Cell-cell contact affects membrane integrity after intracellular freezing

The response of cells to freezing depends critically on the presence of an intact cell membrane. During rapid cooling, the cell plasma membrane may no longer be an effective barrier to ice propagation and can be breached by extracellular ice resulting in the nucleation of the supercooled cytoplasm. In tissues, the formation of intracellular ice is compounded by the presence of cell-cell and cell-surface interactions. Three different hamster fibroblast model systems were used to simulate structures found in organized tissues. Samples were supercooled to an experimental temperature on a cryostage and ice nucleated at the constant temperature. A dual fluorescent staining technique was used for the quantitative assessment of the integrity of the cell plasma membrane. A novel technique using the fluorescent stain SYTO was used for the detection of intracellular ice formation (IIF) in cell monolayers. The cumulative incidence of cells with a loss of membrane integrity and the cumulative incidence of IIF were determined as a function of temperature. Cells in suspension and individual attached cells showed no significant difference in the number of cells that formed intracellular ice and those that lost membrane integrity. For cells in a monolayer, with cell-cell contact, intracellular ice formation did not result in the immediate disruption of the plasma membrane in the majority of cells. This introduces the potential for minimizing damage due to IIF and for developing strategies for the cryoprotection of tissues during rapid cooling.     

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.     

Capillary endothelial cell cultures: phenotypic modulation by matrix components

Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens. When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures. In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times. In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays. Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different. On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane. In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d). Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis. Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture.     

Strong Isotope Effects on Melting Dynamics and Ice Crystallisation Processes in Cryo Vitrification Solutions

The nucleation and growth of crystalline ice during cooling, and further crystallization processes during re-warming are considered to be key processes determining the success of low temperature storage of biological objects, as used in medical, agricultural and nature conservation applications. To avoid these problems a method, termed vitrification, is being developed to inhibit ice formation by use of high concentration of cryoprotectants and ultra-rapid cooling, but this is only successful across a limited number of biological objects and in small volume applications. This study explores physical processes of ice crystal formation in a model cryoprotective solution used previously in trials on vitrification of complex biological systems, to improve our understanding of the process and identify limiting biophysical factors. Here we present results of neutron scattering experiments which show that even if ice crystal formation has been suppressed during quench cooling, the water molecules, mobilised during warming, can crystallise as detectable ice. The crystallisation happens right after melting of the glass phase formed during quench cooling, whilst the sample is still transiting deep cryogenic temperatures. We also observe strong water isotope effects on ice crystallisation processes in the cryoprotectant mixture. In the neutron scattering experiment with a fully protiated water component, we observe ready crystallisation occurring just after the glass melting transition. On the contrary with a fully deuteriated water component, the process of crystallisation is either completely or substantially supressed. This behaviour might be explained by nuclear quantum effects in water. The strong isotope effect, observed here, may play an important role in development of new cryopreservation strategies.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 degrees C/min while the ice nucleation temperature was varied between -3 and -10 degrees C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below -4 degrees C and cell survival exhibits an optimum at a nucleation temperature of -6 degrees C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at -3 degrees C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at -10 degrees C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in alpha-helical structures and a concomitant increase in beta-sheet structures starting at an onset temperature of approximately 48 degrees C.     

Evaluation of feeding and mixing conditions for fractional precipitation of paclitaxel from plant cell cultures

Fractional precipitation based on the difference in solubility of crude paclitaxel dissolved in methanol, to which distilled water must be added, is the most effective method for the pre-purification of paclitaxel. In this study, the effect of the distilled water feeding and mixing method on the efficiency of fractional precipitation and the formation of paclitaxel precipitate were evaluated. When the distilled water was added all at once, the highest purity (∼57.0%) and yield (∼81.0%) were obtained, and a spherical precipitate was formed by the clustering of crystal branches. On the other hand, when the distilled water was added intermittently in several aliquots, the purity and yield tended to decrease with increasing number of additions, and the precipitate took the form of a cross or pentagon with less clustering of branches. Not mixing after the addition of distilled water resulted in high purity (∼57.0%) and the formation of a spherical precipitate that showed increased branching around the nucleus over time. In contrast, when the sample was mixed intermittently after adding distilled water, paclitaxel was obtained in high yield (∼99.7%). Continuously mixing the sample after adding distilled water, however, caused the precipitate crystals to be broken into smaller pieces.     

Protective effect of intracellular ice during freezing?

