CRYOBIOLOGY

28, 279-287 (1991)

Ca2+ Action on the Stability of Egg Phosphatidylcholine Vesicles during FreezeThaw Cycles L. S. BAKAS Institute

de Investigaciones Universidad National

Fisicoqut’micas de La Plata,

Sonicated

AND E. A. DISALVO

Tedricas y Aplicadas (INIFTA), Casilla de Correo 16, Sucursal4,

Facultad de Ciencias Exactas, (1900) La Plata, Argentina

The stability of unilamellar vesicles during freeze-thaw cycles strongly depends on the Ca*+ concentration in the aqueous solution. Experiments performed at equal ionic strengths with Na+ and Ca*+ solutions indicate that the effect observed is specific for Ca’+. This is interpreted to be a consequence of the adsorption of Ca*+ on the vesicle bilayers. The variation of lipid and Ca*+ concentrations indicates that stability is achieved at a particular Ca*+/lipid ratio of 8 mohmol above which vesicles are stable. The stability appears to be mainly conferred by the external Ca*’ in both slow and rapid cycles, independent of the ionic vesicle content. However, internal Ca2+ seems to increase the stability according to the F/T cycle rate to some extent in the absence of Ca*’ in the external solution. 0 1991 Academic Press, Inc.

A significant part of the damage caused to cells during freezing and thawing involves injuries on the membrane structure (20). The nature of these injuries can be explored using multi- and unilamellar liposomes as biomembrane lipid models (21, 22). The different stages the membrane passes through during the freeze-thaw cycles can be studied with an appropriate design of these experimental systems. Thus, details of membrane structure and properties can be studied at each step of the freeze-thaw cycle. For instance, either in a rapid or in a slow freeze-thaw cycle an osmotic unbalance takes place, resulting in an increase in solute concentration inside and outside the cell. The tendency to equilibrate the differences in water chemical potential at both sides of the membrane, especially in slow cycles, promotes a decrease of the cell volume. This shrinkage may induce a spontaneous localized curvature increase (2). Unilamellar vesicles appear to be an experimental system suited to the study of the stability and structural properties of the membranes of high curvature . Received June 2, 1988; accepted June 4, 1990.

Shrinkage of large liposomes by hypertonic stress promotes the formation of small daughter vesicles (2) and defects in the membrane surface (26). These structural changes may affect the bilayer permeability as well as its capacity to adsorb ionic species (7-9). Both consequences have been related to the packing and hydration changes associated with the increase in curvature (5, 6, 16). The packing of the inner lipids in a curved bilayer is different from the outer monolayers (4, 5). Packing and hydration differences between the convex and the concave sides of a sonicated vesicle bilayer affect the strength of Ca*+ adsorption (10). The injury caused by the freezingthawing cycles has been ascribed either to the concentration of salts during ice formation or to a volumetric reduction (11, 24). The latter induces membrane contraction or expansion (27), which in turn would affect the ion adsorption. Thus, ionic adsorption on the lipid interfaces induced by the osmotic stress may affect water activity, volume restrictions, and membrane stability. The stability of curved membranes during the freezing-thawing cycles taken as a 279 OOll-2240/91 $3.00 Copyright All rights

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

280

BAKh

AND

function of the distribution of adsorbable and nonadsorbable ions can be studied using small sonicated vesicles. Among ions of biological interest, Ca2+ presents unique properties with regard to its adsorption on phosphatidylcholine membranes. It preferentially adsorbs on packed gel structures while its interaction with fluid membranes is very weak (18, 19). Thus, it is expected that different distributions of Ca2’ between the surfaces and the bulk aqueous solutions at each side of the bilayer would affect to a different extent membrane stability during freezingthawing cycles. MATERIALS

