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Biomedical subjects

P Mazur

Publications and source records attributed to P Mazur.

At least 73 records · Page 4Linked to original sources
PubMed1984

Freezing of living cells: mechanisms and implications.

Cells can endure storage at low temperatures such as--196 degrees C for centuries. The challenge is to determine how they can survive both the cooling to such temperatures and the subsequent return to physiological conditions. A major factor is whether they freeze intracellularly. They do so if cooling is too rapid, because with rapid cooling insufficient cell water is removed osmotically to eliminate supercooling. Equations have been developed that describe the kinetics of this water loss and permit one to predict the likelihood of intracellular freezing as a function of cooling rate. Such predictions agree well with observations. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration. The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage. Recent experiments, however, indicate that the damage is due more to the decrease in the size of the unfrozen channels. This new view of the mechanism of slow freezing injury ought to facilitate the development of procedures for the preservation of complex assemblages of cells of biological, medical, and agricultural significance.

Animals↗
PubMed1984

Osmotic tolerance of human granulocytes.

Human granulocytes are injured when returned to isotonic conditions after exposure at 0 degree C to hyperosmotic solutions of NaCl or sucrose with osmolalities above 0.6 osmolal. The damage was expressed as a loss of membrane integrity [fluorescein diacetate (FDA) assay] only after 60-90 min incubation at 37 degrees C. Survival after exposure to a 1.4-osmolal solution at 0 degree C was dependent on the extent of subsequent dilution. Dilution to below 0.6 osmolal was damaging, but cells could be returned to near-osmotic conditions provided that the solute concentration was increased again to 0.64 osmolal before the cells were incubated at 37 degrees C. Granulocyte cell volumes were measured under various osmotic conditions by computer-assisted micrometry. The cells did not display a minimum volume but behaved as osmometers over the observed range of 0.2-1.4 osmolal. Granulocyte volume at a given osmolality was independent of whether the cells had first been exposed to a strongly hyperosmotic medium, indicating that no solute loading occurred in hyperosmotic sucrose solutions. Even though the cells did not survive sequential exposure to greater than 0.6 osmolal solutions, subsequent return to isotonicity, and incubation at 37 degrees C, neither cell lysis nor loss in FDA-positive cells occurred after the first two steps. This finding is not consistent with the critical-surface area-increment theory of freezing injury. The mechanism of cell injury in hyperosmotic solutions is thus not known. However, the results show that osmotic stress is potentially a major damaging factor both in the equilibration of cells with protective additives and during freezing and thawing.

Blood Preservation↗
PubMed1984

Toxic and osmotic effects of glycerol on human granulocytes.

Human granulocytes are damaged by exposure to concentrations of glycerol as low as 0.5 M. We therefore investigated the addition of glycerol to granulocytes and its subsequent dilution under various conditions to try to distinguish between toxic and harmful osmotic effects of glycerol. The lesion caused by glycerol at 0 degree C was expressed as a loss of plasma membrane integrity (as visualized by fluorescein diacetate) only after incubation (greater than or equal to 1 h) at 37 degrees C. This damage was not ameliorated when osmotic stress was lessened by reducing the rates of addition and dilution of glycerol to keep the computed cell volume within 80-170% of isotonic cell volume. However, when osmotic stress was reduced further by increasing the temperature of addition and dilution of glycerol from 0 degree C to 22 degrees C, the tolerance of the cells to 1 M glycerol increased somewhat. Reducing exposure to glycerol to 3 min or less at 0 degree C greatly increased survival, but this time was too short to allow glycerol to equilibrate intracellularly. Finally, the presence of extra impermeant solute (NaCl or sucrose) in the medium to reduce the equilibrium cell volume to 60% of isotonic cell volume enabled granulocytes to survive 30-min exposure to 1 M glycerol at 0 degree C, but cells had to remain shrunken during the 37 degrees C incubation to prevent the loss of membrane integrity. Suspensions that contained damaged granulocytes formed aggregates when incubated at 37 degrees C, and these aggregates were responsible for a major fraction of the observed loss in viability.

Blood Preservation↗
PubMed1983

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.

Animals↗
PubMed1983

Interactions of cooling rate, warming rate, glycerol concentration, and dilution procedure on the viability of frozen-thawed human granulocytes.

