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PubMed1987

Ultrastructural preservation of plasma membranes by non-lethal slow freezing to liquid nitrogen temperature.

Secondary hyphae of Lyophyllum ulmarium were shown to tolerate slow freezing, which allowed extracellular freezing, to -196 degrees C. A freeze-fracture study showed that under this non-lethal freezing condition, the plasma membrane of the secondary hyphae did not show any ultrastructural changes as compared with the control, except gross cellular shrinkage. Tertiary hyphae of Lyophyllum ulmarium, on the other hand, were completely injured by slow freezing to -196 degrees C, and the plasma membrane showed distinct intramembrane particle aggregation as a result of direct membrane contact caused by severe cellular deformation. It is suggested that the absence of freezing injury in the secondary hyphae was due to ultrastructural preservation of the plasma membrane, which resulted from avoidance of severe cellular deformation, while occurrence of freezing injury in the tertiary hyphae is considered to be due to ultrastructural changes in the plasma membrane caused by severe cellular deformation.

Cell Membrane↗
PubMedDate not supplied

Use of subambient differential scanning calorimetry to monitor the frozen-state behavior of blends of excipients for freeze-drying.

The purpose of this study was evaluation of mannitol and its blends with other excipients commonly used as freeze-drying cryoprotectants or bulking agents in simulated lyophilization conditions. Subambient differential scanning calorimetry (DSC) has been used as an analytical tool to evaluate the phenomena from both a qualitative and a quantitative point of view. The DSC scans were performed at rates comparable to those generally practicable in industrial lyophilizators. During a simulated freeze-drying process, three crystallization events have been observed on mannitol solutions: the first two on freezing, and the last one on heating. The phenomena are dependent on each other. The addition of other excipients can significantly influence this behavior by specifying the crystallization of mannitol in freezing or by inhibiting it fully. This means that the final physical state of the freeze-dried product (amorphous, crystalline, or partially amorphous/partially crystalline) can be tailored by accurately choosing not only the freeze-drying conditions but also the composition of the solution to be lyophilized. The results point out how the physical state of freeze-dried products can be controlled by an accurate choice of both excipients and their relative ratios, with the possibility of reducing the number of preformulation activities required to design the most appropriate formulation for each new drug candidate.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1993

Impact of freeze substitution on biological electron microscopy.

Considering the increasing necessity for improved preparation techniques in biological electron microscopy as a basis for the identification and localization of cellular substances within the compartments of the cell, this review is focussed on the method of freeze substitution as an important link between the cryofixation (ultrarapid freezing) and resin embedding of biological specimens. The theory and practice of freeze substitution is summarized with particular interest in the physical and thermodynamic as well as in the chemical basis of this technique. A survey of practical aspects of the technical process of freeze substitution concerning the equipment and various protocols successfully applied in biological systems is also given. The main advantage of freeze substitution versus conventional chemical fixation is seen in the maintenance of the hydration shell of molecules and macromolecular structures. This results in an improved fine structural preservation, superior retention of the antigenicity of proteins and decreased loss of unbound, diffusible cellular components. Examples of excellent visualization of the ultrastructure of macromolecular complexes (nucleic acids, extracellular material, membranes etc.), small organisms (bacteria, algae, cyanobacteria and fungi) and large biological samples such as plant and animal tissue as well as the plant-pathogen (fungus) interface and infection structures are presented. Recent data on the molecular characterization of freeze-substituted biological tissue are exemplified with special emphasis on the subcellular detection of soluble components (elements, lipids, proteins and drugs) and the inter-/intracellular localization of proteins including foreign proteins in transgenic plants. The molecular analysis of freeze-substituted specimens is achieved by the combination of low temperature preparation techniques in biological electron microscopy with various detection methods such as X-ray microanalysis, immunocytochemistry and high resolution autoradiography.

Animals↗
PubMed1993

Freeze-substitution for morphological and immunocytochemical studies in insects.

