Viscosities encountered during the cryopreservation of dimethyl sulphoxide systems.

This study determined the viscous conditions experienced by cells in the unfrozen freeze concentrated channels between ice crystals in slow cooling protocols. This was examined for both the binary Me2SO-water and the ternary Me2SO-NaCl-water systems. Viscosity increases from 6.9 ± 0.1 mPa s at -14.4 ± 0.3 °C to 958 ± 27 mPa s at -64.3 ± 0.4 °C in the binary system, and up to 55387 ± 1068 mPa s at -75 ± 0.5 °C in the ternary (10% Me2SO, 0.9% NaCl by weight) solution were seen. This increase in viscosity limits molecular diffusion, reducing adsorption onto the crystal plane. These viscosities are significantly lower than observed in glycerol based systems and so cells in freeze concentrated channels cooled to between -60 °C and -75 °C will reside in a thick fluid not a near-solid state as is often assumed. In addition, the viscosities experienced during cooling of various Me2SO based vitrification solutions is determined to below -70 °C, as is the impact which additional solutes exert on viscosity. These data show that additional solutes in a cryopreservation system cause disproportionate increases in viscosity. This in turn impacts diffusion rates and mixing abilities of high concentrations of cryoprotectants, and have applications to understanding the fundamental cooling responses of cells to Me2SO based cryopreservation solutions.

A simple assay system to monitor the potential for contamination during different stages of cryopreservation.

A simple assay to monitor the potential for contamination during different steps of cryopreservation is described. The assay is based on the contamination of liquid nitrogen using crystals of sucrose hemi-heptahydrate, these are stable in liquid nitrogen, nitrogen vapour and ambient air and can be monitored by a simple assay which allows contamination risks to be evaluated in a direct, rapid manner.

Contaminated liquid nitrogen vapour as a risk factor in pathogen transfer.

Liquid nitrogen in storage containers will gather particulate matter from the atmosphere, or the surfaces of containers placed into it, with time. Some of these accumulating particles may be pathogenic organisms and it can be demonstrated that their viability may be conserved by immersion in the liquid nitrogen. This contamination constitutes a risk to the status of stored samples that can, largely, be avoided by the use of appropriate techniques for sealing sample containers and sterilizing their outer surfaces. The present study uses fungal spores and organic crystals to demonstrate that such particles contained in liquid nitrogen are released back into the environment when nitrogen vapour cools programmable freezers or dry shippers. This demonstrates that storage in the vapour phase above liquid nitrogen still carries a real risk of sample, or facility, contamination. Regardless of the safety of the stored sample, this is a potential source of cross-contamination between repositories or experimental sites, both locally and internationally, that could have serious consequences in clinical and agricultural situations. This study provides evidence to suggest that this possibility, as yet unquantified, should be included in risk analysis of storage protocols for reproductive materials. The risk becomes diverse when, for example, semen and embryos are frozen at an agricultural site and the dry shipper can co-transport spores of contaminating crop plant pathogens to the destination site.

Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation.

The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. An alternative mechanism has been proposed by Morris (2006) that cell damage is a result of an osmotic imbalance encountered during thawing. This paper examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. Horse spermatozoa were cooled at a range of cooling rates from 0.3 to 3,000 degrees C/min in the presence of a cryoprotectant. The ultrastructure of the samples was examined by Cryo Scanning Electron Microscopy (CryoSEM) and freeze substitution, to determine whether intracellular ice formed and to examine alternative mechanisms of cell injury during rapid cooling. No intracellular ice formation was detected at any cooling rate. Differential scanning Calorimetry (DSC) was employed to examine the amount of ice formed at different rate of cooling. It is concluded that cell damage to horse spermatozoa, at cooling rates of up to 3,000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.

Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: Freezing kinetics and storage temperature effects.

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at -196 degrees C and -20 degrees C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (-196 degrees C and -80 degrees C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at -20 degrees C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.

Cryopreservation of murine embryos, human spermatozoa and embryonic stem cells using a liquid nitrogen-free, controlled rate freezer.

A Stirling Cycle Cryocooler has been developed as an alternative to conventional liquid nitrogen controlled rate freezers. Unlike liquid nitrogen systems, the Stirling Cycle freezer does not pose a contamination risk, can be used in sterile conditions and has no need for a constant supply of cryogen. Three types of samples from two species (murine embryos, human spermatozoa and embryonic stem cells), each requiring different cooling protocols, were cryopreserved in the Stirling Cycle freezer. For comparison, cells were also frozen in a conventional liquid nitrogen controlled rate freezer. Upon thawing, the rates of survival of viable cells were generally greater than 50% for mouse embryos and human embryonic stem cells, based on morphology (mouse embryos) and staining and colony formation (human embryonic stem cells). Survival rates of human spermatozoa frozen in the Stirling Cycle freezer, based on motility and dead cell staining, were similar to those of samples frozen in a conventional controlled rate freezer using liquid nitrogen.

