p Journal of Microscopy, Vol. 111, Pt 1, September 1977, pp. 17-34. Revised paper accepted 14 July 1977

Structural and functional aspects of biological freezing techniques

by J. F A R R A NC.TA , . WALTER, H. LEE,G. J . MORRIS*and K. J. CLARKE,* Clinical Research Centre, Harrow, Middlesex, and *Institute of Terrestrial Ecology, Culture Centre of Algae and Protozoa, Cambridge

SUMMARY

The cooling procedures used to prepare samples for ultrastructural examination at low temperatures often differ markedly from those used to recover optimal function of cells on thawing. The implications of these differences are reviewed. Damage and alteration to the structure and function of the cells may be caused by the high concentrations of cryoprotective agents such as glycerol or dimethyl sulphoxide (DMSO) often added to reduce ice crystal artefacts. Under the rapid cooling conditions commonly employed for structural studies, these additives are not cryoprotective; low rates of cooling are necessary for them to be effective. Rapidly cooled cells that contain intracellular ice are only injured during rewarming so their structure may be as yet unaltered by any damaging effects at low temperatures. Most cells able to recover on thawing are grossly shrunken at low temperatures but since they are potentially functional they are of interest structurally. These cryobiological principles are illustrated with freeze-fracture, freeze substitution and functional assays. The cell types chosen were Chlorella sp. and mammalian tissue culture cells. INTRODUCTION

The reason for using freezing as a procedure for fixation is to examine the structure of cells and tissues in their natural state without any effects of chemical fixation and processing. It follows that the freezing itself must not add more artefacts than those it is trying to avoid. For the examination of structure, the general practice has been to cool small samples of cells as rapidly as possible in order to minimize ice crystals that are considered as artefacts in micrographs. With larger samples, the ice has been reduced or avoided by adding high concentrations of so-called cryoprotective agents before rapid cooling. However, the conditions used for the freezing of cells for ultrastructural examination in the frozen state are so different from those used when the recovery of function is the aim, that there is a distinct possibility that the cells have been damaged both functionally and structurally. The purpose of this paper is two-fold. First we will discuss the cryobiological consequences of cooling procedures including those commonly used by microscopists. Secondly, we will consider the relationships between functional recovery, 2

17

J . Farrunt et ai. cryobiological behaviour and structural appearance to see whether it will help in the development of appropriate cooling procedures. EFFECT O F C O O L I N G RATES I N THE ABSENCE OF CRYOI’ROTECTIVE AGENTS

In the absence of cryoprotective additives, the rate of cooling is the primary variable affecting both the structure and funcLion of cells. Figures 1-4 show a series of freeze-fracture micrographs of Chlorella protothcccides (CCAP strain 21117a) cooled over a range of rates. Aliquots of packed cells (10 pl) were placed in a 3 mm diainetsr brass tube and frozen in melting nitrogen. The method of freeze-fracture was similar to that described by Bullivant & Ames (1966). The fractured surfaces were shadowed and replicated at temperatures belou7 123 K with platinum-carbon (at a shadow angle of 20’ to the horizontal) and carbon. The replicas were cleaned in 40°0 hydrofluoric acid followed by ‘Domestos’ bleach, and washed in distilled water. Iiapid cooling produces different thermal profiles across a sample; in aqueous solutions, without high concentrations of cryoprotectants, this results in differences of ice crystal size depending on the depth within the sample (van Venrooij et al., 1975; Nei, 1976; Dempsey & Bullivant, 1976a, b). Comparisons of cellular ultrastructure following freezing at different cooling rates can therefore be made within one sample. At the centre of the frozen sample, where cooling is slowest, large extracellular ice crystals form (Fig. 1). The removal of water from solution produces an external hypertonic medium to which the cells are exposed for long enough during cooling for cellular dehydration to occur (Figs. 1 and 2), under these conditions cells reach low temperatures with little or no intracellular ice formation. Figure 3 shows the effect of more rapid cooling, nearer the surface of the sample, where the cells have less time to lose water osmotically and can only reach equilibrium by the formation of intracellular ice. Finally, cells taken from the periphery of the sample, next to the coolant, were cooled so rapidly that they were unshrunken and no ice crystals could be detected (apparent vitrification) (Fig. 4). A similar effect of different cooling rates on the ultrastructure at low temperatures of unprotected mammalian tissue culture cells (Bank, 1974) and yeast (Moor, 1964; Bank, 1973; Bank & Mazur, 1973) has been reported. How do these different cooling rates affect the survival of cells ? Replicate samples comparable to those depicted in Figs. 1-4 gave a survival after rapid thawing that was indistinguishable from the unfrozen control. This indicates that in this system, shrinkage, intracellular ice and apparent vitrification are all compatible with cellular survival (Morris, 1976a). This finding is not typical however. It is unfortunate for the same reason that the most widely quoted report on the good survival of cells cooled rapidly involves yeast cells (Moor & Muhlethaler, 1963)