Methods of plunge freezing and freeze-substitution (FS) for insect antennae and similar body appendages are described. In these more or less cylindrical specimens, usually a layer below the cuticular surface of 10-15 microns thickness is well preserved without freezing damage, further inwards ice-crystal ghosts of increasing size are encountered, but in the very centre of antennal branches (diameter approximately 80 microns) of the silkmoth, Bombyx mori, freezing damage is usually reduced again. The frost-hardy species, Poecilocampa populi and Boreus hiemalis, exhibit regions free from freezing damage up to 40 microns below the cuticular surface. Secondary freezing damage in silkmoth sensory hairs is observed only after deliberately warming the specimens to -43 degrees C for >> 10 min before FS. Secondary artefacts due to the substitution process are investigated by comparison with freeze-etching and by comparing different FS media and protocols. Methanol is not recommended as a substitution medium for insect specimens. Structures particularly liable to substitution damage are the stimulus-conducting pore tubules of olfactory sensilla and the receptor cell membrane. Extraction of soluble components is more likely with pure organic solvents without added chemical fixing agents and with prolonged substitution at elevated temperatures. Such extraction may also be a possible artefact with soluble antigens in immunocytochemical studies. A review is given of the major achievements attained with these techniques is insect functional morphology and immunocytochemistry.

Animals↗
PubMed1991

Nature of aggregates formed during storage of freeze-dried ribonuclease A.

Ribonuclease A (RNase) has been shown to lose enzymatic activity and to form aggregates when stored in the freeze-dried form at elevated temperature. Polyacrylamide gel electrophoresis showed that the freeze-dried RNase that had lost its enzymatic activity during storage had formed aggregates that were not dissociable using both an anionic detergent and a reducing agent. Isoelectric focusing patterns of freeze-dried RNase before and after storage were quite different. The RNase that had been stored and had aggregated had become more of an acidic protein, while the RNase that was assayed immediately after freeze drying had the same pattern as non-freeze-dried RNase. This evidence indicated that the aggregates were covalently bonded together as a result of some chemical process that occurred during storage of the freeze-dried cake. Amino acid analysis of aggregates formed from RNase freeze dried in pH 10.0 phosphate buffer solutions indicated that the amino acid lysine was instrumental in the formation of the covalent bonds. Asparagine/aspartic acid and glutamine/glutamic acid may have also participated in the bonding of the aggregates.

Amino Acids↗
PubMed1993

Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. I. Enzyme activity and calorimetric studies.

Stabilization of labile proteins during lyophilization requires protection of the protein against both freezing and dehydration stresses. Solutions of 1-10% (wt/vol) polyethylene glycol (PEG) fully protected both lactate dehydrogenase and phosphofructokinase during freezing and thawing, but did not stabilize the proteins during freeze-drying. Thus, with this lyophilization system a second compound could be tested for its capacity to stabilize dried proteins, independent of its ability to provide cryopreservation. In the presence of low concentrations of glucose or trehalose (which alone provided minimal protection) and 1% PEG (wt/vol), almost full enzyme activity was recovered after freeze-drying and rehydration. Differential scanning calorimetry indicated that the PEG was crystalline and the sugars were amorphous in the dried samples. Experiments with lactose and mannitol demonstrated that if these compounds also crystallized during freeze-drying, protein stabilization was reduced or abolished. PEG stabilizes the proteins during freezing, due to preferential exclusion of PEG from the protein's surface. The sugars protect the proteins during dehydration by hydrogen bonding to the dried protein, thus serving as water substitutes. This report provides the first example of stabilization of proteins during lyophilization through separate, specific treatments of the fundamentally different stresses of freezing and dehydration.

Animals↗
PubMed1997

Enhanced permeability of freeze-dried liposomal bilayers upon rehydration.

