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PubMed2006

Involvement of two specific causes of cell mortality in freeze-thaw cycles with freezing to -196 degrees C.

The purpose of this study was to examine cell viability after freezing. Two distinct ranges of temperature were identified as corresponding to stages at which yeast cell mortality occurred during freezing to -196 degrees C. The upper temperature range was related to the temperature of crystallization of the medium, which was dependent on the solute concentration; in this range mortality was prevented by high solute concentrations, and the proportion of the medium in the vitreous state was greater than the proportion in the crystallized state. The lower temperature range was related to recrystallization that occurred during thawing. Mortality in this temperature range was increased by a high cooling rate and/or high solute concentration in the freezing medium and a low temperature (less than -70 degrees C). However, a high rate of thawing prevented yeast mortality in this lower temperature range. Overall, it was found that cell viability could be conserved better under freezing conditions by increasing the osmotic pressure of the medium and by using an increased warming rate.

Calorimetry, Differential Scanning↗

PubMed1994

Alterations in hepatic adrenergic receptor status in Rana sylvatica in response to freezing and thawing: implications to the freeze-induced glycemic response.

In Rana sylvatica, freeze-induced liberation of glucose from hepatic glycogen stores plays a critical role in conferring freeze tolerance. To determine whether an alteration in hepatic adrenergic receptor status, which dictates catecholamine-directed hepatic glycogenolytic responses, is involved in the glycemic response to freezing, hepatic alpha 1, alpha 2, and beta 2 adrenergic receptors and calcium transport were characterized by radioligand and radioisotopic techniques, respectively, in plasma membranes isolated from the livers of control, -2.5 degrees C-exposed, and frozen-thawed frogs. The three adrenergic receptors display marked and different patterns of changes in response to freezing, with two distinct receptor shifts clearly evident. In the control state, the beta 2 adrenergic receptor dominates over the alpha 1 receptor. At 12 h, beta 2 adrenergic receptor dominance intensifies by a receptor shift involving a decrease in the alpha 1 and alpha 2 adrenergic receptors. Coincident with the initiation of the glycemic response, this early shift may be causally related to it. At 24 h, the alpha 1 adrenergic receptor dominates, achieved by a receptor shift involving a decrease in the beta 2 adrenergic receptor and an increase in the alpha 1 and alpha 2 adrenergic receptors. This shift may be related to the maintenance of the glycemic response. Receptor shifts are associated with changes in calcium transport, which accentuate them. The thawed state is characterized by recovery of alpha, but not beta 2, receptor expression correlatable with, and perhaps allowing, a switch to hepatic glycogenesis. The role of thyroid hormone, whose levels are lower in the frozen state, in inducing receptor shifts is discussed.

Animals↗

PubMed1991

Allometry of cooling, supercooling, and freezing in the freeze-tolerant turtle Chrysemys picta.

Although several vertebrates are freeze tolerant, little is known of the relationship between body size and the kinetics of cooling and freezing. We compared these responses for six hatchling and eight adult Chrysemys picta from an Ohio population. All turtles initially recovered from freezing, and all adults, but only two hatchlings (which experienced ice contents of approximately 35%), exhibited long-term survival. Rapid thawing may have compromised hatchling survival. Turtle water content was inversely related to body mass, but we found no significant correlation between the extent of supercooling and body size. Prefreezing and postfreezing cooling rates scaled with body mass to the -0.55 and -0.40 power, respectively, but the latter rate was more than two orders of magnitude slower. Theoretical (assuming 20% bound water) and calorimetric estimates of body ice agreed reasonably well. Ice contents were both body mass and time dependent. The absolute rate of ice formation scaled with body mass to the 0.4 power. Body size strongly influences the freezing response of ectotherms and deserves more attention.

Aging↗

PubMed2004

To freeze or not to freeze: adaptations for overwintering by hatchlings of the North American painted turtle.

