A two-factor hypothesis of freezing injury

Experimental Cell Research 71(1972) 345-355

A TWO-FACTOR HYPOTHESIS OF FREEZING INJURY Evidence from Chinese Hamster Tissue-culture Cells P. MAZUR, S. P. LEIBO and E. H. Y. CHU Biology Division, Oak Ridge National Laboratory, Oak Ridge, Term. 37830, USA

SUMMARY When Chinese hamster tissue-culture cells are frozen in a variety of suspending media, the percentage of cells surviving is maximal at optimum cooling rates, rates that are 2-4 orders of magnitude lower than those used to freeze cells for subsequent processing by the electron microscopy techniques of freeze-cleaving and freeze-substitution. The existence of such optima suggests that at least two factors dependent on cooling rate interact to determine the ultimate survival of a frozen-thawed cell. Other data are consistent with the view that the causes of injury in rapidly and slowly frozen cells are different. First, cells frozen rapidly in 0.4 M solutions of sucrose, glycerol, and dimethyl sulfoxide, or in 0.004 M polyvinylpyrrolidone, are inactivated to a much greater extent by slow warming than are cells frozen slowly in those solutions; that is, cells frozen at rates greater than the optimum are considerably more sensitive to slow warming. Second, the inactivation rate of cells frozen rapidly in glycerol is greater at -40°C than that of cells frozen slowly. Third, the temperatures at which cells are killed as they are slowly frozen are very different from those observed during the slow warming of rapidly frozen cells. The precise nature of the two factors remains uncertain, but indirect evidence suggests that cells cooled slower than optimum are killed by alterations in the properties of the extracellular and intracellular solution induced by ice formation (e.g., high solute concentrations), and that cells cooled faster than optimum are killed by the formation of intracellular ice and its subsequent recrystallization during warming. Such intracellular recrystallization may be a potentially serious source of artifacts in frozen material processed for electron microscopy at temperatures above -6O”C, and perhaps even above - 100°C.

The biological and physical responsesof cells to freezing are responses to the combined effects of lowered temperature, loss of cell water, and changes in the state of water. In these responses, cell membranes play a vital role: their permeability to water strongly influences the physical events that occur intracellularly, and they themselves appear to be the chief site of injury [l]. Because freezing is a perturbant of water and membranes, an understanding of the nature and causes of the resultant injury can possibly contribute to an understanding of the role 23-721804

of these components in the cell at normal temperatures. Freezing is also used to preserve the viability of cells and their constituents, and it is used to stabilize cytological fine structure in electron microscopy. The techniques for the preservation of viability, largely derived empirically, have been successful for many cell types, but by no means all [l]. The techniques for the stabilization of structure (chiefly freeze substitution and freeze-cleaving) have also been successful, in that they produce high quality electron-microscopic images that in Exptl Cell Res 71

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P. Ma.270 et al.

general parallel those observed with more conventional techniques [2]. However, it is difficult to prove that the observed cytological structures remain unperturbed by the freezing itself [3, 4, 4a]. Artifacts introduced by freezing may become especially serious when techniques that produce resolving powers approaching 1 A are developed, for these techniques, some of which require cooling the specimensclose to OX, would preserve structures for which there will be no other electron microscopic standards of comparison [5, 61. Our thesis is that the use of low temperature in preserving both viability and ultrastructure would be aided by an understanding of the mechanisms underlying injury and protection. We submit that knowledge of the conditions under which cells do or do not survive freezing will aid in interpreting the microscopic images of frozen cells, and conversely that the techniques of freeze-cleaving and freeze-substitution will be exceedingly useful in developing and testing theories of freezing injury. One theory, based on studies on yeast and red blood cells, is that injury results from the interaction of two categories of factors [l]. One category consists of alterations in the intra- and extracellular solutions produced by ice formation. The detrimental effect of these alterations increases with decreasing cooling rate, presumably becauseslow cooling results in long exposure times. The other factor is the formation of intracellular ice and its attendant recrystallization during slow warming. Intracellular freezing becomes increasingly likely when cooling is rapid, and the recrystallization of intracellular ice becomesincreasingly likely when subsequent warming is slow. The question remains whether this twofactor hypothesis holds for cells other than yeast and non-nucleated erythrocytes. Initial evidence is that it may be applicable to mouse marrow stem cells [7] and Chinese hamster Exptl

