Preservation of murine embryos in a state of dormancy at 4°C

Philippa M. Wiggins, Jamie Rowlandson, and Alexander B. Ferguson

Department of Medicine, University of Auckland School of Medicine, Auckland 1003, New Zealand


    ABSTRACT
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Abstract
Introduction
Experimental methods
Results
Discussion
References

With the aim of improving preservation of blood products and organs for transplantation, we designed solutions to induce a state of dormancy in cells and tissues at 4°C. The solutions were devoid of combinations of ions (e.g., K+, Rb+, Cs+, and NH+4 with HCO-3, H2PO-4, and Cl-) that are believed to break down low-density water in the entrance compartments of ion channels, resulting in cyclical open states (normal water) and closed states (low-density water). The total osmolality was always 0.29-0.3 osmol/kgH2O, made up of combinations of a di- or trisaccharide, a compatible solute, sodium sulfate, citrate, or chloride, and 1.75 mM CaCl2. The end point was the ability of murine embryos to progress to hatching in culture after preservation in such a solution at 4°C. Embryos hatched after 5 or 6 days in some preservative solutions compared with 1-3 days in most saline solutions; survival was improved by pretreatment with sodium butyrate.

osmosis; water; hydrophobicity; ion channels


    INTRODUCTION
Top
Abstract
Introduction
Experimental methods
Results
Discussion
References

HIBERNATING VERTEBRATES USE many strategies to survive cold in the winter. A common combination (13) is prevention of ion fluxes by freezing of extracellular solutions, using special proteins to nucleate ice, and, at the same time, synthesis of high intracellular concentrations of glucose. We have tried to mimic these strategies, initially without freezing, by inducing in cells a state of dormancy. Methods have been developed, invoking a proposal (15-18, 21-23) that has been shown to describe osmosis and osmotic equilibria in aqueous solutions and gels of polymers better than the classical van't Hoff relation (14). Instead of freezing the extracellular solution, we lowered the temperature to 4°C and closed ion channels to prevent ion fluxes (39, 40); instead of synthesizing intracellular glucose, we pretreated embryos with sodium butyrate. Strengthening of the barrier properties of the lipid bilayer at low temperatures has also contributed to dormancy (20).

Closing of Ion Channels

It is suggested (16, 18) that a channel is closed when its entrance compartment contains viscous low-density water that prevents influx of highly hydrated ions (Na+, Ca2+, Mg2+, and H+). Collapse of that water back to its normal density, viscosity, and solvent properties opens the channel, allowing previously excluded ions in. The selectivity of the channel is determined by a specific filter region, but the open or closed state of the channel is determined by the state of water in the entrance compartment. In the absence of specific channel-opening ligands, these channels open and close spontaneously, the viscous plug of low-density water forming and collapsing cyclically. This behavior of water in small hydrophobic or weakly hydrogen-bonding cavities has been observed on a macroscopic time scale (16) using microporous polyamide beads that resemble proteins in the surfaces that they present to water and resemble ion channel entrance compartments in their pore size (1-2 nm in diameter). These experiments identified channel-opening ions as combinations of cations and anions commonly classed as water structure breakers [K+ or NH+4 together with Cl- and, especially, univalent oxyanions (2, 6)].

Intracellular Low-Density Water

The double layer at a charged polymeric surface is a second site of stressed water. Water inside the double layer has a lower activity than water outside. Again water responds to this state of disequilibrium by expanding where its activity is high and compacting where its activity is low. Thus intracellular water also contains separated regions of high- and low-density water populations. The viscosity of intracellular water and the rate of metabolic processes inside cells are both critically dependent on the proportions of high- and low-density water (19).

Lipid Bilayer and Stressed Water

Water near a lipid bilayer is in a similar state of osmotic stress, as common polar head groups have either a single negative charge (phosphatidic acid and phospatidylinositol), one negative and one positive charge (phosphatidylcholine and phosphatidylethanolamine), or two negative and one positive charge (phosphatidylserine). Each charged group has a counterion in solution so that there is a large excess of solute particles in the double layer. Again, water contracts in the double layer and expands outside it. At the lipid tails, water is in a state of high enthalpy and tends to expand. Where water is doubly stressed because it is both of high enthalpy and of high activity, perturbed by two surfaces, it escapes from its state of stress, the lipid tails come together, and the membrane assembles itself. The greater the degree of energetic stress of water at the lipid surfaces and osmotic stress of water at the charged interfaces, the stronger the stabilization of the bilayers.