Many physiologists believe that hatchling painted turtles (Chrysemys picta) provide a remarkable, and possibly unique, example of 'natural freeze-tolerance' in an amniotic vertebrate. However, the concept of natural freeze-tolerance in neonatal painted turtles is based on results from laboratory studies that were not placed in an appropriate ecological context, so the concept is suspect. Indeed, the weight of current evidence indicates that hatchlings overwintering in the field typically withstand exposure to ice and cold by avoiding freezing altogether and that they do so without benefit of an antifreeze to depress the equilibrium freezing point for bodily fluids. As autumn turns to winter, turtles remove active nucleating agents from bodily fluids (including bladder and gut), and their integument becomes a highly efficient barrier to the penetration of ice into body compartments from frozen soil. In the absence of a nucleating agent or a crystal of ice to 'catalyze' the transformation of water from liquid to solid, the bodily fluids remain in a supercooled, liquid state. The supercooled animals nonetheless face physiological challenges, most notably an increased reliance on anaerobic metabolism as the circulatory system first is inhibited and then caused to shut down by declining temperature. Alterations in acid/base status resulting from the accumulation of lactic acid may limit survival by supercooled turtles, and sublethal accumulations of lactate may affect behavior of turtles after the ground thaws in the spring. The interactions among temperature, circulatory function, metabolism (both aerobic and anaerobic), acid/base balance and behavior are fertile areas for future research on hatchlings of this model species.

Adaptation, Physiological↗

PubMed1996

Cryoprotective effects of cycloinulohexaose on freezing and freeze-drying of liposomes.

Cycloinulohexaose (CF-6) maintained the membrane structure of the liposomes during freeze-thawing, and thus CF-6 was very effective in inhibiting both drug leakage from liposomes and the size change of liposomes during freezing and freeze-drying. As the ratio of CF-6 to lipid increased from 1 to 5, calcein retention in the liposomes increased from 70% up to 85% and a maximum retention was obtained at a ratio of more than 2. The addition of glycerol enhanced the above cryoprotective effect of CF-6 and improved calcein retention up to 90%. Neither detectable leakage of calcein nor particle size change was observed after the storage of liposomes freeze-dried with CF-6 for 6 months at 4 and 25 degrees C.

Calorimetry, Differential Scanning↗

PubMed1991

Post-embedding immuno-electron microscopy with rapid freezing and freeze-substitution techniques for the localization of manganese superoxide dismutase.

Dehydration of specimens with ethanol or acetone makes it impossible to detect manganese superoxide dismutase (Mn-SOD) by immunohistochemistry. To circumvent obstacles and demonstrate localization by post-embedding immuno-electron microscopy, a rapid freezing and freeze-substitution technique was employed using Lowicryl K4M embedding medium. This was effective enough to allow the specific observation of immunogold particles for Mn-SOD on the mitochondria of cardiac muscle cells and WI-38 cells (human normal fetal lung diploid cells). This method preserved the antigen-antibody binding activity of Mn-SOD even after dehydration. Therefore, rapid freezing and freeze-substitution is useful for post-embedding immuno-electron microscopy of Mn-SOD and can further be employed for other antigens previously difficult to detect by conventional methods.

Acrylic Resins↗

PubMedDate not supplied

Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function.

In this study, we evaluated the effects of glutathione (L-gamma-glutamyl-L-cysteinylglycine; GSH) supplementation of the freezing extender on semen parameters during the cooling (2 hours at 5 degrees C) and freezing phases of the cryopreservation process to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we incorporated a new set of functional sperm tests. These included tests of mitochondrial function, inducibility of the acrosome reaction, in vitro penetration (IVP) of oocytes, changes in sulfhydryl group content in membrane proteins, and capacitation status. The main findings emerging from this study were that the addition of GSH to the freezing media resulted in 1) an improvement in percent motility (%MOT) and motion parameters of thawed spermatozoa, as measured by both microscopic analysis and computer-assisted semen analysis (CASA); 2) a higher number of total viable spermatozoa; 3) a higher number of noncapacitated viable spermatozoa; and 4) a decrease in the number of spermatozoa with changes in the sulfhydryl groups in membrane proteins. This protective effect on sperm function was more pronounced with 1 mM of GSH than with 5 mM of GSH.

Acrosome Reaction↗

PubMed2002

Large batch freezing of bull semen: effect of time of freezing and fructose on fertility.