Figs. 1-4. Freeze-fractured cells of C. protothecoides from different regions within a rapidly cooled sample shadowed at 293 K to the horizontal with the direction of shadowing from top to bottom of plate. Fig. 1. Shrunken cells between large ice crystals ( x 8600). Fig. 2. A dehydrated cell ( x 24,000). Fig. 3. Chloroplast from a cell cooled more rapidly containing numerous crystals of intracellular ice ( x 37,800). Fig. 4. Unshrunken cell from the edge of the sample with no apparent intracellular ice ( x 24,000).

18

Structure and function after freezing

19

J . Farrant et al. that are much less sensitive to rapid cooling injury than most cells. The usual picture of survival is that it is a function of the rate of cooling (Fig. 5). This figure shows a diagrammatic representation of cell survival in the absence of added cryoprotectants. If cooling is too slow cells are killed by the combined effects of excessive shrinkage, exposure to hypertonic solutions and reduction in temperature (Farrant & Morris, 1973). If the cooling is more rapid the cells do not shrink but instead form intracellular ice. Under these conditions cell survival is again low. Figure 5 shows finally that very rapid cooling at rates that allow vitrification give an increase in survival. This last effect has been reported for resistant plant cells like yeast (Moor & Muhlethaler, 1963) and Chlorellu (Plattner et ul., 1972), but has not been reported for most cell types. The rates of cooling required are extremely high. In 20°,, gelatin the cooling rate has to be more than lo5 K . s-1 for vitrification Shrunken cells, no ititracellular ice

fl

Vitrification

Slow

Rapid

Very rapid

C o o l m rote

Fig. 5. Diagrammatic representation of the principles underlying the effects of rate of cooling on survival of two cell types following rapid thawing from liquid nitrogen. Cells cooled slowly are damaged by excessive shrinkage and effects of concentrated solutions. Cells cooled more rapidly are damaged as a result of intracellular ice. Optimal survival occurs at intermediate rates of cooling that are dependent on

the cell type. (Luyet, 1966). Also for appreciable survival thawing would have to be equally rapid. Although very high concentrations of cryoprotective additives may reduce the rate of cooling that allows vitrification, this advantage is bought at the expense of imposing toxic effects as will be discussed later. The primary feature of Fig. 5 is that it shows that optimal survival occurs in practice when cells are cooled rapidly enough to avoid excessive shrinkage or solution injury, but not so rapidly that too much intracellular ice is formed. The value of both optimal survival and the cooling rate giving optimal survival, will vary from cell type to cell type (Rapatz & Luyet, 1968; Mazur et al., 1969; Leibo et al., 1970). For example, Fig. 5 also illustrates a different cell type with a much lower survival at its optimal cooling rate. The reasons for these differences between cells lie firstly in their particular sensitivities to shrinkage and hypertonic injury during slow cooling. Secondly, the permeability of the cells to water and their surface to volume ratio determines the rate of cooling at which shrinkage is prevented and intracellular ice forms (Mazur, 1963). It is also important to remember that other cell dependent factors may alter survival following freezing and thawing. 20

Structure and function after freezing

For example, survival can be affected by the stage of the cell cycle (McGann et al., 1972), its state of activation (Knight et al., 1972), the age of culture (Toyokawa & Hollander, 1956; Nag & Street, 1975) and the growth temperature (Sakai & Sugawara, 1973; Morris, 1976b). CRYOPROTECTIVE AGENTS

Toxic efSects Cryoprotective agents may themselves cause irreversible injury both before and after freezing. The damage produced is a function of concentration, time and temperature of exposure and the rate of addition and dilution. Examples of the gross injury to the function of several cell types and tissues by different cryoprotective compounds are presented in Table 1. It is clear that many recipes Table 1. Effects of cryoprotective additives Cell type Mouse lymphocytes