Injury results during freezing when cells are exposed to increasing concentrations of solutes or by the formation of intracellular ice. Methods to protect cells from the damaging effects of freezing have focused on the addition of cryoprotective chemicals and the determination of optimal cooling rates. Based on other studies of innocuous intracellular ice formation, this study investigates the potential for this ice to protect cells from injury during subsequent slow cooling. V-79W Chinese hamster fibroblasts and Madin-Darby Canine Kidney (MDCK) cells were cultured as single attached cells or confluent monolayers. The incidence of intracellular ice formation (IIF) in the cultures at the start of cooling was pre-determined using one of two different extracellular ice nucleation temperatures (-5 or -10 degrees C). Samples were then cooled at 1 degrees C/min to the experimental temperature (-5 to -40 degrees C) where samples were warmed rapidly and cell survival assessed using membrane integrity and metabolic activity. For single attached cells, the lower ice nucleation temperature, corresponding to increased incidence of IIF, resulted in decreased post-thaw cell recovery. In contrast, confluent monolayers in which IIF has been shown to be innocuous, show higher survival after cooling to temperatures as low as -40 degrees C, supporting the concept that intracellular ice confers cryoprotection by preventing cell dehydration during subsequent slow cooling.     

Non-involvement of lysis during sporulation of Bacillus mycoides in distilled water

Washed vegetative cells of Bacillus mycoides obtained and treated under specified conditions have been found to sporulate when shaken in distilled water under specified conditions. Within limitations of the methods, a heat-resistant cell (spore) is produced for each heat-sensitive vegetative cell present initially. Several different experiments designed to detect massive lysis and cell growth during sporulation in distilled water yielded uniformly negative results. Evidence is furnished for the conclusion that a freshly formed spore (heat-resistant cell) weighs considerably less than its progenitor vegetative cell. The observed results are most satisfactorily explained as a direct conversion of a vegetative cell to a spore.     

MEMBRANE SPLITTING IN FREEZE-ETCHING : Covalently Bound Ferritin as a Membrane Marker

The freeze-etch technique was used to observe red blood cell ghosts labeled on both surfaces with covalently bound ferritin. Ferritin molecules were never observed on fracture faces, thus indicating that fracture does not show membrane-surface detail. Subliming away the surrounding ice did expose the ferritin on the membrane surface. These results were consistent with the concept that membranes split during the fracture process of freeze-etching.     

Exposure of N-acetylglucosamine decreases early in dexamethasone-induced apoptosis in thymocytes, demonstrated by flow cytometry using wheat germ agglutinin and pokeweed mitogen.

In the present paper we describe changes in the exposure of oligosaccharides containing N-acetylglucosamine (Glc-NAc) during apoptosis of mouse thymocytes. The structures containing this sugar were probed with fluorescein isothiocyanate-labelled lectins, wheat germ agglutinin and pokeweed mitogen in flow cytometric assays. Both lectins bind to structures containing Glc-NAc. The present report describes experiments in which two different dual-staining techniques were used to simultaneously identify apoptotic cells and measure their lectin exposure. In these experiments, we observed an early and substantial decrease in the exposure of Glc-NAc-containing structures associated with the onset of apoptosis, before or simultaneously with phosphatidylserine exposure. This was followed by an increase in the exposure of Glc-NAc-containing structures after longer incubation times, when a large proportion of cells was demonstrated to have fragmented DNA. These results provide evidence for major changes in the structure of plasma membrane oligosaccharides during apoptosis. The initial decrease may be a by-product of the hydrolysis of glycosphingolipids to yield ceramide for apoptotic signalling or a deliberate process related to the removal of cell adhesion signalling structures, associated with the separation of the apoptotic cell from its neighbours. The later increase in Glc-NAc-containing structures may be the result of the incorporation of internal membranes into the plasma membrane or a deliberate production of prophagocytic signals by a still-functioning Golgi apparatus.     

Dynamics of ice algae and phytoplankton in Frobisher Bay

Summary Vertical and seasonal variations of ice algae and phytoplankton were studied in relation to their physico-chemical environments in Frobisher Bay from 1979 to 1986. The biomass, estimated by both chlorophyll a concentrations and cell counts, was greater in the ice algae than in the phytoplankton in the underlying sea-water during winter and spring. Algal distribution in the sea ice varied vertically and seasonally, while in the underlying water column the phytoplankton distribution was much less variable. The ice algal bloom occurred at the bottom of the ice, particularly in the lower 5 cm during late spring, while the phytoplankton bloom took place at depths between 1 and 10 m during early summer after the ice bloom was over. The community structure of the ice algae changed from pennate to centric diatoms as the ice melted. The centrics dominated through the fall, and then decreased as the pennates increased in dominance when the ice formed again in winter. Species diversity and number were greater in the sea ice than in the seawater, but they were similar vertically within each habitat. The evenness of the species distribution did not vary with ice thickness or water depth. Species composition, abundance and dominance of ice algae and phytoplankton continually change both vertically and seasonally. The differential abilities of the species to attain maximal growth rates under various environmental conditions may result in species succession. Evidence is given for the major role of environmental factors regulating the dynamics of ice algae and phytoplankton.     