Cell Res 71

tissue-culture cells. Our initial experiments with the hamster cells [8] showed that their survival after freezing and rapid thawing, like that of yeast and red cells, depended critically on the cooling velocity. The effects of cooling rates between 1” and 600”C/min were examined, and the lowest and highest rates were both found to be injurious. Maximum survival generally occurred at an intermediate rate, the value of which depended on the particular protective solutes present. The existence of an optimum cooling rate suggestedthat survival is determined by at least two classesof factors oppositely dependent on cooling velocity. The chief purpose of our present studies was to determine in a rigorous fashion whether injury in hamster tissue-culture cells is indeed determined by two distinct classes of factors. The conclusion, drawn from both the viability studies in the present paper and from electron microscope studies in the companion paper [9], is affirmative. The data, furthermore, are consistent with the view that the two factors are (1) altered solution properties and (2) intracellular freezing; and thus suggest that the two-factor hypothesis is applicable to nucleated mammalian cells. Direct evidence on this point will hopefully come from freeze-cleaving studies currently in progress. Certain aspects of the findings are also relevant to the use of freezing in the preservation of cell viability and to its use in the electron microscopy techniques of freeze-cleaving and freeze-substitution. MATERIALS AND METHODS Detailed accounts of the procedures have been given by Mazur et al. [8] and Leibo et al. [7].

Preparation of cell suspensions Cells from the established cell line V79-122Dl were grown in Eagle minimum essential medium (MEM) supplemented with 15 % fetal calf serum. After being disaggregated by trypsinization, the cells were washed once by centrifugation and resuspension in sufficient

Freezing injury in hamster cells Hanks balanced salt solution (HBSS) to yield a suspension containing 1 x IO5 cells/ml. Experimental material was prepared by diluting one volume of the suspension with an equal volume of HBSS with phenol red indicator, or with an equal volume of HBSS containing twice the molar concentration of protective additive desired in the final suspension. Serum was omitted from the suspending medium to simplify the interpretation of the results; however, preliminary results indicate that its absence is not noticeablv detrimental to either control cells or frozenthawed iells. The final concentrations of additives used were: 0.4 and 0.5 M glvcerol. 0.4 and 0.5 M dimethyl sulfoxide @MS0);0.4 anh 0.7 M sucrose, and 0.004 M (15 % w/v) polyvinylpyIrolidone (PVP) (avg. mol.wt = 40 000). The pH of the solutions ranged from 6.8 to 7. (The pH of the PVP, however, had to be adjusted from -4 to 7 before use.) Finally, O.l-ml portions were distributed into freezing tubes (Pyrex, 7 mm OD x 90 mm length).

Controls Three controls were maintained: (1) ‘initial’ controlcells plated immediately after trypsinization and washing; the mean plating efficiency (number of colonies per total number of cells plated x 100) in 24 experiments was 104 %; (2) ‘HBSS’ control; (3) ‘additive’ control. The latter two controls consisted of cells that were suspended in HBSS or HBSS plus additive, respectively, and subjected to all manipulations of the experimental samples, except that they were held at 0°C for the N 5-h duration of the experiment instead of being frozen. The mean plating efficiency of HBSS controls (i.e., those held for 5 h) was 74 %. The mean survivals of the additive controls relative to the HBSS controls were: 0.4 M glycerol, 94 %; 0.5 M glycerol, 92 %; 0.4 M DMSO, 101%; 0.5 M DMSO, 110 %; 0.4 M sucrose, 96 %; 0.7 M sucrose, 93 %; 0.004 M PVP, 100%.

Freezing Some 40 to 120 min after being mixed with additive, the cell suspensions were cooled 1.0” to 1.5”C below the freezing point of the medium, seeded with ice to induce crystallization, and 5 min later either cooled to -196°C at 1.6. 8. 16. 77. 360. or 6OO”Clmin. or cooled at N 1.6”Cim& td desired minimum iemp&atures between -2.5” and - 100°C. We will refer to rates higher than lOO”C/min as ‘rapid’ and to rates of less than lO”C/min as ‘slow’. Samples cooled at 1.6”C/min to -196°C actually were cooled at that rate to about -8O’C and then transferred to liquid nitrogen. (Experiments have demonstrated that varying the temperature of transfer to liquid nitrogen from - 70” to - 100°C has no effect on the percentage survival.) In the case of samples cooled to - 65°C or below, the rates were calculated from the time required to cool from - 10” to -65°C. In the case of samples cooled to final temperatures above -65”C, the rates were calculated from - 10°C to the minimum temperature, except when the minimum temperature

347

was - 10°C or above. The methods used to achieve the various rates are described by Leibo et al. [7].

Storage Except for one series of experiments, samples cooled to minimum temperatures of -75°C or above were held at these temperatures for 1 min or less prior to initiating warming. The samoles in the one exceotion (see fig.-4) were geld in ethanol baths at -40, -50, or - 75°C for up to 60 min. Samples cooled to - 196°C were held at that temperature for l-3 h.

