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PubMed2003

Long-term hematologic reconstitution after autologous peripheral blood progenitor cell transplantation: a comparison between controlled-rate freezing and uncontrolled-rate freezing at 80 degrees C.

BACKGROUND: The most widely used system for peripheral blood progenitor cell (PBPC) cryopreservation is controlled-rate freezing (CRF). Uncontrolled-rate freezing (URF) at -80 degrees C has also been used, but its clinical impact has not been studied sufficiently yet. STUDY DESIGN AND METHODS: Two groups of patients were compared: Group A consisted of 69 patients autotransplanted with PBPCs cryopreserved with CRF; Group B consisted of 192 patients autotransplanted with PBPCs cryopreserved with URF at -80 degrees C. The same cryoprotectant solution and storage system were used. RESULTS: A significant delay of hematologic reconstitution (HR) in the URF group was observed for neutrophils greater than 0.5 x 10(9) per L and for platelets greater than 20 x 10(9) per L and greater than 50 x 10(9) per L; we did not observe any differences in the clinical course. The long-term HR was comparable in the two groups, all patients showed stable engraftment, and no late graft failures were observed. CONCLUSION: Our study confirms that URF is safe and allows sustained long-term engraftment without increasing the risks of transplantation, even though the early engraftment after URF is slower.

Adolescent↗

PubMed1984

Studies on Freezing Injury of Plant Cells: I. Relation between Thermotropic Properties of Isolated Plasma Membrane Vesicles and Freezing Injury.

The thermotropic transition of plasma membrane of Dactylis glomerata was studied by using fluorescence polarization of embedded fluorophore, 1,6-diphenyl-1,3,5-hexatriene. Under the presence of 35% ethylene glycol, reversible thermotropic transitions were observed in isolated plasma membrane vesicles in nearly the same temperature range as the temperature of freezing injury to cells. In liposomes prepared from isolated plasma membranes, however, the thermotropic transitions occurred at much lower temperatures in comparison with those of intact membrane vesicles. Following treatment with pronase, the thermotropic transition also shifted downward.Thus, the thermotropic properties of plasma membranes appeared to be dependent on the membrane proteins. In vitro freezing of the isolated plasma membrane vesicles without addition of any cryoprotectant, such as sorbitol, resulted in an irreversible alteration both in the fluorescence anisotropy values and the temperatures for the thermotropic transition, suggesting an irreversible alteration in the membrane structure, presumably changes in lipid-protein interactions and protein conformation.

Journal Article↗

PubMedDate not supplied

Ultra-rapid freezing and storage of rat embryos in an electric refrigerator at -130 degree C without liquid cryo-agents, with ultra-short exposure in the freezing medium and direct rehydration after thawing.

An ultra-rapid freezing technique for embryos has been developed. This procedure involves: ultra-short exposure to cryoprotectants; freezing of embryos in a metallic powder pre-cooled in an electric refrigerator at -130 degree C; storage of embryos ina refrigerator at -130 degree C; direct rehydration after thawing.

Journal Article↗

PubMed1991

The fine structure of testicular interstitial cells in the mouse. A comparison after conventional double fixation versus rapid freezing followed by freeze substitution.

The interstitial cells in the testis of the mouse have been studied both after conventional double fixation with glutaraldehyde followed by osmium tetroxide and after rapid freezing and cryosubstitution with osmium tetroxide-acetone by light, scanning and transmission electron microscopy. Differences have been found especially concerning the preservation of lipids and the appearance of mitochondria. Varying proportions and different aspects of rough and smooth endoplasmic reticulum were observed corresponding after both preparation methods thus confirming their real existence. Numerous specific contacts are found between macrophages and Leydig cells, indicating a functional interdependence between these two cell types. Though structural preservation is superior after freeze substitution in the outermost zone, artifacts caused by the initial impact, by ice crystal formation, and during substitution must be considered.

Animals↗

PubMed1990

Membrane fusion events during endocytosis in mouse kidney tubule cells detected by rapid freezing followed by freeze-substitution.

Membrane fusion events during endocytosis were examined in renal proximal tubule cells processed by rapid freezing followed by freeze-substitution. An extensive fusion site of the membranes between two apical vacuoles or a vacuole and a lysosome, which appeared as a pentalaminar structure, was not observed in this study, though such a pentalaminar structure has been found in chemically fixed specimens. In vitro experiments using subcellular fractions revealed that the pentalaminar structure did not always represent the initial stage of membrane fusion. Membrane fusion appeared to occur in a specific membrane site, wHere a minute pore connecting the two lumens was formed immediately after the two membranes came into contact with each other. Some pore necks showed an asymmetrical feature which consisted of a sharp corner and a gently curved corner. Enlargement of the pore appears to be mediated by a fibrous structure observed near the sharp corner of the pore neck.

