-5), The cells were then harvested (by pronase or trypsin) and counted in a Coulter Counter. The number of cells grown from the experimental sampIes were expressed as a proportion of the numbers grown from the control cell suspension. Typan
Blue Staining
Cell counts were made in a haemocytometer after dilution in 0.1% trypan blue (in EBSS ) . This method is wideIy used for assessmentof viability on the basis that it is only taken up by highly damaged and leaky cells (2). However, in this investigation where celIs are subjected to high osmotic pressures it is used to indicate increased cell permeability rather than viability. Release of Radiochemicals Cell cultures were incubated with various radiochemicaIs, for up to 18 hr at 37”C, and then harvested and prepared for experiments as described above. During the experiment the release of radiochemicals from the cells into the various saline buffers was measured by adding 10 ml Brays Scintillation fluid to 1 ml of cell-free saline buffer and counting in a Nuclear Enterprise Counter. Gamma counting (chromate and rubidium) of the 1 ml samples was done directIy in a Nuclear Enterprise counter with the gamma head attach-
EFFECT
OF HYPERTONIC
STRESS ON MAMMALIAN
merit, The radioactivity remaining in the cells was also measured after dissolving the cells in 0.2 N NaOH. The values for release of radiochemicals from ceIIs were calculated as a percentage of the tota cpm (cells plus saline buffer). Results in this paper refer to the difference in percentage reIease between control samples (in isotonic buffer ) and the samples from cells subjected to hypertonic concentrations of NaCl. Radiochemicals were obtained from the Radiochemical Centre ( Amersham) and were used as follows: amino acid “C colIection (CFB 103; C7, 8, 9, 10, 15), 0.1 &i/ml for 3 hr; sodium chromate ( Wr ) (CJS. lP), 0.5 &i/ml for 2 hr (9, 10, 11); L-[1-3H]fucose (TRA 366), 0.25 ,&i/ml growth medium for 18 hr; rubidium chloride (eGRb) (RGS IP), 150 pCi/ml of growth medium with 3 x lo8 cells/ml in suspension for 1 hr; [5-H3]uridine (TRA 178), 0.25 pCi/ml growth med,ium 5 hr. The use of these radiochemicaIs to indicate freeze-thaw injury has been described (8). To summarize; amino acid release marks cell destruction with large losses of structural protein; chromate release is from denatured cell protein and can be from both lethal and non-lethal damage (11); fucose labels the carbohydrate component of membrane gIycoproteins; rubidium release is an indicator of cation permeabihty and marks the completion of cehular shrinkage; uridine is an RNA precursor and is thus mainIy cytoplasmic and was found to be released immediately after cell death. Cell Water Volume (CWV) The [‘“Cl thiourea : [3H]mannitol ratio method was used to measure the volume of intracehular fluid (18). The cell suspensions were puIsed with 4 ,&i/m1 of thiourea and 2 &i/m1 of mannito1 for 20 min at 37°C. The cells and suspending saline were separated by centrifugation (15Og/2 min) and the radioactivity of the cells and medium measured in Brays solu-
CELLS
519
tion using a dual label technique in a Liquid Scintillation Counter (Intertechnique SL-30). The medium vohrme was measured accurately by pipette so that the radioactive counts in the medium couId be used to convert cell counts (cpm) into volume. The intracellular voIume was taken as the difference between the apparent thiourea space and the apparent mannitol space ( 18). Cell Diameter Memurements Cells were photographed using a Zeiss photomicroscope ( x500) and cell diameter measurements made from the photographs. The longest diameter, and the diameter at right angles to this was measured in each ceI1 and averaged. Sample size was a minimum of 30 cells. This photographic method enabIed a11measurements to be made within a very short time of each other whilst the cell was under stress in the various hypertonic buffers or immediately after return to isotonicity. Lethal and Sublethal Chromate Release The relationship between RI and tota chromate reIease (PCR) allows the relative amounts of chromate released due to IethaI and sublethal (repairable) damage to be assessed using probabihty theory mathematics in the following equation: In (RI) = K, - Kz (PCR) K1 is a measure of sublethal damage and Kz a measure of the probability of death and PCR-K1 represents the chromate released as a direct consequence of cell death. The derivation and application of this equation has been published (11). RmULTS