Thawing The frozen samples were warmed at rates of about 1, 30, or 1 OOO”C/min (measured between -65” and - 10°C) until they had thawed and warmed to about 5°C. Exact values for individual experiments are given in Results. Warming at N 1‘C/min was carried o;t by placing frozen samples in an ethanol bath precooled to the temperature of the samples and then allowed to warm by contact with room-temperature air. (For samples that had been cooled to - 196”C, the ethanol warming bath was precooled to - 75°C.) Warming at N 3O”C/min was carried out by suspending the frozen samples in room-temperature air, and rapid warming at N 1 OOO”C/minwas achieved by placing the sample tubes in a 35°C water bath for iO-set, ai the end-of which time they had warmed to about 5°C. The thawed samples-were then returned to an ice bath.

Dilution Some 15 to 100 min after thawing, the experimental and control samples were diluted 50-fold in four steps with cold minimal essential medium (MEM) containing 15 % serum. The purpose of carrying out the dilution step-wise was to avoid rapid changes in osmotic pressure. Up to this point in the procedure, all suspensions had been held at 2°C or below (except for a few seconds during thawing).

Survival assay The samples were further diluted with sufficient MEM plus serum at room temperature to make the cell concentrations 200 to 1000 per ml. Then 1 ml from each sample was added to 9 ml of MEM plus serum in each of three 80 mm plastic Petri dishes. After incubation (pH 7.0, 95 % relative humidity, and 37°C) for 7 to 9 days, the colonies were fixed with ethanol, stained with Giemsa solution, and counted. The percentage survival was calculated as the mean ratio of the number of colonies on plates from frozen and thawed samples to the number on the additive control after correcting for the dilution factor. The error bars in the figures represent + one standard deviation of the mean survival, which in most cases was baaed on two or more experiments, each involving duplicate samples plated in triplicate for each dilution. Error bars are omitted from crowded regions of the plots for the sake of clarity. Exptl Cell Res 71

348 P. Mazur et al.

0.7 M SUCROSE T

RESULTS When cells in solutions of HBSS plus protective additive were cooled at various rates to - 196°C and warmed at several rates, the resulting survivals were those shown in fig. 1. The responsesof rapidly warmed cells (upper curve in each set) to cooling rate were similar in the four additives: survivals were low when the cooling rates were low, and they increased with increasing cooling velocity to reach maximum or plateau values at cooling rates ranging from 10” to 3OO”C/min, the exact value depending on the additive. (Cells frozen in HBSS alone also show an optimum cooling rate (350”C/min) for maximum survival, but the maximum is only 25 % [8].) Although rapidly cooled cells exhibited higher viability than slowly cooled cells when warming was rapid, the rapidly cooled cells were considerably more sensitive to slow warming. For example, with DMSO or sucrose as additives (fig. 1a, c), the viability of rapidly cooled cells decreased from about 50% to less than 5 % when the warming velocity was lowered from 1 000” to l”C/min, whereas that of the slowly cooled cells remained unchanged. Similarly, with PVP as Exptl Cell Res 71

Fig. 1. Abscissa: cooling velocity (“C/min); ordinate: % survival.

1020 T/min

Survivalof Chinesehamstercells in variousadditivesaftercoolingto - 196°Cat variousratesandwarming at the ratesshownon the individual curves.Additives:(a) 0.5 M DMSO; (b) 0.5 M glycerol; (c) 0.7 M sucrose; (d) 0.004 M PVP.

the additive (fig. Id), a decreasein warming velocity from 1 lOO”C/min to 29”C/min decimated rapidly cooled cells but did not decreasethe survival of slowly cooled cells. The results in fig. 1 illustrate another feature about the responsesof the cells to freezing, namely, that survival is not correlated with the total time of exposure to subzero temperatures. Cells cooled to - 196°C by both the sequence of rapid cooling-slow warming and the sequence of slow coolingrapid warming spent comparable times between 0” and - 75°C; but the former sequence was considerably more damaging than the latter. Survival

as a function

of temperature

This lack of equivalence with respect to time between 0” and -75°C is also reflected in the lack of equivalence between the effects of temperature in rapidly and slowly cooled cells. If cells cooled faster than optimum and those cooled more slowly than optimum were both killed by the same mechanism, one would expect to see the same relations in both cases between survival and time at various subzero temperatures. That is to say, if we were to subject cells to slow cooling to