Strategy for Design of Preservative Solutions

1) Solutions contain no combinations of channel-opening ions so that influx of NaCl and loss of K+ are minimized. This maintains a high intracellular viscosity that is decreased by NaCl and increased by K+ (19). 2) Solutions contain small solutes that specifically stabilize the lipid bilayer by increasing the osmotic and energetic stress of water. These are trimethylamine oxide (TMAO), betaine, and polyols. They also help keep channels closed. 3) When cells contain high concentrations of NaCl they are pretreated with sodium butyrate to increase the amount of intracellular low-density water, increase intracellular viscosity, and further depress metabolism during storage. 4) Solutions are isotonic with the tissue to be preserved. 5) The storage temperature is 4°C.


    EXPERIMENTAL METHODS
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Abstract
Introduction
Experimental methods
Results
Discussion
References

Preparation of Solutions

Solutions of mixed solutes were prepared from 290 mosmol/kgH2O stock solutions of single solutes. For many of the solutions, vapor pressure varied with temperature; osmolality was therefore measured as freezing point depression in an Advanced Osmometer model 3W, as that was nearest to 4°C at which solutions were used for storage. At the concentrations used, osmolalities were additive so that all mixtures of the stock solutions had the correct osmolality.

Mice

CBA/C57 mice were supplied by the Animal Research Unit of the Auckland Medical School. After weaning at 21 days, the female mice were placed in cages of 10. They were superovulated by intraperitoneal injections given as close to weaning as possible. The first injection of 0.1 ml of 50 IU/ml pregnant mares' serum gonadotropin, manufactured by Intervet and supplied by Pharmaco, was given between noon and 4 PM, and the mice were returned to their cages. Two days later, 0.1 ml of 50 IU/ml human chorionic gonadotropin was injected, between noon and 2:30 PM, and the mice were placed in a cage of intact male stud mice. Embryos were at the eight-cell stage 2 days after mating. Mice were killed by cervical dislocation, placed on their backs, and washed with 70% alcohol. The underside of the mouse was cut open between the hind legs using dissection scissors. The incision was extended up each side of the mouse to the ribs. The flap of skin was folded upwards, and the intestines were moved to one side, exposing the uterus. Oviducts were then located and removed, together with a small part of the uterus. The piece of tissue was placed on the lid of a 90-mm sterile petri dish. Under a microscope, a 30-gauge needle attached to a 1-ml syringe was inserted into the oviduct where it meets the uterus. Flushing solution was forced through the oviduct, washing out all the embryos. Embryos were then counted, graded, and pooled in the flush solution in a 35-mm petri dish on ice while embryos were harvested from other mice. The flushing solution was usually the solution in which they were to be stored, containing 0.1% bovine albumin (Immuno Chemical Products, Auckland, New Zealand) to prevent embryos from adhering to surfaces.

Embryos from up to 10 mice were pooled and held in storage solution at 4°C before being distributed (usually 10 embryos at a time) randomly among various storage regimes. Embryos were transferred from one solution to another using a mouth pipette with which embryos could be delivered with very little carryover (a few microliters) of the first solution into the second.

In early experiments, usually 10 embryos were put into 150 µl in each small petri dish and covered with 3.5 ml of paraffin to keep the embryos in a small volume. In later experiments, paraffin was dropped because it appeared to have toxic effects, and embryos were stored in 150 µl in Eppendorf tubes at 4°C for various time intervals. After storage, embryos were transferred to 200 µl DMEM (no. 31600-034, GIBCO Laboratories, Life Technologies, Gaithersburg, MD) containing penicillin-streptomycin-neomycin (100×; no. 15640-055, GIBCO Laboratories, Life Technologies) and 0.1% bovine albumin and were overlayed with 3.5 ml of paraffin. Dishes were preequilibrated in the incubator for 1 day before use. Embryos were incubated for 2 or 3 days at 37°C. They were then assessed for progress in culture, expressed as the percentage of original embryos that had reached a late blastocyst or hatched blastocyst stage or were still alive. Because there were variabilities among embryos on different occasions, we included as a control embryos stored either in PBS or in a murine medium (OCM) that was PBS containing Ca2+ and Mg2+. Embryo variation followed a pattern that appeared to depend on the age of the mice. Survival of embryos in control solutions was variable over time. Therefore, only internal comparisons with controls were meaningful. There appeared to be a correlation between the numbers of embryos obtained from a superovulated mouse and the quality of those embryos.

