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PubMed1994

1H magnetic resonance imaging of freezing and thawing in freeze-tolerant frogs.

Proton magnetic resonance imaging (MRI) of the processes of freezing and thawing in the wood frog Rana sylvatica provided noninvasive and real-time analysis of the mode of ice propagation through the body of a freeze-tolerant vertebrate. MRI revealed a directional movement of ice from the exterior inward that required several hours to reach completion. Freezing in core organs such as liver, which produces and exports cryoprotectant, and heart, which circulates it, was delayed and occurred well after the organs were surrounded by extraorgan ice. Natural thawing was a very different process; thawing began uniformly throughout the body, but core organs melted more rapidly than peripheral ones, an adaptation that may be key to the early restoration of heartbeat and breathing. The images presented demonstrate the sensitivity and power of MRI and its potential to become a critical monitoring technology in the development of cryopreservation techniques for mammalian organ explants.

Acclimatization↗

PubMed2006

Preservation of C. elegans tissue via high-pressure freezing and freeze-substitution for ultrastructural analysis and immunocytochemistry.

High-pressure freezing (HPF) is capable of converting liquid water, to a depth of approx 0.6 mm, into amorphous ice nearly instantaneously. At midbody, an adult Caenorhabditis elegans hermaphrodite approaches its widest girth of approx 0.1 mm. In theory, an entire living adult animal can be physically immobilized instantly in amorphous ice by HPF, thus, providing a unique opportunity to examine cellular architecture with exquisite spatial preservation. The following chapter will discuss, in detail, procedures for freezing C. elegans under high pressure, for embedding frozen samples in resin after a freeze-substitution step, and for the postembedding immunogold labeling of proteins contained within thin sections of embedded animals. These protocols enable high-resolution analysis of both morphological features and molecular domains within most tissues of C. elegans.

Animals↗

PubMed1992

Formulation and stability of freeze-dried proteins: effects of moisture and oxygen on the stability of freeze-dried formulations of human growth hormone.

This research presents the results of a series of stability studies on freeze-dried formulations of human growth hormone (hGH). Chemical decomposition via methionine oxidation and asparagine deamidation as well as irreversible aggregation are characterized by HPLC. Water sorption isotherms, DSC thermograms, and pulsed proton NMR data are also obtained. No glass transition temperatures are observed in the temperature range of the stability studies. The pulsed NMR data suggest onset of greater mobility in the solid at a water content slightly higher than BET "monolayer" level. Stability of freeze-dried solids at 25 degrees C and 40 degrees C is studied as a function of residual moisture and exposure to oxygen. Formulations with and without a glycine/mannitol excipient system are studied. Significant levels of chemical decomposition and irreversible aggregation occur under most conditions with the effects of residual water content and "headspace oxygen" strongly dependent on the formulation. At low water content with minimal oxygen in the vial headspace, the glycine/mannitol formulation yields optimum stability. However, for either high water content or high oxygen content in the vial, stability of hGH without excipients is superior. The qualitative effect of residual moisture on stability depends on the temperature of the stability study. Generally, the stability of a sample adjusted to a given water content by desorption (during freeze-drying) is identical to the stability of a sample prepared by sorption of water on to a previously highly dried sample.

Calorimetry, Differential Scanning↗

PubMed1990

[Effect of controlled freezing-point storage of hearts--combined effect of controlled freezing-point storage and verapamil].

Controlled Freezing-point Storage (CF Storage) is a new method of preserving foods in minus non-frozen temperature range. Therefore, the authors tried to apply this method to organ preservation. We investigated the effect of controlled freezing-point storage of hearts on ventricular function in isolated perfused rat heart (male, Sprague dawley strain, body weight about 300 g), and the combined effect of controlled freezing-point storage and Verapamil (0.5 mg/l). The hearts were perfused by working heart mode for 10 min, and received infusion of cardioplegic solution which was followed by 4 hours of cardiac arrest. The hearts were stored for 4 hours in Euro-Collins solution at 4 degrees C (4 degrees C group) and minus non-frozen temperature (CF storage group), and were stored for 4 hours in Euro-Collins solution containing Verapamil at minus non-frozen temperature (CF-Ve storage group). Then, the aerobic reperfusion by working heart mode was continued for an additional 30 min. The recovery rate of cardiac output was 33.5%, 62.5% and 74.2% respectively of the preischemic value in 4 degrees C group, CF storage group and CF-Ve storage group. The recovery rate of cardiac output in 4 degrees C group was significantly lower than that in CF storage group and CF-Ve storage group (p less than 0.01), and significant difference was noted between CF storage group and CF-Ve storage group (p less than 0.05). ATP activity after 4 hours of cardiac arrest was 13.9 micrograms/mg, 18.7 micrograms/mg and 18.3 micrograms/mg in 4 degrees C group, CF storage group and CF-Ve storage group respectively. ATP activity after 4 hours of cardiac arrest in 4 degrees C group was significantly lower than that in CF storage group and CF-Ve storage group (p less than 0.05). These data suggest that CF storage of hearts has a protective effect against an ischemic insult upon myocardial cell during hypothermic cardiac arrest, and the best metabolic and functional protection appeared when the hearts were stored in Euro-collins solution containing Verapamil at minus non-frozen temperature.

