Contributions of unfrozen fraction and of salt concentration to the survival of slowly frozen human erythrocytes: Influence of warming rate

The general belief is that slow freezing injury is either the result of exposure to high salt concentrations or the result of excessive cell shrinkage. Increased salt concentration arises as increasing amounts of pure ice precipitate out of solution during freezing and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. Cell shrinkage is an osmotic response to the concentration of external solutes. The consensus has been that the injury is related to the composition of the solution in these channels and not to the amount of residual liquid.Ordinarily, salt concentration and the amount of liquid in the unfrozen channels are reciprocally related; but they can be separated within limits by varying the total concentration of solutes in the suspending medium while holding the mass ratio of additive to salt constant, and by then slowly freezing samples to various subzero temperatures, chosen to produce various molalities of salt, while holding the unfrozen fraction constant, or vice versa. We have recently reported (9) that when human red cells are frozen under these conditions and thawed rapidly, survival is more dependent on the unfrozen water fraction than it is on the salt concentration in that fraction. The present work compares these results with those obtained with slow thawing. While the general conclusion remains unaltered, slowly thawed cells were able to survive the freezing of a higher fraction of extracellular water than were rapidly thawed cells.Calculations were made of the changes in cell volume during the equilibration with glycerol and the subsequent freezing involved in these experiments. Cell size and cell solute concentration were found to be independent of the fraction of unfrozen extracellular water, but cell survival was strongly dependent on that fraction. If applicable to other than human red cells, this finding is likely to require major modifications in current views of slow-freezing injury and its prevention.     

Roles of unfrozen fraction, salt concentration, and changes in cell volume in the survival of frozen human erythrocytes

The cause of slow freezing injury and the basis of the protection by solutes like glycerol are subjects of debate. During slow freezing, cells are sequestered in unfrozen channels between ice crystals that grow by removing pure water from the channels. As a consequence, the solute concentration in the channels rises and the volume of liquid in the channels progressively decreases. The rise in solute concentration, in turn, causes the cells to progressively shrink osmotically. Until recently cryobiologists have ascribed slow freezing injury to either the rise in solute (electrolyte) concentrations in the channels or to the consequent cell shrinkage, rather than to the decrease in the of the channels. Although ordinarily reciprocally coupled, it is possible to separate the composition of the channels from their size, or more precisely from the magnitude of the unfrozen fraction, by suspending cells in NaCl/cryoprotectant solutions in which the mole ratio of the two is held constant, but the molality of the NaCl is allowed to vary below and above isotonic. When human red cells are frozen in such solutions to temperatures that produce given NaCl concentrations (ms), but varying unfrozen fractions (U), survival at low U is found to be strongly dependent on U but independent of ms. At higher values of U, survival becomes inversely dependent on both ms and U. Although cell volume during freezing is independent of the NaCl tonicity in the solution, the cells in the several solutions differ in volume both prior to the onset of freezing and after the completion of thawing. We have now examined and compared the effect of returning the thawed cells to isotonic solutions and isotonic volume or nearly so, and find that there is little change in survival after exposure to low U, but that survival after exposure to high U values exhibits substantially increased sensitivity to ms, a sensitivity that is probably a manifestation of posthypertonic hemolysis. Low values of U were in general attained by the use of solutions with low tonicities of NaCl, and as a consequence cells frozen to low U values had larger volumes prior to freezing than cells frozen to higher U values. The significance of this confounding is discussed.     