Difficulties in the successful freezing of human granulocytes could lie at two levels. One is that critical cryobiological variables have not yet been identified, the other is that the inconsistent results may be due to unusual biological aspects of the cell. This paper is concerned with the former. A prerequisite for the successful freezing of mammalian cells is the ability of the cell to tolerate cryoprotective levels of additive. The additive studied here was glycerol. Based on fluorescent staining with fluorescein diacetate, we found that 1 and 2 M concentrations are in fact chemically toxic at 22 degrees C. Superimposed on this toxicity is some osmotic sensitivity to the removal of the additive by other than slow dilution. The dilution procedure was selected on the basis of computer modeling of the osmotic response of the cells. The model requires a value for the permeability coefficient for glycerol. The value (4 X 10(-5) cm/min) was obtained by measuring the rate of increase of the volume of cells in hyperosmotic glycerol. The response of human granulocytes to freezing to -196 degrees C and thawing in 1 or 2 M glycerol was not unusual. The optimum cooling rate was 1-3 degrees C/min, and cooling at 10 degrees C/min or faster was especially deleterious if warming was slow (1 degree C/min) rather than rapid (188 degrees C/min). The FDA assay showed that some 75% of the cells survived freezing and thawing at optimum rates in 1 or 2 M glycerol; and some 50-60% remained viable after the glycerol had been removed, provided that the cells remained at 0 degrees C. However, granulocytes normally function at 37 degrees C. Because chemotaxis is considered a good assay of normal function, we developed a modified procedure capable of discriminating among random migration, enhanced random migration (chemokinesis), and directed cell migration (true chemotaxis). When frozen-thawed-diluted cells were incubated for 60 min at 37 degrees C, their survival, based both on the FDA assay and on the chemotaxis assay, was zero. In fact, a prior exposure of the cells to 2 M glycerol at 0 degrees C, even in the absence of freezing, resulted in a rapid loss in FDA viability when the cells were subsequently held at 37 degrees C for up to 60 min. Survivals based on FDA are usually reported to be considerably higher than survivals based on functional assays such as chemotaxis or phagocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Preservation↗
PubMed1983

Depression of the ice-nucleation temperature of rapidly cooled mouse embryos by glycerol and dimethyl sulfoxide.

The temperature at which ice formation occurs in supercooled cytoplasm is an important element in predicting the likelihood of intracellular freezing of cells cooled by various procedures to subzero temperatures. We have confirmed and extended prior indications that permeating cryoprotective additives decrease the ice nucleation temperature of cells, and have determined some possible mechanisms for the decrease. Our experiments were carried out on eight-cell mouse embryos equilibrated with various concentrations (0-2.0 M) of dimethyl sulfoxide or glycerol and then cooled rapidly. Two methods were used to assess the nucleation temperature. The first, indirect, method was to determine the in vitro survival of the rapidly cooled embryos as a function of temperature. The temperatures over which an abrupt drop in survival occurs are generally diagnostic of the temperature range for intracellular freezing. The second, direct, method was to observe the microscopic appearance during rapid cooling and note the temperature at which nucleation occurred. Both methods showed that the nucleation temperature decreased from - 10 to - 15 degrees C in saline alone to between - 38 degrees and - 44 degrees C in 1.0-2.0 M glycerol and dimethyl sulfoxide. The latter two temperatures are close to the homogeneous nucleation temperatures of the solutions in the embryo cytoplasm, and suggest that embryos equilibrated in these solutions do not contain heterogeneous nucleating agents and are not accessible to any extracellular nucleating agents, such as extracellular ice. The much higher freezing temperatures of cells in saline or in low concentrations of additive indicate that they are being nucleated by heterogeneous agents or, more likely, by extracellular ice.

Animals↗
PubMed1981

Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes.