Freezing injury in hamster cells 349 various subzero temperatures and then thaw 1I A them rapidly, and find that inactivation oc- 100 .. curs chiefly between, say, -30” and -4X, we would expect that cells subjected to the reciprocal sequence of rapid cooling to -196°C and slow warming to various tem- -GLYCEROL peratures would also be inactivated chiefly between -40 and - 30°C. But this was not the case. As shown in figs 2, 3, the temperatures over which inactivation occurs during the slow cooling of rapidly warmed cells are very different from the inactivation temperatures during the slow warming of rapidly SUCROSE cooled cells. Fig. 2 shows the survival of cells cooled slowly to various temperatures and then immediately warmed rapidly. Fig. 3 shows the relative survivals for the alternative situation, in which cells were first cooled rapidly to -196°C transferred to a -75°C bath, and then warmed slowly to selected Fig. 3. Abscissa: temperature at termination of slow warming (“C); ordinnte: % relative survival. subzero temperatures before being thawed Relative survival of rapidly cooled cells as a function rapidly. (Survival in this latter figure is ex- of the temperature to which they were slowly warmed.

1

I

Cells suspended in HBSS containing 0.4 M concentrations of the indicated media (except 0.004 M PVP) were cooled rapidly to - 196°C at 6OOC/min (-) or 36O”C/min (---), transferred to a dry ice-ethanol bath at -75°C for 1 min, warmed slowly to the indicated temperatures at either 1.6°C/min~ (-; ---) or 4O”C/min* (. . . *), and then thawed rapidly in a 35°C water bath.8 Survivals are normalized so that survival at -75”C=lOO%.

‘i..

0

0.4 M GLYCEROL I I XL-I -20 -40

I -60

1

, -80

+J -100 -196

Fig. 2. Abscissa: minimum temperature (“C); ordinate: % survival. Survival of hamster cells in various additives as a function of the minimum temperatures to which they were slowly cooled. Mean cooling rates between -1O“C and minimum temperatures of -15, -20, - 30, -40, -50, -60, -80, and - 100°C were 2.1, 2.1, 1.8, 1.7, 1.7, 1.7, 1.6, and l.S”C/min, respectively. Warming was rapid (35°C water bath), and was initiated immediately after attainment of the minimum temperature.

pressed in relative terms in order to compare the effect of the various additives on the temperature of inactivation during slow warming. That is, the survival of cells cooled rapidly to -196°C transferred to -75”C, and warmed rapidly to +5”C is taken as 100%. The absolute survivals for this treatment are given in the second column of table 1. As shown in the third column of the l The mean rates from -65°C to -50, -40, - 30, -20, and -10°C were 1.7, 1.7, 1.6, 1.5, and 1.4”C/ min, respectively. * The mean rates from - 65”C, to -40, - 30, and - 10°C were 53, 42, and 28”C/min, respectively. J The approximate warming rates from -65, -50, - 30, and -20°C to - 10°C were 1000,850,650, and 550”C/min, respectively. Exptl Cell Res 71

350 P. Mazur et al.

of hamster cells after rapid cooling and rapid thawing

Table 1. Percentage survivals

cells in DMSO. Furthermore, the temperatures at which 50 % of the cells became inactivated differ widely for cells in glycerol, su% Survivala of cells crose, and DMSO, even though the concentrawarmed rapidly from - 196°C tion of all three additives was 0.4 M. The LT,, ranges from - 15” to - 62°C with To -15” and then Directly slowly cooled cells and from -22” to -49°C Additive to +5”c to +Yc with slowly warmed cells. In addition to demonstrating the different 35.Ok7.0 31.4k7.4 0.4 M glycerol effects of the two sequences of rates, the 17.916.8 0.4 M DMSO 83.2k6.7 0.4 M sucrose results also demonstrate differences in the 10.0+0.3 Cooled at 6OOC/min temperature responsewithin a given sequence. 28.3 k2.9 25.2+ 3.6 Cooled at 36O”C/min 33.lk2.3 28.2k1.9 0.004M PVP Thus, in slowly cooled cells (fig. 2), survival after slow cooling to the lowest temperature a Relative to non-frozen ‘additive’ control. used ( - 196°C) was higher when the additive was PVP than when it was DMSO, in spite table, similar survivals are obtained when the of the fact that the molar concentration of the cells are rapidly warmed directly from - 196” PVP was lOO-fold lower (0.004 vs 0.4 M). to + 5°C.) As summarized in table 2, the rela- Furthermore, the response at intermediate tionship between the type of additive and the temperatures was also different in the two temperature of inactivation differs markedly additives. In DMSO, survivals dropped in the two reciprocal sequences.In no case is rather gradually at temperatures above the median lethal temperature (LT& during -45”C, but then dropped rapidly as the temslow cooling the same as that during slow perature was further reduced to -80°C. In warming. The differences are striking in the PVP, on the other hand, survivals dropped case of sucrose and DMSO. Cells in sucrose rather abruptly above -45”C, but showed show the highest LT,, during slow cooling little change at lower temperatures. (- lYC), but the lowest LT,, during slow In the case of cells rapidly cooled to warming (-49°C). The reverse is true for - 196°C and warmed slowly (fig. 3), the temperatures at which they became inactivated Table 2. Median lethal temperatures for during slow warming were affected by the Chinese hamster cells subjected to slow cooling precise values of both the cooling and the or slow warming in various protective additives warming rates. Thus, for cells frozen in sucrose, a decreasein cooling rate from 600” to Temperaturefor 50 % 360”C/min increased the LT,, of slowly inactivation during warmed cells from -49” to --37°C. And for Additive

