The freezing rate of freeze-etch specimens for electron microscopy

Experiments on the rapid freezing of freeze-etch sized specimens have shown this rate between 0 and ?100 °C to be approximately 1000 °C/sec. This is much faster than the rate of 100 °C/sec estimated by Moor for specimens cooled in liquid Freon 12.Heat transfer from the rapidly immersed specimen and mount appears to be mainly through forced convection. Such a mechanism would make the initial rate highly variable as it would be sensitive to liquid velocity. If this occurs it will be impossible to obtain consistent results for freezingrate studies unless a stable method is evolved for both injecting and containing the specimen.     

Prediction of local cooling rates and cell survival during the freezing of a cylindrical specimen

A finite element numerical model was implemented to simulate the freezing process of an aqueous salt solution in a cylindrical container. Local cooling rates within the container were computed for several defined cooling protocols applied at the boundary. Characteristic cell survival signatures were used to predict the associated local survival rates throughout the system. These calculations show that there are two definite time domains during a typical freezing process: (1) while the surface temperature is changing and (2) after the surface temperature reaches a constant storage value. The calculations also show significant spatial variations in the local cooling rates within the container and considerable local deviation from the volumetric average survival for various simulated freezing protocols.     

Influence of cooling rate on Saccharomyces cerevisiae destruction during freezing: unexpected viability at ultra-rapid cooling rates

The purpose of this work was to study cell viability as a function of cooling rate during freezing. Cooling rate strongly influences the viability of cells during cold thermal stress. One of the particularities of this study was to investigate a large range of cooling rates and particularly very rapid cooling rates (i.e., faster than 20000 degrees C min (-1)). Four distinct ranges of cooling rates were identified. The first range (A(')) corresponds to very slow cooling rates (less than 5 degrees C min (-1)), and results in high cell mortality. The second range (A) corresponds to low cooling rates (5-100 degrees C min (-1)), at which cell water outflow occurs slowly and does not damage the cells. The third range (B) corresponds to rapid cooling rates (100-2000 degrees C min (-1)), at which there is competition between heat flow and water flow. In this case, massive water outflow, which is related to the increase in extracellular osmotic pressure and the membrane-lipid phase transition, can cause cell death. The fourth range (C) corresponds to very high cooling rates (more than 5000 degrees C min (-1)), at which the heat flow is very rapid and partially prevents water exit, which seems to preserve cell viability.     

Hypercapnia increases core temperature cooling rate during snow burial.

Previous retrospective studies report a core body temperature cooling rate of 3 degrees C/h during avalanche burial. Hypercapnia occurs during avalanche burial secondary to rebreathing expired air, and the effect of hypercapnia on hypothermia during avalanche burial is unknown. The objective of this study was to determine the core temperature cooling rate during snow burial under normocapnic and hypercapnic conditions. We measured rectal core body temperature (T(re)) in 12 subjects buried in compacted snow dressed in a lightweight clothing insulation system during two different study burials. In one burial, subjects breathed with a device (AvaLung 2, Black Diamond Equipment) that resulted in hypercapnia over 30-60 min. In a control burial, subjects were buried under identical conditions with a modified breathing device that maintained normocapnia. Mean snow temperature was -2.5 +/- 2.0 degrees C. Burial time was 49 +/- 14 min in the hypercapnic study and 60 min in the normocapnic study (P = 0.02). Rate of decrease in T(re) was greater with hypercapnia (1.2 degrees C/h by multiple regression analysis, 95% confidence limits of 1.1-1.3 degrees C/h) than with normocapnia (0.7 degrees C/h, 95% confidence limit of 0.6-0.8 degrees C/h). In the hypercapnic study, the fraction of inspired carbon dioxide increased from 1.4 +/- 1.0 to 7.0 +/- 1.4%, minute ventilation increased from 15 +/- 7 to 40 +/- 12 l/min, and oxygen saturation decreased from 97 +/- 1 to 90 +/- 6% (P < 0.01). During the normocapnic study, these parameters remained unchanged. In this study, T(re) cooling rate during snow burial was less than previously reported and was increased by hypercapnia. This may have important implications for prehospital treatment of avalanche burial victims.     

Suprazero cooling rate,rather than freezing rate,determines post thaw quality of rhesus macaque sperm