As suspensions of cells freeze, the electrolytes and other solutes in the external solution concentrate progressively, and the cells undergo osmotic dehydration if cooling is slow. The progressive concentration of solute comes about as increasing amounts of pure ice precipitate out of solution and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. The consensus has been that slow freezing injury is related to the composition of the solution in these channels and not to the amount of residual liquid. The purpose of the research reported here was to test this assumption on human erythrocytes. Ordinarily, solute concentration and the amount of liquid in the unfrozen channels are inversely coupled. To vary them independently, one must vary the initial solute concentration. Two solutes were used here: NaCl and the permeating protective additive glycerol. To vary the total initial solute concentration while holding the mass ratio of glycerol to NaCl constant, we had to allow the NaCl tonicity to depart from isotonic. Specifically, human red cells were suspended in solutions with weight ratios of glycerol to NaCl of either 5.42 or 11.26, where the concentrations of NaCl were 0.6, 0.75, 1.0, 2.0, 3.0, or 4.0 times isotonic. Samples were then frozen to various subzero temperatures, which were chosen to produce various molalities of NaCl (0.24-3.30) while holding the fraction of unfrozen water constant, or conversely to produce various unfrozen fractions (0.03-0.5) while holding the molality of salt constant. (Not all combinations of these values were possible). The following general findings emerged: (a) few cells survived the freezing of greater than 90% of the extracellular water regardless of the salt concentration in the residual unfrozen portion. (b) When the fraction of frozen water was less than 75% the majority of the cells survived even when the salt concentration in the unfrozen portion exceeded 2 molal. (c) Salt concentration affected survival significantly only when the frozen fraction lay between 75 and 90%. To find a major effect on survival of the fraction of water that remains unfrozen was unexpected. It may require major modifications in how cryobiologists view solution-effect injury and its prevention.

Erythrocyte Aging↗
PubMed1980

Glycerol permeabilities of fertilized and infertilized mouse ova.

The coefficients of permeability to glycerol, Pglyc, and their temperature-dependence were determined from volume changes of fertilized and unfertilized mouse ova under hyperosmotic conditions. Fertilization caused a significant increase in the value of Pglyc. Unfertilized ova exhibited Pglyc values of 1 X 10(-5), 2 X 10(-6), and 5 X 10(-7) cm/min at approximately 20 degrees C, and approximately 12 degrees C, and approximately 3 degrees C, respectively. Fertilized ova exhibited Pglyc values of 3.4 X 10(-5), 1.3 X 10(-5), and 4.7 X 10(-6) cm/min at apoproximately 20 degrees C, 12 degrees C, and approximately 3 degrees C, respectively. The activation energy of glycerol permeation into fertilized and unfertilized ova was calculated to be 18.7 and 28.4 kcal/mol, respectively. Fertilization caused a rather dramatic increase in glycerol permeability, the major extent of which occurred within about 3 hours after fertilization. Pglyc was also measured at approximately 20 degrees C by isotope uptake. Glycerol uptake calculated indirectly from the volume changes of the ova agreed well with that measured directly by labeled glycerol uptake. It was also found that the zona pellucida fertilized and unfertilized ova did not detectably impede the permeation by glycerol.

Animals↗
PubMed1980

Limits to life at low temperatures and at reduced water contents and water activities.

Liquid water is generally considered an absolute requisite for functional life; consequently, life is expected to function only over the range of temperatures that permit its existence. These limits, however, do not apply to cell survival. Some cells can survive the closest attainable approach to 0 K, and some can survive the loss of over 99% of their water.

Animals↗
PubMed1979

The Viking Mission: implications for life on Mars.

The results of the Viking Biology experiments are best explained by non-biological phenomena: The interaction of the reagents with the materials comprising the regolith. Conditions of water activity, temperature, availability of carbon sources and others in most regions of the planet are too extreme for survival and growth of any known Earth microorganisms. Although the possibility persists that some very unusual form of life is somewhere on that planet the evidence is best interpreted as negative. Even though there is no evidence for current life on Mars, whether or not life ever originated there is not known.

Carbon↗
PubMed1978

Temperature dependence of the survival of human erythrocytes frozen slowly in various concentrations of glycerol.