The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature

We have previously reported [Cryobiology 51 (2005) 29-53] that intracellular ice formation (IIF) in mouse oocytes suspended in various concentrations of glycerol and ethylene glycol (EG) occurs at temperatures where the percentage of unfrozen water is about 6% and 12%, respectively, even though the IIF temperatures varied from -14 to -41 degrees C. However, because of the way the solutions were prepared, the concentrations of salt and glycerol or EG in that unfrozen fraction at IIF were also rather tightly grouped. The experiments reported in the present paper were designed to separate the effects of the unfrozen fraction at IIF from that of the solute concentration in the unfrozen fraction. This separation makes use of two facts. One is that the concentration of solutes in the residual liquid at a given subzero temperature is fixed regardless of their concentration in the initial unfrozen solution. However, second, the fraction unfrozen at a given temperature is dependent on the initial solute concentration. Experimentally, oocytes were suspended in solutions of glycerol/buffered saline and EG/buffered saline of varying total solute concentration with the restriction that the mass ratios of glycerol and EG to salts are held constant. The oocytes were then cooled rapidly enough (20 degrees C/min) to avoid significant osmotic shrinkage, and the temperature at which IIF occurred was noted. When this is done, we find, as previously that the fraction of water remaining unfrozen at the temperature of IIF remains nearly constant at 5-8% for both glycerol and EG even though the IIF temperatures vary from -14 to -50 degrees C. But unlike the previous results, the salt and CPA concentrations in the unfrozen fraction vary by a factor of three. The present procedure for preparing the solutions produces a potentially complicating factor; namely, the cell volumes vary substantially prior to freezing: substantially greater than isotonic in some solutions; substantially smaller in others. However, the data in toto demonstrate that cell volume is not a determining factor in the IIF temperature.     

Relative influence of unfrozen fraction and salt concentration on the survival of slowly frozen eight-cell mouse embryos

This laboratory has previously reported that the survival of frozen-thawed human erythrocytes is determined more by the fraction of the extracellular solution that remains unfrozen than by the salt concentration in that fraction, especially when the cells are frozen at low hematocrit. To determine the extent to which these findings are applicable to nucleated mammalian cells, we have studied the survival of some 3300 mouse embryos as a function of the unfrozen fraction and the concentration of salt in that unfrozen fraction. Also varied in the study was the weight percentage ratio of glycerol to salt. The concentration of embryos in these experiments (i.e., the cytocrit) was so low that embryo-embryo contacts should have been rare during the freezing. As in the case of the red cells at low hematocrit, we find that the survival of slowly frozen eight-cell embryos is not affected by the high concentrations of salt produced by freezing, at least up to 3.3 molal NaCl, and therefore is not affected by the extent to which the cells shrink below their isotonic volume, nor in general is survival influenced by the temperature at which given salt concentrations and unfrozen fractions are attained or by the glycerol concentration at those temperatures. On the other hand, the attainment of low values of the unfrozen fraction (U) is damaging, but the damage appears in part to be due to the fact that low values of U had to be achieved by placing embryos in solutions hypotonic with respect to NaCl, which caused their volume to be greater than isotonic prior to freezing.     

On the mechanism of injury to slowly frozen erythrocytes.

When cells are frozen slowly in aqueous suspensions, the solutes in the suspending solution concentrate as the amount of ice increases; the cells undergo osmotic dehydration and are sequestered in ever-narrowing liquid-filled channels. Cryoprotective solutes, such as glycerol, reduce the amount of ice that forms at any specified subzero temperature, thereby controlling the buildup in concentration of those other solutes present, as well as increasing the volume of the channels that remain to accommodate the cells. It has generally been thought that freezing injury is mediated by the increase in electrolyte concentration in the milieu surrounding the cells, rather than reduction of temperature or any direct action of ice. In this study we have frozen human erythrocytes in isotonic solutions of sodium chloride and glycerol and have demonstrated a correlation between the extent of damage at specific subzero temperatures, and that caused by the action at 0 degrees C of solutions having the same composition as those produced by freezing. The cell lysis observed increased directly with glycerol concentration, both in the freezing experiments and when the cells were exposed to corresponding solutions at 0 degrees C, showing that the concentration of sodium chloride alone is not sufficient to account quantitatively for the damage observed. We then studied the effect of freezing in anisotonic solutions to break the fixed relationship between solute concentration and the volume of the unfrozen fraction, as described by Mazur, P., W. F. Rall, and N. Rigopoulos (1981. Biophys. J. 653-675). We confirmed their experimental findings, but we explain them differently. We ascribe the apparently dominant effect of the unfrozen fraction to the fact that the cells were frozen in, and returned to, anisotonic solutions in which their volume was either less than, or greater than, their physiological volume. When similar cell suspensions were subjected to a similar cycle of increase and then decrease in solution strength, but in the absence of ice (at 20 degrees C), a similar pattern of hemolysis was observed. We conclude that freezing injury to human erythrocytes is due solely to changes that occur in the composition of their surrounding milieu, and is most probably mediated by a temporary leak in the plasma membrane that occurs during the thawing (reexpansion) phase.     

