Vol. 276, Issue 2, C291-C299, February 1999
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
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ABSTRACT |
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,
H2PO4, 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
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INTRODUCTION |
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.
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EXPERIMENTAL METHODS |
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.
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RESULTS |
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.
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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.
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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
SO24 is relatively low (18.8 J · K1 · mol1)
compared with that for the typical channel-opening ions,
K+ (102.5 J · K1 · mol1)
and NO3 (146.6 J · K1 · mol1)
(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 HSO4,
which has a high positive entropy of hydration, displacing the
equilibrium H+ + SO24 
HSO4. 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
HSO4 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.
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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
HSO4 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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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DISCUSSION |
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
HSO4 and Cl but made an excellent
preservative solution with citrate.
Na+, on the other hand, impaired
survival when the concentration of
HSO4 increased, but embryos
survived well with Na+ and
citrate, Cl, or
SO24.
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.
| |
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Abstract
Introduction