Some embryos were pretreated at room temperature with PBS in which some NaCl had been replaced by sodium butyrate. NaCl in PBS was sequentially replaced with sodium butyrate at concentrations from 5 to 138 mM, keeping the osmolality constant at 0.29 osmol/kgH2O. After pretreatment for various times, embryos were transferred to preservative solution at 4°C.


    RESULTS
Top
Abstract
Introduction
Experimental methods
Results
Discussion
References

Nonelectrolyte Solutions

The first solutions used contained only nonelectrolyte mixtures in various ratios and combinations, together with 1.5-2 mM calcium chloride or calcium sulfate. These were not particularly successful solutions, but embryos maintained their morphology in the light microscope and some were still alive after 3 days at 4°C and at least some hatched. Solutions contained a large solute (sucrose, lactose, trehalose, or raffinose) that seemed to be necessary to maintain the integrity of the zona pellucida, together with a smaller molecule, a compatible solute, sugar, or polyol (TMAO, betaine, sarcosine, glucose, mannose, fructose, galactose, ribose, sorbitol, inositol, or taurine). None of these solutes could cross membranes passively, because they were either too big or too hydrophilic. These were combined in various ratios (1.4:1 to 2.0:1), with the larger molecule always in excess. The best combination was that of raffinose and TMAO in a molar ratio of ~1.6:1. Figure 1 shows the percent survival of embryos stored in solutions of these two solutes in various ratios, comparing them with PBS. After 3 days at 4°C, 25-75% of stored embryos hatched or nearly hatched in culture. They performed better than those in PBS. Comparison with PBS is rather arbitrary, because the two sets of conditions are so different: in PBS at 4°C, channels still open and close spontaneously; Na+, Ca2+, K+, and Cl- diffuse passively across membranes; and Na+ and Ca2+ are actively transported outward and K+ is actively transported inward. The Na+ gradient drives glucose in, and the solution is oxygenated. Anabolism and catabolism both continue more slowly than at 37°C and are probably not as evenly balanced. In the preservative solutions, presumably, there is no flux of ions across membranes and residual metabolism is slower.


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Fig. 1.   Survival of embryos following storage in solutions containing a range of osmolal ratios of raffinose and trimethylamine oxide (TMAO) together with 1.75 mM CaCl2; total osmolality was 0.29 osmol/kgH2O. Percentage that progressed to hatching or near hatching [hatched blastocysts and late blastocysts (HB + LB)] and percentage still alive but not progressed as far are shown. Storage was for 1 (A), 2 (B), or 3 (C) days. C, control.

Dependence on Ca2+ in medium. Figure 2 shows the biphasic dependence of survival of embryos on Ca2+ in solutions that were otherwise devoid of electrolytes. Embryos were stored for 2 or 3 days in 0.29 osmol/kgH2O solutions of raffinose and TMAO at an osmolar ratio of 1.6:1 containing CaCl2 at concentrations from 0 to 2 mM. There was no survival even after 2 days in the absence of Ca2+. At both 2 and 3 days, the best survival was in solutions containing between 1.5 and 2 mM CaCl2. In subsequent experiments, we used 1.75 mM CaCl2. Although there appeared to be better survival with 0.5 mM, concentrations on either side of 0.5 mM fell sharply, suggesting that it might not be a real effect. The higher concentration range with many compositions appeared to be the most stable.


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Fig. 2.   Percentages of embryos that hatched after storage in a 0.29 osmol/kgH2O solution of raffinose and TMAO in an osmolar ratio of 1.6:1 together with 0-2 mM CaCl2. [Ca2+], Ca2+ concentration.

