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PubMed2002

[Freezing of umbilical blood cells in mechanical freezers (-80 degrees C)].

Umbilical cord blood (UCB) contains hematopoietic stem cells capable of reconstituting hematopoiesis in the recipient. This property has prompted search for methods to store such cells in liquid nitrogen (-196 degrees C) and offer them for future use in HLA-matched patients. UCB cells are often collected in hospitals remote from storage facilities. Immediate freezing is important to prevent apoptosis which develops relatively quickly after isolation from cord blood. Investigations into the freezing of bone marrow cells have corroborated the feasibility of uncontrolled rate freezing and storage of cells in a mechanical freezer at -80 degrees C. The aim of the present study was to apply this approach to UCB cells and to determine optimal freezing conditions as measured by progenitor cell survival. The highest survival rate among UCB cell species subjected to mechanical freezing was demonstrated by mononuclear cells enriched for the CD34+ subpopulation. The presence of CD34- mononuclear cells (monocytes, lymphocytes) and erythrocytes in the cell suspension reduced progenitor cell survival and proliferation potential. Moreover, it was found that CD34+ cells from UCB can be frozen in an uncontrolled manner. Optimal results were achieved with 10% DMSO and 70% serum. The results were unaffected by the type of medium used (RPMI, Dulbecco, Iscove). In conclusion, uncontrolled rate freezing of UCB cells is an acceptable method prior to transport of the cells in dry ice to a central banking station and storage in liquid nitrogen until needed.

Cell Count↗
PubMed1991

Aldehyde fixation causes membrane vesiculation during platelet exocytosis: a freeze-substitution study.

Despite a plethora of reports on the ultrastructure of secretory granule release by exocytosis, the release of coagulant activity from stimulated platelets is still being attributed to membrane vesiculation. Membrane vesiculation and the formation of myelin figures have been shown to be artifacts of glutaraldehyde GA fixation. Cells fixed by direct osmium or rapid freezing are free of such structures. Yet there is still doubt that rapid freezing interferes with vesiculation process. This study has addressed this issue by examining: (1) whether freezing and freeze-substitution affects membrane vesiculation, (2) whether paraformaldehyde-fixation also induces the phenomenon, and (3) whether the aldehyde concentration is of influence. Aldehyde fixation was carried out prior to impact freezing and freeze-substitution. In thrombin-stimulated platelets, membrane vesiculation and myelin figures were found. Glutaraldehyde induced multivesicular structures, paraformaldehyde or low aldehyde concentrations only blebs on the platelet surface. The membrane vesicles were in continuity with the cytoplasmic matrix. Unstimulated platelets did not show vesiculation or myelin figures. Control samples, without aldehyde fixation, showed instead of membrane vesiculation, granule fusion with the plasmalemma, or, instead of myelin figures, compound granules. This confirms that membrane vesiculation and the formation of myelin figures are artifacts induced by the failure of aldehydes to arrest lipid mobility within membranes undergoing rapid changes in structure. Although the presence of membrane vesiculation and myelin figures in platelets indicates that exocytotic processes were occurring at the moment of aldehyde fixation, the finding of membrane vesiculation in aldehyde-fixed platelets does not indicate a separate type of exocytosis.

Aldehydes↗
PubMed1989

The application of cryo-ultramicrotomy and freeze-substitution in immuno-gold labelling of hybrid proteins in Escherichia coli. A comparison.

To study the possible effects of chemical fixation upon antigenicity and structural preservation, the subcellular localization of LamB-LacZ hybrid proteins in Escherichia coli K-12 strains pop3234 and pop3299 was investigated both by cryo-ultramicrotomy and freeze-substitution. Immuno-gold labelling of sections of freeze-substituted bacteria showed the same localization of the hybrid protein as found after cryo-ultramicrotomy. The efficiency of labelling of the accumulated form of the hybrid protein was lower after freeze-substitution whereas the efficiency of labelling of the membrane-bound form showed no difference. Different fixatives and Lowicryl resins had no clear effect on the label-efficiency but the complex substitution medium, containing osmium tetroxide, uranyl acetate and glutaraldehyde, in combination with the apolar Lowicryl HM20 gave the best sectioning properties and membrane contrast. For this specific problem, although the somewhat better preservation after freeze-substitution, cryo-ultramicrotomy is to be favored since it is much less time-consuming, there are no freezing problems, ultrastructural preservation is sufficient and the theoretical benefits of freeze-substitution are not expressed.