Large-scale batch freezing of bull semen should be done in a processing schedule that yields the highest fertility and when it can be fitted efficiently into the work schedule. Conflicting reports have appeared on survival and fertility of bull sperm frozen within a few hours of semen collection or on the next day. To study this problem, a factorially arranged experiment with semen from 10 bulls was conducted, comparing whole milk-glycerol semen extender with and without fructose, and semen frozen in 0.5-ml straws after 4 versus 18 h of equilibration at 5 degrees C. Both fructose and 18 h of equilibration resulted in a small but significant improvement in freeze-thaw survival of sperm. A field trial followed with replicated semen collections from nine bulls processed in a whole milk-glycerol control extender frozen after 4 h of equilibration versus the addition of 1.25% (wt/vol) fructose to whole milk glycerol divided to freeze sperm after 4 and 28 h of equilibration. Semen from these bulls was used to inseminate 14,775 first-service cows. The 56-d nonreturn rates obtained for these three treatments were 74.7, 74.3, and 73.9%, respectively. As there was no difference in fertility, it would appear that programs to freeze sperm in whole milk extenders the same day of collection or the day after semen collection should yield equivalent results.

Animals↗

PubMed1992

[Fine structure of the guinea pig organ of Corti revealed by a quick-freeze, freeze-substitution method].

A quick-freeze, freeze-substitution method was applied to the guinea pig organ of Corti. Many similarities were apparent between the conventionally fixed samples and the freeze-substituted samples, including the lateral membrane system of the cochlear outer hair cells consisting of the lateral plasma membrane, pillars, cytoskeletal lattice and subsurface cysternae. However, some ultrastructural differences were found in the cells prepared by the freeze-substitution method. Electron-dense mosaic structures were found in the lumen of the tubules of the subsurface cysternae. Tangential sections of the tubules of the subsurface cysternae showed spirally wound parallel rows of electron-dense materials. In a rectangularly cut membrane with a trilaminar structure, the electron-dense materials appeared to be triangular protrusions from the outer leaflet of the unit membrane. These structures suggest that the subsurface cysternae may play an important role in the contraction of cochlear outer hair cells.

Animals↗

PubMed1992

Scanning electron microscopy of freeze-dried preparations: relationship of morphology to freeze-drying parameters.

The structure of the freeze-dried plug of several biological preparations was examined using scanning electron microscopy (SEM). A variety of carbohydrate preparations were frozen at two different cooling rates of 150 degrees C per minute and 1 degree C per minute. After lyophilization, residual moisture analysis demonstrated that under conditions of rapid freezing a drier product could be obtained. SEM observations showed the lyophilised plug structure of rapidly frozen material consisted of a fine amorphous meshwork, while material frozen slowly consisted of a leafy amorphous material. Freeze-dried mannitol preparations were shown to be of a crystalline nature. In most instances, rapidly frozen material was easier to reconstitute after lyophilization. SEM of proteinaceous preparations which had been frozen at differing rates also showed a variety of different appearances which were probably related to the direction of ice growth. During ice growth formation the migration of buffers and salts towards the surface may occur. Localised high salt/buffer areas may cause deterioration of the product and reduce shelf life. SEM observations were made on the freeze-dried plug of preparations which had collapsed during the lyophilization process. Typically, the basal region of the plug had a more solid structure than the open meshwork of successfully dried material.

Biological Products↗

PubMed1978

Freezing of rat lymphocytes. V. The effect of suppressor cells in phytohaemagglutinin-stimulated spleen cell cultures before and after freeze-thawing.

Spleen lymphocyte proliferation, as measured by 3H-thymidine incorporation induced by phytohaemagglutinin (PHA), was increased after freeze-thawing with 10% dimethyl sulphoxide. Depletion or intoxication of macrophages in fresh spleen cell preparations also increased lymphocyte proliferation in response to PHA. On the other hand, freezing of macrophage-depleted spleen cell suspensions lowered 3H-thymidine uptake of stimulated cultures. At concntrations above 3%, macrophages added to cultures of fresh purified lymphocytes showed a dose-dependent inhibitory effect on the PHA response, and fresh macrophages were more inhibitory than frozen-thawed macrophages. Purified lymphocytes mixed with 10% macrophages showed a higher response after freeze-thawing. It is concluded that macrophages suppress the lymphoproliferative response to PHA in rat spleen cell cultures, and that these macrophages are more sensitive than lymphocytes to the present freeze-thaw process.