One widely accepted explanation of injury from slow freezing is that damage results when the concentration of electrolyte reaches a critical level in partly frozen solutions during freezing. We have conducted experiments on human red cells to further test this hypothesis. Cells were suspended in phosphate-buffered saline containing 0-3 M glycerol, held for 30 min at 20 degrees C to permit solute permeation, and frozen at 0.5 or 1.7 degrees C/min to various temperatures between -2 and -100 degrees C. Upon reaching the desired minimum temperature, the samples were warmed at rates ranging from 1 to 550 degrees C/min and the percent hemolysis was determined. The results for a cooling rate of 1.7 degrees C/min indicate the following: (a) Between 0.5 and 1.85 M glycerol, the temperature yielding 50% hemolysis (LT50) drops slowly from -18 to -35 degrees C. (b) The LT50's over this range of concentrations are relatively independent of warming rate. (c) With glycerol concentrations of 1.95 and 2.0 M, the LT50 drops abruptly to -60 degrees C and to below -100 degrees C, respectively, and becomes dependent on warming rate. The LT50 is lower with slow warming at 1 degree C/min than with rapid. With still higher concentrations (2.5 and 3.0 M), there is no LT50, i.e., more than 50% of the cells survive freezing to-100 degrees C. Results for cooling at 0.5 degrees C/min in 2 M glycerol were similar except that the LT50s were some 10-20 degrees C higher. A companion paper (Rall et al., Biophys. J. 23:101-120, 1978) examines the relation between survival and the concentrations of salts produced during freezing.

Cell Survival↗
PubMed1978

Physical-chemical basis of the protection of slowly frozen human erythrocytes by glycerol.

One theory of freezing damage suggests that slowly cooled cells are killed by being exposed to increasing concentrations of electrolytes as the suspending medium freezes. A corollary to this view is that protective additives such as glycerol protect cells by acting colligatively to reduce the electrolyte concentration at any subzero temperature. Recently published phase-diagram data for the ternary system glycerol-NaCl-water by M. L. Shepard et al. (Cryobiology,13:9-23, 1976), in combination with the data on human red cell survival vs. subzero temperature presented here and in the companion study of Souzu and Mazur (Biophys. J.,23:89-100), permit a precise test of this theory. Appropriate liquidus phase-diagram information for the solutions used in the red cell freezing experiments was obtained by interpolation of the liquidus data of Shepard and his co-workers. The results of phase-diagram analysis of red cell survival indicate that the correlation between the temperature that yields 50% hemolysis (LT(50)) and the electrolyte concentration attained at that temperature in various concentrations of glycerol is poor. With increasing concentrations of glycerol, the cells were killed at progressively lower concentrations of NaCl. For example, the LT(50) for cells frozen in the absence of glycerol corresponds to a NaCl concentration of 12 weight percent (2.4 molal), while for cells frozen in 1.75 M glycerol in buffered saline the LT(50) corresponds to 3.0 weight percent NaCl (1.3 molal). The data, in combination with other findings, lead to two conclusions: (a) The protection from glycerol is due to its colligative ability to reduce the concentration of sodium chloride in the external medium, but (b) the protection is less than that expected from colligative effects; apparently glycerol itself can also be a source of damage, probably because it renders the red cells susceptible to osmotic shock during thawing.

Buffers↗
PubMed1978

Physiological response of Neurospora conidia to freezing in the dehydrated, hydrated, or germinated state.

This study concerned the response to freezing of Neurospora crassa conidia in four different states: air-dry, hydrated in water, hydrated in Vogel medium lacking only sucrose, or hydrated in complete Vogel medium. All hydrated conidia were incubated in one of the above media for various times before freezing and were then washed and frozen in distilled water. Viability was estimated by three techniques, and the agreement among them was good. Hydration of air-dry conidia was found to be very rapid and, once hydrated, the conidia were much more sensitive to rapid freezing than they were before hydration. Rapidly cooled conidia survived freezing to a much higher extent when the warming rate was rapid than when it was slow; slowly cooled conidia showed little or no dependence on the warming rate. This sensitivity to rapid cooling and slow warming was attributed to the effects of intracellular ice. The sensitivity to freezing could be reversed by dehydrating the conidia in vacuo before freezing; thus, it was concluded that the presence or absence of water is the determining factor in the initial sensitivity due to freezing. In water, the sensitivity remained constant from 2 min to 15 days after hydration. Although conidia hydrated in growth medium lacking sucrose remained metabolically inactive, their sensitivity to rapid freezing decreased as a function of time in the medium before freezing. The reason for this decreased sensitivity is not understood. Conidia hydrated in complete growth medium (i.e., containing sucrose) became metabolically active and, after the initial sensitivity associated with hydration, became increasingly more sensitive to freezing as a function of their time in the medium. Drying itself was deleterious to metabolically active conidia, and those that survived dehydration did not exhibit a large absolute increase in resistance to subsequent freezing. The increase in sensitivity to freezing and to drying seems associated with the presence of metabolic activity; however, the precise cause of the sensitization remains obscure.

Culture Media↗