Factors contributing to inactivation of isolated thylakoid membranes during freezing in the presence of variable amounts of glucose and NaCl.

During freezing of isolated spinach thylakoids in sugar/salt solutions, the two solutes affected membrane survival in opposite ways: membrane damage due to increased electrolyte concentration can be prevented by sugar. Calculation of the final concentrations of NaCl or glucose reached in the residual unfrozen portion of the system revealed that the effects of the solutes on membrane activity can be explained in part by colligative action. In addition, the fraction of the residual liquid in the frozen system contributes to membrane injury. During severe freezing in the presence of very low initial solute concentrations, membrane damage drastically increased with a decrease in the volume of the unfrozen solution. Freezing injury under these conditions is likely to be due to mechanical damage by the ice crystals that occupy a very high fraction of the frozen system. At higher starting concentrations of sugar plus salt, membrane damage increased with an increase in the amount of the residual unfrozen liquid. Thylakoid inactivation at these higher initial solute concentrations can be largely attributed to dilution of the membrane fraction, as freezing damage at a given sugar/salt ratio decreased with increasing the thylakoid concentration in the sample. Moreover, membrane survival in the absence of freezing decreased with lowering the temperature, indicating that the temperature affected membrane damage not only via alterations related to the ice formation. From the data it was evident that damage of thylakoid membranes was determined by various individual factors, such as the amount of ice formed, the final concentrations of solutes and membranes in the residual unfrozen solution, the final volume of this fraction, the temperature and the freezing time. The relative contribution of these factors depended on the experimental conditions, mainly the sugar/salt ratio, the initial solute concentrations, and the freezing temperature.     

Influence of cell concentration on the contribution of unfrozen fraction and salt concentration to the survival of slowly frozen human erythrocytes

We have shown previously that the survival of human red blood cells during slow freezing at 2% hematocrit is dependent more on the magnitude of the unfrozen fraction than on the salt concentration in that unfrozen fraction. In parallel, first Nei and more recently Pegg and colleagues have shown that survival is affected by the hematocrit of the suspension. Freezing at hematocrits above 30% becomes increasingly damaging. The present studies were designed to see whether there is a link between the two phenomena. Cells were suspended at nominal hematocrits of 0.4, 2, 8, 40, or 60% in five test solutions of glycerol-NaCl. The test solutions were of such composition that when frozen to a specified temperature, the magnitude of the unfrozen fraction differed but the NaCl concentration (ms) remained constant. At low hematocrits (0.4 to 8%), red cell survival was dependent predominantly on the unfrozen fraction and was relatively independent of the salt concentration in that fraction. This we term the "rheological" effect because injury appears to be related to interaction with the ice walls and perhaps is due to shearing forces or cell deformation. But at high hematocrits (40 or 60%), cell survival became dependent on both the unfrozen fraction and the salt concentration in that fraction. When freezing occurs at high hematocrits, increasing numbers of cells are presumably brought into contact with their neighbors. Furthermore, they are increasingly shrunken cells, for the progressive removal of liquid water, which is responsible for the crowding, also causes a rise in ms and the consequent osmotic shrinkage of cells. Our data suggest that at unfrozen fractions above those producing injurious rheological forces, the tight packing of less shrunken cells (i.e., high hematocrit, low ms) and the extensive shrinking of loosely packed cells (high ms, low hematocrit) are both quite innocuous. Injury becomes substantial only when extensively shrunken cells are brought into close contact (i.e., high ms, high hematocrit). At high hematocrit the cells occupy a substantial fraction of the unfrozen space, and the water that they lose during slow freezing adds substantially to the volume of extracellular ice. Accordingly, we defined other measures of unfrozen fraction that include these perturbations. However, we found that the conclusions on the relation between survival, unfrozen fraction, and hematocrit were not affected by the method of expressing the unfrozen fraction. Freezing at high hematocrit to high ms and low values of unfrozen fraction is one way to produce contact between shrunken cells at low temperatures.(ABSTRACT TRUNCATED AT 400 WORDS)     