Animals↗

PubMed1977

[Rapid freezing of biologic tissue. Measurement of temperature and rate of freezing by thin-layer thermocouple].

An apparatus for rapid freezing of biological tissues by contact with a copper block cooled by liquid helium has been devised to reduce the contamination of copper block surface. It prevents the precooling of the specimen while going through the layers of cold helium gas surrounding the copper block and reduces the quantity of helium necessary for freezing. It also enables one to obtain easily reproducible results from freezing by immersion in liquid coolants. Freezing rates are measured directly, related to the specimen thickness, by a thin film thermocouple; its low thermal inertia gives speed measurements of the order of 100,000 degrees C/s.

Animals↗

PubMed1991

Assessment of ultrastructure in isolated cochlear hair cells using a procedure for rapid freezing before freeze-fracture and deep-etching.

Separated cochlear outer hair cells and isolated strips of organ of Corti containing hair cells and supporting cells have been rapidly frozen before freeze-fracture and deep-etching by immersion of samples sandwiched between two copper plates into liquid nitrogen-cooled propane: isopentane. Assessment of this procedure has shown that no significant freezing damage occurs. The ultrastructure of the hair cells revealed by freeze-fracture of these non-chemically fixed preparations was generally very similar to that seen in fixed material. This indicates that the processing of cochlear tissue normally used for electron microscopy produces few obvious structural artefacts. It also demonstrated that procedures for isolating cochlear hair cells generally do not affect cell structure significantly. However, some isolated hair cells did show abnormalities within the membranes of the lateral cisternae. Such membrane alterations, which would not be identified by light microscopy, occurred to a variable extent but were more commonly present after prolonged periods in maintenance medium. Deep-etching of the preparations to examine extracellular features around stereocilia revealed clearly lateral cross-links between stereocilia. However, tip-links could not be positively identified in either unfixed or prefixed preparations.

Animals↗

PubMed1974

Freezing of Nonwoody Plant Tissue: II. Cell Damage and the Fine Structure of Freezing Curves.

Temperature recordings of the freezing of plant tissues include two plateaus or regions of reduced slope. During the second of these, small positive spikes were observed. When a completely frozen tissue was thawed and refrozen, neither the second plateau nor the spikes were recorded. Both were present, however, if the initial freezing had been terminated before the second plateau had been reached. The spikes appear to represent the release of heat of crystallization during the freezing of individual cells. Such a freezing and thawing cycle destroys the ability of the cells to remain supercooled in the presence of the ice that is formed as the first plateau is recorded.

Journal Article↗

PubMed1979

Freezing Injury and Phospholipid Degradation in Vivo in Woody Plant Cells: III. Effects of Freezing on Activity of Membrane-bound Phospholipase D in Microsome-enriched Membranes.

Freeze-thawing of microsome-enriched membranes from living bark tissues of black locust trees, especially those from less hardy tissues, caused a drastic increase in sensitivity to Ca(2+) and a complete loss of the regulatory action of Mg(2+) in membrane-bound phospholipase D activity with endogenous (membrane-bound) substrates. Also, the freeze-thaw cycle made phospholipase D in these membranes more resistant to digestion by proteases. Thus, the regulatory properties of the membrane-bound phospholipase D seem to be dependent on the nature of the membranes and on the interaction between the enzyme and membranes as well. The alteration of regulatory properties by freezing was protected by sucrose, at lower concentrations, and more effectively for membranes from hardy tissues than for membranes from less hardy tissue. Addition of partially purified soluble phospholipase D to the reaction system containing membranes caused only a slight stimulation of the degradation of endogenous phospholipids. Phospholipid degradation in vivo during freezing of less hardy tissue may be catalyzed mainly by the bound enzyme. Disintegration of the tonoplast, however, besides releasing soluble phospholipase D into the cytosol, would release organic acids (lowering the pH) and free Ca(2+). Both factors would stimulate drastically the membrane-bound phospholipase D, causing degradation of membrane phospholipids.