Bacterial Proteins↗
PubMed1995

Freezing influences the healing of rabbit medial collateral ligament autografts.

Connective tissue allografts are treated commonly with deep freezing to facilitate their storage in tissue banks and to reduce their immunogenicity. The influence of freezing on the healing of these grafts is unknown. The effect of an aggressive freezing protocol on ligament graft healing in a nonimmunogenic autograft model was investigated. The femoral-medial collateral ligament-tibial complex of the rabbit knee was transplanted orthotopically into the site from which it had just been harvested. The fresh group had their grafts rinsed in normal saline, and the frozen-thawed group had their grafts repetitively frozen-thawed in liquid nitrogen immediately before reimplantation. The healing of the ligament complexes were evaluated mechanically at intervals as long as 1 year after transplantation. Repeated freezing had little effect on the initial mechanical behavior of the graft complexes but was found to be detrimental to subsequent graft incorporation. The frozen-thawed grafts were weaker than the fresh grafts overall, and this effect persisted over time. The failure load and stress of the frozen-thawed grafts reached only 87% and 75% of that of the fresh grafts after 48 weeks. Because the initial mechanical effects of freezing were minimal, subsequent effects on graft healing must be attributed to factors other than gross structural injury. Despite showing almost no signs of damage to bone-ligament-bone grafts initially, freezing procedures may have important subsequent biologic consequences that may influence the clinical success of stored connective tissue grafts used in ligament reconstruction.

Animals↗
PubMed1993

[Festination and freezing].

Akinesia, the core symptom of parkinsonism, is a comprehensive term and a disorder characterized by poverty, slowness and also freezing of movement. Parkinson (1817) in his original essay pointed out the festinating gait. Festination is a tendency to speed up in parallel with a loss of normal amplitude of repetitive movement (petit pas, micrographia and inaudible speech). Freezing is a breakdown of repetitive voluntary movement emerging through festination or suddenly. Freezing of gait occurs either at the start (start hesitation), through festination of gait or suddenly when turning or going through the narrow doorway. Narabayashi and the present author reported the first 2 cases with pure akinesia or freezing symptom without rigidity or tremor and unresponsive to L-DOPA therapy as a new condition at the 14th annual meeting of this Society, 1973. Kinésie paradoxal was always accompanied by this type of akinesia. HVA level in the cerebrospinal fluid was not below normal. The author suggested that the main pathological structures of the condition are different from Parkinson's disease. L-threo-DOPS, a synthetic norepinephrine (NE) precursor, had a mild-to-moderate effect on some cases with freezing, and the NE hypothesis for freezing was proposed (Narabayashi). 35 cases with this condition have been known in our clinic to date. Slowly progressive supranuclear ophthalmoplegia appeared later in some patients, several of whom were clinically and two were pathologically diagnosed as progressive supranuclear palsy. The nosological position and responsible lesion sites of this condition are discussed.

Gait↗
PubMed2004

Immobilization of nonviral vectors during the freezing step of lyophilization.