Slow coolinga Slow warmingb

0.4 M sucrose 0.4 M glycerol 0.004M PVP 0.4 M DMSO

- 15°C -30 -30 -62

- 49°C -35 -43 -22

a Warming was rapid. Seecaption to fig. 2 for description of procedure. b Cooling was rapid. Seecaption to fig. 3 for description of procedure. Exptl Cell Res 71

cells frozen in glycerol, an increase in warming rate from 1.5” to about 40”C/min increased the LT,, from -35” to -13°C. This latter upward shift suggested that the inactivation

might be time dependent as well as temperature dependent, and made it of interest to determine the kinetics of inactivation

stant temperatures.

at con-

Freezing injury in hamster cells

351

no observable inactivation of cells held at -75°C and only a slow loss in viability at -50°C. On the other hand, the rate of inactivation at -40°C was high, a result that is not surprising in view of the abrupt loss in survival that occurred when cells in glycerol were warmed at l.S’C/min to above -40°C (fig. 3). This very rapid decline in the viability of cells that had been rapidly cooled to - 196°C and warmed to and held at -40°C (fig. 4, solid curve) contrasts with the much slower decline of cells that had been slowly cooled to and held at -40°C (fig. 4, dashed curve). DISCUSSION Two factors in freezing ir@ry

Most of the plots of survival versus cooling rate (fig. 1) exhibit optimum cooling rates for maximum survivals, especially when warming is slow. As mentioned in the introduction, Fig. 4. Abscissa: time (min); ordinate: % survival. this suggeststhat survival is determined by at Survival of hamster cells in 0.4 M glycerol as a least two classesof factors oppositely dependfunction of time at - 40, - 50, or - 75°C. The samples in the solid curves labeled ‘rapid cool’ were cooled ent on cooling rate. The deleterious effect of at 6OO”Cjmin to - 196°C. They were then transferred one class increases with increasing cooling to baths at - 40,’ - 50, or - 75”C, and held at these temperatures for various times before being thawed velocity, and the deleterious effect of the rapidly. The samples in the dashed curve labeled other class decreaseswith increasing cooling ‘slow cool’ were cooled at 1.6”C/min to -4O”C, held at -40°C for the indicated times, and then thawed velocity. The optimum occurs at those cooling rapidly. rates where the combined effects of both classes of events are minimal. This conclusion Effect of time at various temperatures on that the causesof injury in rapidly and slowly inactivation cooled cells are different is supported by The three solid curves in fig. 4 show the effects several other observations: of time at - 75, - 50, or - 40°C on cells that (1) Cells cooled to - 196°C at rates above were suspended in 0.4 M glycerol and inilOO”C/min (‘rapidly’ cooled cells) are much tially cooled rapidly to - 196°C. There was more sensitive to slow warming than are cells cooled at rates lower than lO”C/min (‘slowly’ 1 Warming to -40°C was carried out in two ways. In most cases samples were transferred directly from cooled cells) (fig. 1). Furthermore, time spent - 196°C to a bath at - 4O”C, but in one case the during cooling does not produce the same samples were transferred from -196” to a bath at -75°C and then warmed at 2”C/min to -40°C and effects as an equivalent time spent during held. Since the two methods yielded similar results, warming; i.e., the sequence of rapid cooling and since the curve in fig. 3 shows no inactivation for cells in 0.4 M glycerol during slow warming below and slow warming is generally much more -4O”C, the results for the two methods have been injurious than the reciprocal sequence of pooled.