Animals↗

PubMedDate not supplied

Retrospective comparison of freeze and non-freeze myopic epikeratophakia.

We conducted a retrospective study of 20 eyes of 20 selected patients with preoperative myopia of -14 to -28 diopters who were treated with myopic epikeratophakia and followed for 8 weeks. Seven eyes were treated with frozen-lyophilized donor lenticules by the Kaufman-McDonald technique and 13 eyes were treated with freshly prepared donor lenticules using the Barraquer-Krumeich-Swinger (BKS) technique. All lenticules were sutured into a circular keratotomy without keratectomy. Postoperatively, all eyes had a refraction between -4.00 and +2.00 D, and there was no meaningful difference in the refractive outcome of the two groups. The freeze-lyophilized technique showed a greater overall loss of best spectacle corrected visual acuity, although no eyes in the entire series lost more than two lines of Snellen visual acuity compared to preoperative values. Uncorrected visual acuity after surgery was 20/40 or better in 5% of each group. The rate of postoperative epithelialization was slower in the freeze-lyophilized group, but all eyes were re-epithalialized by 3 weeks after surgery. No lenticules had to be removed for complications, but there was one case of epithelial ingrowth into the interface that required a second operative procedure. Our clinical experience with this small group of eyes shows that both techniques of myopic epikeratophakia can safely and effectively reduce high amounts of myopia, but that the refractive outcome is less predictable than desired. The technical requirements for the non-freeze (BKS) technique are high, but the rate of visual recovery is faster.

Adult↗

PubMedDate not supplied

An ultrastructural study of the nasal mucosa by rapid freezing and freeze-substitution.

We demonstrated the ultrastructures of the nasal mucosa of the rat by rapid freezing and freeze substitution with OsO4. Liquid helium was used for the rapid freezing. The depth of the good preservation was approximately 20 micron below the cut surface of the tissue. The characteristic features of the nasal mucosa obtained here are as follows: 1) The cross-section profiles of the cilium were round and smooth. 2) Mitochondrial matrix was electron dense and a few dense granules were observed in it. 3) Secretory granules of the goblet cells were enclosed by a limited membrane. 4) Extracellular space was of variable widths. 5) Cytoplasmic filaments were well preserved.

Animals↗

PubMed1988

Retention and structure of extracellular matrix in early chick embryos after quick-freezing and freeze-substitution.

Early chick embryos, stages 11 to 14, were isolated, quick-frozen by immersion in isopentane/propane cryogen (-185 degrees C) and freeze-substituted for study by scanning electron microscopy. Emphasis was placed on the extracellular matrix (ECM) in the axial region of the segmental plate and developing somites. Ultrarapid freezing, followed by delicate freeze-substitution, immobilizes and retains much more ECM than chemical fixatives that include tannic acid (TA). The matrix on the dorsal surface of the neural tube is preserved as delicate filaments which are expressed bilaterally over the tube in a dorso-ventral orientation. These parallel primary ridges of ECM have a spacing of 1 to 3 micron, forming grooves on the wall of the neural tube. Interrupting this pattern are funnel-shaped ridges about 80 to 100 micron apart along the neural tube. The ridges become decorated with cross-bridges creating a dense lattice in the region of somite development, to the extent that a basal lamina composed of dense fibrillar network and amorphous mats of matrix accumulates on the lateral wall of the neural tube. Heavy strands and fenestrated lamellae of ECM interconnect the neural tube, notochord and somites, and attach the overlying epithelium to the upper surface of the somites. The pattern of ECM is complimentary to the migratory pathways of ventrally migrating neural crest cells and is the basis for suggesting that a physical substratum influencing the direction of neural crest cell migration is an idea that should be revived.