Chlorella emersoni

Schistosomula ( Schistosoma mattheei) Smooth muscle (guinea-pig taenia coli) Carrot tissue culture cells

Additive DMSO (looo) D M S O (lo",,) PVP (lSoo) DMSO (looo) Glycerol (looll) Methanol (looo) D M S O (1200)

Tem- Timeof perature exposure (OK) bin) 293K 20 273K 20 293 K 5 293 K 5 293 K 5 293 K 5 293K 60

Methanol (17 500) 273 K

Survival ("") 50

70 >95 74 48 82 i30

10

100

D M S O (30",,) D M S O (30",,)

310K 266K

120 120

0 100

Glvcerol 15" n ) D M S O (50,J' D M S O (looii)

293K 293K 293K

120 120 120

55 ..

70 60

References Thorpe et al. (1976) Morris (1976a)

James & Farrant (1976) James & Farrant (in preparation) Elford (1970) Nag & Street (1975)

for freezing for ultrastructural examination use potentially damaging concentrations of these additives, e.g. 15" glycerol for 20 min followed by 25" glycerol for 30 min at 277K (Stolinski & Breathnach, 1975) and 25O,, glycerol for 20min at 277K (Bullivant, 1973). It is possible that these and similar techniques will cause functional injury to the cells and alterations to the structure. More subtle effects both functional and structural may occur at lower concentrations. Injury caused by cryoprotective agents is due, either to direct chemical toxicity, or with compounds that enter cells damage may result from osmotic swelling during the dilution of the additive during and after thawing. If the injury is of this latter type it has not yet become manifest at the low temperature itself and will not alter the structural appearance. Another problem with cryoprotective additives is that they may induce reversible structural alterations to cells. Examples include modifications to the appearance of the lipid components of membranes (Buckingham & Staehelin, 1969; Costello & Gulik-Krzywicki, 1976) and membrane particles; this latter effect is the subject of current discussion (Kirk & Tostesen, 1973; McIntyre et al., 1974; Martinez-Palomo et al., 1976; Scott et al., 1977; Breathnach et al., 1976; Stolinski & Breathnach, 1977).

Low molecular weight permeant additives One of the most important findings concerning additives is that their cryoprotective properties vary with the rate of cooling (Rapatz & Luyet, 1968). Figure 6

21

J . Farrant et al.

I ~

With DMSO or glycerol (eel 10%~

Slow

Rapid

Very rapid

Coolinq rate

Fig. 6. Diagrammatic representation of the principles underlying the effect of DMSO or glycerol on the survival of a cell type at different rates of cooling. The cryoprotective additives only increase cellular survival when cooling rates are slow. With rapid cooling they have no effect and a t some intermediate rates of cooling their presence is detri-

mental.

shows a generalized diagrammatic representation of survival over a wide range of cooling rates both in the presence and absence of a cryoprotective additive. It can be seen that the injury caused during slow cooling is reduced or minimized by the incorporation of glycerol or dimethylsulphoxide (DMSO) before freezing. These low molecular weight compounds rapidly penetrate most cells. The most likely reason for their protective action is that when they are present less ice forms at any temperature during freezing than in their absence (Lovelock, 1953). This has a dual effect. First, the cells are exposed during freezing to less extreme levels of the increased concentration of the other solutes present (usually salts). As an example, when freezing in saline (0.15 M), cells are exposed to a salt concentration of over 30 times normal by 252 K in the absence of DMSO, but to a concentration of about 4-5 times the normal level by the same subzero temperature if DMSO (looo v/v) is present in the medium before freezing (Farrant et al., 1967). The second physical effect of the additives on the cells is that there will be less shrinkage at any temperature during freezing, because a proportion of the impermeant extracellular solute has now been replaced with a permeant additive. Both effects help to explain why glycerol and DMSO protect cells against injury at low rates of cooling. Totally different results are obtained when cells are cooled rapidly. Figure 6 illustrates that not only are these low molecular weight additives like DMSO and glycerol unable to protect cells from injury during rapid freezing but there is a range of high cooling rates close to the optimal rate where the incorporation of the additive may lower the survival (Rapatz & Luyet, 1968). Why should this be? At intermediate rates of cooling, some shrinkage of the cells will occur; in the presence of penetrating additives there will be less shrinkage allowing more ice to form within the cells thus increasing injury. Figure 6 also indicates that a cryoprotectant may reduce the cooling rate at which apparent vitrification can