Mixed Electrolyte-Nonelectrolyte Solutions

Recovery of embryos following storage at 4°C was improved by the addition of electrolytes. K+ and other channel-opening cations were easily avoided, but it was difficult to exclude all channel-opening anions. Because, even in as small a volume as the entrance compartment of an ion channel, macroscopic electroneutrality must be conserved, both an anion and a cation are required for channel opening. An anion with a high enough partition coefficient would be effective even with Na+, a cation that partitions preferentially into normal water. All univalent anions, with their high positive entropies of hydration (the criterion for preferential partition into low-density water) (18), were avoided. The first electrolyte-containing solution consisted of mixtures of TMAO and Na2SO4 with 1.75 mM CaSO4. The conventional entropy of hydration of SO2-4 is relatively low (18.8 J · K-1 · mol-1) compared with that for the typical channel-opening ions, K+ (102.5 J · K-1 · mol-1) and NO-3 (146.6 J · K-1 · mol-1) (12). The result is illustrated in Fig. 3. Previous experiments had shown that 290 mosmol/kgH2O TMAO was not a good storage solution. This is confirmed in Fig. 3. Na2SO4 was almost as good as the control solution for 1 day, but embryos did not survive or progress after storage for much longer than that. Figure 3 is remarkable for the deleterious effect of just 10% TMAO on survival of embryos. This had to be a specific interaction between Na2SO4 and TMAO because subsequent experiments (see Fig. 4) showed that, when both were diluted with raffinose, survival following storage was high. The methyl groups of TMAO induce a zone of low-density water that selectively accumulates HSO-4, which has a high positive entropy of hydration, displacing the equilibrium H+ + SO2-4 right-left-harpoons  HSO-4. This made available during storage a powerfully channel-opening anion that, apparently, could take Na+ with it. The impairment of survival then followed, with fluxes of ions stimulating pumps that rapidly used up residual internal energy. Survival was increased at pH 8.5, when the concentration of dissociation of HSO-4 decreased.


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Fig. 3.   Survival of embryos following preservation in solutions made from mixtures of equiosmolar (0.29 osmol/kgH2O) solutions of TMAO and Na2SO4 to which 1.75 mM CaSO4 was added. Means ± SE; duplicates contained 10 embryos each. Control solution was 70% raffinose-TMAO and ~30% Na2SO4 with 1.75 mM CaCl2 (70/30a). Preservation was for 1 (A), 2 (B), or 3 (C) days.

When this experiment was repeated using K2SO4 and TMAO, even after only 1 day there was no survival at all except in 100% TMAO (i.e., the combination of K+ and HSO-4 destroyed the barrier to ion fluxes).

Raffinose-TMAO and sodium sulfate. The harmful effects of TMAO on Na2SO4 were largely abolished when it was diluted with raffinose. Figure 4 shows results of storing embryos in mixtures of equiosmolar solutions of raffinose-TMAO (ratio 1.6:1) and Na2SO4. Neither 100% Na2SO4 nor 100% raffinose-TMAO was as effective as a solution consisting of ~70% raffinose-TMAO and ~30% Na2SO4. Like all solutions used, this one contained 1.75 mM CaCl2. It became one of the most-used solutions for other applications and was given the code 70/30a.


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Fig. 4.   Survival of embryos following preservation in solutions made from mixtures of equiosmolar (0.29 osmol/kgH2O) solutions of raffinose-TMAO (ratio 1.6:1) and Na2SO4 to which 1.75 mM CaSO4 was added. Means ± SE; duplicates contained 10 embryos each. Preservation was for 1 (A), 2 (B), or 3 (C) days.

Raffinose-TMAO and potassium citrate. The citrate ion is too hydrophobic to partition selectively into low-density water. It was tried as a safe anion to pair with K+, partly to test the proposal that both anion and cation were needed to open channels. Figure 5 shows that 20 or 30% isosmolar potassium citrate added to raffinose-TMAO (ratio 1.6:1) was at least as good as 70/30a, confirming both that citrate does not partition selectively into ion channels and that K+ is ineffective as a channel opener in the absence of a suitable anion.