Efect of Increasing Hyptertonicity on MRC-5 Cells (Fig. I ) CelI size and release of various radiochemicals were measured as MRC-5 cells were subjected to increasing concentrations of sodium chloride in EarIe’s Buffered Salts
520
J. B. GRIFFITHS
Solution (EBSS) ranging from isotonic (0.18 M NaCl) to 2.40 M NaCI. The results, summarised in Fig. 1, show that MRC-5 cells rapidly shrink and Iose water down to a minimum value at 0.64 M NaCl. During this period there was virtualIy no release of radiochemicals or loss of cells. Increasing the NaCl molarity above 0.64 M caused a gradual increase in celI dmiameter and cell water vohnne, some release of rubidium, chromate and amino acids but very little release of fucose or uridine. There was approximately 15% loss of ce1I.s and this occurred between 0.8 and 1.6 M NaCl and was accompanied by the loss of radiochemicals. Cell viabiIity, which fell from 95% at 0.8 to 14% at 1.6 M NaCI, was mea-
NoCt
sured by trypan bIue staining and was, therefore, a better measure of cell permeability changes than actual ability of the cell to recover and resume growth (2, 9, 12), The morphological appearance of the cells was normal to 0.84 M NaCl but: at higher concentrations the cells lost their regular spherical shape and some apparently dead celIs appeared. There was very little evidence of complete cell destruction. The cells that were assumed to be dead were enIarged and “ghost-like” with a definite form, with and without very large vesicles, and often with denatured cell material adhering to one part of the cell. This morphoIogica1 appearance perhaps expIains the low release of fucose (eel1
CONCENTRATION
IMI
FIG. 1. Effect of subjecting MRC-5 cells to hypertonic concentrations of NaCl. V-V, cell water volume; T-T, cell diameter (V, viable cells; D, dead cells); X-X, total celI count; I--I, percentage viability. Percentage release of chromate (O-O), rubidium ( l -e ), uridine ( O--O), amino acids ( A-A ) and fucose ( A--A ).
EFFECT
OF HYPERTONIC
STRESS QN MAMMALIAN
CELLS
521
0.6 NaCt CONCENTRATON
ItAl
FIG. 2. Effect of returning MRC-5 cells to isotonic NaCl from various hypertonic concentrations of NaCl. V-V, cell water volume; V-7, cell diameter; X-X, total cell count; W-W, percentage viability; @- -8 recovery index. Percentage release of chromate (O-O), rubidium ( l -e ), uridine ( O-C! ), amino acids ( A-A ), and fucose ( A-A ).
shape maintained) and uridine (stiI1 within the vesicles) and relatively higher release of chromate and amino acids from the denaturation of the ceI1. Efiect of Returning MRC-5 Cells from Hypertonic to Isotonic Condith.s (Fig. 2) The cells were returned slowly and stepwise from their final hypertonic concentration to isotonic EBSS. This involved an S-fold increase in volume of all cell suspensions which caused some damage even to the cells which were kept in isotonic EBSS throughout the experiment. This damage was indicated by the initial release of amino acids, uridine and chromate
in Fig. 2 as well as a slight decrease in cell water voIume and diameter. AIthough ceI1 size increased on return to isotonic conditions it was stiI1 smaller than norma in the ceIIs subjected to NaCl concentrations up to 0.4 M. Above this salt concentration the cells returned to near their original volume and this was accompanied by additional release of chromate, amino acids and fucose. The morphological appearance of the cells remained good after returning from 0.4 M NaCl but at higher molarities they began to deteriorate so that after exposure to 0.8 M NaCl aII cells were adversely affected, showing heavy blebbing and loss of cell material. Cells exposed to 1.6 M N&l or above were extremeIy dam-
522
J, B. GRIFFITBS
aged with extensive granulation, vesicIes and protrusions but mainly intact. The cell loss from 2.4 M NaCl was less than 40% and this occurred mainly after returning from NaCl molarities of 0.8 to 1.2. Recovery index (RI) and cell viability (by trypan blue staining) declined at a rapid but steady rate to very low values after exposure to 2.4 M NaCI. The additional release of radiochemicals that did occur due to returning the cells to isotonic conditions occurred after exposure to concentrations above 0.8 M NaCl, the most significant being amino acids, chromate and uridine. There was very little release of
NoC4
FIG.
