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PubMedDate not supplied

[Use of hydroxyethyl starch as a cryoprotective medium during platelet storage at low temperatures].

The effects of HES and HES + DMSO used as cryoprotective media for storage of human platelets in liquid nitrogen and vapor phase of liquid nitrogen were studied. Solution of 6% and 15% HES with molecular weight ranging from 65,000 to 250,000, and 10% DMSO were used. The criteria accepted for evaluation of the efficiency of these cryoprotective media were: 1. platelet counts, 2. participation of platelets in the processes of hemostasis measured in vitro by the ability of platelets to release adenine nucleotides (ATP + ADP) after thrombin stimulation. It was found that 15% HES is a more effective cryoprotective medium than 6% HES. The use of 15% HES + 10% DMSO gave similar results as the use of 10% DMSO alone.

Blood Cell Count↗
PubMed1981

[Comparison between cryoprotective potentials of glycerol, dimethylsulphoxide, dimethylformamide, and ethylene glycol in cryopreservation of human semen (author's transl)].

The cryoprotective effects of dimethylsulphoxide, dimethylformamide, and ethylene glycol in definite concentrations of 3.5, 7.0, and 12.0 per cent were compared with glycol action in cryoprotective preservation of human semen. Motility behaviour in response to laboratory stress (due to incubation at + 40 degrees C) as well as respiratory activation quotients for succinate, digitonin, and 2.4-dinitrophenol were used as criteria of assessment. The cryoprotective effect of glycerol was in no single instance surpassed by any of the other compounds, with the concentrations tested.

Cryoprotective Agents↗
PubMed1998

Effect of different concentrations of cryoprotectant and extender on the hatching of indian major carp embryos (Labeo rohita, catla catla, and cirrhinus mrigala) stored at low temperature

Rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) embryos were examined for hatching success after short-term (8-13 h) storage at low temperature (4 degreesC) using three concentrations of three cryoprotectants, methanol (1, 2, and 3 M), dimethyl sulfoxide (1.282, 1.923, and 2.564 M, equal to 10, 15, and 20%), and glycerol (1.087, 1.63, and 2.174 M, equal to 10, 15, and 20%), separately as well as together with a single concentration of sucrose (0.5 M). The aim was to determine the toxicity of cryoprotectants for fish embryos at 4 degreesC. Of the cryoprotectants used, methanol was found to be most suitable for low-temperature storage in the refrigerator. The hatching rate of embryos was significantly higher when sucrose was added to methanol when compared with methanol alone. Of the three concentrations of methanol tested, survival was maximal at 2 M in the presence of 0.5 M sucrose (rohu, 57.5 +/- 5.24; catla, 47.5 +/- 5.24; and mrigal, 32. 5 +/- 5.24). Both rohu and catla embryos survived with either 1 or 2 M methanol, with or without the addition of sucrose, but the addition of sucrose was essential for survival of mrigal at 1 and 2 M methanol. Copyright 1998 Academic Press.

Journal Article↗
PubMed2002

Simple cryoprotection and cell dissociation techniques for application of the comet assay to fresh and frozen rat tissues.

The single-cell gel electrophoresis (comet) assay has been widely used for genotoxicity studies in cell cultures, but its use in solid tissues is hindered by problems in isolation of cells and in cryopreservation techniques. Here, we used minced liver tissues from rats to compare a homogenization technique for isolation of nuclei with a collagenase digestion method (300 units/g liver at 37 degrees C for 20 min) for isolation of intact cells for subsequent comet assay We found that collagenase digestion was preferred to the homogenization technique in fresh tissues, but neither method prevented the extensive DNA damage caused by cryopreservation (-85 degrees C for 72 h). To minimize this damage, minced liver (1.0 g) and kidney (0.5 g) tissues were added to 20 ml of pre-cooled 10% glycerol or 10% dimethylsulfoxide (DMSO). We showed that cryoprotection with DMSO (-85 degrees C for 72 h and 3 weeks), and to a slightly lesser extent with glycerol (72 h), followed by collagenase digestion led to satisfactory recovery of liver cells with little or no DNA strand breakage. We then used DMSO as a cryoprotective agent to optimize the amount of collagenase and its incubation time in frozen liver and kidney tissues. We showed that the collagenase digestion at 150units/g liver and 300units/g kidney for 10 min produced highest cell numbers and minimal DNA strand breaks. We also validated these procedures by injection (i.p.) of rats with a known renal carcinogen, ferric nitrilotriacetate (Fe/NTA). We showed that Fe/NTA strongly induced DNA strand breaks in both rat liver and kidney, while no DNA strand breakage occurred in these tissues from the control rats. In addition, no significant differences in strand breaks were found between fresh tissues and tissues treated with DMSO during freezing at - 85 degrees C for 72 h. Thus, the cryoprotection and the cell dissociation techniques developed here are satisfactory for preparing both fresh and frozen tissues for comet assay. These simple techniques are expected to expand greatly the usefulness and efficacy of the assay.