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Fig. 5.   Survival of embryos following preservation in solutions made from mixtures of equiosmolar (0.29 osmol/kgH2O) solutions of raffinose-TMAO (ratio 1.6:1) and tripotassium citrate to which 1.75 mM CaCl2 was added. Control was preservation solution 70/30a. Poststorage culture was for 3 days. Means ± SE; duplicates contained 10 embryos each. Preservation was for 3 (A), 4 (B), or 5 (C) days.

Raffinose-TMAO and sodium citrate. When K+ in the above solution was replaced with Na+, survival of embryos was considerably improved. Figure 6 shows that this was one of the most successful preservative solutions. Again, ~30% isosmolar ions seemed optimal. These results show an extreme example of the variability of controls at different times. Internal comparisons with controls show that sodium citrate was a better solute than potassium citrate.


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Fig. 6.   Survival of embryos following preservation in solutions made from mixtures of equiosmolar (0.29 osmol/kgH2O) solutions of raffinose-TMAO (ratio 1.6:1) and trisodium citrate to which 1.75 mM CaCl2 was added. Control was preservation solution 70/30a. Poststorage culture was for 3 days. Preservation was for 1 (A), 2 (B), 3 (C), or 4 (D) days.

Raffinose-TMAO and NaCl. Although Cl- in conjunction with K+ collapses low-density water, NaCl does not (21). Figure 7 shows that combinations of raffinose, TMAO, and NaCl made good preservative solutions for embryos, especially in the region of 20-50 osmolar % NaCl.


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Fig. 7.   Survival of embryos following preservation in solutions made from mixtures of equiosmolar (0.29 osmol/kgH2O) solutions of raffinose-TMAO (ratio 1.6:1) and NaCl to which 1.75 mM CaCl2 was added. 70/30, control solution 70/30a. Poststorage culture was for 3 days. Preservation was for 3 (A), 4 (B), or 5 (C) days.

TMAO and NaCl. Raffinose was found to be unnecessary in NaCl-containing solutions. The solution of 30% NaCl and 70% TMAO, given the code name 70/30b (Fig. 8), was subsequently used very successfully in many applications. It was only successful, however, when there was little carryover from the endogenous K+-containing medium into the preservative solution. Because embryos were transferred from one solution to another with only microliters of solution, they survived very well. A rat heart, perfused with cold 70/30b, stored, and reperfused with warm Krebs solution also survived well (P. M. Wiggins and N. S. Fernando, unpublished observations). Some blood cells, on the other hand, diluted 1:1 with 70/30b, failed to survive, presumably because the activity of KCl in the resulting mixed solution was too high.


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Fig. 8.   Survival of embryos following preservation in solutions made from mixtures of equiosmolar (0.29 osmol/kgH2O) solutions of TMAO and NaCl to which 1.75 mM CaCl2 was added. Means ± SE. Poststorage culture was for 3 days. Control solution was preservation solution 70/30a. P, PBS. Preservation was for 2 (A), 3 (B), 4 (C), or 5 (D) days.

Pretreatment of Embryos With Sodium Butyrate

Cells that have relatively high concentrations of NaCl contain predominantly high-density, reactive fluid water that promotes enzyme activity and accelerates diffusion (19). Their survival in storage can therefore be expected to be limited because residual metabolism is high and energy stores are rapidly exhausted. It has been proposed that cells are activated to perform a biological function (e.g., to grow) by influx of ions such as Na+, which make the intracellular solution more fluid; they then return to a resting viscous state when ions are pumped out across the plasma membrane or back into intracellular stores (18, 19). Ideally, cells should be put in preservative solution at the moment when they have just returned to their resting state. Because this is not possible in practice, the alternative strategy of converting intracellular high-density water into low-density water was tried. Embryos were pretreated at room temperature with PBS in which some NaCl had been replaced by sodium butyrate. All concentrations of butyrate (5, 10, 15, 20, 35, 70, and 140 mM butyrate) and times of pretreatment (5, 10, 15, 20, and 30 min) gave dramatic improvement in embryo survival. This is illustrated in the single example of Fig. 9. Other data are not shown because they were so similar. Survival in raffinose-TMAO solutions and in 70/30a were improved with pretreatment. Survival in PBS was also enhanced. Survival after pretreatment in PBS without butyrate, on the other hand, was impaired.