Cryopreservation of horse semen under laboratory and field conditions using a Stirling Cycle freezer.

A Stirling Cycle freezer has been developed as an alternative to conventional liquid nitrogen controlled rate freezers. Horse semen samples were cooled in 0.25 ml straws and 15 ml bags in the Stirling Cycle freezer under laboratory conditions and as a portable device, powered from a car battery. For comparison, straws were frozen in a conventional liquid nitrogen controlled rate freezer. Upon thawing, motility and viability of samples frozen in the Stirling Cycle freezer were not significantly different when compared to samples frozen in the liquid nitrogen freezer. Unlike liquid nitrogen systems, the Stirling Cycle freezer does not pose a contamination risk, can be used in sterile conditions and has no need for a constant supply of cryogen. The freezer has potential for use in veterinary and genetic conservation applications.

Rapidly cooled human sperm: no evidence of intracellular ice formation.

BACKGROUND: The cellular damage that human spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. Alternatively, the cell damage may be the result of an osmotic imbalance encountered during thawing. This article examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. METHODS: In this study, human spermatozoa were cooled at a range of cooling rates from 0.3 to 3000 degrees C/min. The ultrastructure of the samples was examined by cryo scanning electron microscopy and freeze substitution to determine whether intracellular ice formed during rapid cooling and to examine alternative mechanisms of cell injury during rapid cooling. RESULTS: No intracellular ice formation was detected at any cooling rate. Freeze substitution of cells that had been cooled at 3000 degrees C/min and then slowly warmed showed that the cells had become plasmolysed and had evidence of membrane damage. CONCLUSIONS: Cell damage to human spermatozoa, at cooling rates of up to 3000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.

Improved methods for controlled rapid cooling of cell suspensions.

Simple, reproducible, methods of achieving rapid rates of cooling in the range 100 degrees C min(-1) to 1000 degrees C min(-1) are described for straws and cryovial. These methods use the direct contact of straws or cryovials with pre-cooled granules or plates as the heat sink. Liquid nitrogen may be adsorbed into suitable material, for example activated charcoal, zeolites and molecular sieves as the matrix to achieve rapid cooling. Controllable rapid rates of cooling may also be attained by using non-adsorbants. The rate of cooling may be modified by changing the adsorbant material, the size of the adsorbant granules and the temperature of the adsorbant.

A novel approach to sperm cryopreservation.

Human spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been achieved by the standard approaches of cryobiology. Conventional approaches to cryopreservation impose a linear change of temperature with time; however, the stresses that cells encounter during cryopreservation are all non-linear with time. In this paper it is shown that improved methods of cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes instead of imposing a linear temperature reduction. Several treatments were compared: control of solidification to achieve constant ice formation with time was more damaging than the standard linear reduction in temperature. However, treatments which followed a chosen non-linear concentration profile, referred to as 'controlled concentration' allowed recovery of almost all the cells which were motile before freezing. The biophysical basis of these different responses was examined using the cryostage of a scanning electron microscope and freeze substitution and it was found that, surprisingly, all samples of spermatozoa in the frozen state were neither osmotically dehydrated nor had any visible intracellular ice. Viability on thawing did not appear to correlate with conventional theories of cellular freezing injury, which suggests that for human spermatozoa other factors determine viability following freezing and thawing.

Cytokine dysregulation associated with exam stress in healthy medical students.

The mechanisms of stress-related immune alterations have not been fully elucidated. Cell-mediated immune responses as well as antibody and certain cytokines are reported as being suppressed during times of high stress. However, the role of suppression vs dysregulation has not been established in human stress models. The effect of exam stress on regulatory cytokines in 16 healthy medical students was assessed by measuring type-1 (IFN-gamma) and type-2 (IL-10) cytokines from 72-h PHA/PMA-stimulated PBMC 4 weeks before and 48 h after exams. Results demonstrated decreased IFN-gamma accompanied by increased IL-10 during exam stress that resulted in a decreased IFN-gamma:IL-10 ratio. There was a significant correlation between the cytokine response to PHA/PMA and number and subjective adjustment to daily hassles. Additionally, students who reported greater levels of loneliness also reported greater numbers of and poorer subjective adjustment to hassles. The differences were consistent in both males and females but did not correlate with AM cortisol levels. Additionally, when individuals were grouped into high vs low preexam hassle levels, the type-1/type-2 shift in the IFN-gamma:IL-10 ratio occurred in the low hassles group only. These data suggest that psychologically stressful situations shift type-1/type-2 cytokine balance toward type-2 and result in an immune dysregulation rather than overall immunosuppression. This may partially explain the increased incidence of type-2-mediated conditions such as increased viral infections, latent viral expression, allergic/asthmatic reactions, and autoimmunity reported during periods of high stress.