Journal Article↗

PubMed1989

Relative sensitivity of photosynthesis and respiration to freeze-thaw stress in herbaceous species : importance of realistic freeze-thaw protocols.

The relative effect of a freeze-thaw cycle on photosynthesis, respiration, and ion leakage of potato leaf tissue was examined in two potato species, Solanum acaule Bitt. and Solanum commersonii Dun. Photosynthesis was found to be much more sensitive to freezing stress than was respiration, and demonstrated more than a 60% inhibition before any impairment of respiratory function was observed. Photosynthesis showed a slight to moderate inhibition when only 5 to 10% of the total electrolytes had leaked from the tissue (reversible injury). This was in contrast to respiration which showed no impairment until temperatures at which about 50% ion leakage (irreversible injury) had occurred. The influence of freeze-thaw protocol was further examined in S. acaule and S. commersonii, in order to explore discrepancies in the literature as to the relative sensitivities of photosynthesis and respiration. As bath cooling rates increased from 1 degrees C/hour to about 3 or 6 degrees C/hour, there was a dramatic increase in the level of damage to all measured cellular functions. The initiation of ice formation in deeply supercooled tissue caused even greater damage. As the cooling rates used in stress treatments increased, the differential sensitivity between photosynthesis and respiration nearly disappeared. Examination of agriculturally relevant, climatological data from an 11 year period confirmed that air cooling rates in the freezing range do not exceed 2 degrees C/hour. It was demonstrated, in the studies presented here, that simply increasing the actual cooling rate from 1.0 to 2.9 degrees C/hour, in frozen tissue from paired leaflet halves, meant the difference between cell survival and cell death.

Journal Article↗

PubMed2006

Comparison of two freezing protocols in an open freezing system for cryopreservation of rat ovarian tissue.

AIM: To compare two freezing protocols in an automatic open-vessel freezing system for cryopreservation of rat ovarian tissue. METHODS: Ovarian tissue was transplanted heterotopically into the neck muscle, either without cryopreservation (group 1, n = 6) or with cryopreservation after equilibration with 1.5 mol/L dimethyl sulfoxide and propanediol (protocol A, group 2, n = 6) or 1.5 mol/L ethyl glycol (protocol B, group 3, n = 6). The ovarian tissue was examined with LIVE/DEAD fluorescent viability staining and histologically after isotransplantation. RESULTS: The healthy follicular loss (intact oocyte and >50% granulosa cells alive) due to cryopreservation was 15.5% with protocol A and 12.2% with protocol B. Histological examination showed follicles in all developmental phases in all groups: group 1, 35.5 +/- 5.7/mm(2) (mean +/- SD); group 2, 16.0 +/- 5.0/mm(2); group 3, 17.3 +/- 5.7/mm(2). The differences between groups 1 and 2 and between groups 1 and 3 were significant (P < 0.001). The difference between groups 2 and 3 was not significant (P = 0.33). CONCLUSIONS: These results demonstrate that the use of an open freezing system with both freezing protocols allows cryopreservation of rat ovarian tissue with equally good survival rates.

Animals↗

PubMed1984

New observations on the ultrastructure of mammalian conducting airway epithelium: application of liquid propane freezing, deep etching, and rotary shadowing techniques to freeze-fracture.

Freshly isolated, viable hamster tracheal epithelium was rapidly frozen in a jet of liquid nitrogen-cooled propane followed by freeze-fracture, deep etching, and rotary shadowing. Examination of the replicas by transmission electron microscopy revealed characteristic features of both ciliated and nonciliated cells, profiles of tight junctional complexes, and two distinct types of membrane particle complexes. The findings of this study suggest that rapid freezing of viable biological specimens coupled with freeze-fracture, deep etching, and rotary shadowing may provide a useful approach to achieving some additional perspectives of cell structure and function. Since the experimental protocol avoided any use of chemical fixation and processing procedures generally employed in electron microscopy, this study also validates for airway epithelium the details of fine structure observed using more conventional ultrastructural methods.

Animals↗