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Fig. 9.   Percentage of embryos that hatched following storage in PBS, in raffinose-TMAO, or in 70/30a with or without pretreatment with 70 mM sodium butyrate in PBS at room temperature for 10 min: a, 70/30a without pretreatment; b, PBS without treatment; c, 70/30a pretreated with sodium butyrate; d, raffinose-TMAO without pretreatment. Means ± SE. Culture was for 2 days.

Sodium butyrate and culture for 3 days. Survival of embryos stored in 70/30a without pretreatment equalled that of butyrate-treated embryos when they were cultured for 3 instead of 2 days. Presumably butyrate decreased the loss of energy stores so that embryos were able to grow and hatch more quickly. Figure 10, compared with Fig. 9, illustrates the difference between culture for 2 and 3 days. In experiments cultured for 3 instead of 2 days, the large differences between embryos that were alive and embryos that had hatched disappeared, indicating that embryos that had not hatched in 2 days either proceeded to hatch in 3 days or died.


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Fig. 10.   Survival of embryos after 3 days of culture following storage in 70/30a without pretreatment, PBS without pretreatment or 70/30a with 3 different times of pretreatment (in min) with 70 mM sodium butyrate. Means ± SE. Preservation was for 1 (A), 2 (B), 3 (C), 4 (D), or 5 (E) days.

Bacteria Do Not Grow in Preservative Solutions

Storage of cells and tissues at 4°C in preservative solutions is reliable only if the solutions inhibit growth of microorganisms. Volumes (10 ml) of solution or nutrient broth were separately inoculated with 105 cells/ml of either Escherichia coli or Staphylococcus aureus. Cultures were then placed at 4°C for 28 days. At 7, 14, 21, and 28 days, samples of each culture were inoculated on blood agar plates and bacterial numbers determined. Although large increases in bacterial numbers for both organisms were observed in broth cultures over the 28-day period, there were no increases in numbers of E. coli or S. aureus in preservative solution cultures. Similar experiments using a stored suspension of platelets and two microorganisms (Yersinia enterocolitica and Listeria monocytogenes) that are known to grow in nutrient broth at 4°C also gave negative results.


    DISCUSSION
Top
Abstract
Introduction
Experimental methods
Results
Discussion
References

At 4°C in preservative solutions, metabolism was suppressed but not eliminated; embryos continued to use energy. Nevertheless, the solutions are useful for considerable improvement in safe short-term preservation of tissues at 4°C and have also been successful for freezing without conventional cryoprotectants.

Specificity of Solutes and Their Interactive Relationship With High- and Low-Density Water

Closing of ion channels. The experimental results are consistent with the proposal that cations and anions that accumulate selectively into low-density water open ion channels. If channels were open in all solutions, survival in 70/30b would always be impossible as NaCl, CaCl2, and water diffused freely into cells, stimulating ATPases, activating other enzymes, and rapidly depleting ATP. We observed that cells that began storage with high concentrations of NaCl and water (e.g., tumor cell lines) survived poorly in any preservative solution. But survival of less active cells in 70/30b and other Na+-containing solutions was good in the absence of K+ and lightly hydrated univalent anions.

The experiments highlight the need for macroscopic electroneutrality in even small aqueous cavities: K+ impaired survival in the presence of HSO-4 and Cl- but made an excellent preservative solution with citrate. Na+, on the other hand, impaired survival when the concentration of HSO-4 increased, but embryos survived well with Na+ and citrate, Cl-, or SO2-4.

Roles of TMAO and other compatible solutes. Trimethylamines (betaine and TMAO), taurine, and polyols have important physiological functions (3, 5, 8, 9, 24) that appear to be a consequence of their amphiphilic character and the way in which they partition rather evenly between high- and low-density water at both charged and hydrophobic surfaces. A consequence is that the hydrophobic moiety of the molecule is in contact with low-density water and the hydrogen bond that is lost is stronger than normal, necessitating formation of very low density water or escape of water and aggregation of surfaces. In the preservation experiments, the solutes were confined to the extracellular solution, where they stabilized the bilayer, which is more tightly constrained by escape of highly stressed water and is a correspondingly better barrier. The solutes also helped to close ion channels.