The "unfrozen fraction" hypothesis of freezing injury to human erythrocytes: a critical examination of the evidence

This paper examines the experimental evidence presented by Mazur and his colleagues to support their hypothesis that the survival of slowly frozen human red blood cells is primarily dependent on the fraction of water that remains unfrozen, rather than on the high concentrations of sodium chloride produced by the formation of ice. This hypothesis is in direct conflict with the general belief that freezing injury under such conditions is caused by the concentration of solutes in the solution surrounding the cells: if the "unfrozen fraction" hypothesis is true, then much of the evidence supporting that belief must be dismissed as mere coincidence. We have reexamined Mazur's data, and have suggested an alternative explanation--that cells which are initially suspended in solutions that are not isotonic differ in their susceptibility to subsequent freezing and thawing, shrunken cells being more resistant and swollen cells more susceptible than normal cells. If this is true then the data can be explained without invoking a direct effect of the unfrozen fraction, solely on the basis of changes in the concentration of the solution surrounding the cells. We cite other experimental evidence, obtained in the absence of freezing, that red blood cells do indeed possess the required property. We further argue that the known effects of variations in cooling and warming rate, and in hematocrit, are able to account for the features observed by Mazur and his colleagues in their three published studies.     

Extra- and intracellular ice formation in mouse oocytes

The occurrence of intracellular ice formation (IIF) during freezing, or the lack there of, is the single most important factor determining whether or not cells survive cryopreservation. One important determinant of IIF is the temperature at which a supercooled cell nucleates. To avoid intracellular ice formation, the cell must be cooled slowly enough so that osmotic dehydration eliminates nearly all cell supercooling before reaching that temperature. This report is concerned with factors that determine the nucleation temperature in mouse oocytes. Chief among these is the concentration of cryoprotective additive (here, glycerol or ethylene glycol). The temperature for IIF decreases from -14 degrees C in buffered isotonic saline (PBS) to -41 degrees C in 1M glycerol/PBS and 1.5M ethylene glycol/PBS. The latter rapidly permeates the oocyte; the former does not. The initial extracellular freezing at -3.9 to -7.8 degrees C, depending on the CPA concentration, deforms the cell. In PBS that deformation often leads to IIF; in CPA it does not. The oocytes are surrounded by a zona pellucida. That structure appears to impede the growth of external ice through it, but not to block it. In most cases, IIF is characterized by an abrupt blackening or flashing during cooling. But in some cases, especially with dezonated oocytes, a pale brown veil abruptly forms during cooling followed by slower blackening during warming. Above -30 degrees C, flashing occurs in a fraction of a second. Below -30 degrees C, it commonly occurs much more slowly. We have observed instances where flashing is accompanied by the abrupt ejection of cytoplasm. During freezing, cells lie in unfrozen channels between the growing external ice. From phase diagram data, we have computed the fraction of water and solution that remains unfrozen at the observed flash temperatures and the concentrations of salt and CPA in those channels. The results are somewhat ambiguous as to which of these characteristics best correlates with IIF.     

Effect of hypertonicity on survival of unprotected human cultured cells following freezing and thawing

An investigation was carried out on the post-thaw survival of unprotected human heteroploid EUE cells, either maintained in isotonic medium (0.137 M NaCl) or adapted to hypertonicity (0.356 M NaCl) and frozen in medium with an increased concentration of NaCl. A fivefold increase in the survival fraction of the adapted cells in comparison with the unadapted ones was observed when cells were frozen in isotonic medium. When cells were frozen in hypertonic medium (0.356 M NaCl), the two cell types exhibit comparable survival values. The results are discussed, with special attention to cell defense mechanisms against freezing injury.     