22

Stmctzire and function after freezing be achieved. However, for any significant effect, such high concentrations of additives would have to be used (e.g. 30-50",, w w solute; Luyet & Rasmussen, 1968) that toxic effects would be encountered, except in unusual cases. There is thus a common but fundamental misconception of the role of cryoprotective additives in structural studies. Since these compounds reduce the amount of ice during freezing and this effect is important both for allowing the recovery of function on thawing and also for avoiding ice crystal artefacts at low temperatures, there is a belief that both of these objectives can be realized simultaneously. However, for cryoprotection the additive concentration has to be low to avoid toxicity and the cooling must be slow. In contrast, to prevent ice crystal artefacts at low temperatures, concentrations of additives should be high and cooling rapid. In most systems these conditions are mutually exclusive. In other words, so-called cryoprotective compounds are not cryoprotective when cooling is rapid. It is, however, also clear that optimally preserved cells may contain some intracellular ice when both cooling rate (Bank, 1974) and two-step procedures (Farrant et d.,1977) are used. Because some ice is present, thawing must be especially rapid for optimal recovery in these latter cases. Further complications occur when it is necessary to carry out a sublimation step as in freeze etching. With the low vapour pressure of glycerol, mainly water is removed leaving progressively more concentrated solutions of glycerol; DMSO is removed more easily. It might be worth considering using other more volatile low molecular weight neutral solutes, for example methanol. This compound has been demonstrated to be non-cytotoxic and also to be cryoprotective, within limits at some rates of cooling (Meryman, 1968; Rapatz, 1973; Ashwood-Smith & Lough, 1975; James & Farrant, 1976).

Low molecular weight iEnperfizeant additives Another group of low molecular weight compounds, including sucrose, can protect cells without permeating them. Sucrose can reduce the amounts of ice and therefore protect cells from injury at low rates of cooling. With more rapid cooling, however, in contrast to DMSO and glycerol, there is a small range of intermediate cooling rates at which sucrose does protect cells (Rapatz & Luyet, 1965), presumably by hindering the formation of a damaging amount of intracellular ice by preshrinking the cell before freezing. However, at non-toxic concentrations sucrose is not effective as a cryoprotective agent at the high rates of cooling used in most ultrastructural studies. A further complication is that compounds like sucrose, although initially impermeant, have been demonstrated to enter cells during freezing and thawing (Daw er a]., 1973). High molecular weight imperineant additives The second group of non-permeant cornpounds are the high molecular weight ones like dextran, hydroxyethyl starch (HES) and polyvinylpyrrolidone (PVP). Because of their high molecular weight they do not shrink the cells appreciably before freezing begins. For a long time it was thought that they could not act like the low molecular weight compounds in reducing the exposure of the cells to high ionic strength conditions during freezing. However, it is now clear that as freezing progresses and their concentration increases, these polymers behave as if they were of progressively lower molecular weight (Farrant, 1969). This can explain their beneficial effects in protecting slowly cooled cells against injury, but their protective effects during rapid freezing are less than those of sucrose (Rapatz & Luyet, 1965). PVP has been demonstrated to reduce the size of ice crystals

23

J . Farrant et al. during freeze-etch techniques and by remaining extracellular does not affect the sublimation of ice from intracellular spaces (Franks & Skaer, 1976). ULTRASTRUCTURAL APPEARANCE AND CELL SURVIVAL