Animals↗

PubMed1990

The lamina suprachoroidocapillaris of the guinea pig choroid confirmed by the rapid freezing and freeze-substitution method.

There is a compact layer (the lamina suprachoroidocapillaris) between the vascular and the choroidocapillaris layer of the guinea pig and rat choroids. The specimens were treated with the rapid freezing and freeze-substitution method. The choroidal melanocytes and their processes were exclusively built up of several layers, and the arterioles and venules passed through this layer. Numerous nerve terminals were seen throughout this layer, which contained small or large-size synaptic vesicles with dense or light core. These results may facilitate further understanding of the microcirculation in the choroidal architecture.

Animals↗

PubMed2000

Ultrastructure of sea urchin calcified tissues after high-pressure freezing and freeze substitution.

The improvements brought by high-pressure freezing/freeze substitution fixation methods to the ultrastructural preservation of echinoderm mineralized tissues are investigated in developing pedicellariae and teeth of the echinoid Paracentrotus lividus. Three freeze substitution (FS) protocols were tested: one in the presence of osmium tetroxide, one in the presence of uranyl acetate, and the last in the presence of gallic acid. FS in the presence of osmium tetroxide significantly improved cell ultrastructure preservation and should especially be used for ultrastructural studies involving vesicles and the Golgi apparatus. With all protocols, multivesicular bodies, suggested to contain Ca(2+), were evident for the first time in skeleton-forming cells. FS in the presence of gallic acid allowed us to confirm the structured and insoluble character of a part of the organic matrix of mineralization in the calcification sites of the tooth, an observation which modifies the current understanding of biomineralization control in echinoderms.

Animals↗

PubMed1996

Effects of blanching, freezing and freeze-drying on the fatty acid contents of green beans.

GC-FID was used to monitor changes over time in palmitic, stearic, arachidic, oleic, linoleic and linoleic acid contents of green beans subjected to various preservation treatments. In beans stored in polyethylene bags at -22 degrees C without prior blanching, all fatty acid contents dropped appreciably within the first month of storage, regardless of whether the beans had been hand- or vacuum-packed. In beans which had been freeze-dried then stored at room temperature in an airtight container, polyunsaturated fatty acid contents dropped appreciably only after 2 months.

Chromatography, Gas↗

PubMed1980

The vitelline layer of the sea urchin egg and its modification during fertilization. A freeze-fracture study using quick-freezing and deep-etching.

Eggs of the sea urchin Strongylocentrotus purpuratus were quick-frozen, freeze fractured, and deep-etched to reveal the detailed structure of the vitelline layer (VL), an extracellular coat. The VL consisted of a network of fibers lying in sheet raised 20 nm off the plasma membrane and connected to it by a series of short processes. Sperm attached to the fibers of this sheet and upon fertilization the VL rose off the egg surface to form the fertilization envelope (FE). By 1 min postinsemination (p.i.), the FE had become augmented by a new set of smaller fibrils, and the original fibers of the VL appeared to be undergoing degradation. The FE exhibited casts of microvilli the VL had once covered. These were rounded at 1 min p.i., but by 2 min they had become angular and coated with an orderly array of repeating macromolecular units. In areas between casts, the coating process was slower; incomplete rows of units were seen at 5 min p.i. and complete rows at 10 min. Deep-etching of FE isolated from eggs by homogenization and differential centrifugation showed that both top and bottom surfaces were coated. The coat pattern was made up of 17.5-nm wide rows of parallelogram-like units that repeated every 12.2 nm along the row axis. Units in adjacent rows were in register to produce a secondary axis 76 degrees from the row axis. The results of this and previous studies suggest that the coating process plays a major role in "hardening" the FE to produce a tough barrier that protects the early embryo from chemical and mechanical injury.

Animals↗

PubMed1975

Freeze-fracturing and freeze-etching of cardiac myosin filaments.

Myofilament structure was studied in freeze-etch replicas of unfixed, glycerinated beef cardiac muscle. The information which is revealed depends upon the direction of metal shadowing in relation to the filament axis. Shadows oblique to this axis reveal that the outer surface of a longitudinal half of a thick filament comprises three, sometimes four, rows of myosin molecules. These molecules are generally assembled in a braided manner with both left and right-handed helical components. Occasionally a more parallel to the myofilament axis reveal cross-bridges linking thick and thin filaments. These bridges are readily detectable by optical diffraction techniques, giving an axial bridge spacing of approximately 40 nm. In unetched preparations cross bridges appear as vertical rows of beads. In all replicas the effects of plastic deformation of proteins must be considered.