Numerical Study of Cell Cryo-Preservation: A Network Model of Intracellular Ice Formation

In this study, a new intracellular ice formation network model, coupled with an improved cell dehydration model has been developed. The non-uniform dehydration of the cell during freezing is simulated with moving boundary condition. Internal cell structures like cell nucleus are taken into consideration. The IIF network model is developed from classic diffusion limited IIF model in order to simulate spatial ice growth pattern inside cells. Simulation results suggest that cell nuclear plays a significant role in cryo-dehydration and would affect water/CPA concentration gradient inside the cell. At the same time, the ice growth pattern of exogenous IIF hypothesis is examined in the model. It is consistent with our previous experiments, in which we witnessed the intracellular ice first grown into the nucleus before spreading to the whole intercellular space. According to this model, the water concentration difference between nucleus and cytoplasm during cryo-dehydration could partly explain why ice crystal in the nucleus grows faster. However, it is not the dominate factor. Higher diffusion coefficient in cell nucleus might play a more important role in the phenomenon.     

An hypothesis for survival of spermatozoa via encapsulation during plane front freezing

Plane front freezing presents the possibility of encapsulating individual cells in the ice phase. The cells may also be pushed ahead of the plane front ice interface, as is always the case for conventional dendritic freezing, where the cells are pushed ahead of the thickening dendrite arms. Cells which are encapsulated during freezing are exposed to hypotonic liquid (pure water) initially upon thawing, while cells which are pushed into the last liquid to freeze are exposed to hypertonic liquid upon thawing. Some exposure to hypertonic intercellular liquid prior to freezing may be required to build up the salt and CPA content in the intracellular liquid and thereby avoid intracellular ice formation at the given cooling rate. Encapsulation of cells by a plane front ice interface should result in three regions of cell survival in the sample: an initial region of cell death due to intracellular ice formation, a final region of cell death due to overexposure to hypertonic intercellular liquid, and an intermediate region of cell survival, where neither damage mechanism has operated to a lethal level. An advantage of plane front freezing over dendritic freezing is that the regions of cell survival and death should be geometrically separate in the sample, rather than mixed at the dendritic microstructural level, as is the case for dendritic freezing. Samples containing populations with very high or very low survival rates for spermatozoa could be obtained by simply cutting up the frozen sample.     

Structure of a second crystal form of Bence-Jones protein Loc: strikingly different domain associations in two crystal forms of a single protein

We have determined the structure of the immunoglobulin light-chain dimer Loc in a second crystal form that was grown from distilled water. The crystal structure was determined to 2.8-A resolution; the R factor is 0.22. The two variable domains are related by local 2-fold axes and form an antigen binding "pocket". The variable domain-variable domain interaction observed in this crystal form differs from the one exhibited by the protein when crystallized from ammonium sulfate in which the two variable domains formed a protrusion (Chang et al., 1985). The structure attained in the distilled water crystals is similar to, but not identical with, the one observed for the Mcg light-chain dimer in crystals grown from ammonium sulfate. Thus, two strikingly different structures were attained by this multisubunit protein in crystals grown under two different, commonly used, crystallization techniques. The quaternary interactions exhibited by the protein in the two crystal forms are sufficiently different to suggest fundamentally different interpretations of the structural basis for the function of this protein. This observation may have general implications regarding the use of single crystallographic determinations for detailed identification of structural and functional relationships. On the other hand, proteins whose structures can be altered by manipulation of crystallization conditions may provide useful systems for study of fundamental structural chemistry.     

Reorganization of the Golgi apparatus of epithelial cells in the frog bladder in stimulation of water transport by antidiuretic hormone

Ultrastructural changes of Golgi apparatus of frog urinary granular cells at antidiuretic hormone (ADH) stimulation of water transport were studied. During a short-time ADH action (5 min) the fragmentation of the complex on single dictyosomes and dilution of certain cisternae is discovered. A conclusion is made that the granular cell giant vacuoles may originate from the Golgi cisternae. It is suggested that the microtubules may be involved in the translocation of dictyosomes and migration of formed vacuoles. The quantity of microtubules increases during ADH action very significantly. Moreover, the involvement of the Golgi apparatus is shown in the maintenance of the cell membrane balance due to budding of tubular structures from transcisternae and shuttling between luminal and vacuolar membranes.     