Animals↗

PubMed1981

In situ liquid propane jet-freezing and freeze-etching of monolayer cell cultures.

A procedure for in situ liquid propane jet-freezing of unfixed and unglycerinated monolayer cell cultures is described. We have used the method to study monolayer cultures of rat Sertoli cells and human monocytes. The described procedure gives a cooling rate during specimen freezing high enough to yield adequately frozen specimens even in the absence of any cryoprotectant. Use of the procedure therefore eliminates artefacts usually caused by cell fixation and addition of cryoprotectives.

Animals↗

PubMed2005

Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports.

The aim of this study was to improve the freezing protocol of bull sperm, by investigating the influence on sperm viability after freeze/thawing of different freezing medium components, as well as the effect of cooling rates in the different stages of the cooling protocol, in single factor experiments. The experimental variables were: (1) salt-based versus a sugar-based medium (Tris versus sucrose); (2) glycerol concentration; (3) detergent (Equex) concentration; (4) presence of bicarbonate; (5) rate of cooling from 22 degrees C to holding temperature (CR1); (6) holding temperature (HT); (7) rate of cooling from holding temperature to -6 degrees C (CR2); (8) rate of cooling from -10 to -100 degrees C (CR3). All experiments were performed using five bulls per experiment (three ejaculates per bull). Sperm motility after freezing and thawing was assessed by CASA system, and sperm membrane integrity was assessed by flow cytometry. Sucrose-based medium did not offer a clear significant benefit compared to Tris medium. The concentration of Equex that gave the best results in Tris-based media group and sucrose-based media group was in a range between 2-7 and 4-7 g/l, respectively. In both media groups, a glycerol concentration of 800 mM was the best in any post-thaw viability parameters. In the Tris media group, the presence of bicarbonate had a negative effect on sperm viability. CR1 and CR2 had no significant effect on any of the post-thaw sperm viability parameters, but a CR1=0.2 degrees C/min and CR2=4 degrees C/min appeared to give better results in both media. The holding temperature (HT) that gave the best results was found to be in the range of 5-9 degrees C. There was a significant disadvantage of using a low CR3 of 10 degrees C/min, while 150 degrees C/min appeared to be the best cooling rate for either medium.

Acrosome↗

PubMed2004

Freeze fracture direct imaging: a new freeze fracture method for specimen preparation in cryo-transmission electron microscopy.

In this paper, we present a new freeze fracture method for specimen preparation for transmission electron microscopy frozen samples. We call it freeze fracture direct imaging (FFDI) because it is a hybrid of conventional freeze fracture electron microscopy (FFEM) and cryo-transmission electron microscopy (cryo-TEM), combining elements of the fracture technique with direct imaging. Like in FFEM, the sandwich method is used to prepare the sample in a protected fashion. However, after the sample is vitrified and fractured, it is not shadowed but directly imaged. The new technique avoids some experimental artifacts produced by the blotting procedure in conventional cryo-TEM. It relies, though, on occasional fractures transparent to the electrons. The advantageous features are demonstrated by a comparison between conventional cryo-TEM and FFDI micrographs of vesicular solutions. The second outstanding advantage over conventional cryo-TEM is the fact that it is now possible for the very first time to directly image oil-rich mixtures films which normally would dissolve in the cryo-medium ethane. Micrographs of pure oil and of oil-rich microemulsions clearly prove the reliability of the FFDI technique as well as its enormous potential.

Journal Article↗

PubMed1993

Inheritance of freezing resistance in tuber-bearing Solanum species: evidence for independent genetic control of nonacclimated freezing tolerance and cold acclimation capacity.