Animals↗
PubMed2006

Cryoprotective effects of saccharides on the freeze-drying of sonicated vesicles of the novel lipid A analog, E5531.

A sonicated dispersion of the novel lipid A analog, E5531, was feeze-dried in the presence of various additives such as saccharides and polyalcohols, and their cryoprotective effects were investigated. Fusion of the vesicles was examined by measuring fluorescence energy transfer and size distribution. Cryoprotective ability differed among the addtive species. The addition of polyalcohols led to considerable fusion. Although monosaccharides, similar to disaccharides, completely prevented the fusion of the vesicles during lyophilization, they showed far less ability to retain the entrapped calcein in the vesicles compared to disaccharides. Differential scanning calorimetry heating profiles of vesicles that had been lyophilized with various additives were obtained. Disaccharides and monosaccharides again resulted in markedly different thermal properties of the vesicles. This variety in cryoprotective ability of saccharide species can be attributed to differences in their interaction with the E5531 head group.

Calorimetry, Differential Scanning↗
PubMed1974

Cryoprotectant-induced redistribution of intramembranous particles in mouse lymphocytes.

This study demonstrates, by freeze fracture, clustering of intramembranous particles caused by cryoprotectant treatment of intact unfixed mouse lymphoid cells. Both T and B cells react in a similar fashion, while similar clustering of particles is not observed in some other cell types. The intramembranous particles can be aggregated by incubating unfixed cells in glycerol or dimethylsulfoxide (DMSO) before freezing. The aggregation phenomenon is dependent on the length of time of exposure and the concentration of the cryoprotectants. Further, the cells remain viable and the cryoprotectant-induced clustering is completely reversible. Prefixation of glycerol-treated cells with glutaraldehyde prevents the formation of these particle clusters, and unfixed nonglycerinated cells show no clusters. Thin sections of cells exposed to glycerol or DMSO without previous fixation exhibit bizarre membrane alterations and numerous other degenerative changes. These observations stress the importance of prefixation of lymphoid cells before exposure to glycerol or DMSO, as well as indicate that the membrane characteristics of certain cell types may be probed by glycerol treatment of unfixed cells.

Animals↗
PubMed1979

Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L.

Proline is an effective cryoprotectant for the storage of cultured cells of Zea mays L. in liquid N(2). Increased freeze tolerance can be achieved by pregrowth for 3 to 4 days in medium containing proline. Cells cryoprotected with proline have an increased recovery potential when compared with cells cryoprotected with dimethylsulfoxide and glycerol. They also show a reduced postthaw viability loss and greater tolerance of a range of postthaw culture conditions. It is suggested that the mechanism of action of proline may be similar to that in its putative role of conferring protection against natural stresses. It may be protecting the cell against solution effects caused by dehydration during freezing. These findings are discussed in relation to other freeze tolerance enhancing treatments.

Journal Article↗
PubMed1980

Cryoprotection by glucose, sucrose, and raffinose to chloroplast thylakoids.

Differential cryoprotection is afforded to chloroplast thylakoids against freeze-induced uncoupling of cyclic photophosphorylation by equimolar concentrations of glucose, sucrose, and raffinose. This differential protective effect appears to be due to nonideal activity-concentration profiles exhibited by the sugars during freezing. When cryoprotection is analyzed as a function of the mole fraction of NaCl to which the membranes are exposed during freezing, the pattern of protection to cyclic photophosphorylation and its component reactions is not dependent upon the chemical identity of the protective solute. Cryoprotective efficiency of glucose, sucrose, and raffinose can be accounted for by proposing an activity dependent alteration in the freezing environment rather than specific solute-membrane interactions.