Until now, studies on the protection of liposomes against freeze-drying damage have mainly focused on the bilayer integrity during the freezing or drying step of this process. Here, we investigated the bilayer permeability of freeze-dried, lyoprotected liposomes to a nonbilayer interacting compound after rehydration, by monitoring the leak-in kinetics of externally added carboxyfluorescein (CF). The results showed that freeze-drying and rehydration of DPPC:DPPG 10:1 liposomes with sucrose in- and outside the vesicles caused a temporary increase in the bilayer permeability for CF, which leveled off after approximately 20 h. The amount of CF/mol phospholipid which leaked into the vesicles increased with vesicle size (range 0.1-1 micro m) / lamellarity. Reduction of the number of bilayers in 1-1 micro m) vesicles enhanced the permeability to CF after freeze-drying and rehydration. The presence of CHOL decreased CF leak-in rates into 1 micro m MLVs consisting of DPPC:DPPG 10:1, but not into 0.1-micro m unilamellar vesicles. In the absence of sucrose similar leak-in profiles as a function of time were found after rehydration, suggesting that repacking processes of the bilayer were responsible for the enhanced permeability after freeze-drying and dehydration both with and without sucrose. The effect of size and lamellarity on the CF leak-in correlated with the retention of encapsulated CF after freeze-drying and rehydration, but no correlation was found with the effect of lipid composition. Both small (0.1 micro m) lyoprotected liposomes made of DPPC:DPPG 10:1 and DPPC:DPPG:CHOL 10:1:4 were highly permeable during the rehydration step itself. The results indicate that, despite the presence of the lyoprotectant, "repacking" of the bilayer components takes place both during and after rehydration. This eventually leads to regaining of its barrier function.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

A histological analysis of liver injury in freezing storage.

As part of a more extensive study on the use of high subzero freezing for cryopreservation of mammalian livers we have tried to single out the effects of freezing and thawing on tissue damage. We compared the morphology of livers after freezing and thawing with what we considered an optimal high subzero cryopreservation protocol with the morphology of livers preserved under the same thermal conditions and in the same solution in a supercooled state, without freezing. The results show that while hepatocytes survive high subzero cryopreservation, detachment of endothelial cells occurs in every freezing experiment. On the other hand, the endothelial cells in livers that are not frozen are intact. This suggests that endothelial cell damage is caused by freezing and may be an important factor in high subzero freezing cryopreservation of the liver.

Animals↗
PubMed1992

A molecular marker to select for freezing tolerance in Gramineae.

We isolated, and expressed in Escherichia coli, a gene (Wcs120) that is strongly induced during cold acclimation of wheat. The gene product was purified and used to produce antibodies. Immunoblotting experiments with the anti-WCS120 antibody identified several cold-induced proteins named FTMs for Freezing Tolerance Markers since they are associated with the development of freezing tolerance. This protein family was found to be coordinately regulated specifically by low temperature, highly hydrophilic, stable to boiling, and to have a pI above 6.5. The accumulation kinetics during the acclimation period indicated a positive correlation with the capacity of each genotype to develop freezing tolerance. Accumulation of the proteins was higher in the freezing-tolerant genotype than in the less tolerant one. In addition, their accumulation was more pronounced in the crown and leaf tissues compared with roots, confirming a relationship to the capacity of the different tissues to develop freezing tolerance. Analysis of different species (eight monocots and four dicots) indicated that this protein family is specific for freezing-tolerant cereals. The antibody did not cross-react with any of the non-cereal species examined. The anti-FTMs antibody represents a potential tool for breeders to select for freezing tolerance traits in the Gramineae.

Adaptation, Physiological↗
PubMed1976

[Influence of temperature and rate of freezing of bovine muscle on the subcellular distribution of some mitochondrial enzyme (author's transl)].