3. Effect
of subjecting
water volume; V-7, Percentage ( A-A
CHO
release of chromate ( O-O fucose ( A-A ).
) and
CONCENTRATION
cells
cell diameter;
fucose and this correlated with the relatively small cell loss. Analysis of the RI and chromate release data using the probability equation, In (RI) =K1 -I& (PCR), where K1 is a measure of the non-lethal damage to cells (11) showed that in this experiment there was virtually no non-lethal chromate release (K, = 1.0). The other instance where all the chromate reIease was lethal was during slow cooling of cells after exposure to temperatures between 0 and -4O”C, i.e. the period of thermal shock, dehydration and shrinkage in the cooling cycle.
X-X,
to hypertonic
total
), rubidium
IMI
concentrations of cell count; H-m, ( e-0 ), uridine
NaCI. V-V, cell percentage viability. ( 0-U ), amino acids
EFFIXT
OF HYPERTONIC
STRESS ON MAMMALIAN
CELLS
523
FIG. 4. Effect of returning CHO cells to isotonic NaCl from various hypertonic concentrations of NaCl. V-T/, cell water volume; V-7, cell diameter; X-X, total celI count; m-m, percentage viability; m--m, recovery index. Percentagerelease of chromate (O-O), rubidium ( 0 -@ ), uridine (O--O), amino acids ( A-A ) and fucose ( A-A ).
Effect of Increasing Hypertonicity on CHO Cells (Fig. 3) This experiment was carried out in an identical manner to that described for MRC-5 celIs (in Fig. 1) for comparative purposes as CHO and MRC-5 cells have been shown to have different behavioural patterns during freeze-thaw cycIes (S-11). However, there were many similarities in their behavior in hypertonic NaCI. The degree of shrinkage to a minimum value at 0.64 M NaCI was identical but CHO cells remained at this reduced level whilst the NaCI moIarity was increased to 1.2 or 1.6 before abruptly rising to normal (contro1) values. The only other difference between the two cell lines was that CHO cells re-
leased a significantly higher amount of uridine and a much higher quantity of rubidium than MRC-5 cells, The morphological appearance of the celIs rapidly changed from their normal smooth spherical shape to become smaller and crinkly (accompanied by a small amount of fucose reIease) at 0.4 M NaCl. The cells then became very “angular” (at 0.8 M NaCl) and this was followed by dense granulation and denaturation (at 1.2 M NaCl) and finally swollen “ghost-like” cells and signs of cell disruption (at 1.2 to 2.4 M NaCI). Effect of Returning CHO Cells from Hypertonic to Isotonic Conditions (Fig. 4) CHO cells remained smaller than control cells to a similar degree to MRC-5
J. B. GRIFFlTHS
524
index and percentage chromium release of concentrations of NaCl before and after returning to isotonic NaCl. Recovery index; W--II DMSO-free; O-0, 10% DMSO. Percentage chromium release: at hypertonic NaCl concentrations, l -a (DMSO-free) and O-O (10% DMSO); after returning to isotonic NaCl from various hypertonic concentrations -A--A (DMSO-free) and A--A (10% DMSO). FIG.