O.’Ib

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352 P. Mazur et al.

slow cooling and rapid warming, even when brane of cells prevents the growth of ice the total times required to complete the se- crystals from the extracellular medium into quencesare comparable. the cell interior [12]. As a result, cells tend (2) The temperatures at which hamster to become supercooled, even in the presence cells are inactivated during slow cooling bear of external ice. Since supercooled water has a no apparent relation to the temperatures at higher vapor pressure than ice, water tends which rapidly cooled cells are inactivated to flow out of the cell and freeze externally during slow warming (figs 2, 3; table 2). as cooling progresses. If the cooling rate is (3) Cells held at -40°C after being rapidly sufficiently low or the permeability of the cooled to - 196°C undergo more rapid inac- cell to water sufficiently high, the cell can tivation than cells held at -40°C after being dehydrate rapidly enough to avoid extensive slowly cooled to that temperature. supercooling. But if the cooling rate is too (4) Finally, the morphology of rapidly high or the permeability to water too low, the frozen cells differs from that of slowly frozen water in the cells becomesincreasingly supercells when the thawed cells are examined by cooled, and eventually freezesintracellularly. These qualitative statements have been exelectron microscopy [9]. pressed quantitatively [19], and the resulting calculations indicate that a cell with the dimenPossible nature of the two factors sions and the water permeability of a mamOur working hypothesis is that (1) rapidly malian leucocyte ought to undergo intracelcooled cells are killed by the formation of lular freezing when the cooling rate exceeds intracellular ice during cooling and its subse- about SO”C/min [20]. If one could assume quent growth by recrystallization during that the water permeability of Chinese hamster cells is similar to that of leucocytes, this warming (especially during slow warming); that (2) slowly cooled cells are killed by the calculated value of SO”C/min for the cooling relatively long exposure to the major altera- rate required to produce intracellular ice tions produced in extracellular and intra- would be consistent with our present findings cellular solutions by the conversion of water and with the two-factor hypothesis just to ice. These alterations, or solution effects, stated. (3) The finding that rapidly cooled cells include the concentration of solutes, dehydration, changes in pH, and the precipitation of are more sensitive to slow warming than are slowly cooled cells applies to yeast and other solutes. cells as well as to hamster cells [7, 121. In The basis of this hypothesis is the following: (1) Yeast and red cells also show optimum yeast, there is evidence to support the view cooling rates for maximum survival. In these that the higher sensitivity to slow warming is two cases there is microscopic and other associated with the recrystallization of intraevidence that cells cooled at supraoptimal cellular ice [ll, 13, 211. One way to obtain rates do in fact contain intracellular ice [lO- direct evidence on the relations between cool141,and there is evidence in the red cell that ing rate, intracellular freezing, and recrystalinjury at suboptimal rates is associated di- lization is to examine frozen hamster cells by rectly or indirectly with the high concentra- the technique of freeze-cleaving. Moor [l I] tions of electrolytes produced by ice forma- carried out such a study on yeast, and Bank & Mazur have initiated a comparable study on tion [15-181. (2) Above about - 10°C the surface mem- hamster cells. Exptl

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Freezing injury in hamster cells

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cooled at 8”C/min (fig. la), and it does not explain why slow warming at l”C/min has In terms of the hypothesis just discussed, our no effect on cells cooled at 1.6”C/min in interpretation of the curves in fig. 1 is the 0.5 M DMSO or 0.7 M sucrose (fig. la, c), following: Cells cooled at l”C/min show less but is decidedly detrimental to cells cooled at than maximum survival, the nonsurvivors 1.6”C/min in 15% PVP (fig. 1d). being killed by alterations in the solution brought about by ice formation. The per- Effect of trypsinization centage that survive varies from 1 to 45 % The trypsin used to disaggregate tissue-culand depends on the ability of specific addi- ture cells is known to affect the surfaces of tives to protect against these solution effects. cells, but there is little knowledge as to Increasing the cooling rate to above l’C/ whether it affects their response to freezing. min at first generally increases survival. It Robinson [Zla] has compared the effect of does so becausefaster cooling produces short- plating medium on the viability of slowly er exposure to the altered solutions. Some- frozen Chinese hamster cells that have been where between 10” and lOO”C/min, however, trypsinized with that of small aggregates of this beneficial effect of faster cooling begins cells frozen on coverslips. Although the to be counteracted by the formation of intra- trypsinized cells were slightly more sensitive, cellular ice in some of the cells. The effect of the results were comparable. In the present intracellular ice on the survival curve depends studies, all that is known about the effect of on how much ice is formed in how many trypsin is that (1) the plating efficiency of cells, and it depends critically on the rate of control cells is 100% immediately after trypwarming. Cells that are warmed at the highest sinization and 74 % some 5 h later; (2) the rate used (N 1 OOO”C/min)show the least evi- addition of protective compounds does not dence of a clear optimum. This is because decrease the plating efficiency; and (3) the rapid warming minimizes the growth of intra- trypsinized cells can be frozen to - 10” or cellular ice, and therefore allows a fraction of -20°C with little loss in viability (fig. 2). rapidly cooled, internally frozen, cells to survive. (The ability of rapid warming to Implications for the design of lowprevent the potentially deleterious effects of temperature experiments intracellular ice and to obscure the existence In order to design experiments to investigate of an optimum cooling rate has been clearly mechanisms of freezing injury, one needs to demonstrated in yeast [8, 111).On the other realize that such injury is caused by at least hand, when the cells are warmed slowly, two classes of events; one also needs to recrystallization can occur, and intracellular know the cooling rates over which each class ice exerts its full lethal consequences. The exerts its effects so as to be able to select effect of slow warming on the survival curves rates at which only one class is operative. is, thus, to sharpen the optimum and move Thus, in the present study, the slowly cooled it to a lower cooling velocity. samples in figs 2 and 4 were cooled at about Although this interpretation accounts for 2”C/min, a rate that is well below optimum the main features of the curves, it leaves (see fig. 1) and, therefore, a rate that should unanswered a number of the details. It does involve only one of the factors (i.e., solution not, for example, explain the minimum sur- effects). However, the numerical values of vival observed when cells in 0.5 M DMSO are suboptimal, optimal, and supraoptimal rates