Journal Article↗
PubMed2005

Cryoprotection by urea in a terrestrially hibernating frog.

The role of urea as a balancing osmolyte in osmotic adaptation is well known, but this 'waste product' also has myriad other functions in diverse taxa. We report that urea plays an important, previously undocumented role in freezing tolerance of the wood frog (Rana sylvatica), a northern woodland species that hibernates terrestrially in sites where dehydration and freezing may occur. Wood frogs inhabiting an outdoor enclosure accumulated urea to 65 mmol l-1 in autumn and early winter, when soil moisture was scarce, but subsequently urea levels fell to approximately 2 mmol l-1 as the availability of environmental water increased. Laboratory experiments showed that hibernating R. sylvatica can accumulate at least 90 mmol l-1 urea under relatively dry, warm conditions. During experimental freezing, frogs synthesized glucose but did not accumulate additional urea. Nevertheless, the concentrations of urea and glucose in some tissues were similar. We tested urea's efficacy as a cryoprotectant by measuring lysis and lactate dehydrogenase (LDH) leakage in samples of R. sylvatica erythrocytes frozen/thawed in the presence of physiological levels of urea or other osmolytes. In conferring protection against freeze/thaw damage, urea was comparable to glycerol and as good as or better than glucose, cryoprotectants found in freeze-tolerant frogs and other animals. Urea treatment also improved the viability of intact tissues frozen in vitro, as demonstrated by post-thaw measures of metabolic activity and LDH leakage. Collectively, our findings suggest that urea functions both as an osmoprotectant and a cryoprotectant in terrestrially hibernating amphibians.

Adaptation, Physiological↗
PubMedDate not supplied

Addition of cryoprotective agent into biological tissue through minimally invasive injection.

In this article, a minimally invasive injection method to enhance the addition of cryoprotective agent into the target biological tissues or organs to be cryopreserved was studied. Compared with the traditional treatment through immersion and permeation, direct injection could be much more effective and faster. To illustrate the benefit of this approach, a mathematical model for the addition of cryoprotective agent was established and experiments were performed to test the differences given by the two methods. Both the theoretical and the experimental results demonstrate that the minimally invasive injection method for enhancing the addition of cryoprotective agent is a promising substitution for the commonly used immersing method.

Coloring Agents↗
PubMed1983

Refractive keratoplasty. Tissue dyes and cryoprotective solutions.

The cryoprotective dye solution KM-26 (0.25% kiton green, 8% glycerin, and 4% dimethyl sulfoxide), which is presently used for all lamellar refractive surgical procedures, was evaluated and compared to other dyes that can stain corneal tissue. With the use of canine and human eye bank tissue, we found kiton green and light green to produce the least tissue alteration and to destain readily. Glycerin with or without dimethyl sulfoxide or dye was deleterious to the endothelium in the area under the keratectomized recipient bed. On the basis of our findings, glycerin and dimethyl sulfoxide should be deleted from the cryoprotective solution and a 0.1% concentration of light green in balanced salt solution or M-K medium should be used and the frozen tissue should be thawed in a balanced salt solution or M-K medium to eliminate potential damage to the recipient corneal endothelium.

Animals↗
PubMed2006

Cryoprotection with L- and meso-trehalose: stereochemical implications.

Two unnatural stereoisomers of alpha,alpha-trehalose (L- and meso-trehalose) were synthesized and evaluated as cryoprotectants in order to determine the functional consequences of relative or absolute stereochemistry on their physicochemical properties. Adherent yeast cell cultures were frozen in 10% solutions of D-, L-, and meso-trehalose for periods of 7-28 days, then evaluated by a MTT viability assay. D- and L-trehalose were equally effective in maintaining high rates of cell survival, thus demonstrating the absence of chiral discrimination at the carbohydrate-lipid interface, whereas meso-trehalose was inferior in cryoprotection efficacy. Differential scanning calorimetry revealed a difference in the glass transition temperatures (Tg) of D- and meso-trehalose of nearly 75 degrees C. This can be attributed to differences in conformational behavior, as portrayed by torsional energy maps for rotation about the glycosidic bonds of D- and meso-trehalose. We conclude that the biostabilizing properties of alpha,alpha-trehalose depend on relative stereochemical factors, but are independent of absolute stereochemical configuration.