Direct observation of cold-shock effects in ram spermatozoa with the use of a programmable cryomicroscope.

Cryoinjury in individual ram spermatozoa was investigated in cells cooled at 10 degrees C/min on a programmable cryomicroscope. In physiological buffer and cryoprotective media, there was a smooth decline in sperm swimming speed with decreasing temperature; cooling in buffer caused a marked decline in the proportion of cells displaying forward progression, especially once the temperature fell below 16 degrees C. Spermatozoa cooled in the presence of rhodamine 123, a mitochondrial-specific dye, showed that abolition of sperm motility by cold shock in buffer was not due to mitochondrial inactivation. Temperature decline through the region of 10 degrees C caused a number of spermatozoa in buffer to undergo a sudden asymmetric bending of the flagellum in the region of the midpiece. Ultrastructural studies suggest that this was caused by an unstable, asymmetric membrane lesion. Spermatozoa cooled in the presence of cryoprotective media showed better recovery of motility after rewarming and failed to exhibit the bending effect described above.

The effect of low temperature on Antarctic endolithic green algae.

Laboratory experiments show that undercooling to about -5 degrees C occurs in colonized Beacon sandstones of the Ross Desert, Antarctica. High-frequency temperature oscillations between 5 degrees C and -5 degrees C or -10 degrees C (which occur in nature on the rock surface) did not damage Hemichloris antarctica. In a cryomicroscope, H. antarctica appeared to be undamaged after slow or rapid cooling to -50 degrees C. 14CO2 incorporation after freezing to -20 degrees C was unaffected in H. antarctica or in Trebouxia sp. but slightly depressed in Stichococcus sp. (isolated from a less extreme Antarctic habitat). These results suggest that the freezing regime in the Antarctic desert is not injurious to endolithic algae. It is likely that the freezing-point depression inside the rock makes available liquid water for metabolic activity at subzero temperatures. Freezing may occur more frequently on the rock surface and contribute to the abiotic nature of the surface.

Cold shock injury in animal cells.

Cold shock injury (damage to cell structure and function arising from a sudden reduction in temperature) was for many years considered a phenomenon peculiar to certain cell-types. Only in recent years has it become apparent that widely different cell-types manifest cold shock injury. Thus, cold shock appears to be a more general phenomenon, differences between cell-types being quantitative (in the rate of cooling and temperature range at which injury is sustained) rather than qualitative. Loss of particular cell functions depends on cell-type, but reflects the underlying structural and biochemical damage which has been inflicted by rapid cooling. In particular, membranes lose their selective permeability with the result that many cellular components are released including lipids, proteins and ions. Additionally, sodium and calcium gain access to the interior of the cell. Consequent upon this initial disruption, metabolic activities are diminished and further secondary changes ensue. The possible mechanisms of cold shock injury include membrane thermo-tropism and protein denaturation. Susceptibility to cold shock is influenced by membrane composition, and much experimental evidence points to particular involvement of membrane lipids. One hypothesis implicates lipid phase changes in a cooling rate dependent loss of membrane integrity. Other recent hypotheses invoke biophysical concepts and cytoarchitectural features as considerations in a better understanding of cold shock.

Effect of osmotic stress on the ultrastructure and viability of the yeast Saccharomyces cerevisiae.

Exposure of the yeast Saccharomyces cerevisiae to hypertonic solutions of non-permeating compounds resulted in cell shrinkage, without plasmolysis. The relationship between cell volume and osmolality was non-linear; between 1 and 4 osM there was a plateau in cell volume, with apparently a resistance to further shrinkage; beyond 4 osM cell volume was reduced further. The loss of viability of S. cerevisiae after hypertonic stress was directly related to the reduction in cell volume in the shrunken state. The plasma membrane is often considered to be the primary site of osmotic injury, but on resuspension from a hypertonic stress, which would have resulted in a major loss of viability, all cells were osmotically responsive. The effects of osmotic stress on mitochondrial activity and structure were investigated using the fluorescent probe rhodamine 123. The patterns of rhodamine staining were altered only after extreme stress and are assumed to be a pathological feature rather than a primary cause of injury. Changes in the ultrastructure of the cell envelope were examined by freeze-fracture and scanning electron microscopy. In shrunken cells the wall increased in thickness, the outer surface remained unaltered, whilst the cytoplasmic side buckled with irregular projections into the cytoplasm. On return to isotonic solutions these structural alterations were reversible, suggesting a considerable degree of plasticity of the wall. However, the rate of enzyme digestion of the wall may have been modified, indicating that changes in wall structure persist.