Interpretation rate

of plots of survival vs cooling

Exptl Cell Res 71

354 P. Mazur et al.

depend on the type of cells and on the additive. For example, a cooling rate of lOO”C/min is supraoptimal for yeast cells in water [13], whereas it is suboptimal for red cells in blood [20, 221. Implications for the preservation of cells

Until recently, most cryobiologists have stated that the optimum cooling rate for cells is l”C/min in the presence of protective additives, and that to be protective an additive must be present in high molar concentration and must permeate the cell [17, 18, 231. Our findings are not consistent with these views. The optimum cooling rate for Chinese hamster cells in four commonly used additives is not l”C/min; it ranges from 10” to 300”C/min. Moreover, protection does not appear to be well correlated either with the molarity of the additive or its ability to permeate. Thus, we see from figs 1 and 2 that 0.004 M PVP protects cells cooled slowly to - 196°C somewhat better than does 0.4 or 0.5 M DMSO even though its molarity is only l/lOOth that of the DMSO and even though it probably does not permeate [8, 241. (PVP is actually somewhat polydisperse, with 90 Y., of the molecules having molecular weights higher than about 15 000 and less than 60 000 [25, 25a]. The value of 0.004 M thus represents an average value corresponding to the average molecular weight of 40 000.) Parenthetically, it should be noted that the cooling rates and additives reported here only produce high survivals when warming is rapid (with the exception of 15 % PVP). Other concentrations and combinations of additives, however, can reduce this critical dependency on warming rate [24, 261. Implications for electron microscopy

The cooling rates used in the microscopy techniques of freeze-cleaving and freezesubstitution (5 OOO”--50 OOO”C/min) [27, 281 Exptl

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are 2 to 4 orders of magnitude higher than those generally found to yield optimum survival (lo-lOO”C/min). There is evidence that even these very high rates do not eliminate artifacts, at least in freeze-substituted specimens [4a]. Moreover, it is clear that cells cooled at rates far above optimal are in a highly susceptible state, a state so susceptible that injury during thawing can be avoided only by using the highest possible warming rates, if it can be avoided at all. For example, survivals of very rapidly cooled yeast remain relatively high after thawing only if the warming velocity is of the order of 40 OOO”C/min. Decreasing the warming rate to 1 400”C/min decreasessurvival by two orders of magnitude [13, 291. Presumably this high sensitivity to warming rate reflects the fact that the smaller the intracellular ice crystals produced by rapid cooling, the more likely they are to grow by recrystallization during warming. The smaller an ice crystal the higher its surface-free energy relative to planar ice, and the faster it will grow at a given subzero temperature [12, 301.We suggestthat thisdependence of the rate of recrystallization on crystal size and temperature may be the explanation for the fact that cells in sucrose cooled at 600”C/min underwent inactivation at a lower temperature during slow warming than did cells cooled at 360”C/min (fig. 3); i.e., the faster cooling rate produced smaller crystals. The observation that inactivation of rapidly cooled hamster cells can occur at temperatures as low as -60°C (fig. 3) raises warning signals to those who use freezing techniques in electron microscopy, and who hold cells above - 100°C during processing (e.g., most freeze-substitution techniques and especially ethylene glycol substitution at N -50°C as reported by Pease[31]). If cells that have been cooled at 6OO”C/min can be killed by being warmed to -6O”C, nonlethal cellular modifications may occur at lower temperatures; and