AND

METHODS

Egg yolk phosphatidylcholine was obtained from eggs using standard methods and purified through a silica gel 60 column (15). Purity of collected fractions was checked by thin-layer chromatography (elution solvent chloroform:methanol:water, 65:25:5). A single spot was detected under iodine vapors in all cases. 5-carboxyfluorescein (CF) was from Eastman-Kodak and purified through an LH20 Sephadex column (25). Merocyanine 540 (MC540) and octadecylrhodamine were obtained from Molecular Probes, Inc. All other chemicals were reagent grade from Mallinkrodt (Argentina). Vesicles were prepared by sonication of liposomes obtained by dispersing a dry lipid film in the desired salt concentrations. All solutions were buffered with 10 mM TrisHCl (pH 7.4). Sonication was performed in a bath sonicator until a clear solution was obtained (usually 30 min) under N2 and below 25°C. Cooling and Warming Procedures Sample vesicle volumes of 1 ml, to minimize temperature gradients inside the sample, were maintained in glass tubes of comparable thickness. Slow cooling was performed by submerging successively the sample tubes in -4, -20, and - 180°C

DISALVO

baths. Fast cooling processes were achieved by submerging the sample tubes in a liquid air bath (- ISO‘C). Warming up of frozen samples was carried out in both a fast and a slow way. Slow processes were achieved by keeping the samples in air at room temperature (warming rate about 0.3”Clmin). Fast warming was performed in a 25°C water bath (ca. S”C/min). Turbidity of samples before and after each freeze-thaw cycle was determined measuring the absorbance at 450 nm in a Hitachi double-beam spectrophotometer. Merocyanine 540 assays were performed adding an MC540 aliquot to vesicle suspensions to obtain a final concentration of about lop5 M. Dye partitions between aqueous and membrane phases were determined by the 5671500 absorbance ratio. Absorbance at 567 nm corresponds to dye in the membrane phase and at 500 nm to dye in water. This ratio changes when the same lipid concentration is present as multilamellar liposomes or as unilamellar vesicles (17) because it depends on the amount of accessible lipid interfaces. Leakage was measured using vesicles trapping 50 mM CF. At concentrations higher than 10 mM, this dye is selfquenched. When liberation or dilution takes place a net increase in fluorescence is observed (25). Vesicles were prepared by sonicating a coarse liposome dispersion loaded with the CF solution. Outer CF was eliminated by elution through a Sephadex G-50 column. Leakage of fluorophore was determined in a M2000 Perkin-Elmer spectrofluorometer at excitation and emission wavelengths of 472 and 520 nm, respectively. The 100% CF trapping was determined by adding 50 ~1 of a 10% (v/v) Triton X-100 solution. Assays with octadecylrhodamine were performed by mixing an aliquot of vesicles containing octadecylrhodamine at 5% mol/ mol ratio with another aliquot of vesicles without fluorophore. At that ratio, octadecylrhodamine is self-quenched and the

STABILITY

DURING

281

FREEZE-THAW

mixing of lipids of the labeled and unlabeled populations gives rise to an increase in fluorescence (13). All assays were performed in duplicate using two different batches of vesicle preparations. Asymmetric Distribution of Ca2+ between the Inner and the Outer Media Vesicles prepared in Ca2’ or Na+ solutions were dialyzed extensively against Na+ or Ca2+ solutions of equal normality to replace the external media. The elimination of Ca2+ was completely achieved by adding EDTA to the dispersion. The absence of external Ca2+ after dialysis was verified by the formation of the Ca2+murexide complex. For experiments in which several conditions were tested all samples belonged to the same vesicle preparations. Each condition was assayed by duplicate for each batch of vesicles. Different batches of vesicles were tested in each case. RESULTS

Turbidity increase, leakage of vesicle content, lipid exchange, and formation of larger particles are well-known consequences of the fusion occurring between acidic phospholipid vesicles triggered by Ca2+ or by freezing-thawing of phosphatidylcholine vesicles. In order to test the intluence of the presence of Ca” on the latter phenomenom in connection with the rate of the freeze-thaw cycles, the four methods were assayed using unilamellar liposomes (sonicated vesicles) as a model system. Turbidity of egg yolk phosphatidylcholine vesicle suspensions increases as a function of the low rate freezing-thawing cycles when Ca2+ is present in the solution. In contrast, few changes in turbidity are found with the same type of vesicles during rapid cycles (Fig. 1). Under the same conditions, a redistribution of lipids among the suspended particles