Sperm become most sensitive to cold shock when cooled from 37 °C to 5 °C at rates that are too fast or too slow; cold shock increases the susceptibility to oxidative damage owing to its influence on reactive oxygen species (ROS) production, which are significant stress factors generated during cooling and low temperature storage. In addition, ROS may be a main cause of decreased motility and fertility upon warming. They have been shown to change cellular function through the disruption of the sperm plasma membrane and through damage to proteins and DNA. The objective of this study was to determine which cryopreservation rates result in the lowest degree of oxidative damage and greatest sperm quality. In the rhesus model, it has not been determined whether suprazero cooling or subzero freezing rates causes a significant amount of ROS damage to sperm. Semen samples were collected from male rhesus macaques, washed, and resuspended in TEST-yolk cryopreservation buffer to 100 × 10⁶ sperm/mL. Sperm were frozen in 0.5-mL straws at four different combinations of suprazero and subzero rates. Three different suprazero rates were used between 22 °C and 0 °C: 0.5 °C/min (slow), 45 °C/min (medium), and 93 °C/min (fast). These suprazero rates were used in combination with two different subzero rates for temperatures 0 °C to −110 °C: 42 °C/min (medium) and 87 °C/min (fast). The different freezing groups were as follows: slow-med (SM), slow-fast (SF), med-med (MM), and fast-fast (FF). Flow cytometry was used to detect lipid peroxidation (LPO), a result of ROS generation. Motility was evaluated using a computer assisted sperm motion analyzer. The MM and FF treated sperm had less viable (P < 0.0001) and motile sperm (P < 0.001) than the SM, SF, or fresh sperm. Sperm exposed to MM and FF treatments demonstrated significantly higher oxidative damage than SM, SF, or fresh sperm (P < 0.05). The SM- and SF-treated sperm showed decreased motility, membrane integrity, and LPO compared with fresh semen (P < 0.001). Slow cooling from room temperature promotes higher membrane integrity and motility post thaw, compared with medium or fast cooling rates. Cells exposed to similar cooling rates with differing freezing rates were not different in motility and membrane integrity, whereas comparison of cells exposed to differing cooling rates with similar freezing rates indicated significant differences in motility, membrane integrity, and LPO. These data suggest that sperm quality seems to be more sensitive to the cooling, rather than freezing rate and highlight the role of the suprazero cooling rate in post thaw sperm quality.     

Theoretical analysis of the ice crystal size distribution in frozen aqueous specimens.

To estimate theoretically how suited different freezing techniques are for freezing of freeze-etch specimens, it is necessary to know the relationship between specimen cooling rate and the resulting average ice crystal size. Using a somewhat simplified theoretical analysis, we have derived the approximate ice crystal size distribution of nonvitrified frozen aqueous specimens frozen at different cooling rates. The derived size distribution was used to calculate the relationship between relative change in average ice crystal size, (delta l/l), and relative change in specimen cooling rate delta (dT/dt)/(dT/dt). We found this relationship to be (delta l/l) = -k X delta (dT/dt)/(dT/dt) where k = 1.0 when specimen solidification takes place at about -6 degrees C, and k congruent to 1.3 when it takes place at about -40 degrees C.     

Cryopreservation of cornea: a low cooling rate improves functional survival of endothelium after freezing and thawing

AIM: To investigate the influence of low cooling rates on endothelial function and morphology of corneas frozen with propane-1,2-diol (PROH). METHODS: Rabbit corneas, mounted on support rings, were exposed to 1.4mol/l (10% v/v) PROH, seeded to initiate freezing, and cooled at 0.2 or 1 degrees C/min to -80 degrees C. Corneas were frozen immersed in liquid or suspended in air. After being held overnight in liquid nitrogen, corneas were warmed at 1 or 20 degrees C/min. After stepwise removal of the cryoprotectant, the ability of the endothelium actively to control corneal hydration was monitored during normothermic perfusion. Morphology was assessed after staining with trypan blue and alizarin red S, and by specular microscopy during perfusion. RESULTS: Functional survival was achieved only after slow cooling (0.2 degrees C/min) with the cornea immersed in the cryoprotectant medium, and rapid warming (20 degrees C/min). These conditions also gave the best morphology after freezing and thawing. CONCLUSION: Cooling rates lower than those typically applied to cornea improved functional survival of the endothelium. This result is in accord with previous observations showing the benefit of low cooling rates for cell monolayers [CryoLetters 17 (1996) 213-218].     

The impact of freezing during maturation on storage products in canola seeds

Mature canoia ( Brassica napus cv. Westar) seeds contain large quantities of the storage proteins cruciferin and napin and storage lipids rich in C18: 1 and C18:2 fatty acids. Both the quantity and quality of these products are altered by freezing during development. Further, the response to freezing changes during seed development. The effects include decreased fatty acid chain elongation, altered fatty acid unsaturation, higher lipid levels and lower protein levels. In addition, seeds in the pivotal moisture range (55%) may be predisposed to precocious germination, which is then inhibited by a lack of adequate seed moisture. The results indicate that freezing imparts its effect in two ways. Initially, there is a freezing (low temperature) component and this is followed by rapid desiccation of the seeds. Although most responses probably result from a combination of the stresses, it appears that inhibition of fatty acid chain elongation is caused by the freezing component and the gradual inhibition of storage protein accumulation is a result of accelerated seed desiccation.     

Interaction of rate of latent heat removal during freezing and rates of subsequent cooling and warming on survival of the blood protozoan, Babesia rodhaini

Babesia rodhaini parasites in murine blood containing 1.5 m DMSO were frozen at two rates, as judged by the duration of the “freezing plateau”, then cooled to ?196 °C and rewarmed at two rates to detect interactions between the duration of the plateau and rates of subsequent cooling and rewarming. Infectivity tests showed that fast and slow freezing (plateau times of about 1 sec and 30 sec, respectively) had similar effects on parasite survival when cooling was at 130 °C/min and warming was at 800 °C/min. However, when either the cooling rate was increased to 3500 °C/min or the warming rate was decreased to 2.3 °C/min, fast freezing decreased parasite survival more than did slow freezing. It is suggested that fast freezing accentuated the damaging effects of fast cooling and slow warming by increasing intracellular ice formation.     