Effects of countercations at charged surfaces. Ca2+ is the most effective countercation for lipid bilayers. It is held close to the phospholipid head groups so that the double layer is thin and the osmotic stress is high. Water has a strong tendency to escape, and the bilayer is tightly folded. Li+ is a poor replacement. When we used Li2SO4 with raffinose and TMAO to replace the highly successful Na2SO4, very few embryos survived even for 1 day. Li+ at a concentration of 48 mM must have displaced enough Ca2+ from the double layers of lipids to decrease the osmotic stress of water and loosen the bilayer structure. The success of Na+ as a component of a preservative solution and the failure, for different reasons, of K+ and Li+, the ions most closely resembling it, illustrate the subtlety of ion-water interactions in the presence of hydrophobic and charged surfaces.

Maximizing intracellular low-density water. Embryos are actively growing cells, presumably oscillating between active states, in which influx of Na+ and cascades of phosphorylation have increased the fluidity of intracellular compartments, and resting states, in which ion gradients have been restored, viscosity is high, and metabolism is slow (19). Survival in a preservative solution is maximal when all cells are resting at the moment of coming into contact with the solution. Thus bone marrow stem cells (unpublished observations) survive for weeks instead of days at 4°C. Survival of active cells that are growing (embryos) or contracting (heart cells) can be prolonged if the amount of low-density intracellular water is increased before storage. This can be achieved either by decreasing intracellular Na+ before storage or by introducing into cells small solutes that induce low-density water around polymers (e.g., the protein-stabilizing solutes betaine and TMAO) (3, 5, 8, 9, 24).

The most successful pretreatment of embryos was with sodium butyrate, which is known to have important physiological effects (1, 10). For example, fermentation of dietary fiber in the colonic lumen produces short-chain fatty acids, including butyrate, which inhibits the development of a malignant phenotype (7).

The mechanism of action of sodium butyrate. Cooke and Macknight (4) showed that acetic acid diffused passively into renal cortical cells from an acetate-containing solution and dissociated into an acetate ion and an H+. The Na+/H+ exchanger exchanged H+ for Na+, and the Na+-K+-ATPase exchanged Na+ for K+. The result was an increase in intracellular potassium acetate and water. Presumably, butyrate in the extracellular solution did the same: pretreatment of embryos resulted in accumulation of potassium butyrate, which was unable to leave cells during storage. The butyrate ion has a hydrophobic moiety that, by itself, partitions strongly into high-density water, and a carboxyl moiety that, together with K+, partitions strongly into low-density water. Because electroneutrality must be maintained, the ion pair partitions fairly evenly between high- and low-density populations of water at the charged surface of proteins and between normal and low-density water at hydrophobic-hydrophilic surfaces. Thus potassium butyrate behaves as a compatible solute inside cells, increasing intracellular viscosity and slowing metabolism. Embryos thus loaded with potassium butyrate used up their ATP more slowly during storage and could grow and progress after only 2 days of culture.

In conclusion, solutions that were designed to suppress metabolism at 4°C appear to have been successful, giving support to the underlying assumptions, most of which have been directly tested before only in nonliving systems of aqueous gels. Other applications include preservation of rat hearts, human platelets, and human and murine bone marrow cells at 4°C and freezing of platelets and bone marrow cells without cryoprotectants, using the same principles of manipulation of high- and low-density water.


    ACKNOWLEDGEMENTS

Present addresses: A. B. Ferguson, Genesis Research and Development Corp., 1 Fox St., Parnell, PO Box 50, Auckland 1001, New Zealand; J. Rowlandson, AC Nielsen, 129 Hurstmere Rd., Takapuna, Auckland 1309, New Zealand.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and present address of P. M. Wiggins: Genesis Research and Development Corp., 1 Fox St., Parnell, PO Box 50, Auckland 1001, New Zealand.

Received 8 July 1998; accepted in final form 22 October 1998.


    REFERENCES
Top
Abstract
Introduction
Experimental methods
Results
Discussion
References

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3.   Burg, M. B., E. D. Kwon, and E. M. Peters. Glycerophosphocholine and betaine counteract the effect of urea on pyruvate kinase. Kidney Int. Suppl. 57: S100-S104, 1996[Medline].

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Am J Physiol Cell Physiol 276(2):C291-C299
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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