The response of multilamellar liposomes to freezing and thawing

The use of liposomes as a model system for investigating the mechanism of freezing injury was investigated. Modification of the liposome phospholipid and cholesterol content allows a correlation to be made between the composition of a membrane system and its response to the stresses of freezing and thawing. The data on phase transitions are contradictory in the sense that liposomes become more sensitive to freezing injury following treatments which both increase or decrease phase transition temperature. In contrast the effect of cholesterol in sensitizing membranes to the stresses of freezing and thawing appears to be more fundamental. Direct cryomicroscope observations of liposomes during slow cooling indicate that they are osmotically active at low temperatures and upon thawing morphological alterations to the membranes occur. The response of liposomes following cooling at a range of rates to ?196 °C and the effects of cryoprotective additives are similar to those observed with many cell types. These results indicate that liposomes are a valid model for investigating the biochemistry of membrane damage induced by the stresses of freezing and thawing.     

Water transport and cell survival in cryobiological procedures.

Living cells may be cooled to 77 K (liquid nitrogen) either to destroy them selectively or to store them for long periods. Water transport across the cell membranes during freezing and thawing is a primary factor determining whether the cells survive. These water movements are controlled by phase changes both intracellular and extracellular and by other factors such as the nature of any cryoprotective agent present, and the rates of cooling and thawing. The relation between cooling procedure, water transport and cell survival is discussed. In particular, the crucial r?le of dilution shock is emphasized: this is the damage to cells induced during the dilution that occurs both as ice melts during rewarming and when any cryoprotective additives are removed after thawing. Apart from the usefulness of understanding these processes for maximizing preservation or controlling selective destruction, the diverse responses of cells to different combinations of water transport and temperature changes appear likely to provide basic information on the properties of cell membranes.     

Cryomicroscopy of liposome sytems as simple models to study cellular freezing response

Liposomes represent an attractive model system to study the freeze/thaw-induced alterations in biological membranes primarily because liposomes may be custom-manufactured for a particular investigation.This paper has reviewed briefly the degree of flexibility possible in terms of manufacturing liposomes with desired membrane composition and intraliposomal markers.Earlier research using liposomes as a model to study the freezing response of biological cells demonstrated their usefulness in this respect. Combining these earlier results with new results obtained in our laboratory, many analogies between the responses of biological cells and liposomes have been demonstrated. These analogies are summarized in Table 3.It is significant to note that in virtually every category thus far examined the relevant cryobiological behavior of biological cells can be mimicked qualitatively (and in some cases quantitatively) by a pure lipid membrane system. This is not to say that protein components of the membrane are insignificant. They are likely to be quite important in some cases. The liposome model system offers some interesting possibilities of examining the relative importance of lipids and proteins in model systems.Cryomicroscopic observation of liposome systems represents a promising approach to an improved understanding of membrane-related phenomena which occur during freezing and thawing. Especially interesting are the observations of membrane instabilities (“popping”) which appear to be induced osmotically.Real-time observation of well-defined membrane bilayer systems subjected to computer-controlled freeze/thaw protocols should lead to valuable insights into the nature of membrane freezing injury in the future.     

The cryopreservation of liposomes. 2. Effect of particle size on crystallization behavior and marker retention.

Liposome dispersions (bilayer composition Phospholipon 100H/dicetylphosphate (molar ratio 10:1) dispersed in 10 mM Tris buffer) are frozen in a differential scanning calorimeter. In the cooling curves of the dispersions a heat-flow below -40 degrees C is observed. This heat-flow is due to the crystallization of maximally supercooled water. Evidence is provided that at this temperature, defined as the homogeneous nucleation temperature, part or all encapsulated water in the liposomes crystallizes. At a cooling rate of 10 degrees C/min only for small liposomes with particle sizes below approximately 0.2 micron the internal volume crystallizes at the homogeneous nucleation temperature. After a freezing/thawing cycle of the liposomal dispersions retention of the water-soluble marker carboxyfluorescein (CF) was significantly better if crystallization of the encapsulated volume occurred at the homogeneous nucleation temperature. Up to 55% retention of CF in dispersions with mean vesicle sizes below 0.2 micron was found after storage for 45 min at -50 or -75 degrees C. Only relatively small particle size alterations were found in comparison with the original mean particle sizes after a freezing/thawing cycle with storage for 45 min at -50 or -75 degrees C. Independent of particle size, dispersions stored for 45 min at -25 degrees C showed low CF retention (less than 10%) after thawing. For most of the liposome dispersions stored at -25 degrees C, large particle size alterations compared to the original particle sizes were observed after a freezing/thawing cycle.     