Before considering the practical problems of freezing both for ultrastructural and functional survival, let us consider two methods that have been tried in an attempt to solve the problem without the addition of cryoprotectants. Vitrijication Ideally this would be the best method. If cooling were sufficiently rapid no ice crystals would form and no artefacts would be seen; provided warming is equally rapid, survival should be high. The avoidance of visual artefacts has been done with ‘spray frozen’ suspensions of cells or liposomes (Bachmann & Schmitt, 1971; Plattner et al., 1972; Ververgaert et al., 1973; Lang et al., 1976) or at the surfaces of tissue frozen by the van Harreveld method (van Harreveld & Crowell, 1962; Dempsey & Bullivant, 1976a, b). However, only small regions of samples (10 pm diameter for spray freezing, and 10-12 pm layer of tissue by the van Harreveld method) can be considered either vitrified or at least frozen without ice crystals large enough to detect. Correlative experiments demonstrating the protection given to the cells have not been done; because of the high rate of thawing required, they would be hard to do in practice. High pressure freezing The application of high pressure has a similar effect to the incorporation of a cryoprotective additive in that the amount of ice present during freezing is reduced (Moor & Riehle, 1968; Moor, 1971). The problems are also similar in that the application of high pressure has been shown to exert cytotoxic effects (Taylor, 1960). Progressive depression of freezing point using cryoprotectant There is one other totally different way of reaching low temperatures without ice, particularly with large samples. This involves the progressive increase in the concentration of cryoprotective agent (e.g. DMSO) during cooling (Farrant, 1965; Farrant et ai., 1967). Because the concentrations are increased only during cooling, the toxic effects of these high concentrations at temperatures above 273 K are avoided and good recovery is obtained both for ultrastructure and function. To our knowledge this approach has not been tried for structural studies at low temperatures. The use of this method will, however, depend on the maintenance of permeability to the cryoprotectant by the cells in question throughout the cooling. Rapid freezing The main topic we have to consider is the relationship between survival and ultrastructure after rapid freezing, since this is the most commonly used freezing method for structural studies at low temperatures. In order to illustrate comparisons between structure and function following rapid cooling we have frozen a mammalian tissue culture cell line derived from Chinese hamster fibroblasts. Survival was assessed by the colony forming capacity of these cells on thawing (McGann & Farrant, 1976), and ultra-structural examination at low temperatures was done using a freeze-substitution technique that has already been described (Walter et al., 1975). Figure 7 shows the appearance of cells cooled directly into liquid nitrogen. The

24

Structure and function after freezing sample was 0.1 ml frozen in a glass tube and the rate of cooling was approximately 473 K . min--l. Although this rate is by no means as high as those usually used when freezing for structural examination, it is rapid enough to kill the cells. It can be seen that after freeze-substitution these cells contain numerous intracellular ice cavities of varying size throughout the nucleus and cytoplasm. Comparison with unfrozen cells fixed for electron microscopy in the conventional way (Fig. 8) shows that the cooling was sufficiently rapid for the cells to remain unshrunken. When the ice crystal cavities are of small size as in Fig. 7, the ultrastructural appearance following freeze-substitution is comparable with that of an unfrozen cell (Fig. 8). Despite this, the cells were invariably dead on rewarming giving no

7

Fig. 7. Electron micrograph of Chinese hamster fibroblast freeze-substituted at 193K after rapid cooling (473K/min) in DMSO (5y/;v/v) to 77K. Despite the presence of numerous intracellular ice cavities, note the unshrunken appearance and close similarity to the unfrozen cell (Fig. 8) ( x 10,500). Scale bar=3 pn.

25

J . Farrant et al. colonies following culture. The presence of DMSO (5",, viv) did not help the survival since, as already discussed, the cooling was too rapid for this compound to be cryoprotective. Although these and other results indicate that almost all cells cooled rapidly for ultrastructural examination are dead on thawing, there is now much evidence that the injury associated with intracellular ice in rapidly cooled cells is not brought about by the formation of this ice within the cells during cooling but occurs only during thawing. Ice per se is not damaging. Micrographs such as that of Fig. 7 can thus be considered to be of cells that are as yet undamaged since they have been cooled but not yet thawed. This point is so important that it is worth providing clear evidence that cells containing intracellular ice are injured during rewarming and not during cooling. Although some work has already been done (see Mazur et aZ., 1970), we have recently re-investigated this. It is not possible to provide a

8

Fig. 8. Electron micrograph of an unfrozen Chinese hamster fibroblast that has been exposed to DMSO ( 5 O , v/v) at room temperature for 60 min before fixation in glutaraldehyde ( 1 . 2 5 O I)followed by osmic acid (2';;) ( x 7500). Scale bar=3 pm.