2 3 N’ Cycles FIG. 1. Effect of freezing and thawing (F/T) cycles on the turbidity of egg phosphatidylcholine vesicle dispersion. F/T cycles were performed at two different rates. Fast (0) and slow (0) (see Materials and Methods). Lipid concentration was 5 mg/ml and vesicles were prepared in 0.1 N CaCl,. 0

1

was observed, using octadecylrhodamine as a membrane probe (Fig. 2). Fluorescence increase obtained during slow cycles indicates that turbidity variations involve a lipid exchange between vesicle populations. This lipid redistribution takes place affecting dramatically the permeability barrier, as indicated by the leakage of CF (Fig. 3). In order to know the type of particles resulting after vesicle disruption, experiments with MC540 were performed. The relative amount of vesicles and liposomes after each cycle can be quantitatively determined by the 567/500 absorbance ratio using MC540. This ratio is lower for a multilamellar dispersion than for a sonicated vesicle suspension, at equal lipid concentrations. When plotted as a function of known relative amounts of liposomes and vesicles, that ratio provides a standard curve to establish the liposome/vesicle ratio at each cycle of the process (Fig. 4A). Figure 4B shows that the 567/500 ratio, for Ca2’ vesicles, decreases with the number of cycles at low rate. No variations are obtained with

BAKAS

AND

DISALVO

N-Cycles

3. Effect of F/T cycles on leakage of 5carboxyfluorescein. Vesicles of egg phosphatidylcholine (total concentration, 5 mg/ml) containing 50 mM CF were alternatively frozen and thawed in slow (0) and fast (0) cycles. Leakage of CF was followed by an increase in fluorescence. Total trapping was obtained after adding 10 ~1 Triton X-100 to the dispersion. FIG.

0

2

1

3

NCycles

FIG. 2. Effect of F/T cycles on the transference of lipid molecules between vesicles, as measured by the dequenching of octadecylrhodamine (Rl8). Vesicles containing 5% mol/mol Rl8/phospholipid were mixed with a vesicle suspension without the fluorophore. The ratio between labeled and nonlabeled populations was 1:4. Results are expressed as the percentage of dequenched rhodamine after the F/T cycles (fast F/T cycles (0) and slow FiT cycles (0)). One hundred percent dequenching was measured by vesicle disruption with Triton X-100.

fast rate cycles. Percentages of liposomes appearing after each low and rapid cycles are shown in Table 1. They were calculated by using the standard curve of Fig. 4A. The results of Figs. l-4 indicate that vesicles prepared in 0.1 h4 CaCl, are stable during fast freezing-thawing cycles. This effect is specific for vesicles prepared in Cazf solutions. Data in Fig. 5 show that vesicles prepared in NaCl concentrations, equivalent to those in CaCl, in Figs. l-4 for the same lipid concentration, are disrupted during the rapid cycles. In addition, fast cycles promote disruptions of the vesicles when the lipid concentration is increased at a constant Ca2+ concentration (Fig. 6).

In accordance with these results, vesicles prepared in decreasing Ca2+ concentrations at constant lipid concentrations are susceptible to disruption during rapid cycles (Fig. 7). In these cases, the preparations of vesicles was performed in Tris buffer without Na+ with the Ca2+ concentrations indicated in the figures. The comparison of the data for zero Ca2’ in Fig. 7 with those obtained in the presence of 0.1 N NaCl (Fig. 5) shows that the ionic strength slightly increases the stability. However, the differences obtained at equal ionic strengths with Ca2’ and Naf show that the effect of Ca2’ is much more pronounced. Thus, it is clear that vesicles prepared in Ca2+ are more stable during the rapid cycles than those prepared at the same Nat/lipid ratio. It can be deduced from Figs. 6 and 7 that vesicles are stable during rapid cycles when the Ca”/Iipid ratio is above 8 mol/mol.