Osmotic response of individual cells during freezing. I. Experimental volume measurements

The osmotic response of yeast to freezing was measured as a function of cooling rate and degree of extracellular supercooling. Thirteen experimental trials were conducted on a cryomicroscope in which incremental size changes of individual cells were recorded photographically, and the corresponding volume variations were measured using a digital computer image analysis algorithm. Plots were obtained of normalized cell volume as a function of temperature. Cellular dehydration during freezing was progressively inhibited with increasing values of cooling rate and extracellular supercooling. Normalized cell volume changes were not a function of the relative initial cell size. A constant volume plateau occurred for conditions under which intracellular ice formation was expected.     

A Cryomicroscopic Study of Cylindrocystis brebissonii De Bary and Two Species of Micrasterias Ralfs (Conjugatophyceae, Chiorophyta) during Freezing and Thawing

The changes in morphology of the unicellular algae Cylindrocystisbrebissonii and two species of Micrasterias during freezingand thawing were observed on a light microscope fitted witha temperature controlled stage. At slow rates of cooling extensiveshrinkage of the protoplast was observed. The response of thecell wall varied with cell-type. In C. brebissonii plasmolysiswas not observed and the cell wall and protoplast shrank together.In Micrasterias the cell wall did not contract and a distinctplasmolysis was observed. Following freezing to and thawingfrom –25?C cells of C. brebissonii were non-viable butremained osmotically responsive. Cooling at faster rates inducedintracellular ice formation in all cell-types. The criticalrate of cooling varied with cell-type and was determined bycell volume and suface area. Intracellular gas bubbles wereobserved during thawing following both rapid and slow cooling. Following cooling in dimethylsulphoxide cells of C. brebissoniiwere protected against freezing injury. The recovery on thawingfrom –196?C being determined by the rate of cooling, anoptimum rate of 1?C min^–1 was observed. During slow ratesof cooling (<2?C min^–1) cells remained unshrunken,at faster rates (10?C min^–1) the loss of cell viabilitywas related to osmotic shrinkage during cooling rather thanto nucleation of intracellular ice. Intracellular ice formationwas observed only following significantly faster rates of cooling(>20?C min^–1). Key words: Cylindrocystis, Micrasterias, cryomicroscopy, freezing injury     

A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing

The changes in morphology of Penicillium expansum Link and Phytophthora nicotianae Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage. Viability of 0.5-1.0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 degrees C in the presence or absence of glycerol. In P. expansum shrinkage occurred in all hyphae at rates of cooling of less than 15 degrees C min-1; at faster rates intracellular ice nucleation occurred. The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18 degrees C min-1 to 55 degrees C min-1. This species was particularly resistant to freezing injury and recovery was greater than 60% at all rates of cooling examined. At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated. In contrast, during the cooling of Ph. nicotianae in the growth medium, shrinkage occurred and no samples survived on thawing from -196 degrees C. However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 degrees C min-1 the loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.     