The development of nonviral vectors as commercial therapeutics will require formulations that are sufficiently stable to allow shipping and storage for prolonged periods. Given the well-known instability of these systems as aqueous suspensions, it would be desirable to develop lyophilized formulations that are resistant to shipping stress and can be stored for extended periods at ambient temperatures. Previous studies have shown that aggregation and structural changes resulting in reduced transfection rates can occur during the freezing step of lyophilization. While it has been clearly demonstrated that freezing-induced damage is promoted by vector crowding that results from the reduced volume of unfrozen solution, the precise mechanism of damage has yet to be fully elucidated, i.e., damage may occur due to ice formation and/or during incubation in the frozen state. In this study, we investigate the time- and temperature-dependence of damage during freezing and demonstrate that aggregation can occur while frozen vector suspensions are incubated at a constant temperature. Aggregation is not seen during incubation at temperatures below T(g)', and can also be avoided above the glass transition temperature under some conditions. Our data are consistent with a model describing the mobility of vectors in the unfrozen sucrose solution being sufficiently restricted such that inter-particle interactions are prevented in the frozen state. Furthermore, the protection achieved during freezing at temperatures above T(g)' is applicable to a complete lyophilization cycle (i.e., freezing and drying), and provides stabilization at higher primary drying temperatures.

Animals↗
PubMed1980

Ultrastructure of spores of Rhizopus nigricans after repeated freezing and thawing shocks.

Disintegration of nuclear envelopes is the only ultrastructural change detectable by freeze-etching in dormant spores of the mould Rhizopus nigricans, both dry and swollen, subjected to repeated freezing and thawing. The increase of the number of freeze-inactivated spores corresponds well with the increase of the number of damaged nuclei. This fact led us to formulate a hypothesis that the structure of the nucleus is the primary target of the freezing or thawing damage. As other biomembranes are not damaged it may be assumed that the disintegration of the nuclear membrane is probably secondary. No changes in ultrastructure of metabolically activated spores could be detected, in spite of the fact that the spores lost their germinative ability. Thus, the mechanism of the freeze injury may be different in dormant and growing spores.

Cytoplasm↗
PubMed2006

A new approach for freezing of aqueous solutions under active control of the nucleation temperature.

An experimental setup for controlled freezing of aqueous solutions is introduced. The special feature is a mechanism to actively control the nucleation temperature via electrofreezing: an ice nucleus generated at a platinum electrode by the application of an electric high voltage pulse initiates the crystallization of the sample. Using electrofreezing, the nucleation temperature in pure water can be precisely adjusted to a desired value over the whole temperature range between a maximum temperature Tn(max) close to the melting point and the temperature of spontaneous nucleation. However, the presence of additives can inhibit the nucleus formation. The influence of hydroxyethylstarch (HES), glucose, glycerol, additives commonly used in cryobiology, and NaCl on Tn(max) were investigated. While the decrease showed to be moderate for the non-ionic additives, the hindrance of nucleation by ionic NaCl makes the direct application of electrofreezing in solutions with physiological salt concentrations impossible. Therefore, in the multi-sample freezing device presented in this paper, the ice nucleus is produced in a separate volume of pure water inside an electrode cap. This way, the nucleus formation becomes independent of the sample composition. Using electrofreezing rather than conventional seeding methods allows automated freezing of many samples under equal conditions. Experiments performed with model solutions show the reliability and repeatability of this method to start crystallization in the test samples at different specified temperatures. The setup was designed to freeze samples of small volume for basic investigations in the field of cryopreservation and freeze-drying, but the mode of operation might be interesting for many other applications where a controlled nucleation of aqueous solutions is of importance.

Cryopreservation↗
PubMed1997

Characterization of the glycation of albumin in freeze-dried and frozen human serum.

Human serum albumin (HSA) in fresh frozen and freeze-dried serum reference materials was examined by mass spectrometry and a variety of affinity chromatography techniques. The relative molecular mass distribution of HSA in fresh frozen serum was found to be identical to that of an HSA standard. However, the HSA in the freeze-dried reference serum exhibited a relative molecular mass distribution that was shifted to higher mass, broader, and substantially more heterogeneous than that of HSA in fresh frozen serum. A proteolytic cyanogen bromide digestion of the HSA from freeze-dried serum contained adducts approximately 162 u higher in mass than digest fragments 124-298 and 447-548, suggesting glycation. The presence of glycation on fragments 124-298 and 447-548 correlates with the known sites of HSA glycation. Glycation was further confirmed by the mass spectral analysis of the retained and unretained fractions from glycoaffinity chromatography of HSA from freeze-dried serum. The relative molecular weight of the HSA in the retained fraction indicated the presence of a doubly glycated species. The chemical heterogeneity of Cys-34, the site of the only free thiol in HSA, was examined and found not to be a substantial source of molecular mass heterogeneity for HSA from either fresh frozen of freeze-dried serum.