Calorimetry, Differential Scanning↗
PubMed1976

Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs.

Frozen mouse eggs were examined to determine the effects of low temperatures, concentration of cryoprotective agents and cooling rates on their survival, fertilizability in vitro and subsequent development. Dimethyl sulfoxide administered at 1.5 M concentration was found to be the most effective cryoprotective agent. Upon thawing, 51% and 56% of the eggs appeared to be normal after having been cooled at 0.33 degrees C/min to -30 degrees C and -50 degrees C, but only 18% of the eggs appeared to be normal after having been cooled at the same rate to -75 degrees C. When eggs were cooled at 0.33 degrees C/min to -45 degrees C and the cooling rate increased to 1 degree C/min from -45 degrees C to -75 degrees C, 44% and 72% appeared normal upon thawing. Of the normal eggs fertilized in vitro from C3H mice, 65% cleaved to the 2-cell stage and 24% of the 2-cell eggs developed into blastocysts. Following the transfer of 17 blastocysts into three recipient mice, one mouse delivered three normal young.

Animals↗
PubMed1983

Survival of mouse embryos frozen-thawed slowly or rapidly in the presence of various cryoprotectants.

Eight-cell mouse embryos were frozen by using glycerol, ethylene glycol, or erythritol as the cryoprotectant. The samples were slowly cooled (0.5 degrees C/min) to temperatures between -10 and -79 degrees C before direct transfer to liquid nitrogen (-196 degrees C) and thawed slowly (approximately 20 degrees C/min) or rapidly (approximately 500 degrees C/min). The highest survival rates of embryos in 1 and 2 M glycerol were obtained after transfer to -196 degrees C from -40 to -79 degrees C, regardless of the thawing rate (72 to 90%), except for those of embryos thawed slowly in 2 M glycerol, in which case highest survival rates were obtained after transfer from -70 and -79 degrees C (78 to 82%). With slow thawing, survival of embryos in 1.2 M ethylene glycol reached maximal levels at transfer temperatures between -50 and -79 degrees C (82 to 89%) and with rapid thawing at transfer temperatures between -40 and -79 degrees C (84 to 90%). The highest survival rates of slowly thawed embryos in 0.6 M erythritol were obtained after transfer to -196 degrees C from -35 and -40 degrees C (53 to 55%) and of rapidly thawed embryos after transfer from -30 degrees C (46%). These results show that the transfer temperatures to liquid nitrogen, at which the highest survival rates of mouse embryos are obtained, vary with the cryoprotectant and the thawing rate. It appears that with glycerol and ethylene glycol mouse embryos are less sensitive to the thawing rate when slowly cooled to lower subzero temperatures before rapid cooling.

Animals↗
PubMed1992

Vitrification of mouse oocytes using short cryoprotectant exposure: effects of varying exposure times on survival.

The effects on oocyte viability of varying the duration of exposure to cryoprotectants before rapid cooling to -196 degrees C were examined, using the vitrification protocol of Nakagata. A very short exposure (15 sec) was found to be optimal, resulting in an overall rate of development from vitrified oocytes to hatching blastocysts of 31.8%. Very high rates of survival (77-89%) of oocytes exposed to the cryoprotectant media, but without the vitrification, together with extreme variability in results between straws in the vitrified groups, suggest that losses in viability during vitrification may result from ice damage during devitrification of the medium.

Animals↗
PubMed1993

Effects of various cryoprotective agents and membrane-stabilizing compounds on bull sperm membrane integrity after cooling and freezing.