Cryopreservation and biophysical properties of articular cartilage chondrocytes

In order to successfully cryopreserve articular cartilage chondrocytes, it is important to characterize their osmotic response during the cryopreservation process, as the ice forms and the solutes concentrate. In this study, experimental work was undertaken to determine the osmotic parameters of articular cartilage chondrocytes. The osmotically inactive volume of articular cartilage chondrocytes was determined to be 44% of the isotonic volume. The membrane hydraulic conductivity parameters for water were determined by fitting a theoretical water transport model to the experimentally obtained volumetric shrinkage data; the membrane hydraulic conductivity parameter L(Pg) was found to be 0.0633 microm/min/atm, and the activation energy E, 8.23 kcal/mol. The simulated cooling process, using the osmotic parameters obtained in this study, suggests a cooling rate of 80 degrees C/min for the cryopreservation of the articular cartilage chondrocytes of hogs. The data obtained in this study could serve as a starting point for those interested in cryopreservation of chondrocytes from articular cartilage in other species in which there is clinical interest and there are no parameters for prediction of responses.     

Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing

Diluents containing sonicated liposomes of purified phosphatidylserine (PS), phosphatidylcholine (PC) with varying fatty acyl chain lengths and double bonds and cholesterol (CH) alone or in combination, or egg yolk lecithin were evaluated for protection of bull sperm during cold shock produced by rapid cooling from 25 to 0 degrees C and during freezing and thawing. Bull semen was washed twice and diluted to 50 X 10(6) sperm/ml in diluents containing no lipid, 0.5 or 5 mM sonicated lipid or 20% egg yolk and plunged into ice water to cold shock the sperm. Sperm so treated were frozen using conventional methods. The percentage of progressively motile sperm (MS) was estimated prior to cooling, after cold shock, and after freezing and thawing. Lipids with fatty acyl chains of less than 12 carbons were toxic to sperm cells. Phosphatidylserine alone or in combination with PC or CH, but not PC or CH alone, protected sperm from cold shock as well as did egg yolk lecithin liposomes or egg yolk. Liposomes of PS/PC or PS/CH were not better than PS in protecting sperm from cold shock. Lipid concentrations of 0.5 mM were more effective than liposomes at 5 mM in protecting sperm during freezing and thawing. During freezing, PS alone or in combination with PC partially protected sperm, but only PS/CH was as effective as egg yolk in protecting sperm from freeze-thaw damage. It is concluded that defined diluents, particularly those containing PS, may be useful in studies of cryobiology of spermatozoa.     

Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane

Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.     

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)     

Effect of hydration on the water content of human erythrocytes.

An ideal, hydrated, nondilute pseudobinary salt-protein-water solution model of the RBC intracellular solution has been developed to describe the osmotic behavior of human erythrocytes during freezing and thawing. Because of the hydration of intracellular solutes (mostly cell proteins), our analytical results predict that at least 16.65% of the isotonic cell water content will be retained within RBCs placed in hypertonic solutions. These findings are consistent not only with the experimental measurements of the amount of isotonic cell water retained within RBCs subjected to nonisotonic extracellular solutions (20-32%) but also with the experimental evidence that all of the water within RBCs is solvent water. By modeling the RBC intracellular solution as a hydrated salt-protein-water solution, no anomalous osmotic behavior is apparent.     

Freezing response and optimal cooling rates for cryopreserving sperm cells of striped bass, Morone saxatilis

This study explored the optimization of techniques for sperm cryopreservation of an economically important fish species, the striped bass Morone saxatilis. The volumetric shrinkage or the water transport response during freezing of sperm cells was obtained using a differential scanning calorimeter (DSC) technique. Water transport was obtained in the presence of extracellular ice at a cooling rate of 20 degrees C/min in two different media: (1) without cryoprotective agents (CPAs), and (2) with 5% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of length of 22.8 microm and diameter 0.288 microm and was assumed to have an osmotically inactive cell volume (V(b)) of 0.6 V(0), where V(0) is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined water transport data, the best fit membrane permeability parameters (reference membrane permeability to water, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) were determined and ranged from L(pg)=0.011-0.001 microm/min-atm, and E(Lp)=40.2-9.2 kcal/mol). The parameters obtained in this study suggested that the optimal rate of cooling for striped bass sperm cells in the presence and absence of DMSO range from 14 to 20 degrees C/min. These theoretically predicted rates of optimally freezing M. saxatilis sperm compared quite closely with independent and experimentally determined optimal rates of cooling striped bass sperm.     

Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing

Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze-substitution and freeze-quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.     

Maple sap uptake, exudation, and pressure changes correlated with freezing exotherms and thawing endotherms

Sap flow rates and sap pressure changes were measured in dormant sugar maple trees (Acer saccharum Marsh.). In the forest, sap flow rates and pressure changes were measured from tap holes drilled into tree trunks in mature trees and sap flow rates were measured from the base of excised branches. Excised branches were also brought into the laboratory where air temperature could be carefully controlled in a refrigerated box and sap flow rates and sap pressures were measured from the cut base of the branches.

Under both forest and laboratory conditions, sap uptake occurred as the wood temperature declined but much more rapid sap uptake correlated with the onset of the freezing exotherm. When sap pressures were measured under conditions of negligible volume displacement, the sap pressure rapidly fell to −60 to −80 kilopascals at the start of the freezing exotherm. The volume of water uptake and the rate of uptake depended on the rate of freezing. A slow freezing rate correlated with a large volume of water uptake, a fast freezing rate induced a smaller volume of water uptake. The volume of water uptake ranged from 0.02 to 0.055 grams water per gram dry weight of sapwood. The volume of water exuded after thawing was usually less than the volume of uptake so that after several freezing and thawing cycles the sapwood water content increased from 0.7 to 0.8 grams water per gram dry weight.

These results are discussed in terms of a physical model of the mechanism of maple sap uptake and exudation first proposed by P. E. R. O'Malley. The proposed mechanism of sap uptake is by vapor distillation in air filled wood fiber lumina during the freezing of minor branches. Gravity and pressurized air bubbles (compressed during freezing) cause sap flow from the canopy down the tree after the thaw.

     

Stabilization of liposome bilayers to freezing and thawing: Effects of cryoprotective agents and membrane proteins

Lipid bilayer vesicles (liposomes) with and without glycoprotein incorporated into the membranes were tested for stability during freezing and thawing, in presence and absence of the cryoprotective agents (CPA) glycerol and dimethyl sulfoxide. Changes in turbidity and loss of energy transfer between fluorescent probes present in the bilayers were used to estimate membrane integrity.Freezing caused a 30 to 40% destruction of protein-free liposomes, in absence of CPA. CPA at 10 to 20% concentration prevented such losses, but at higher concentrations destabilized liposomes even without freezing. Protein-containing liposomes suffered no loss on freezing in absence or presence of CPA at moderate concentrations.Lowering of the storage temperature of frozen samples within the range of ?5 to ?27 °C increased the freeze damage. Slower rates of cooling and warming caused a slightly greater loss.The results are interpreted in terms of the liquid mosaic model for lipid bilayers. CPA at higher concentrations destabilize bilayers by dissolving phospholipids. At moderate concentrations, however, they prevent the damaging effect of dehydration of the lipid on freezing. Proteins appear to stabilize bilayers by providing increased hydration at the membrane surface, and by additional hydrophobic binding in the membrane interior.     

Effect of cooling and rewarming rates on glycerolated human erythrocytes.

Human erythrocytes suspended in a sodium-free buffered salt solution containing glycerol in 1 m concentration (1 part of packed cells to 4 parts buffered salt solution) were frozen by slow, moderately rapid, or very rapid cooling to various subzero C temperatures. The frozen specimens, after a 5-min storage period at a given temperature, were thawed at low, moderately high, or very high rates. The hemolysis in the frozen and thawed samples was measured by a colorimetric determination of the hemoglobin released from the damaged cells. At ?10 °C, the highest freezing temperature employed, nearly 100% recovery of intact erythrocytes was obtained irrespective of the cooling and rewarming conditions. The extent of the hemolysis after exposure to lower freezing temperatures depended upon the cooling and rewarming conditions. Moderately rapid and very rapid freezing to, and thawing from temperatures below ?40 °C permitted significantly higher recoveries of intact cells than the other freezing/ thawing combinations. In the temperature range ?15 to ?30 °C the combination slow cooling and slow rewarming afforded maximum protection. Very rapid freezing/ slow thawing was the most damaging combination throughout the entire freezing range. The results were interpreted in part by a conventional two-factor analysis, lower cooling rates allowing concentrated salts to determine hemolysis, higher cooling rates destroying the cells by intracellular freezing. Apparent anomalies were explained in terms of a generalized “thermal/osmotic” shock according to which the erythrocytes were subject to greater hemolysis the higher the rates of cooling and/or warming.     