Samples of bovine muscle were frozen at -5 degrees, -10 degrees, -20 degrees, -40 degrees, -60 degreese, -80 degreese, and -196 degrees C at slow (about 1 degrees/10 min) and high freezing rate (maximum 1 degrres/0.05 min) and subsequently thawed at room temerature. The changes in the extractable activity ("total activity") of the enzymes aconitase (AC), fumarase (FU) succine dehydrogenase (SDH), glutamic oxaloacetic transaminase (GOT), the mitochondrial isozyme of the COT (GOTM) and glutamate pyruvate transaminase (GPT) by freezing and thawing were studied. Neither the temperature nor the rate of freezing influenced the total activity of these enzymes. The subcellular distribution of these enzymes was investigated by determination of the enzyme activities in the muscle press juice. Freezing at -5 degrees C hat only little influence, but between -10 degrees and -40 degrees or -60 degrees C the release of AC, FU, GOTM and GPT from the mitochondria into the sarcoplasma increased with falling temperature, apparently by increasing damage of the mitochondria. The extent of this effect was not significantly influenced by the rat of freezing. Between -60 degrees and -196 degrees C no further rise of this effect was observed. A release of SDH did not occur at all conditions of freezing. It is suggested that the damage of muscle mitochondria by freezing and thawing is mainly due to a dehydration process.

Aconitate Hydratase↗
PubMed1990

Water transport and estimated transmembrane potential during freezing of mouse oocytes.

The kinetics of water transport and the changes in transmembrane potential during freezing of mouse oocytes in isotonic phosphate buffered saline (PBS) were simulated using thermodynamic models. The permeability to water at 0 degree C, Lpg, and the activation energy, ELp, of metaphase II mouse oocytes from B6D2F1 mice were determined to be 0.044 +/- 0.008 micron/min-atm and 13.3 +/- 2.5 kcal/mol during freezing at 2 degrees C/min. The inactive cell volume was determined to be 0.214 with a correlation coefficient of 0.995, indicating that the oocytes closely follow the ideal Boyle-van't Hoff relation. The mean value of the oocyte diameter was 79.41 +/- 4.62 microns. These results were used to predict the behavior of mouse oocytes under various freezing conditions. The effect of the cooling rate on the cell volume and cytoplasm undercooling was investigated. The changes in transmembrane potential were also investigated during freezing of mouse oocytes. The computer simulations showed that at the beginning of the freezing process (-1 degrees C), the fast growth of ice in the extracellular solution causes a sharp increase of the membrane potential. It is predicted that the change in membrane potential is substantial for almost all cooling rates. Estimations show that values as high as -90 mV may be reached during freezing. The hyperpolarization of the membrane may cause orientation of the dipoles within the membrane. For membrane proteins with 300 debye dipole moment, the theoretical prediction suggests that the percentage of dipoles aligned with the membrane potential increases from 16% at 0 degrees C prior to freezing to 58% at -8 degrees C after seeding of the external ice followed with a cooling at 120 degrees C/min.

Animals↗
PubMed1999

Viability and thermal stability of a strain of Saccharomyces cerevisiae freeze-dried in different sugar and polymer matrices.

The viability and thermal stability of a freeze-dried yeast strain were studied in relation to some physical properties of the matrices in which the cells were freeze-dried. Samples of inoculum with solutions of the matrix components [polyvinylpyrrolidone (PVP), maltose, trehalose, maltodextrins, or mixtures of maltodextrin and trehalose] and controls without matrices were freeze-dried and then equilibrated at several relative humidities. Viability was determined before and after freeze-drying and after heat treatment (100 min at 70 degrees C). Freeze-drying with trehalose, PVP, maltose or 1.8-kDa maltodextrin, and mixtures of maltodextrin/trehalose increased viability in comparison with controls. The 3.6-kDa maltodextrin was ineffective at protecting the cells during freeze-drying. The glass transition temperature (Tg), which depends on moisture content, was indicated as a possible factor to determine the stability of labile materials. Protective effects of the excipients during thermal treatment were analysed in relation to the physical changes (collapse or structural shrinkage) which were dependent on the Tg of the systems. The presence of a certain amount of amorphous disaccharides during freeze-drying and heating was found to be a critical factor for ensuring cell viability, which was protected even in rubbery (above Tg) matrices.