STABILITY

DURING

283

FREEZE-THAW

r" 1.0 -

0.0

I 0

I 50

I

I

1CO

% Liposomes

0 0

I

1

I

1

2

3

N’ Cycles

FIG. 4. Determination of liposome/vesicle percentage during slow and fast F/T cycles using Merocyanine 540 (MC540). (A) Standard curve showing the dependence of the A,,,IA,, absorbance ratio with the relative amount of liposome and vesicle. MC540 (lo-’ M final concentration) was added to suspensions with a total lipid concentration of 0.2 mg/ml. Absorbances at 567 nm (monomer band in membrane) and 500 nm (monomer band in water) and their ratio were determined at each liposome/ vesicle ratio. (B) Effect of slow and fast F/T cycles on the MC540 ratio (A,,,/A,,). Vesicles were frozen and thawed by slow (0) and fast (0) cycles. MC540 was added to the suspension after thawing. Absorbances were determined at 20°C.

The effect of Ca*+ present on the inner or the outer side of the bilayer was studied comparatively in low and fast rate F/T cycles. In these cases, only the results of one cycle were taken into account because the asymmetry of the media is not preserved after the release in the first cycle (Table 2). It is observed that protection is due mainly to external Ca* + on both Ca* + - or Naf-containing vesicles in the slow and fast FR cycles. However, internal Ca*+ seems to be TABLE

1

Percentage of Liposomes Appearing after Low and Rapid Freeze-Thaw (F/T) Cycles of Sonicated Vesicles Percentage liposomes No. cycles

Rapid FfI

Slow F/T

0

20 15 25

10 60 75 85

1 2 3

10

slightly effective in the two cycles when external Ca*+ is absent. In contrast, Na+ vesicles are more unstable during the low rate F/T cycle than during the rapid one. Another difference shown in the Table is that Ca*’ vesicles dispersed in Na+ solutions are more unstable than Naf vesicles under the same conditions. DISCUSSION

The turbidity increase observed during the slow cycles (Fig. 1) is caused by the vesicle disruption into large liposomes (Fig. 4). This process involves leakage of the content and lipid exchange among vesicles (Figs. 2 and 3). The mechanism of this particle transformation can be explained by assuming a close contact among the vesicles at some stage of the cycle. Through that contact transitions to larger and more ordered “mesophase” structures are commonly observed at high concentrations (low water content). Both attractive and repulsive forces between aggregates can lead to such phase transitions (14).

284

BAKh

AND

DISALVO

0 25mg/mI

,120

0.000

-

0060

-

0.040

-

21 ,5

,000

3

x 5 A 75 0 lo

l

” I. .I

0

l

/

2 G .z 5 k-

.040 0

? -_ 0

_.

I 1 N-Cycles

I

2

I

3

FIG. 5. Effect of fast F/T cycles on vesicles prepared at equal ionic strengths. 0.1 N Ca (0); 0.1 N Na (0). All the solutions were prepared in 10 n&f Tris buffer, pH 7.4. Total lipid concentration was 5 mg/ml.