APPLICATIONS OF FREEZE-SUBSTITUTION TO ELECTRON MICROSCOPE STUDIES OF INVERTEBRATE OOCYTES

A modified freeze-substitution process is described which gives a low percentage (less than 5 per cent) of preparations of invertebrate eggs which appear to be ice crystal-free at the resolution of the electron microscope. The mitochondria show no membranes in these preparations but can be recognized by internal spaces with the size and the distribution of the cristae. The Golgi bodies resemble those seen with diffusion fixatives, but the limiting membranes are here double; that is, they appear to be a triple-layered sandwich with two outer dark approximately 25A layers and an inner light layer of the same thickness. The endoplasmic reticulum is clearly present and resembles that seen with diffusion fixatives. Here again, the limiting membranes are double with the same dimensions as those in the Golgi bodies. The membranes of the Golgi bodies and ER are seen after permanganate but not lead hydroxide staining. The hyaloplasm (or "cell sap") is crowded with 150 to 200A particles and these are also seen lining the ER membranes. In general, the structures as seen with the present technique show considerable similarity to those seen with diffusion fixatives.     

Polarization of plasma membrane glycoconjugates in amphibian epidermis during metamorphosis

Summary Wheat germ agglutinin (WGA) binding sites have been examined in tadpole epidermal cells at the level of both light and electron microscopy using the WGA-ovomucoid-gold technique. In premetamorphic tadpoles the reaction was observed on the plasma membranes of epithelial cells showing a gradient from inner to outer membranes. These glycoconjugates were polarized during development, and at the end of metamorphic climax they were only located in plasma membranes of stratum corneum. The existence of an apical cell surface coat is needed to facilitate the absorption of water through the adult epidermis. The possible implications of this polarization process are discussed.     

Heat and mass transfer scale-up issues during freeze drying: II. Control and characterization of the degree of supercooling

This study aims to investigate the effect of the ice nucleation temperature on the primary drying process using an ice fog technique for temperature-controlled nucleation. In order to facilitate scale up of the freeze-drying process, this research seeks to find a correlation of the product resistance and the degree of supercooling with the specific surface area of the product. Freeze-drying experiments were performed using 5% wt/vol solutions of sucrose, dextran, hydroxyethyl starch (HES), and mannitol. Temperature-controlled nucleation was achieved using the ice fog technique where cold nitrogen gas was introduced into the chamber to form an “ice fog”, there-by facilitating nucleation of samples at the temperature of interest. Manometric temperature measurement (MTM) was used during primary drying to evaluate the product resistance as a function of cake thickness. Specific surface areas (SSA) of the freeze-dried cakes were determined. The ice fog technique was refined to successfully control the ice nucleation temperature of solutions within 1°C. A significant increase in product resistance was produced by a decrease in nucleation temperature. The SSA was found to increase with decreasing nucleation temperature, and the product resistance increased with increasing SSA. The ice fog technique can be refined into a viable method for nucleation temperature control. The SSA of the product correlates well with the degree of supercooling and with the resistance of the product to mass transfer (ie, flow of water vapor through the dry layer). Using this correlation and SSA measurements, one could predict scaleup drying differences and accordingly alter the freeze-drying process so as to bring about equivalence of product temperature history during lyophilization.     

Plant Freezing and Damage

Imaging methods are giving new insights into plant freezingand the consequent damage that affects survival and distributionof both wild and crop plants. Ice can enter plants through stomataand hydathodes. Intrinsic nucleation of freezing can also occur.The initial growth of ice through the plant can be as rapidas 40 mm s^-1, although barriers can limit this growth. Onlya small fraction of plant water is changed to ice in this firstfreezing event. Nevertheless, this first rapid growth of iceis of key importance because it can initiate further, potentiallylethal, freezing at any site that it reaches. Some organs andtissues avoid freezing by supercooling. However, supercooledparts of buds can dehydrate progressively, indicating that avoidanceof freezing-induced dehydration by deep supercooling is onlypartial. Extracellular ice forms in freezing-intolerant as wellas freezing-tolerant species and causes cellular dehydration.The single most important cause of freezing-damage is when thisdehydration exceeds what cells can tolerate. In freezing-adaptedspecies, lethal freezing-induced dehydration causes damage tocell membranes. In specific cases, other factors may also causedamage, examples being cell death when limits to deep supercoolingare exceeded, and death of shoots when freezing-induced embolismsin xylem vessels persist. Extracellular masses of ice can damagethe structure of organs but this may be tolerated, as in extra-organfreezing of buds. Experiments to genetically engineer expressionof fish antifreeze proteins have not improved freezing toleranceof sensitive species. A better strategy may be to confer toleranceof cellular dehydration.Copyright 2001 Annals of Botany Company Freezing, dehydration, infrared video thermography, low temperature scanning electron microscopy, NMR micro-imaging     

Unmasking of a potassium leak in resealed human red blood cell ghosts

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0° C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.