Frost or winter survival is regarded as a complex trait with polygenic inheritance. Two major components of this survival in crop plants are freezing tolerance in the nonacclimated state and cold acclimation capacity. To date researchers have not distinguished the two components as separate heritable traits. The mode of inheritance of these two traits was investigated in F1 and backcross populations of two wild diploid potato species (Solanum commersonii and Solanum cardiophyllum) exhibiting extremes of freezing tolerance and acclimation capacity. Precise assessment of these two traits allowed distinction of small but significant differences among genotypes. The two traits were not correlated in segregating populations, suggesting independent genetic control. Analyses of generation means indicate that all of the variance for acclimation capacity and a major proportion of the variance for the nonacclimated freezing tolerance can be best explained by an additive-dominance model with both traits being partially recessive. Recovery of parental phenotypes in limited populations suggests that both traits are controlled by relatively few genes. To our knowledge this is the first study demonstrating independent genetic control of the two main traits associated with frost or winter survival. Our results show that it should be possible to incorporate these traits from wild germ plasm into cultivated crop plants by independent selection. These results help explain the lack of progress in improving winter survival through field selection. Furthermore, our study demonstrates relative simplicity of the inheritance of cold acclimation, thus providing avenues for understanding the link between biochemical and genetic aspects of low-temperature stress in crop plants.

Journal Article↗

PubMedDate not supplied

Effects of a freezing event during hibernation on further survival, reproduction and growth in the partially freezing tolerant land snail Helix aspersa muller (Gastropoda: helicidae).

Tolerance of ectothermic animals to freezing is often estimated by assessing survival a few days after the treatment. However, in the long term, ice formation in the body tissues can affect survival, as well as reproductive capability and growth. The land snail Helix aspersa survives only short durations with ice in its tissues, to a lethal limit of 40 to 60 % of its body water frozen. Adult and immature snails were treated during their winter dormancy period to a freezing event above this limit; their survival was observed both in the short and long term, as well as their ability to reproduce (adults) and grow (immature snails). Treated snails were compared with a control group, which was not frozen. No difference appeared in the survival, reproduction and growth of control and frozen snails. This study confirms partial freezing tolerance in this population of Helix aspersa.

Animals↗

PubMedDate not supplied

Quick freezing of mouse embryos: freezing of inbred strains and 2- and 4-cell embryos by vitrification.

Murine embryos of mice of four different inbred strains and one hybrid strain were evaluated for their ability to survive quick freezing by post-thaw in vitro development. The embryos were transferred to an equilibration medium [10% 1,2-propanediol and 20% glycerol in modified PBS (mPBS)] for 10 minutes and frozen in a vitrification medium (25% glycerol and 25% 1,2-prapanediol in mPBS) by direct lowering into liquid nitrogen. Following thawing at 30 degrees C, dilution in 1 M sucrose in mPBS and washing in mPBS the embryos were cultured, and development was evaluated 24-28 hours later. The number of fertilized eggs obtained by superovulation differed among the strains. The survival rates evaluated by in vitro cultivation of the post-thawed inbred embryos varied from 50-85% depending on the genotype, whereas the normal live offspring from transfer of frozen-thawed embryos to recipient females confirms that the quick freezing method is an applicable method for storage of genetically defined mouse strains and stocks. The quick freezing technique was applied on 4- and 8-cell (day-3) mouse embryos of hybrids. The in vitro development of frozen thawed 4- and 8-cell embryos (23% and 21% respectively) was found to be significantly lower than that of frozen thawed morulae (89%). Permeation in glycerol-solutions before equilibration significantly increased survival of 4- and 8-cell embryos (66% and 77% respectively). By the use of dimethylsulfoxid (DMSO) in the permeation solutions an even higher survival rate was obtained in the cryopreservation of 8-cell mouse embryos (95%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Effects of freezing and freeze-drying on the biomechanical properties of rat bone.

The effects of various preservation techniques on the biomechanical properties of bone have been investigated in a rat model. Freezing of the specimens to -20 degrees C, -70 degrees C, and -196 degrees C did not adversely affect the strength of long bones tested in torsion or of vertebral bodies tested in compression. Freeze-drying did not markedly affect the compression properties of the vertebral specimens; however, it did produce a significant deleterious reduction in the torsional strength of the long bones.

Animals↗

PubMed1998

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution.