Animals↗

PubMed1984

Design and application of a freeze-fracture stage with a cleaver device for Balzers 'freeze-etching' apparatus.

A fracturing mechanism in the form of a double-wedged cleaver, used previously on other freeze-fracture machines, has been incorporated in the Balzers BAF 400 D apparatus. The design of the stage, with the cleaver, is explained and its performance and advantages are outlined. By minimizing mechanical damage to the exposed surface of the frozen specimen and reducing contamination by effective shrouding, an improvement in the quality of the replicas is achieved. The suitability of this mechanism for the fracturing of tough tissues such as skin is also outlined.

Animals↗

PubMed1992

Short fixation-shock freezing and freeze-drying versus chemical fixation and dehydration: computer assisted image analysis of morphological variables and immunogold labeling density on pituitary secretory granules.

We have performed a computer assisted image analysis evaluation of the effects of two preparation protocols on morphological variables and immunolabeling density of secretory granules using rat adenohypophysis as a model. Glutaraldehyde (GA) fixation for 2 hr and chemical dehydration was compared with short GA fixation (15 min) followed by cryofixation and freeze-drying (CF-FD). The 2 hr GA fixed specimens showed spherical nuclei and secretory granules regardless of their size, contrasting with the more irregularly shaped nuclei and secretory granules after short GA-CF-FD. In the latter group of specimens a correlation could be found between smaller nuclear areas and more irregular shapes. The differences in morphological variables between the two preparation protocols might be due to a better protein stabilization and a reduced collapse of macromolecules after the 2 hr GA fixation, strengthened by the chemical dehydration. The specific immunolabeling with antiserum to growth hormone was much greater but more varied after short time GA-CF-FD than after long GA fixation. Epon surface topography over the granule area also differed: smooth after short time GA-CF-FD and furrowed after long GA fixation. Our results, demonstrating important differences in morphological parameters and immunolabeling density between two common preparation protocols, seem critical for more reliable interpretation in quantitative immunoelectron microscopy. We also emphasize the need for computer assisted image analysis and measurements in immunoelectron microscopy to ensure objective evaluations.

Animals↗

PubMed1986

Freeze-fracture studies of plasma membranes of astrocytes in freezing lesions.

Epileptic foci are frequently associated with a proliferation and hypertrophy of fibrous astrocytes. Such proliferating astrocytes, which are involved in scar formation and are referred to as reactive, have been characterized by a number of morphological changes involving the cytoplasmic organelles and nucleus. With the development of freeze-fracture techniques, which allow for the splitting and analysis of the macromolecular structure of biological membranes, another morphological characteristic could be used to define reactive astrocytes. This characteristic is the alteration in the normal number and distribution of intramembranous particles. Specifically, there is an increase in the number of orthogonal arrays of intramembranous particles, called assemblies, in the plasma membrane of reactive astrocytes. Besides the presence of assemblies in the plasma membranes of astrocytes, similar particle arrays are also found in the central nervous system (CNS), in cell membranes of ependymal cells, in Müller cells near the inner limiting membrane of the retina, and in satellite cells in spinal ganglia. Assemblies are concentrated in the plasma membranes of astrocytic processes that form the glia limitans at the outer surface of the brain and the perivascular sheath around parenchymal blood vessels in the adult mammalian CNS. Plasma membranes of perineuronal astrocytic processes and astrocytic cell bodies have few, if any, assemblies. The factor responsible for this polarity of assemblies in the normal CNS is unknown. However, this polarity is lost when astrocytes respond to various types of injuries to the CNS. It has been postulated that gliosis, resulting in an epileptic scar, may be a response of cortical astrocytes to changes in the local cellular environment and that this reactivity of astrocytes may be related to plasma membrane changes. However, little is known about the initiation of the astrocytic response or the involvement of the astrocytic plasma membrane. Because of the presence of assemblies, astrocytic plasma membranes are an ideal subject for a morphological investigation of alterations of the cell membrane. Assemblies have a distinctive structure and changes in their number and distribution can be readily identified. Also, since there is polarity of the assemblies in astrocytic plasma membranes, such that their number is greatest in subpial and perivascular areas and least in the perineuronal regions, it is easy to identify when assemblies are added or lost from the astrocytic plasma membranes of a particular brain region.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Application of glycerol to freezing bovine pancreas. Part 3. Effects of freezing rate and glycerol addition on changes in pancreas proteins.