5. Effect of 10% DMSO
on recovery
MX-5 and CHO cells subjectedto hypertonic
cells except that they did not return to norma size until after being subjected to 1.2 M NaCI (compared to 0.64 M for MRC-5 cells), Recovery Index (RI) began to decline significantIy after exposure to molarities greater than 0.8 M NaCl. Return to isotonicity caused large additional release of amino acids, fucose and chromate, but not of rubidium or uridine. A comparison between Figs. 2 and 4 show that the release of rubidium and uridine was very much higher in CHO ceIIs but fucose and amino acids were only slightly higher and chromate reIease was less than for MRC-5 ceIIs. Chromate release (PCR = 17) was mainly lethal, although there was some
non-lethal release (Ki = 2.5). Morphological changes occurred after exposure to molarities above 0.64 when the 1~11sbecame larger and more dense with extensive vesicle formation and some cell Ioss (about 30%). Effect of DMSU on Cells Subjected to Hypertonic NaCl Concentrations (Fig. 5) Recovery index and percentage chromate reIease of MRC-5 and CHO cells subjected to increasing concentrations of NaCl, and after dilution back to isotonic EBSS in the presence and absence of 10% DMSO are shown in Fig. 5. Recovery index was always lower at each hypertonic NaCl con-
EFFECT
OF HYPERTONIC
STRESS ON MAMMALIAN
centration in the absence of DMSO but only became really significantly different after exposure to molarities of 1.2 and above. The addition of DMSO had the effect of delaying chromate release from 0.4 to 0.64 M NaCl at hypertonic salt concentrations but above 16 M NaCl there was very Iittle difference in chromate release with and without DMSO. After the cells had been brought back to isotonic concentration DMSO only had its main protective effect on MRC-5 cells exposed to above 1.6 M NaCl. CHO cells were very significantly protected by DMSO during their return from all hypertonic concentrations up to 2.4 M NaCl. The results in Fig. 5 definitely show DMSO to have a protective effect on cells subjected to hypertonic stress. An analysis of chromate release in the absence of DMSO (using the equation In RI = Kr - KZ PCR) showed a change in the relationship between RI and PCR as the hypertonicity increased, MRC-5 cells subjected to 0.16 to 0.40 M NaCl showed a similar K, to that in the presence of DMSO (K, increased from 1.0 to 1.7) but after exposure to between 0.64 and 2.4 M NaCl the non-IethaI chromate release became much greater (Kr = 9.0). SimilarIy CHO celIs after exposure to 0.16 to 0.8 M NaCI had a K1 of 7.5 (compared to 2.5 with DMSO) but this increased to 17.5 after exposure to 1.2 to 2.4 M NaCl. DISCUSSION
There is extensive information about the effect of osmotic stress on erythrocytes (4, 6, 7, 15, 16, 19, 21, 22, 25) but very few data pertaining to this aspect of the cooling cycIe for mammalian cell lines. This present study was carried out to investigate the responses of such cell lines during hypertonic stress at a constant temperature and to compare this with similar data obtained during freeze-thaw experiments (8). The first conclusion to be made from the data in this nauer is that cell shrinkage
CELLS
525
(dehydration) occurs extensiveIy as the molarity of NaCI increases from isotonic (0.16 M) to 0.64 M. The minimum cell size is reached at 0.64 M and both MRC-5 and CHO cells show an identical degree of size change (46-45s ) . This was not predictable as the two cell lines are quite dissimiIar both in morphology and in growth potential. The second conclusion is that shrinkage is not damaging to either cell line as there was no radiochemical reIease or loss of ceIIs during this period. This confirms various conclusions drawn from studies with erythrocytes on shrinkage at constant temperatures (3, 5, 23). Radiochemical release occurs after cells reach their minimum size and are subjected to further increases in hypertonicity, InitialIy rubidium is released followed by chromate, amino acids and, in CHO cells only, uridine and this is accompanied by a gradual increase in ceI1 size from the minimum value at 0.64 M NaCl. Rubidium is a marker of the intracellular space occupied by potassium (1) and its release marks the Ioss of membrane integrity and permeability to cations (3). This increase in membrane permeabibty is also indicated by the increase in trypan blue staining and subsequent sweIling of cells. Thus damage occurs when the cells can shrink no further resulting in some Ioss of viability and ceII destruction; perhaps due to salt denaturation of protein (13) or disruptive sweIIing of cells, Some discrimination between obviousIy dead cells and probable viabIe celIs could be made by microscopical examination and this showed that the dead cells were significantly larger (Fig. 1). CHO cells retain their minimum voIume at greater NaCl molarities than MRC-5 cells and lose more rubidium and uridine demonstrating that they have a higher degree of membrane leakiness and are able to withstand a greater Ioss of cytopIasmic constituents before crossing the threshold for recovery. When this happens cell swelling occurs, vesicles on the cell surface are
526
J, B. GRIFFITHS
FIG. 6. A comparison of radiochemical release by CHO and MRC-5 cells subjected to various hypertonic concentrations of KaC1 and after their return to isotonic NaCI. A, amino acids; C, chromate; F, fucose; R, rubidium; U, uridine. ZZ release at stated NaCl concentrations; 0, extra release on return to 0.16 M NaCI.