Permeability of the human erythrocyte to glycerol in 1 and 2 m solutions at 0 or 20 °C

There are increasing numbers of exceptions to a central tenet in cryobiology that low-molecular-weight protective solutes such as glycerol must permeate cells in high concentration in order to protect them from freezing injury. To test this supposition, it is necessary to determine the amount of solute that has permeated a cell prior to freezing. The amount in human red cells was estimated from the flux equation $\text{ds}\text{dt}\text{= P}{}_{\mathrm{<mtext>\gamma </mtext>}}\text{A}$[(activity external solute) — (activity internal solute)]. Solving the equation required knowledge of P_γ the permeability constant for the solute. Estimates of P_γ for glycerol were made in two ways: (i) by measuring the time to 50% hemolysis of human red cells suspended in 1 or 2 m solutions of glycerol that were hypotonic with respect to NaCl, and (ii) by measuring the time required for red cells in 1 or 2 m solutions of glycerol in isotonic saline-buffer to undergo osmotic shock upon tenfold dilution with isotonic saline-buffer. The measurements were made at 0 and 20 °C. The values of P_γ were about 2.5 × 10^?4 cm/min at 20 °C and about 0.9 × 10^?4 cm/min at 0 °C. The difference corresponds to an activation energy of 7.2 kcal/mole. These values of P_γ are 100 to 600 times higher than those for glycerol permeation in the bovine erythrocyte. The values of P were relatively unaffected by whether calculations were based on classical or irreversible thermodynamics and by the choice of concentration units in the flux equations. Calculations of the kinetics of glycerol entry using these P values showed that the concentration of intracellular glycerol reaches 90% of equilibrium in 1.2 min at 0 °C and in 0.6 min at 20 °C. The osmolal ratio of intracellular glycerol to intracellular nonpermeating solutes reaches 90% of equilibrium in 7 min at 0 °C and in 3.2 min at 20 °C.     

Colligative and non-colligative freezing damage to thylakoid membranes

When spinach thylakoid membranes were frozen in vitro in solutions containing constant molar ratios of cryotoxic to cryoprotective solute, maintenance of functional integrity strongly depended on initial osmolarities. Optimum cryopreservation of cyclic photophosphorylation was observed when the membranes were suspended in solutions of intermediate osmolarities (approx. 50–100 mM NaCl, 75–150 mM sucrose). Both higher and lower initial osmolarities were found to result in decreased cryopreservation. In the absence of added salt, more than 100 mM sucrose were needed for full cryopreservation of the membranes. When thylakoids were frozen in solutions containing low concentrations of NaCl (2 mM), the ratio of sucrose to salt necessary to give full protection was high (up to 50). When the salt concentration was about 60 mM, ratios as low as 1.5 were sufficient for maintaining membrane integrity. This ratio increased again, as the initial NaCl concentration was increased beyond 60 mM. During freezing, proteins dissociated from the membranes, and the amount of the released proteins was correlated linearly with inactivation of photophosphorylation. The gel electrophoretic pattern of proteins released at low initial osmolarities differed from that of proteins released at high initial osmolarities. Cryopreservation was also found to depend on membrane concentration. Concentrated membrane suspensions suffered less inactivation than dilute suspensions. The protective effect of high membrane concentrations was particularly pronounced at high initial solute concentrations. It is proposed that damage at low initial osmolarities is caused predominantly by mechanical stress and by osmotic contraction/expansion. Damage at high initial osmolarities is thought to be caused mainly by solute effects. Under these conditions, both the final volume of the unfrozen solution in coexistence with ice and the membrane concentration affect membrane survival by influencing the extent of the loss of membrane components through dissociation reactions. Membrane protection by sugars is caused by colligative action under these circumstances.     

Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake

AIM: To evaluate the effect of modifications of water activity (aw 0. 996-0.92) of a molasses medium with different solutes (glycerol, glucose, NaCl, proline or sorbitol) on growth, intracellular water potentials (psi(c)) and endogenous accumulation of polyols/sugars in the biocontrol yeast Candida sake. METHODS AND RESULTS: Modification of solute stress significantly influenced growth, psi(c) and accumulation of sugars (glucose/trehalose) and polyols (glycerol, erythritol, arabitol and mannitol) in the yeast cells. Regardless of the solute used to modify aw, growth was always decreased as water stress increased. Candida sake cells grew better in glycerol- and proline-amended media, but were sensitive to NaCl. The psi(c) measured using psychrometry showed a significant effect of solutes, aw and time. Cells from the 0.96 aw NaCl treatment presented the lowest psic value (- 5.20 MPa) while cells from unmodified media (aw = 0. 996) had the highest value (- 0.30 MPa). In unmodified medium, glycerol was the predominant reserve accumulated. Glycerol and arabitol were the major compounds accumulated in media modified with glucose or NaCl. In proline media, the concentration of arabitol increased. In glycerol- and sorbitol-amended media, the concentration of glycerol rose. Some correlations were obtained between compatible solutes and psi(c). CONCLUSIONS AND SIGNIFICANCE: This study demonstrates that subtle changes in physiological parameters significantly affect the endogenous contents of C. sake cells. It may be possible to utilize such physiological information to develop biocontrol inocula with improved quality.     

Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest

Phase diagrams are of great utility in cryobiology, especially, those consisting of a cryoprotective agent (CPA) dissolved in a physiological salt solution. These ternary phase diagrams consist of plots of the freezing points of increasing concentrations of solutions of cryoprotective agents (CPA) plus NaCl. Because they are time-consuming to generate, ternary diagrams are only available for a small number of CPAs. We wanted to determine whether accurate ternary phase diagrams could be synthesized by adding together the freezing point depressions of binary solutions of CPA/water and NaCl/water which match the corresponding solute molality concentrations in the ternary solution. We begin with a low concentration of a solution of CPA+salt of given R (CPA/salt) weight ratio. Ice formation in that solution is mimicked by withdrawing water from it which increases the concentrations of both the CPA and the NaCl. We compute the individual solute concentrations, determine their freezing points from published binary phase diagrams, and sum the freezing points. These yield the synthesized ternary phase diagram for a solution of given R. They were compared with published experimental ternary phase diagrams for glycerol, dimethyl sulfoxide (DMSO), sucrose, and ethylene glycol (EG) plus NaCl in water. For the first three, the synthesized and experimental phase diagrams agreed closely, with some divergence occurring as wt% concentrations exceeded 30% for DMSO and 55% for glycerol, and sucrose. However, in the case of EG there were substantial differences over nearly the entire range of concentrations which we attribute to systematic errors in the experimental EG data. New experimental EG work will be required to resolve this issue.     

Freezing damage of bovine erythrocytes: Simulation using glycerol concentration changes at subzero temperatures

When a cell is frozen and thawed, it is exposed to (i) lowered temperature, (ii) increased solute concentration during freezing, and (iii) decreased solute concentration during thawing. Without actually freezing the cells, an attempt has been made to simulate physical-chemical changes to which bovine erythrocytes are exposed when frozen and thawed in glycerol solutions. Experimentally, the study consisted of suspending erythrocytes in 1, 2, or 3 glycerol at 20 °C for various times and then exposing them to each of several dilution sequences. The dilution sequences were: (i) transfer from the initial glycerol concentration at 20 °C into the same concentration at −5 °C, (ii) transfer into an increased glycerol concentration at 20 °C, (iii) transfer into an increased followed by a decreased glycerol concentration at 20 °C, (iv) transfer into an increased glycerol concentration at −5 °C, and (v) transfer into an increased followed by a decreased glycerol concentration at −5 °C. This last sequence is analogous to the exposure that cells undergo at subzero temperatures to increased solute concentration during freezing and decreased solute concentration during thawing. This dilution sequence yielded a survival pattern very similar to that obtained when bovine erythrocytes are frozen and thawed, and thus does appear to mimic freezing damage. It is concluded that a major factor in freezing damage is the extent to which a cell must shrink or swell to achieve osmotic equilibrium at subzero temperatures in partially frozen or thawed solutions.     