Three decades of structural analysis have produced the view that the kinetochore in vertebrate cells is a disk-shaped structure composed of three distinct structural domains. The most prominent of these consists of a conspicuous electron opaque outer plate that is separated by a light-staining electron-translucent middle plate from an inner plate associated with the surface of the pericentric heterochromatin. Spindle microtubules terminate in the outer plate and, in their absence, a conspicuous corona of fine filaments radiates from the cytoplasmic surface of this plate. Here we report for the first time the ultrastructure of kinetochores in untreated and Colcemid-treated vertebrate somatic (PtK1) cells prepared for optimal structural preservation using high-pressure freezing and freeze substitution. In serial thin sections, and electron tomographic reconstructions, the kinetochore appears as a 50-75 nm thick mat of light-staining fibrous material that is directly connected with the more electron-opaque surface of the centromeric heterochromatin. This mat corresponds to the outer plate in conventional preparations, and is surrounded on its cytoplasmic surface by a conspicuous 100-150 nm wide zone that excludes ribosomes and other cytoplasmic components. High magnification views of this zone reveal that it contains a loose network of light-staining, thin (<9 nm diameter) fibers that are analogous to the corona fibers in conventional preparations. Unlike the chromosome arms, which appear uniformly electron opaque, the chromatin in the primary constriction appears mottled. Since the middle plate is not visible in these kinetochore preparations this feature is likely an artifact produced by extraction and coagulation during conventional fixation and/or dehydration procedures.

Animals↗

PubMed2006

Morphological analysis of lamellar structures in mouse type II pneumocytes by quick-freezing and freeze-drying with osmium tetroxide vapor-fixation.

The lamellar body is a membranous structure periodically laminating in vesicles that is known as the most distinctive feature of type II pneumocytes by conventional preparation methods for transmission electron microscopy. The quick-freezing and freeze-drying method, followed by osmium tetroxide vapor-fixation (QF-FD-OsV), was performed to examine the in situ morphology of the lamellar body in type II pneumocytes of living mouse lungs. Typical lamellar structures were rarely seen in vesicles of the type II pneumocytes, but amorphous components and dispersed stripes were often detected in the vesicles, as revealed by the QF-FD-OsV method. To clarify how the lamellar body was formed during the conventional preparation steps, lung tissues of mice were treated with different fixation procedures, such as immersion-fixation with osmium tetroxide or perfusion-fixation with glutaraldehyde followed by osmium tetroxide, in combination with alcohol dehydration or QF-FD-OsV. In addition to lamellar bodies of type II pneumocytes in the specimens with alcohol dehydration, some lamellar structures were also formed even with the QF-FD-OsV method. These findings suggest that the labile lamellar body is easily modified and formed during both chemical fixation and alcohol dehydration steps.

Animals↗

PubMed1994

An improved microscope stage for direct observation of freezing and freeze drying.

A microscope stage for observation of freezing and freeze drying is described. The stage uses thermoelectric (Peltier) heaters configured in two stages, with circulating fluid as a heat sink on the high temperature side. Lowest attainable sample temperature is about -47 degrees C. Principal advantages of this system are closed-loop control of stage temperature, rapid response to changes in temperature set point, and improved documentation of experiments by use of a video recorder system with a character generator which allows display of sample identity and temperature. Accuracy of measuring the sample temperature in the field of view was validated by comparing observed values of eutectic melting with published values for a series of solutes with eutectic temperatures in the range from -2 degrees C to -32 degrees C. Good agreement was obtained throughout this range.

Freeze Drying↗

PubMed2003

Cytochemistry of gastric parietal cells with high-pressure freezing followed by freeze-substitution.

Gastric parietal cells were examined for changes in their ultrastructure and distribution of the proton pump during feeding and fasting states in rats. The fundic glands from rats fed ad libitum or fasted with free access to water were cryofixed using high-pressure freezing followed by freeze-substitution in acetone containing osmium or acrolein and then embedded in Epon 812 or Lowicryl K4M resin, respectively. Excellent ultrastructural preservation was achieved. During the feeding state, intracellular canaliculi and numerous microvilli were well developed, while tubulovesicles were poorly developed. In contrast, during the fasting state, the microvilli in the narrowed space of the intracellular canaliculi were tightly packed and the tubulovesicles were enlarged. Ultrathin sections were immunostained with antibodies against the alpha- and beta-subunits of the proton pump, H+ x K(+)-ATPase, using the immunogold method. The labelling was strong and clearly localized in comparison with that obtained using the conventional chemical-fixation method. Each subunit was localized on the membrane of the microvilli, intracellular canaliculi and tubulovesicles. The distribution of subunit proteins varied between the two states. During ad libitum feeding, the immunolabelling was localized strongly on the membranes of the microvilli and intracellular canaliculi. In contrast, the labelling was strong on the tubulovesicle membrane in the fasting state. The results obtained with each anti-subunit antibody by H+ x K(+)-ATPase immunostaining revealed differences in distribution and labelling density between the feeding and fasting states.

Animals↗