Effects of cooling and freezing on pH of semen extender

The pH change of 10 different buffering systems with temperature ranging from room to 5 °C was examined; three were conventional buffers which included phosphate yolk, citrate yolk, and skim milk. Seven were Good's buffers with egg yolk which included TES, TRIS, BES, MOPS, PIPES, MES, and TEST. The pH of the three conventional buffers did not change with decreasing temperature, but Good's buffers showed an increase in pH with decreasing temperature from room to 5 °C. The pH change due to temperature was measured for TEST buffer solution with and without 20% egg yolk containing 2 or 6% of five different cryoprotective compounds. The pH at 5 °C was significantly higher than at room temperature. The addition of egg yolk and/or cryoprotective compound did not alter the pH significantly during cooling, even though a slight drop in pH was noted with the addition of egg yolk indicating that the change in pH is primarily due to the buffer. The pH of TEST yolk buffer (pH 7.2 at room temperature) was measured continuously from 37 °C to below freezing (?18 °C). The pH increased with decreasing temperature to 8.0 ± 0.2 from 37 to ?14 °C at which point it dropped abruptly to pH 6.5 ± 0.2.     

Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos

Two studies were conducted to evaluate the influence of cryoprotectant, cooling rate, container and cryopreservation procedure on the post-thaw viability of sheep embryos. In Study 1, late morula- to blastocyst-stage embryos were exposed to 1 of 10 cryoprotectant (1.5 M, glycerol vs propylene glycol)-plunge temperature treatments. Embryos were placed in glass ampules and cooled at 1 degrees C/min to -5 degrees C, seeded and further cooled at 0.3 degrees C/min to -15, -20, -25, -30 and -35 degrees C before rapid cooling by direct placement in liquid nitrogen (LN(2)). Post-thaw embryo viability was improved (P<0.01) when embryos were cooled to at least -30 degrees C before LN(2) plunging. Although there were no overt differences in embryo viability between cryoprotectant treatments (each resulted in live offspring after embryo transfer), there was a lower (P<0.01) incidence of zona pellucida damage using propylene glycol (4%) compared to glycerol (40%). In Study 2, embryos were equilibrated in 1.5 M propylene glycol or glycerol or a vitrification solution (VS3a). Embryos treated in propylene glycol or glycerol were divided into ampule or one-step((R)) straw treatments, cooled to -6 degrees C at 1 degrees C/min, seeded, cooled at 0.5 degrees C/min to -35 degrees C, held for 15 minutes and then transferred to LN(2). Embryos vitrified in the highly concentrated VS3a (6.5 M glycerol + 6% bovine serum albumin) were transferred from room air to LN(2) vapor, and then stored in LN(2). Propylene glycol- and glycerol-treated embryos in straws experienced lower (P<0.05) degeneration rates (27%) and yielded more (P<0.05) hatched blastocysts (73 and 60%, respectively) at 48 hours of culture and more (P<0.05) trophoblastic outgrowths (67 and 53%, respectively) after 1 week than vitrified embryos (47, 40 and 20%, respectively). In vitro development rate for VS3a-treated embryos was similar (P>0.10) to that of ampule controls, which had fewer (P<0.05) expanded blastocysts compared to similar straw treatments. Live offspring were produced from embryos cryopreserved by each straw treatment (propylene glycol, 3 of 7; glycerol, 1 of 7; VS3a, 2 of 7). In summary, freeze-preservation of sheep embryos was more effective in one-step straws than glass ampules and propylene glycol tended to be the optimum cryoprotectant. Furthermore, these findings demonstrate, for the first time, the biological competence of sheep embryos cryopreserved using the simple and rapid procedure of vitrification.     

The effect of specimen geometry on the mechanical behaviour of trabecular bone specimens.

The effect of specimen geometry on the mechanical behaviour of trabecular bone specimens was studied by non-destructive uniaxial compression to 0.4% strain using cylindrical specimens with different sizes and length-to-diameter ratios, and by comparing cubic and cylindrical specimens with the same cross-sectional area. Both the length and the cross-sectional area of the specimen had a highly significant influence on the mechanical behaviour (p less than 0.0001). Within the actual range of length (2.75-11.0 mm) the normalized stiffness (Young's modulus) was related nearly linearly to the specimen length. This dependency on specimen length is suggested to be caused mainly by structural disintegrity of the trabecular specimens near the surface. The normalized stiffness (Young's modulus) was also positively correlated to the cross-sectional area. This dependency on cross-sectional area is probably due to friction-induced stress inhomogeneity at the platen-specimen interface. A cube with side length 6.5 mm or a cylindrical specimen with 7.5 mm diameter and 6.5 mm length are suggested as standard specimens for comparative studies on trabecular bone mechanics.