Analysis of “solution effects” injury: Rabbit renal cortex frozen in the presence of dimethyl sulfoxide

Slices of rabbit renal cortex were frozen in 0.64 or 1.92 M dimethyl sulfoxide (Me₂SO) to various subzero temperatures, thawed, and assayed for viability. Salt and Me₂SO concentrations were calculated and correlated with the injury taking place during freezing. In separate experiments, slices were treated with NaCl or Me₂SO in concentrations sufficient to simulate the exposure brought about as a result of freezing. The effects of these treatments on cortical viability were compared with the results of freezing to equivalent concentrations of either NaCl or Me₂SO. The results show that whereas slices will tolerate exposure to at least six times the isotonic concentration of NaCl at 0 °C, they are unable to tolerate even three times the isotonic salt concentration when frozen in 1.92 M Me₂SO. They can, however, tolerate 3 × NaCl when frozen in 0.64 M Me₂SO. Freezing damage did not depend upon the amount of ice formed per se, since slices frozen in the low concentration of Me₂SO tolerated removal of about 75% of the initial fluid content of the system, whereas slices frozen in 1.92 M Me₂SO did not tolerate an identical removal of unfrozen solution. It was found that treatment of slices with high concentrations of Me₂SO at subzero temperatures in accordance with Elford's application (14) of Farrant's method (20) produced damage which correlated approximately with the damage observed when the same concentrations of Me₂SO were produced by freezing. It is concluded that most of the damage caused by freezing in 1.92 M Me₂SO is produced either directly or indirectly by Me₂SO. Possible mechanisms for this injury are discussed.     

Cryoprotection of mammalian cells in tissue culture with polymers; possible mechanisms

Studies were undertaken to more clearly define the mechanism of cryoprotection by polymers. Significant cryoprotection of Chinese hamster cells in tissue culture was found in the presence of hydroxyethyl starch (HES), polyvinylpyrrolidone (PVP), and dextran. The addition of PVP to the medium after thawing did not increase the survival of these cells. The presence of PVP in the medium was shown to have no effect on the transport mechanism for alanine in unfrozen cells. The source of freeze-thaw injury did not appear to be due to a direct effect on this transport mechanism. Several physical parameters of polymeric solutions were monitored at subzero temperatures. The freezing point depression was found to increase dramatically at higher polymer concentrations. Tests on the NaCl concentration in the liquid fraction of partially frozen solutions showed that the increase in salt concentration with decreasing temperature was similar in the presence of 10% PVP or 2.5% DMSO, two agents which gave similar cryoprotection at these concentrations. NMR studies showed that polymers could retain water in the liquid state at temperatures as low as −35° C, and that the remaining water was highly structured. The cryoprotective properties of polymers appear to reside in their ability to alter the physical properties of solutions during the freezing process rather than in direct effects on cell membranes.     

Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium--a revisit

We have previously reported that intracellular ice formation (IIF) in mouse oocytes suspended in glycerol/PBS solutions or ethylene glycol (EG)/PBS solutions and rapidly cooled to −50 °C or below occurs at temperatures where a critical fraction of the external water remains unfrozen [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53; P. Mazur, I.L. Pinn, F.W. Kleinhans, The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature, Cryobiology 54 (2007) 223-233]. For mouse oocytes in PBS or glycerol/PBS that fraction is 0.06; for oocytes in EG that fraction was calculated to be 0.13, more than double. The fractions unfrozen are computed from ternary phase diagrams. In the previous publication, we used the EG data of Woods et al. [E.J. Woods, M.A.J. Zieger, D.Y. Gao, J.K. Critser, Equations for obtaining melting points for the ternary system ethylene glycol/sodium chloride/Water and their application to cryopreservation., Cryobiology 38 (1999) 403-407]. Since then, we have determined that ternary phase diagrams for EG/NaCl/water synthesized by summing binary phase data for EG/water NaCl/water gives substantially different curves, which seem more realistic [F.W. Kleinhans, P. Mazur, Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest, Cryobiology 54 (2007) 212-222]. Unfrozen fractions at the temperatures of IIF computed from these synthesized phase diagrams are about half of those calculated from the Woods et al. data, and are in close agreement with the computations for glycerol; i.e., IIF occurs when about 92-94% of the external water is frozen. A parallel paper was published by Guenther et al. [J.F. Guenther, S. Seki, F.W. Kleinhans, K. Edashige, D.M. Roberts, P. Mazur, Extra-and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes, Cryobiology 52 (2006) 401-416] on IIF in oocytes of the frog Xenopus. It too examined whether the temperatures of IIF were related to the unfrozen fractions at those temperatures. It also used the Woods et al. ternary phase data to calculate the unfrozen fractions for EG solutions. As reported here, once again the values of these unfrozen fractions are substantially different from those calculated using synthesized phase diagrams. With the latter, the unfrozen fractions at IIF become very similar for EG and glycerol.     