Blood Specimen Collection↗
PubMed1998

Stabilization of lactate dehydrogenase following freeze thawing and vacuum-drying in the presence of trehalose and borate.

PURPOSE: The purpose of this work was to investigate the effects of trehalose and trehalose/sodium tetraborate mixtures on recovery of lactate dehydrogenase (LDH) activity following freeze-thawing and centrifugal vacuum-drying/rehydration. The storage stability of LDH under conditions of either high relative humidity or high temperature was also studied. METHODS: LDH was prepared in buffered aqueous formulations containing trehalose alone and trehalose/"borate" mixtures. Enzymatic activity was measured immediately following freeze-thawing and vacuum-drying/rehydration processes, and also after vacuum-dried formulations were stored in either high humidity or high temperature environments. Also, glass transition temperatures (Tg) were measured for both freeze-dried and vacuum-dried formulations. RESULTS: The Tg values of freeze-dried trehalose/borate mixtures are considerably higher than that of trehalose alone. Freezing and vacuum-drying LDH in the presence of 300 mM trehalose resulted in the recovery of 80% and 65% of the original activity, respectively. For vacuum-dried mixtures, boron concentrations below 1.2 mole boron/ mole trehalose had no effect on recovered LDH. After several weeks storage in either humid (100% relative humidity) or warm (45 degrees C) environments, vacuum-dried formulations that included trehalose and borate showed greater enzymatic activities than those prepared with trehalose alone. We attribute this stability to the formation of a chemical complex between trehalose and borate. CONCLUSIONS: The high Tg values of trehalose/borate mixtures offer several advantages over the use of trehalose alone. Most notable is the storage stability under conditions of high temperature and high relative humidity. In these cases, formulations that contain trehalose and borate are superior to those containing trehalose alone. These results have practical implications for long-term storage of biological materials.

Borates↗
PubMed2001

Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa.

Chromosome stability was maintained in mouse spermatozoa after freeze-drying or freezing without cryoprotection in a simple Tris.HCl buffer containing EGTA (50 mM) and NaCl (50 mM). The ability of spermatozoa to activate oocytes spontaneously was not destroyed by freeze-drying or freezing without cryoprotection in this solution. Embryos derived after injecting oocytes with sperm heads from rehydrated freeze-dried and from thawed spermatozoa developed normally. Provided the DNA integrity of the sperm nucleus is maintained, embryos can be generated by the intracytoplasmic sperm injection technique (ICSI) from severely damaged spermatozoa that are no longer capable of normal physiological activity. This procedure was effective for preserving spermatozoa from strains (C57BL/6J, 129/SvJ, and BALB/c) in which the fertility of spermatozoa frozen conventionally is extremely poor. The technique provides an effective means of storing mouse spermatozoa from many different inbred, mutant, and transgenic strains for biomedical research.

Animals↗
PubMed1972

Energy-linked ultrastructural transformations in isolated liver mitochondria and mitoplasts. Preservation of configurations by freeze-cleaving compared to chemical fixation.