During low rate cycles an increase in the concentration of vesicles may be promoted as a consequence of the extra vesicular ice formation. As in rapid cycles vesicle concentration would not be expected, the probability of vesicle-vesicle contact is lower. This accounts for the difference observed in stability between both cycles. This is supported by the experiments of Fig. 6, which show that rapid cycles affect vesicle stability when lipid concentration is increased. However, vesicles turned unstable (Fig. 7) when they were frozen and thawed several times in a decreasing Ca*’ concentration at low lipid concentration. This observation may be explained assuming that in all the concentrations assayed a high number of vesicle collisions was produced. An increase in Ca* + concentration produced when the solution freezes can be an effective factor in disrupting the vesicles. However, this interpretation would not explain data in Fig. 6, which indicate that sta-

1 N-Cycles

2

3

FIG. 6. Effect of lipid concentration on the turbidity after fast F/T cycles. Vesicles were prepared at different egg phosphatidylcholine concentrations (2.5 mg/ml (O), 5 mg/ml (x), 7.5 mg/ml (A), and 10 mg/ml (0)). In all cases Ca” concentration inside and outside the vesicles was 0.1 N.

bility decreases when the Ca*+/lipid ratio is also decreased. According to Figs. 6 and 7 there is a specific Ca*+/lipid ratio above which vesicles are stable. A third possibility could account for the different behaviors observed during rapid or slow cycles on sonicated vesicles prepared with Na+ or Ca*‘. The efficiency factor modulating the success of each vesicle-vesicle encounter to form a larger aggregate would be related to the “preparation” of the membrane in each freezing process. During low rate cycles the formation of ice in the extravesicular millieu would promote a water outflux. Hence, vesicles tend to shrink. This vesicle collapse would promote packing and curvature defects which would be different for Na+ and Ca*’ vesicles. As Ca*’ vesicles are larger than those prepared in NaCl at equivalent concentrations (9), they have a greater chance to decrease their volume during the low rate cycle by osmotic shrinkage. This would give rise to a change in Ca*+ concentration inside the vesicle and to a simultaneous packing increase of the membrane. In this direction, previous re-

STABILITY

.2cc

DURING

‘F

l

0 0.25 N cd* x 0.10 A 0.05 00

l

/

A60

2 % I%80

N-Cycles

7. Effect of ionic strength on the turbidity after fast F/T cycles. Vesicles were prepared in different Ca2+ concentration solutions (0 N(O), 0.05 N(A), 0.1 N (x), and 0.25 N (0)). Lipid concentration was 5 mg/ml. FIG.

sults have shown by means of fluorescence anisotropy that a rigidity increase of the bilayer is promoted by the Ca*’ inner concentration in hypertonic solutions (Bakas and Disalvo, in press). TABLE 2 Effect of the Internal and the External Ca2+ Concentration on the Stability of Unilamellar Vesicles during a Low and Fast Rate F/T Cycle Vesicle content

Dispersing solution

NaCl CaCl, CaCl, NaCl

NaCl NaCl CaCl, CaCl,

Fast rate cycle 0 19.6 * 3.2 96.8 5 1.9 95.5 f 2.3

Low rate cycle 28.8 21.1 81.3 79.9

+ 0.4 If: 4.7 k 0.3 2 1.4

Note. All the solutions were 0.1 N in the indicated salt in 10 mM Tris buffer pH 7.4. Percentage of stability was obtained taking the turbidity increase in rapid FiT cycle in Nat vesicle dispersed in NaCl as the reference of minimum of stability. Data correspond to the average of two different batches of vesicles and duplicates for each condition.

FREEZE-THAW

285

It must be stressed that the Ca*+ binding on the inner monolayer is stronger than that on the outer one. Thus, Ca*+ distribution between the surface and the solution would be different at each side of the membrane (10). The asymmetric distribution of Ca*’ between the membrane faces has been shown to affect membrane stability (9, 23). On the other hand, in rapid cycles water outflux is much slower than the freezing of the solution. So both inner and outer solutions are frozen without any osmotic stress. Under these conditions a rapid freezing nucleation of ice in the inner media has been postulated (11). In this case, Ca*’ at each side is increased simultaneously. In addition, as far as packing is concerned, the quality of the surfaces would remain unchanged. Therefore, the Ca*+ adsorption on the bilayer would be similar at both sides. Hence, stability would depend on the Ca*+/lipid ratio at each membrane interface. It must be noted that this stability is specifically conferred by Ca*+. This is clear from the comparison of the results obtained with Tris buffer, Tris buffer with 0.1 N NaCl, and Tris buffer with 0.1 N CaCl, shown in Figs. 5 and 7. The effectiveness of the preservation method depends on the ionic atmosphere at each side of the membrane. In our case, Ca*+ on the outer side confers stability regardless of the F/T rate (see Table 2). However, the effect on stability of the F/T rate is also dependent on the Ca*+ content of the vesicle (Table 2), although to a much lesser extent. The protection of external Ca*+ does not significantly depend on the Ca*’ content of the vesicle. It is possible that the electrical charge conferred by Ca*’ adsorption could be involved in this behavior. Closely related to these results, it has been shown that phosphatidylserine bilayers are disrupted in low rate freezing cycles. The effect has been ascribed to a dehydration that promotes phase transforma-