26

Structure and function after freezing direct demonstration that injury occurs during the rewarming of rapidly cooled unshrunken fibroblasts since they contain so much ice that none of the cells survive whatever the warming procedure. However, partially protected cells can be used to show the effect of different rates of thawing. We have used the two-step method of cooling to protect the Chinese hamster tissue culture cells. This method consists of interrupting rapid cooling by a period at a constant subzero temperature before resuming the rapid cooling by plunging the sample to the storage temperature (e.g. into liquid nitrogen). Figure 9 shows the survivals obtained when Chinese hamster fibroblasts are frozen in DMSO (5",, v/v) by being held for 10 min at 248 K before rapid cooling into liquid nitrogen. As can be seen from Fig. 9, a high survival of 81(',, was obtained when cells were thawed rapidly from 77 K. Examination of the appearance of the cells after freeze-substitution at 193 K (point A in Fig. 9) is 89O/o

8 I O/o

9% 4

!

-25"C 10'

-i

+

Fig. 9. Survival ( f > ( l ) of Chinese hamster fibroblasts frozen in DMSO (5O, v/v) by a two-step method (248 K for 10 min). This protected cells from injury, provided thawing was rapid, but the cells were severely damaged by 5 min at 248 K during rewarming. Points A and B represent the ultrastructural examination of the cells following freeze-substitution at 193 K depicted in Figs. 10 and 11 respectively.

given in Fig. 10. It is clear that this cell is highly shrunken and the micrograph shows little evidence of any intracellular ice cavities. A similar observation of no detectable ice on freeze-substitution was made by Rebhun & Sander (1971) when cells were shrunk by exposure to a hypertonic environment. However, although the grossly misshapen cells like that shown in Fig. 10 are recoverable in a functional sense on rapid thawing, they are killed both following slow thawing or by being held for 5 min at 248 K during rewarming (see Fig. 9). If cells so held are recooled to 77 K rapidly and then freeze-substituted at 193 K (point B in Fig. 9) it is observed (Fig. 11) that although they remain shrunken, ice crystal cavities are now present. This indicates that the cells cooled by the two-step method (Fig. 10) contain intracellular ice nuclei too small to detect by freezesubstitution. In addition, the severe injury to the cells following thawing procedures that allow the intracellular ice to recrystallize, is strong evidence that the injury associated with intracellular ice occurs only during rewarming.

27

J . Farrant et al. T h e presence of intracellular ice can also be demonstrated by freeze-fracture. Figure 12 shows chloroplasts from Clzlorella cells rapidly cooled in DMSO 5",, v v. No evidence for intracellular ice can be seen. Following recrystallization for 5 min at 248 K and rapid return to 77 K, numerous crystals of ice could be seen within the cell (Fig. 13) This indicates that rapid cooling in DMSO reduces ice crystal size rather than inhibiting nucleation. Despite a report of structural changes to ice crystals at 77 K (Lehmann & Schulz, 1976), there is no evidence for biological deterioration at this temperature. Although microscopists might feel that thawing is irrelevant to the structure at low temperatures, there may often be unintentional periods of rise in temperature within the ultrastructural technique. Figure 14 shows the effect of rewarming from 77 K to different temperatures on the survival of fibroblast cells frozen in DMSO (5",, v/v). As can be seen, warming to any temperature of 228 K or above is potentially harmful to the recovery of functional cells. Other work indicates that only a few seconds rewarming are needed to kill the cells even when they are

Fig. 10. Electron micrograph of Chinese hamster fibroblast freeze-substituted at 193 K after two-step cooling (Point A, Fig. 9). This potentially functional cell shows a typical shrunken appearance without any evidence of intracellular ice cavities ( x 15,000). Scale bar = 3 pm.

28

Structure and function afte-r freezing still below 243 K. During different low temperature procedures there are several occasions when the risk of rewarming is great. Examples include the wiping away of isopentane or other coolant from rapidly cooled samples, the re-embedding of spray frozen specimens and freeze-substitution itself. These are potentially risky procedures, if any rewarming is allowed to occur. When cells are rapidly cooled for ultrastructural examination the actions of cryoprotective agents can be summarized as follows: (a) at high cooling rates it is extremely probable that the additives will not be cryoprotective; (b) the additives will reduce or prevent ice crystal artefacts; (c) too high a concentration of additive may damage or alter cells both structurally and functionally.