Freeze Drying↗
PubMed1996

Preparation of bank bone using defatting, freeze-drying and sterilisation with ethylene oxide gas. Part 1. Experimental evaluation of its efficacy and safety.

We devised a method of sterilising bone allografts which consists of defatting in chloroform and methanol, freeze-drying and sterilisation with ethylene oxide gas. The purpose of defatting and freeze-drying was to facilitate subsequent sterilisation by eliminating the barrier to diffusion of the gas into bone, to lower residual levels of ethylene oxide and its toxic by-products, to eliminate alloantigens and to make storage possible at room temperature. The efficacy and safety of the method were evaluated by testing the sterilisation of infected bone from 6 patients with active chronic osteomyelitis, the penetration of ethylene oxide into human femoral heads treated by this or by freeze-drying or freeze-thawing, and the desorption of ethylene oxide and its toxic by-products from pieces of bone treated by these methods. All the samples of infected bone tested negative for bacteria after treatment. The gas penetrated into the central area of the femoral heads in a few hours. Residual levels of ethylene oxide and its toxic by-products were much lower in the treated bone than in freeze-dried or freeze-thawed bone, and decreased quickly in flowing air. Prior defatting and freeze-drying facilitated penetration of ethylene oxide into bone during sterilisation and the desorption of ethylene oxide and its toxic by-products after sterilisation. Preparation under clean, but not sterile, conditions and storage at room temperature make bone banking more practical and efficient.

Bone Transplantation↗
PubMed2000

Cold acclimation and cryoprotectants in a freeze-tolerant Antarctic nematode, Panagrolaimus davidi.

Panagrolaimus davidi is a freeze-tolerant Antarctic nematode which survives extensive intracellular freezing. This paper describes the development of culture techniques which provide clean samples, with a high degree of freeze tolerance and in sufficient quantities for the analysis of potential cryoprotectants. Cultures grown at 20 degrees C survived a short-term freezing stress but survival declined with the time spent frozen. Acclimation of cultures at 5 degrees C enhanced the long-term survival of freezing. Starvation, however, reduced the nematode's ability to survive short-term freezing. The principal cryoprotectants detected by gas chromatography were trehalose and glycerol. The levels of trehalose, but not those of glycerol, increased significantly after acclimation. Trehalose may stabilise membranes and protect them against the dehydrating effects of the osmotic stresses resulting from freeze concentration effects but other factors, such as recrystallisation inhibition, may be involved in long-term survival.

Acclimatization↗
PubMed2005

HIF-1alpha involvement in low temperature and anoxia survival by a freeze tolerant insect.

Winter survival for many insect species relies on the ability to endure the freezing of extracellular body fluids. Because freezing impedes oxygen delivery to tissues, one component of natural freeze tolerance is a well-developed anoxia/ischemia resistance. The present study explores the responses of the hypoxia-inducible factor-1alpha (HIF-1alpha) to cold, freezing and anoxia exposures in the freeze tolerant goldenrod gall fly larva, Eurosta solidaginis. Reverse transcription-PCR was used to quantify hif-1alpha transcript levels; transcripts were significantly elevated by approximately 70% in chilled (3 ( composite function)C), frozen (-16 ( composite function)C) and thawed (returned to 3 ( composite function)C) insects, compared with 15 ( composite function)C controls. Transcripts also rose by approximately 3-fold in insects given anoxia exposure under a nitrogen gas atmosphere. Cold and freezing exposure also elevated HIF-1alpha protein content in the larvae and HIF-1alpha levels increased over the winter months in insects sampled from an outdoor population; levels peaked in February at 2.1-fold higher than in September. A partial sequence of HIF-1alpha that covers the bHLH and PAS domains of the protein was obtained from E. solidaginis and sequence analysis revealed that this segment shared 62% identity overall with Drosophila melanogaster HIF-1alpha and higher percent identities within specific domains: 76% within the bHLH domain and 70% within the PAS domain. The data provide the first documentation of a potential role for HIF-1 in regulating the expression of genes that can aid freezing survival in a cold-hardy animal.