An investigation was carried out in which microsamples of isolated rat liver mitochondria and freshly prepared mitoplasts in defined energy states were freeze-cleaved. Parallel microsamples were fixed with osmium tetroxide and with glutaraldehyde followed by osmium tetroxide as previously used in this laboratory for the preservation of energy-linked mitochondrial configurations. The details of the orthodox configuration of energized mitochondria and the condensed configuration of de-energized mitochondria, as revealed previously by chemical fixation, are confirmed in this report for nonfixed, freeze-cleaved mitochondria. The precise agreement in preservation of configuration obtained by the physical fixation of rapid freezing and by chemical fixation establishes unequivocally that mitochondria undergo energy-linked ultrastructural transformation between the condensed and the orthodox configurations which are thus natural structural states related to the metabolic activity of the mitochondrion. Configurations observed by freeze-cleaving and by chemical fixation reveal that mitoplasts also undergo a specific and dramatic ultrastructural transformation with the induction of oxidative phosphorylation. The transformation appears to be isovolumetric and therefore is thought to be mediated through energized conformational activity in the surface electron-transport membrane of the mitoplast. Passively swollen, spherical, osmotically active mitoplasts could not be fixed rapidly enough by chemical fixatives as normally used without altering the spherical form. In this special case preservation of configurational form required rapid freezing or chemical fixatives of low osmolar concentration.

Aldehydes↗
PubMed1981

Cryofixation of monolayer cell cultures for freeze-fracturing without chemical pre-treatments.

A cryofixation method is presented which gives excellent ultrastructural preservation of monolayer cell cultures without any chemical pretreatments. Rat hepatocytes in primary culture were used in this study. The equipment needed is inexpensive and easy to manufacture. Cells are grown on a usual tissue culture support material (Thermanox plastic sheets). For cryofixation, samples are prepared essentially by a combined sandwich-cryogen-jet technique, 3 mm large discs are punched out and sandwiched with Cu- or Au-object holders of little mass; a 15 micrometer spacer is put in between. The viability of the cells is not impaired by the manipulations before freezing. The sandwich sample is quickly frozen by shooting a propane jet from a simple pressure chamber on to the metal object holder. The relevant parameters were optimized by parallel freeze-fracture analyses of 5% glycerol as a model system and by thermocouple measurements. Sandwich samples are then mounted in an appropriate double replication specimen table for further analysis by freeze-fracturing. It is possible to obtain a certain selectivity of the fracture plane with regard to apical, lateral or basal aspects of the cell layer. Alternatively, disc samples can be processed by chemical fixation methods (including freeze substitution to determine the freeze-fracture plane), since the support material Thermanox is insensitive to organic solvents and easy to cut. In each case the cells remain attached to their substratum throughout the whole procedure. Thus, the ultrastructural data can be directly correlated with parallel functional analyses obtained from the same cell cultures.

Animals↗
PubMed1986

Study of the conditions necessary for propane-jet freezing of fresh biological tissues without detectable ice formation.

The performance of a commercial double-propane-jet freezer (Balzers QFD 101) has been assessed, for rapid freezing of fresh tissues in freeze-etch work. Samples of diaphragm muscle and intestinal villi were frozen between copper sheets, with a spacer to give 20-30 microns thickness of tissue. Fracture cuts were made with the Balzers BAF 400 freeze-etch microtome within 5-10 microns of a freezing face (i.e. a tissue face in contact with the copper sheets of the frozen sandwich). After some modifications to the QFD 101, replicas showing no evidence of ice were obtained of muscle cells, although for intestinal epithelial cells some evidence of ice formation was found. Infiltration with 5% glycerol or dimethylsulphoxide improves the depth of good freezing. Results and problems arising from such infiltration are briefly discussed.

Animals↗
PubMed1976

The physico-chemical basis for the freeze-drying process.

To the extent that the final form and quality of a freeze-dried product depends on the way the freeze-drying is conducted, an understanding of the many factors involved is most important. The numerous effects of the design and mode of operation of the freeze-drying equipment on the course of the process need to be known, as do the properties intrinsic to the material to be freeze-dried. Much can be learned and predicted from the study of the "supplemented phase diagram", a series of experimental plots describing the equilibrium and the non-equilibrium phase behavior of the system in question. Such diagrams map and distinguish eutectic and amorphous phase behavior. Further information is available from gravimetric studies allowing the construction of "desorption isotherms", the plots describing the loss of sorbed water accompanying the sublimation of ice, frequently termed "secondary drying". These plots relate the water retained by the product to the "water activity", or relative humidity at different temperatures. Observations in the freeze-drying microscope contribute additional information, in that they reveal the actual course of the process at the microscopic level. These and other laboratory findings facilitate the analysis and comparison of pilot-plant and commercical scale processing experiences. Where scientific and engineering factors appear to interrelate, the nature and extent of the interdependence can often be determined.