formed but there is no widespread lysis at hypertonic NaCl concentrations. After subjecting the cells to hypertonic NaCl they were then brought back into isotonic EBSS, a process analogous to the warming phase of freeze-thaw cycles, Cells did not return to their control (normal) size until after exposure to 0.64 (MRC-5) or 1.2 M NaCl (CHO). The recovery index became significantly reduced only after the cells had’ begun to swell back to control size, and this was followed by considerabIe radiochemical release. This is summarized in Fig. 8 so that the extra reIease caused by the return to isotonicity can be more easily seen. There was no further release of rubidium as could be predicted if this is a marker of shrinkage, but uridine release occurred in MRC-5 celIs at this stage rather than during increasing hypertonicity as was the case with CHO cells. The results are indicative of MRC-5 celIs succumbing to hypertonic stress at a low NaCl molarity (0.64 or below) and becoming denatured (presumably by saIt damage). Thus dilution back to isotonic conditions
causes litle extra damage, as shown by the Iow release of chromate, amino acids and fucose (Fig. 6). CHO ceIIs, on the other hand, are able to withstand molarities of at least 1.2 M NaCI, possbiIy due to the protective leakage of cytoplasmic constituents (e.g., rubidium). However, they are prone to dilution damage resulting in n higher amino acid, chromate and fucose release than MRC-5 cells. Microscopical examination reveals more vesicle formation, disruptive swelling and loss of cell material from CHO than from MRC-5 ~11s. It should be noted that CHO cells with an RI of 0.11 after exposure to 2.4 M NaCl are far more stressed than during a freezethaw cycle with an RI of 0.53 after cooling at 200”C/min. This RI occurs after exposure to 1.2 M NaCl which, in the presence of DMSO, is probabIy greater than the NaCl concentration at -40°C during a freeze-thaw cycle (21). A stated aim of this investigation was to determine if freeze-thaw data on radiochemical release ( 8) could be interpreted in terms of hypertonic stress.A comparison
EFFECT
OF HYPERTONIC
STRESS ON MAMMALIAN TABLE
CELLS
527
1
Comparative Effects of Freeze-Thaw Cycles Using Various Cooling Rates to - 196°C Exposure to 2.4 M NaCl and Return to Isotonic NaCl (0.16 M) After Exposure to 2.4 M NaCl on the Recovery Index and Percentage Release of Radiochemicals from CHO and MRC-5 Cells MRC-5 NaCl
RI Radiochromate Rubidium Uridine Fucose Amino acids
eonc.
(M)
cells Cooling
CHO rate
(DC.hin)
2.4
2.4 to 0.164
1.7
25
20.5 16 6.5 4.5 14.5
0.08 30.5 16 17.5 7.5 21
0.91 10.5 40.5 15.5 5.5 9
0.29 23 42.5 27 15.5 14.5
b 200
0.03 57 31 41.5 41 35
NsCl
mm.
(M)
cells Cooling
rate
(T/minP
2.4
2.4 to 0.m
10
25
13.5 36.5 30 4 7.5
0.11 24 36.5 33 13.5 25.5
0.95 11.5 43.5 5 4 5
0.91 9.5 42 16 10 9
200
0.53 31 40 24 21.5 20.5
a Exposed to 2.4 M NaCl and then returned to 0.16 lf NaCl. 1 Cooling rate data hasbeenpreviouslyprablished (8).