286

BAKh

AND

tions (3). In these types of membranes, bound Ca*+ will be the major determinant of the collapse processes. In addition, this would depend mainly on the size of the interacting vesicles (1). Ca2+ is an ion unevenly distributed between the cell and the environment and between the cytoplasm and the organelles (12). It has also been reported that microorganisms grown in the presence of Ca*+ have a higher resistance to freeze and thaw than those grown in Na+ (28). In conclusion, vesicles are stable during rapid cycles when they contain Ca*+ . However, Na+ vesicles are unstable during both rapid and slow cycles. The difference in responses of both membrane systems can be ascribed to the Ca*+/lipid ratio at membrane interfaces of the vesicle bilayer. This opens the possibility that stability obtained at different freeze-thaw rates would be intimately linked to Ca*’ distribution at membrane interfaces. This distribution might, in turn, be sensitive to the osmotic stress occurring at the membrane level.

DISALVO

5.

6.

7.

8. 9. 10.

11. 12. 13.

ACKNOWLEDGMENTS

is a member of the Research Career of the Consejo National de Investigaciones Cientlficas y Tecnicas de la Rephblica Argentina (CONICET). L.S.B. is the recipient of a fellowship of the Comisi6n de Investigaciones Cientificas de la Provincia de Buenos Aires (CIC B.A.) This work was supported by funds from CONICET, CIC B.A., and SECYT.

14.

E.A.D.

REFERENCES

1. Bentz, J., Nir, S., and Wilschut, J. Mass action kinetics of vesicle aggregation and fusion. ColZoids Surf. 6, 333-363 (1983). 2. Boroske, E., Elwenspoek, M., and Helfrick, W. Osmotic shrinkage of giant egg-lecithin vesicles. Biophys. J. 34, 95-109 (1981). 3. Caffrey, M. The combined and separate effects of low temperature and freezing on membrane lipid mesomorphic phase behavior: Relevance to cryobiology. Biochim. Biophys. Acta 896, 123-127 (1987). 4. Chrzeszczyk, A., Wishnia, A., and Spuriger, C. S., Jr. Evidence for cooperative effects in

15.

16. 17.

18.

19.

20.