Cryomicroscope investigation and thermodynamic modeling of the freezing of unfertilized hamster ova

Thermodynamic computer modeling was used to predict the freezing response of single-celled unfertilized hamster ova. The cell membrane transport characteristics were investigated, using a microscope diffusion chamber system. The mean osmotically inactive cell volume was determined to be 21.6% of the initial cell volume. An overall mean value of 0.8 +/- 0.1 micron3/micron2.min.atm (= 18 +/- 2.5 micron/sec) was determined for the membrane hydraulic coefficient, Lp. The effect of the extracellular solute concentration on Lp was determined at room temperature (approximately 23 degrees C). A thermodynamic computer model was used to predict the cell response to freezing. The predicted response was compared to the actual volumetric response observed during freezing on a temperature-controlled cryomicroscope conduction stage. The effect of the cooling rate on the nucleation temperature of unprotected ova and protected ova suspended in a 1.5 M DMSO solution was investigated. Overall mean nucleation temperatures of -13 and -57.1 degrees C were observed for unprotected and protected ova, respectively, where the mean nucleation temperature for protected ova was strongly cooling rate dependent.     

Theoretical aspects of canine semen cryopreservation

Changes in canine sperm cells during freezing and thawing can cause damage to the cells resulting in cell death. No standardized freezing or thawing method appears to be ideal for all dogs and all ejaculates, because intrinsic variations in properties such as osmotic sensitivity between sperm cells from different dogs and ejaculates makes the cellular response to cryopreservation unpredictable according to the normal physics of cryobiology. Research in canine semen cryopreservation is difficult because the low ejaculate volume makes multiple comparisons from a single ejaculate difficult. True fertility data is also very limited on cryopreserved canine ejaculates. Despite this, the cottage industry that has evolved to cryopreserve dog sperm has been very successful using empirically derived methods that accommodate most ejaculates. Therefore, the practitioner must follow the recommendations supplied by the freezing center to achieve the best potential results.     

Cryopreservation of platelets using trehalose: The role of membrane phase behavior during freezing

In blood banks, platelets are stored at 20–24°C, which limits the maximum time they can be stored. Platelets are chilling sensitive, and they activate when stored at temperatures below 20°C. Cryopreservation could serve as an alternative method for long term storage of platelet concentrates. Recovery rates using dimethyl sulfoxide (DMSO) as cryoprotective agent, however, are low, and removal of DMSO is required before transfusion. In this study, we have explored the use of trehalose for cryopreservation of human platelets while using different cooling rates. Recovery of membrane intact cells and the percentage of nonactivated platelets were used as a measure for survival. In all cases, survival was optimal at intermediate cooling rates of 20°C min^?1. Cryopreservation using DMSO resulted in high percentages of activated platelets; namely 54% of the recovered 94%. When using trehalose, 98% of the platelets had intact membranes after freezing and thawing, whereas 76% were not activated. Using Fourier transform infrared spectroscopy, subzero membrane phase behavior of platelets has been studied in the presence of trehalose and DMSO. Furthermore, membrane hydraulic permeability parameters were derived from these data to predict the cell volume response during cooling. Both trehalose and DMSO decrease the activation energy for subzero water transport across cellular membranes. Platelets display a distinct lyotropic membrane phase transition during freezing, irrespective of the presence of cryoprotective agents. We suggest that concomitant uptake of trehalose during freezing could explain the increased survival of platelets cryopreserved with trehalose. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012