In this study attempts were made to improve the survival rates of bull spermatozoa after freezing/thawing and to clarify the importance of certain agents to the cryopreservation of spermatozoa. For that purpose the standard freezing extender was modified by the addition of different concentrations of various cryoprotectants and membrane-stabilizing agents: glycerol, 1,2-propanediol, polyvinylpyrrolidone, sucrose, egg yolk, lipid vesicles, and bovine serum albumin (BSA). Sperm membrane impermeability toward H33258 was employed as the parameter for sperm integrity during cooling and after freezing/thawing. Exclusion of glycerol from the extender did not significantly affect sperm integrity. Replacing 6% glycerol by 6% 1,2-propanediol resulted in reduced sperm survival, whereas replacement of glycerol by 62.5 mM sucrose slightly improved survival rates. Addition of 5 or 10% polyvinylpyrrolidone (either or not in combination with 0.5 M sucrose) significantly reduced sperm integrity. Excluding egg yolk from the extender caused a serious decrease of sperm survival after both cooling and freezing. The cryoprotection offered by egg yolk could not be mimicked by dioleoylphosphatidylcholine (DOPC) vesicles or DOPC/phosphatidic acid/cholesterol vesicles in concentrations up to 29 or 9 mM, respectively. However, the freezing extender containing 6.5 mM DOPC vesicles in combination with 6% BSA yielded results which did not significantly differ from those obtained with the standard extender; higher vesicle concentrations combined with BSA might produce even better results. Further research on the cryopreservation of bovine spermatozoa should focus on membrane stabilization since the membrane-stabilizing compounds yield more promising results than the ice-preventing agents.

Animals↗
PubMed1993

Cryoprotective effect of egg yolk in cryopreservation of porcine embryos.

The objective of the present study was to assess the effect of egg yolk supplementation in solutions for either cooling or freezing of porcine embryos. In Experiment 1, porcine embryos collected on Day 3 to 7 (Day 0 = onset of estrus) were allocated to 12 experimental groups according to their developmental stages (four-cell to compact morula, early blastocyst to blastocyst, expanded blastocyst, and hatched blastocyst) and the concentrations of egg yolk (0, 5, and 10% (v/v)) for cooling to 0 degrees C. Whereas no embryos ranging from four-cell to blastocyst survived, the 10-min exposure to 0 degrees C despite egg yolk supplementation, expanded blastocysts and hatched blastocysts allocated to egg yolk-supplemented groups survived cooling to 0 degrees C. In Experiment 2, porcine expanded and hatched blastocysts collected on Day 6 or 7 were assigned to six experimental groups according to their developmental stages and the type of cryoprotectant used (10% glycerol, 11% Me2SO, and 10% propylene glycol) for freezing at -196 degrees C. All freezing solutions contained either 5 or 10% egg yolk. The embryos were equilibrated with one of the freezing solutions, cooled from 25 to -7 degrees C at 1 degree C/min, seeded at that temperature, then cooled to -36 degrees C at 0.3 degree C/min, and finally plunged into liquid nitrogen for storage. The frozen embryos were thawed by immersion in 37 degrees C water. After the cryoprotectants were removed by dilution, the embryos were cultured for 48 h to evaluate their viability.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Mechanisms of cryoinjury and cryoprotection in split-thickness skin.

Successful cryopreservation of tissues will ultimately require a more detailed understanding of how the in situ environment modifies cell responses during cooling and warming. Low-temperature responses of porcine split-thickness skin and isolated basal keratinocytes were compared after various cooling protocols and in the presence and absence of cryoprotectants. Recovery was assessed by measuring oxygen consumption kinetics in skin and tetrazolium reduction in isolated cells. Freeze substitution was used to visualize ice nucleation and growth in skin. The results indicated that the time required for diffusion of water in split-thickness skin delayed osmotic responses in the basal keratinocytes and resulted in increased intracellular and intercellular ice formation. Rapid cooling (-200 degrees C/min) in the presence of cryoprotectants resulted in a reduction in the number of cells containing ice and the size of the intercellular ice crystals and an increase in tissue recovery. These observations support other reports which suggest that cell-to-cell and cell-to-substrate interactions are sensitive sites for cryoinjury. A practical recommendation from this study is that high recovery of split-thickness skin may be achieved with protocols using high cooling rates.

Animals↗