Changes in protein solubility, amino nitrogen and activity of proteases in bovine pancreas frozen at -70 and -18 degrees C after soaking in various aqueous glycerol solutions were studied. Reduction of freezing temperature causes decrease of value in all studied parameters. Additionally, an inhibitory effect of glycerol on protease activity was demonstrated.

Animals↗

PubMed1985

Electron-microscopic study of the collagen fibrils of the rat tail tendon as revealed by freeze-fracture and freeze-etching techniques.

The ultrastructure of the collagen of rat tail tendon was investigated by the freeze-fracture technique. Collagen fibers were pretreated with the digestive enzymes, alpha-amylase, elastase and collagenase to remove matrix substances. Some of the samples were etched for 20 min. Fibrils had an average diameter of 318 +/- 12 nm and a banded structure with a mean periodicity of 64.2 +/- 0.9 mm; the banding was most marked in alpha-amylase/elastase-treated specimens, although the periodicity was independent of pretreatment. Microfibrils were well-displayed following alpha-amylase/elastase and collagenase pretreatments. A difference in the diameters of microfibrils was, however, observed between etched specimens (8.3 +/- 0.3 nm) and those prepared by other experimental methods (11.4 +/- 0.5 nm). In replicas of collagenase-treated and etched specimens, the interconnecting filaments in the interfibrillar region formed a network that was continuous with the microfibrils of collagen fibrils. The diameter of the interconnecting filaments was the same as that of microfibrils. Microfibrillar bundles were observed in the interfibrillar region.

Amylases↗

PubMed1985

Ultrastructural observations on the cytoarchitecture of axons processed by rapid-freezing and freeze-substitution.

The structure and organization of axons in the cervical connective of wild-type Drosophila fruit flies were examined in anticipation of studies of various neurological mutants. Dissected flies were rapid-frozen from the living state against a copper block cooled with liquid helium, freeze-substituted, and prepared for electron microscopic examination of thin sections. These cryotechniques showed new details of the structure of cell organelles and cytoplasm in Drosophila axons. The cytoplasmic matrix of axons and glia consists of a material with a fine granular texture enmeshed in a three-dimensional meshwork of short, fine filaments which vary in shape, size and electron density. No neurofilaments are present, but bundles of microtubules are interwoven into the filamentous matrix of the axoplasm. The round wall of microtubules (27 nm overall diameter) is composed of twelve cylindrical protofilaments with a typical substructural periodicity. Mitochondria frequently make contact with microtubules in both axons and glial processes. A thin layer of electron-dense filamentous matrix, which appears to be an axonal basal lamina, contacts most of the axonal exoplasmic surface, especially that of axons where they are surrounded by processes of glial cells, but is scant wherever single axons are contiguous. Thus, an axonal basal lamina occupies the constricted spaces around axons, where extracellular K+ accumulates during neural activity.

Animals↗

PubMed1985

Application of rapid freezing followed by freeze-substitution acrolein fixation for cytochemical studies of the rat stomach.

A method involving rapid freezing followed by substitution fixation was developed, using acrolein as a fixative. This was then applied to several cytochemical stainings, and showed well preserved and clear cell structures. Membranes were apparently negatively stained and the ultrastructure of mitochondria, rough endoplasmic reticulum and Golgi apparatus was clearly discernible. The mitochondrial and cytoplasmic matrices were stained rather densely compared with routine chemically fixed preparations, implying a good preservation of matrix substances. Periodic acid-thiocarbohydrazide-silver proteinate staining was applied to the present method. The mucous granules of surface covering epithelial cells indicated fine staining of bipartite structure and the Golgi apparatus of mucous cells showed clear staining differences based on polarity. Postembedding lectin-ferritin and immunocytochemical stainings were applicable to the present preparations and stable stainings of secretory granules were obtained. A low temperature embedding material, Lowicryl K4M, was also examined. The cell preservation of these samples was not as good as those embedded in Epon, but the rough endoplasmic reticulum and Golgi apparatus of chief cells were stained with anti-pepsinogen antibody as were the secretory granules. The present method was also applicable to light microscopy.

Acrolein↗

PubMed1985

Vesicular profiles in frog perineural cells preserved by rapid-freezing and freeze-substitution.

Vesicular profiles in perineurial cells of frog peripheral nerves were examined in tissues preserved by rapid-freezing or by conventional chemical fixation. Tissues were processed immediately after removal from the animal or after remaining in Ringers for several hours. Vesicular profiles were present in perineurial cells in all experimental groups, demonstrating that they are not an artifact of chemical fixation. However, variations in their morphology correlated with the different preservation techniques.

Animals↗