11

Fig. 11. Electron micrograph of Chinese hamster fibroblast freezesubstituted at 193 K after two-step cooling and recrystallization (Point B, Fig. 9). This cell is shrunken but also contains recognizable

ice cavities of varying size (arrows). This suggests that cells similar to that shown in Fig. 10 contain ice nuclei too small to detect by freeze-substitution ( x 11,500). Scale bar = 3 pm.

29

J. Farrant et al.

Figs. 12 and 13. Freeze fractured Chlorella cells after rapid cooling in DMSO (5% w/v). Fig. 12: after the initial rapid cooling there are no apparent intracellular ice crystals ( x 63,300). Fig. 13: cells rewarmed to 248 K for 5 min and then cooled rapidly again showing recrystallization of ice within the cells ( x 35,000).

T OC

0

-20

I

I

-40

-60

-

196 "C

-/i-

T(Ti

Fig. 14. Effect of rewarming to different temperatures (T) on survival of Chinese hamster fibroblasts cooled by a two-step procedure (248 K for 10 min in DMSO 5", v/v). After the rewarming temperature survivals were assessed after thawing directly (N) or after return to liquid nitrogen and thawing (P). At rewarming temperatures above 228 K survival decreased markedly.

30

Structure and .function after freezing If one is aware of these problems, the rapid cooling of cells may still provide the best procedure for ultrastructural examination. If it is imperative to use additives to help to avoid ice crystal artefacts, it is essential to use low concentrations to avoid toxic problems. One example of this is that if too high a concentration of DMSO or glycerol is used (e.g. 20",,) it has sometimes been necessary to use a chemical fixative before freezing in order to stabilize cellular structures that would otherwise be altered by the damaging actions of the so-called protective compounds (Gilula et al., 1975). Such a manoeuvre, although understandable, invalidates the whole concept of using freezing to fix cells in a natural state. A more appropriate use of rapid freezing (in the absence of glycerol) is to study the effects on membrane particles of different fixatives before freezing (Parish, 1975). As we have seen clearly, most rapidly cooled cells ( e g Fig. 7) have a close similarity to the ultrastructure of the chemically fixed unfrozen material but are dead on rewarming, whereas the deformed and shrunken cells obtained following two-step cooling (Fig. 10) are potentially viable after rapid thawing. Despite the gross deformation of the structure of these latter cells, it would seem to be extremely worth while to study their fine structure at low temperatures for they (unlike the more familiar unshrunken cells) are able to survive on thawing. CONCLUSIONS

We can conclude that an awareness of the possible effects of different cooling regimes on cell survival may be of more importance to the microscopist than any single recipe. I n general, toxic changes or structural alterations may be induced by using cryoprotective additives in concentrations high enough to prevent ice crystal artefacts and this should be avoided. Cryoprotective additives are only protective under specific cooling conditions (usually at low rates of cooling). With more rapid freezing, these additives not only do not protect but they can be positively harmful. Rewarming to temperatures that may permit the recrystallization of intracellular ice should be prevented. Finally, provided no rewarming has occurred, the presence of some intracellular ice in rapidly cooled cells does not indicate that any damage has yet occurred to these cells when they are examined ultrastructurally at low temperatures. The presence of the ice does, however, indicate that the cells may be damaged on thawing. Judicious use of cryoprotective additives (in non-toxic concentrations, i.e. usually less than 10" ,,) may reduce ice crystal artefacts but is unlikely to protect the cells in a functional sense unless cooling rates are low. If cooling is slow or the two-step method is used, severely shrunken cells are observed at the low temperatures but these cells will survive with the appropriate thawing conditions. The problem confronting those wanting to use freezing to observe cells in their natural state is first to avoid adding artefacts by the use of chemical fixatives or excessive concentrations of cryoprotective additives. The problem is then to choose between two categories of cooling procedures. The first is to observe rapidly cooled unshrunken cells that may contain varying amounts of intracellular ice and cannot be recovered alive after thawing. The knowledge that injury only occurs during rewarming may be helpful in this respect. The second category of cooling procedures that has hitherto been almost totally neglected for structural studies involves slow or two-step methods that produce shrunken deformed cells at the lowest temperature. These cells, however, can be thawed in a living state. The choice has to be made each time for each set of experimental conditions. ACKNOWLEDGMENT

We would like to thank Miss F. E. Barclay for excellent technical assistance. 31

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