Aerobiosis↗
PubMed1986

Controlled rate freezing of human marrow in a constant temperature cooling gradient in air.

A new instrument which utilizes a computer controlled freezing platform moving in a constant air temperature gradient generated over liquid nitrogen (LN2) was evaluated for cryopreservation of human marrow. Marrows were placed horizontally on the freezing platform which was suspended over LN2 in a cylindrical freezing chamber. The platform was raised or lowered to maintain a predetermined fixed cooling rate in response to temperature monitored and recorded by the computer from a thermocouple placed at platform level. Separate freezing programs were created for different marrow volumes. The viability of normal marrow was tested in vitro before and after freezing. Recovery of marrow cells after freezing and thawing, as measured by cell counts and CFU-GM assays, were the same for the constant air gradient instrument as for a conventional freezing instrument. Thirteen patients received autologous marrow transplants utilizing marrow cryopreserved in the constant air gradient instrument and engraftment results were indistinguishable from those obtained for marrow cryopreserved with a conventional instrument.

Bone Marrow Cells↗
PubMed1990

Elevated frequencies of micronuclei in cultured fibroblasts after freezing and thawing.

Spontaneous micronuclei (MN) were determined in 69 fibroblast lines in the first subculture after cryoconservation. 45 cultures (65%) showed micronuclei in the normal range (less than 10 MN/500 cells), but 24 (35%) exhibited an elevation up to 60 MN/500 cells. In order to determine whether freezing and thawing is responsible for the enhanced MN level we studied the persistence of elevated MN in 10 cell cultures after freezing and thawing, and found that the MN levels returned to normal after 3 subcultures. In addition, the micronucleus formation before and after freezing was investigated in 2 newly established cell cultures obtained from probands whose cells had a particularly high number of MN. Both cultures had regular MN levels before freezing but a more than doubled number of MN when frozen samples were recultivated. This effect of the freezing procedure was confirmed with 5 separate biopsies obtained from the same donor. Since a freezing-related increase in MN was constantly observed in cells from some individuals but not in others, it might be possible that it is a constitutive trait which causes cultured fibroblasts of some individuals to be hypersensitive to the freezing and thawing procedure. It is also noteworthy that fibroblasts from 8 out of 12 probands with a familial malignant melanoma exhibited this phenomenon.

Biopsy↗
PubMed1994

A morphological and immunolabelling study of freeze-substituted human and simian immunodeficiency viruses.

Rapid freezing, freeze substitution and low temperature embedding were used to obtain resin-embedded specimens of HIV and SIV for morphological and immunolabelling studies, with particular emphasis on the 'lateral bodies' and p6 protein. HIV- or SIV-infected cells were fixed in 3% paraformaldehyde and cryoprotected with 0.5 M sucrose. Cells were applied to pieces of Whatman No 1 filter paper and impact-frozen onto a liquid nitrogen cooled copper block. Specimens were freeze-substituted at -90 degrees C using one of three different media: (a) absolute methanol, (b) methanol containing 0.5% uranyl acetate, and (c) methanol containing glutaraldehyde, osmium tetroxide and uranyl acetate. Specimens substituted in methanol and uranyl acetate showed both good structural preservation and retention of antigenicity. We found that the use of filter paper for supporting the specimen was an important factor in obtaining good freezing rates and was more practical than freezing mixtures of cells and gelatin. When compared with specimens prepared by conventional fixation and embedding, freeze-substituted virus particles showed a greater uniformity of shape and size and were more dense in appearance. Distinct 'lateral bodies' were not observed in freeze-substituted viruses. The viral protein p6 was widely distributed in the centre of mature virus particles.

Animals↗