Crystallization↗
PubMed1992

A stimulus timing device for capturing fast physiologic events by quick-freezing.

A timing device was designed that, in conjunction with an impact type of quick-freezing apparatus and an externally-triggerable stimulus generator, allows the application of an electrical stimulus to a muscle preparation at a selected time interval before quick-freezing and the measurement of the interval with submillisecond precision. This is needed for stopping fast physiological events in calcium release and excitation-contraction coupling and allows studying the morphological parameters (by freeze-fracture and freeze-substitution) and elemental distributions (by x-ray microanalysis) as a function of time after stimulation. The device should be adaptable for use with most equipment designed for quick-freezing electrically excitable tissue by impact on a cold solid surface.

Animals↗
PubMed1988

[Methods of quick-freezing and cryosubstitution of biological tissues in analytical ion microscopy].

The localization and relative concentration of diffusible ions (Li+, K+) or bound but easily displaceable ions (Ca2+) have been studied in myocardium prepared by rapid-freeze fixation and compared with results from chemically prepared samples. Contrary to chemical fixation which gives diffused potassium imaging, cryofixation enables elemental precise mapping in cellular compartment. Bound displazable external calcium, extracted by chemical fixation, is visualized after rapid-freeze fixation. Its localization is distinguished by a bright cellular outline, which corresponds to the glycocalix. Intracellular calcium is less emissive and has the same distribution and concentration whatever the sample preparation. Very movable and present at low concentration in tissues, lithium is localized after rapid-freeze fixation. Those qualitative and quantitative results show the adequation of rapid freeze fixation and freeze substitution to the study of diffusible ions by means of analytical ion microscopy.

Animals↗
PubMed2000

Ultrastructure of the echinoderm cuticle after fast-freezing/freeze substitution and conventional chemical fixations.

The cuticles of the pedicellaria primordia in the sea urchin Paracentrotus lividus and of the tube foot disk in the sea star Asterias rubens were preserved by different methods, viz., glutaraldehyde fixation followed by osmium tetroxide postfixation, glutaraldehyde-ruthenium red fixation followed by osmium tetroxide-ruthenium red postfixation, and two fast freezing / freeze substitution methods (FF/FS). The gross ultrastructure of the cuticle as well as the influence of the preservation method on this ultrastructure were identical for the two tissues studied. The cuticle ultrastructure was poorly preserved after glutaraldehyde fixation / osmium tetroxide postfixation. Its preservation was improved after ruthenium red was added in the fixative and postfixative, but the best preservation was consistently achieved using FF/FS. Both low-pressure freezing (plunge freezing) and high-pressure freezing were tested, the latter giving seemingly better results. With these methods, the cuticle appeared to be composed of a proximal lower cuticle, an intermediate upper cuticle, and a distal fuzzy coat. In particular, cryoimmobilization methods emphasized or revealed the occurrence of a well-developed fibrillar lower cuticle in the pedicellaria, the complexity of the upper cuticle which consisted of several zones, and the importance of the usually poorly preserved fuzzy coat that is actually the thickest layer of the cuticle. These observations bring new insights on the functions of the cuticle, and particularly of the fuzzy coat. According to its preservation characteristics, the fuzzy coat presumably consists mostly of proteoglycans. This composition could give it shock absorption and antifouling properties. Furthermore, its important thickness also implies that molecules detected by the short sensory cilia must diffuse through and could be selected by the fuzzy coat.

Animals↗