of radiochemical release in hypertonic and shock than CHO cells, The data in this freeze-thaw experiments (Table I) can paper suggest that they are able to shrink only provide circumstantial evidence, par- to the same degree as CHO cells but are ticularly for fast-frozen cells where, if unable to withstand increases in osmohypertonic damage occurs, this must be Iarity at their minimum volume and theremanifested during thawing ( 5). Rubidium fore lose membrane integrity at relatively release from MRC-5 ceIIs is considerably low NaCl molarities (0.4-0.64 M NaCl). lower in the hypertonic experiments and is During freezing MRC-5 cells show a far not fohowed by uridine release as in freez- greater release of membrane labels than ing experiments (S), CHO ceils show a far CHO cells which would allow early inhigher rubidium release, almost equivaIent tracelluIar ice formation and explain the to that released in freeze-thaw experiments extremely low RI of 0.03. CHO cells would and this is followed by uridine, as in freez- on the evidence of this hypertonic investiing experiments. The low level of fucose gation, be able to withstand molarities release indicates that only minimal mem- 1ikeIy to be met during a freezing experibrane destruction occurs, which also cor- ment and therefore be ,better able to rerelates with the freeze-thaw data in that duce intracellular ice formation to a minifucose is only released after exposure to mum. In fact the data indicate that they temperatures below -40°C (i.e. after are more likely to be damaged during reshrinkage is complete) except during con- warming (dilution). The fact that all ditions in which intracellular ice formation chromate release is lethal during both hyoccurs (8). A limited amount of ceIlular pertonic stress (with DMSO present) and materia1 is lost, as indicated by chromate after exposure to temperatures between 0 and amino acid reIease, but this was about and -40°C indicate the stress may be the level that occurs during freeze-thaw similar for both conditions, experiments with SIOW cooling rates DMSO was included in all these ex(TabIe 1). periments for comparative purposes with Thus freeze-thaw stress causes a higher the freeze-thaw data (Table 1). The rerelease of radiochemicals than hypertonic stress from MRC-5 cells indicating a much sults sum’marized in Fig. 5 include some hypertonic experiments carried out in the greater susceptibility to cold ( thermal)
528
J. B. GRIFFITHS
absence of DMSO and cIearly demonstrate a protective effect by DMSO, partiruIarly for CHO cells which show a very high chromate reIease in the absence of DMSO during their return to isotonic EBSS. As there was not such a difference in RI values with and without DMSO after the cells had been subjected to high NaCl concentrations the data can be extrapoIated with some confidence to conclude that DMSO prevents disruptive swelling and disintegration of cells, and therefore aids the cells to maintain their integrity whilst they are being forced to swell under high osmotic pressure and, more especiaIIy, when they are being returned to isotonic conditions. This conclusion is substantiated by the fact that much of the extra chromate release in the absence of DMSO was non-lethal, i.e. the extra release was not correlated to extra cell death but to the disruption of dead cells. The effect of DMSO (10% ) was to delay the release of chromate and loss of RI to slightly higher NaCl molarities. In the presence of 10% DMSO (data in Figs. I-4) considerabIe cell shrinkage occurs and cation leak (measured by rubidium loss) started at Iower NaCl concentrations than 0.75 M (25) or 0,8 M (16) or even higher concentrations (6, 7) quoted for erythrocytes.
used for erythrocytes but not for nucIeated mamamlian cell lines. The findings were that considerable ceII shrinkage occurred, with a minimum size at 0.6 M NaCl, but that this caused no cell injury or death. Injury, measured by cation leakage and release of membrane and cytoplasmic labels occurred whilst the cell was swelling after reaching its minimum volume. MRC-5 cells succumbed at reIatively Iow salt concentrations and became denatured, CHO cells withstood far high saIt concentrations but were then damaged during dihrtion back to isotonic conditions. Comparison of the data obtained from hypertonic stress experiments and freeze-thaw experiments showed many similarities for CHO cells and indicated that the cell membrane could withstand high salt concentrations both at constant and changing temperatures but were prone to injury on dilution back to isotonic conditions. MRC-5 ceIIs were shown to be very prone to cold shock and the results indicated that they probably succumb to damage and death during the hypertonic phase of cooling rather than thawing thus explaining their much lower survival from freeze-thaw experiments than CHO cells. The influence of DMSO in delaying cell damage to higher salt concentrations and lessening disruptive swelling during dilution were aIso demonstrated.
SUMMARY
The effect of subjecting the mammalian cell Iines MRC-5 and CHO to hypertonic salt concentrations (0.16 to 2.4 M) and returning them to isotonic conditions was investigated. Parameters for measuring cell size, viability and release of radiochemical markers were used to determine the relative susceptibilities of the two cell lines to hypertonic stress and the relative effects of increasing and decreasing hypertonicity. The aim of this study was to determine how great a role hypertonic stress plays in freeze-thaw damage of mamalian cells. This type of study has been extensiveIy
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