the binding of polyvalent surfaces. Biochim. Biophys. Actu 648, 2-8 (1981). Cornell, B. A., Middlehurst, J., and Separovic, F. The molecular packing and stability within highly curved phospholipid bilayers. Biochim. Biophy. Acta 598, 405-510 (1980). Disalvo, E. A., and de Gier, J. Contribution of aqueous interphases to the permeability barrier of lipid bilayers for non-electrolytes. Chem. Phys. Lipids 32, 3-7 (1983). Disalvo, E. A. Divalent cations promote transversal perturbations in lipid bilayers of sonicated vesicles. Bioelectrochem. Bioenerg. 11(2-3), 145-154 (1984). Disalvo, E. A. Thermodynamic factors determining the barrier properties of lipid bilayers. Chem. Phys. Lipids 38, 385-397 (1985). Disalvo, E. A. Leakage from egg phosphatidylcholine vesicles induced by Ca2+ and alcohols. Biochim. Biophys. Acta 905, 9-16 (1987). Disalvo, E. A., and Bakas, L. S. Stoichiometry of the binding of Ca2+ on the inner and the outer monolayers of sonicated vesicle bilayers. Bioelectrochem. Bioenerg. 20, 257-267 (1988). Franks, F. “Biochemistry and Biophysics at Low Temperatures.” Cambridge University Press, London/New York, 1985. Gordon, L. M., and Saverheber, R. D. In “The Role of Ca2+ in Biological Systems” (Anghileri, Ed.). CRC Press, Boca Raton, FL, 1982. Hoekstra, D., De Boer, T., Klappek, K., and Wilschutt, J. Fluorescence methods for measuring the kinetics of fusion between biological membranes. Biochemistry 23,5675-5681(1984). Israelachvili, J. N. “Intermolecular and Surface Forces.” Academic Press, London, 1985. Kates, M. “Techniques of Lipidology: Isolation Analysis and Identification of Lipids” (T. Work, ed.). Amer. Elsevier/North-Holland, New York/Amsterdam 1972. Lawaczek, R. On the permeability of water molecules across vesicular lipid bilayers. .Z. Membr. Biol. 51, 229-261 (1979). Lelkes, P. I., and Miller, I. R. Perturbations of membrane structure by optical probes: I. Location and structural sensitivity of Merocyanine 540 bound to phospholipid membranes. .Z. Membr. Biol. 52, 1-15 (1980). Lis, J. L., Lis, W. T., Parsegian, V. A., and Rand, R. P. Adsorption of divalent cations to a variety of phosphatidylcholine bilayers. Biochemistry 20, 1771-1777 (1981). McLaughlin, A., Grathwohl, C., and McLaughlin, S. The adsorption of divalent cations to phosphatidylcholine bilayer membranes. Biochim. Biophys. Acta 513, 338-357 (1978). Meryman, H. T., Williams, R. J., and Douglas,

STABILITY

21. 22.

23. 24.

25.

DURING

M. S. J. Freezing injury from “solution effects” and its prevention by natural or artiticial cryoprotection. Cryobiology 14, 287-302 (1977). Morris, G. J., and Clarke, A. (Eds.) “Effects of Low Temperatures on Biological Membranes.” Academic Press, New York, 1981. Morris, G. J. The response of liposomes to various rates of cooling to - 196°C: Effect of phospholipidcholesterol ratio. Cryobiology 19,215218 (1982). Papahadjopoulos, D., and Ohki, S. Stability of asymmetric phospholipid membranes. Science 164, 1075-1077 (1969). Rall, W. F., Mazur, P., and Souzu, H. Physical Chemical Basis of the protection of slowly frozen human erythrocyte by glycerol. Biophys. J. 23, 101-120 (1978). Ralston, E., Hjelmellan, L. M., Klausner, R. D., Weinstein, J. W., and Blumenthal, R. Carbox-

FREEZE-THAW

287

ifluorescein as a probe for liposome-cell interactions: Effect of impurities and purification of the dye. Biochim. Biophys. Acta 649, 133-137 (1981). 26. Senisterra, G. A., Disalvo, E. A., and Gagliardino, J. J. Osmotic dependence of the lysophosphatidylcholine lytic action on liposomes in the gel state. Biochim. Biophys. Acta 941, 26&270 (1988). 27. Steponkus, D. L., Wolfe, J., and Dowgert, M. F. Stresses induced by contraction and expansion during a freeze-thaw cycle: A membrane perspective. In “Effects of Low Temperatures on Biological Membranes” (G. J. Morris and A. Clarke, Eds.), pp. 307-322. Academic Press, New York, 1981. 28. Wright, C. T., and Klaenhammer, T. Survival of Lactobacillus bulgaricus during freezing and freeze-drying after growth in the presence of calcium. J. Food Sci. 48, 773-777 (1983).