Freezing injury in hamster cells

both lethal and nonlethal modification may occur at lower temperatures in cells subjected to the much higher cooling velocities generally used in electron microscopy. Moor [ll], for example, found that when rapidly cooled yeast cells were subjected to slow warming, recrystallization of intracellular ice became visible in freeze-cleavedspecimenssome 10°C below the temperature at which cell viability began to be affected. The more the resolving power of this technique (and of others involving freezing) improves, the greater becomes the danger that small degreesof such recrystallization can introduce significant artifacts. We thank Patricia A. Brimer for her expertness in preparing cell suspensions and carrying out the plating assays. This work was sponsored by the United States Atomic Energy Commission under contract with Union Carbide Corporation. Preliminary reports on the research were presented at the 1970 meetings of the Society for Cryobiology, Baltimore, Md.

REFERENCES 1. Mazur, P, Science 168 (1970) 939. 2. Bullivant, S, Some biological techniques in electron microscopy (ed D F Parsons) p. 101. Academic Press. New York (1970). 3. Weinstein, k S & Someda, K, Cryobiology 4 (1967) 116. 4. Ghosh, B K, Lampen, J 0 & Remsen, C C, J bact 100 (1969) 1002. 4~. Rebhun, L I & Sander, G, Am j anat 130 (1971) 1, 17. 5. Fernandez-Moran, H, Proc natl acad sci US 56 (1966) 801. 6. Welton, T A, Proc 26th ann meeting of the Electron Microscope Society of America, p. 338 (1968). 7. Ieibo, S P, Farrant, J, Mazur, P, Hanna, M G, Jr & Smith, L H, Cryobiology 6 (1970) 315. 8. Mazur, P, Farrant, I, Leibo, S P & Chu, E H Y, Cryobiology 6 (1969) 1.

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9. Bank, H & Mazur, P, Exptl cell res 71(1972) 441. 10. Mazur, P, J bact 82 (1961) 662. Moor, H, Z Zellforsch 62 (1964) 546. ::: Mazur, P, Cryobiology (ed H T Meryman) p. 213. Academic Press, New York (1966). 13. Mazur, P & Schmidt, J, Cryobiology 5 (1968) 1. 14. Rapatz, G L, Menz, L J & Luyet, B J, Cryobiology (ed H T Meryman) p. 139. Academic Press, New York (1966). 15. Lovelock, J E, Biochim biophys acta 10 (1953) 414. 16. Farrant, J & Woolgar, A E, The frozen cell. Ciba Foundation symposium (ed G E W Wolstenholme & M O’Connor) p. 97. Churchill, London (1970). 17. Meryman, H T, Nature 218 (1968) 333. 18. - The frozen cell. Ciba Foundation symposium (ed G E W Wolstenholme & M O’Connor) p. 51. Churchill, London (1970). 19. Mazur, P, J gen physiol 47 (1963) 347. 20. Mazur. P. Problems in blood transfusion. Blood and bone’marrow preservation, part 3, Proc 1lth toner int sot blood transfusion. Sydney. Aug 24-29, 1966 (ed L Hollander) p. 764: S. Karger, Base1and New York (1968). Published as Bibliotheca haematologica, no. 29, part 3. :!a- Mazur, P, Biophys j 1 (1961) 247. LIP. Robinson, D M, Cryobiology 6 (1970) 573. 22. Rapatz, G, Sullivan, J J & Luyet, B, Cryobiology 5 (1968) 18. 23. Lovelock, J E, Biochim biophys acta 11 (1953) 28. 24. Mazur. P. Leibo. S P, Farrant J, Chu, E H Y, Hanna; M G, Jr’& Smith, L H, The frozen cell. Ciba Foundation symposium (ed G E W Wolstenholme & Maeve O’Connor) p. 69. Churchill, London (1970). 25. Miller, L E & Hamm, F A, J phys them 57 (1953) 110. 25a. Ashwood-Smith, M J, & Warby, C, Cryobiology 8 (1971) 377. 26. Mazur, P, Leibo, S P & Chu, E H Y, Cryobiology 6 (1970) 581. 27. Moor, H & Mtihlethaler, K, J cell biol 17 (1963) 609. 28. Rebhun, L I, Fed proc 24 no. 2, part 3, suppl. 15 (1965) S-217. 29. Anderson, N G, Green, J G & Mazur, P, Nat1 cancer inst monogr 21 (1966) 415. 30. Kingery, W D, J appl phys 31 (1960) 833. 31. Pease, D C, J ultrastruct res 21 (1967) 98. Received August 2, 1971 Revised version received October 19, 1971

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