Cell inactivation and membrane damage after long-term treatments at sub-zero temperature in the supercooled and frozen states

The survival of cells subjected to cooling at sub-zero temperature is of paramount concern in cryobiology. The susceptibility of cells to cryopreservation processes, especially freeze-thawing, stimulated considerable interest in better understanding the mechanisms leading to cell injury and inactivation. In this study, we assessed the viability of cells subjected to cold stress, through long-term supercooling experiments, versus freeze-thawing stress. The viability of Escherichia coli, Saccharomyces cerevisiae, and leukemia cells were assessed over time. Supercooled conditions were maintained for 71 days at -10 degrees C, and for 4 h at -15 degrees C, and -20 degrees C, without additives or emulsification. Results showed that cells could be inactivated by the only action of sub-zero temperature, that is, without any water crystallization. The loss of cell viability upon exposure to sub-zero temperatures is suggested to be caused by exposure to cold shock which induced membrane damage. During holding time in the supercooled state, elevated membrane permeability results in uncontrolled mass transfer to and from the cell maintained at cold conditions and thus leads to a loss of viability. With water crystallization, cells shrink suddenly and thus are exposed to cold osmotic shock, which is suggested to induce abrupt loss of cell viability. During holding time in the frozen state, cells remain suspended in the residual unfrozen fraction of the liquid and are exposed to cold stress that would cause membrane damage and loss of viability over time. However, the severity of such a stress seems to be moderated by the cell type and the increased solute concentration in the unfrozen fraction of the cell suspension.     

The effect of initial tonicity on freeze/thaw injury to human red cells suspended in solutions of sodium chloride

Human red blood cells, suspended in solutions of sodium chloride, have been frozen to temperatures between -2 and -14 degrees C and thawed, and the extent of hemolysis was measured. In parallel experiments, red cells were exposed to similar cycles of change in the composition of the suspending solution, but by dialysis at 21 degrees C. The tonicity of the saline in which the cells were initially suspended was varied between 0.6x isotonic and 4x isotonic; some samples from each experimental treatment were returned to isotonic saline before hemolysis was measured. It was found that the tonicity of the saline used to suspend the cells for the main body of the experiment affected the amount of hemolysis measured: raising the tonicity from 0.6x to 1x to 2x reduced hemolysis, both in the freezing and in the dialysis experiments, whereas raising the tonicity further to 4x reversed that trend. There was little difference between the freeze/thaw and the dialysis treatments for the cells suspended in 1x or 2x saline, whether or not the cells were returned to isotonic conditions. However, the cells suspended in 0.6x saline showed greater damage from freezing and thawing than from the comparable change in the composition of the solution, whether or not they were returned to isotonic conditions. Cells that were suspended in 4x saline and exposed to changes in salt concentration by dialysis showed less hemolysis when they were assayed in the 4x solution than cells that had received the comparable freezing/thaw treatment, but when the experiment included a return to isotonicity, the two treatments gave similar results. Returning the cells to isotonic saline had a negligible affect on the cells in 0.6x and 1x saline, but caused considerable hemolysis in the 2x and 4x samples, more so after dialysis than after freezing and thawing. We conclude that cells suspended in 0.6x and 4x saline behave differently from cells suspended in 1x and 2x saline and hence that cells suspended in a range of solutions of differing initial tonicity should not be treated as a homogeneous population. We argue that an effect of the unfrozen fraction of water (U) cannot be distinguished, within the framework of these freeze/thaw experiments alone, from an effect of initial tonicity, and that the biphasic nature of the correlation between haemolysis and U makes a causal connection improbable.(ABSTRACT TRUNCATED AT 400 WORDS)