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At least 19 recordsLinked to original sources
PubMed1978

[The influence of cryoprotective solutions on the ultrastructure of rabbit corneal endothelium (author's transl)].

A series of rabbit corneae was preserved at -196 degrees C in liquid nitrogen using five different cryoprotective solutions: (1) 5% dextran T 40, (2) 10% polyvinylpyrrolidone, (3) 5% dextran T 40 + 10% polyvinylpyrrolidone + dimethyl sulfoxide, (4) 25% rabbit albumin + 10% polyvinylpyrrolidone, (5) 25% rabbit albumin + dimethyl sulfoxide. After thawing the corneae, the influence of the different cryoprotective solutions on the ultrastructure of corneal endothelium was evaluated and compared with fresh corneae. The results were as follows: (1) Using 5% dextran T 40 or 10% polyvinylpyrrolidone or 25% rabbit albumin as cryoprotective solutions marked intracellular damage. (2) Adding dimethyl sulfoxide to the solutions cryopreservation appeared to be more successful at the cellular basis, despite the freezing and thawing procedure mediated moderate intracellular changes. Cryopreservation including dimethyl sulfoxide renders better conditions for successful corneal grafting in the light of ultrastructural aspects.

Albumins↗
PubMed2018

Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants.

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

Cell Survival↗
PubMed1977

Polymeric cryoprotectants in the preservation of biological ultrastructure. II. Physiological effects.

A study has been made of the physiological effects of three non-penetrating polymeric cryoprotective agents on sixteen different plant and animal cells and tissues. The cryoprotectants, when used at concentrations at which they are effective in preventing ice-crystal formation, generally have a lower toxicity to cells and tissue than similar concentrations of glycerol. The relatively low toxicity of these substances suggests that they would be more suitable as cryoprotectants for morphological and analytical studies than the commonly used low molecular weight compounds.

Cell Survival↗
PubMedDate not supplied

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

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

Blood Cell Count↗
PubMed1976

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

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

Animals↗
PubMed1976

The influence of different freezing procedures and different cryoprotective agents on the immunological capacity of frozen-stored lymphocytes.

The influence of a series of factors on the frozen storage of lymphocytes was investigated. The cells were frozen using different freezing programmes, using the cryoprotectants dimethyl sulphoxide and glycerol in different concentrations in the freezing medium, and with variations in the period of exposure of cells to cryoprotectants before freezing and after thawing. Cell viability was evaluated by quantitative measurements of the cell-mediated immune response after stimulation with phytohemagglutinin, pokeweed mitogen, concanavalin A, and allogenic cells in mixed lymphocyte cultures. The factors investigated were found to have an important effect on the immune response, so that careful investigation and exact specification of the freezing system are necessary before frozen cells are used in blast-transformation tests. The best freezing programme had a duration of approximately 40 min with a smooth progression through the temperature range where phase transition takes place. The optimum dimethyl sulphoxide concentration in this programme was 8--10%. Dimethyl sulphoxide had no toxic effect on the cells, and no equilibration period was necessary prior to freezing. An equilibration period of 15 min with 10% glycerol was even better than the optimum programme with dimethyl sulphoxide.

Blood Preservation↗
PubMed1978

Plasma clearance and renal excretion of erythrocyte cryoprotectant hydroxyethylated amylopectin.

The value of low molecular weight-hydroxyethylated amylopectin (cryo-HES) as an extracellular cryoprotectant has been demonstrated in vitro. It is important that details of the intravascular persistence and urinary excretion be determined to compare with data already available as other grades of HES and with data on transfusion of cryo-HES cryoprotected blood. Following a single 400 ml (14% solution) infusion in man, the intravascular clearance of cryo-HES was well described mathematically by the equation: y = 3.30+6.49e-0.15kappa. The plasma concentration of cryo-HES fell to half its peak value in approximately 9.6 h. Approximately 20% of the total infused cryo-HES was excreted in the urine during the first post-injection hour, and 50% by 72 h. The ESR was not altered significantly by the presence of this material. The present study indicates that cryo-HES is eliminated rapidly and may thus be safe for transfusion to recipients of frozen blood.

Adult↗
PubMed1977

Polymeric cryoprotectants in the preservation of biological ultrastructure. III. Morphological aspects.

Two high molecular weight polymers, polyvinylpyrrolidone (PVP) and hydroxyethyl starch (HES), have been used as cryoprotectants for preparing specimens to be freeze fractured. Solutions of 25% (w/w) suppress the formation of intracellular ice in single cells and tissue blocks from both plants and animals to the extent that fine structural details of the cell can be elucidates. The mode of action of these cryoprotectants, together with the structures they reveal and the peculiar advantages attached to their use, is discussed.

Cryoprotective Agents↗
PubMed1976

Freeze-fracture replication of organized tissue without cryoprotection.

Fresh pieces of rat liver and pancreas were rapidly frozen without prior chemical fixation or cryoprotection, and replicated folloing freeze-fracture. Replicas revealed small peripheral areas free of ice crystals or damage and, within such areas, general ultrastructural morphology was essentially similar to that seen in conventionally processed material. On fracture faces of plasma and nuclear membranes a population of less prominent particles in addition to conventional membrane-associated particles was seen, and smooth areas devoid of particles of any type were seen on some nuclear membranes. These smooth areas did not appear to be similar to smooth areas allegedly arising as artifacts of conventional processing. Tight junctions and gap junctions appeared as they do in cryoprotected specimens. The results provide a base-line for assessing the possible effects of processing steps or agents on the ultrastructure of organized tissues as revealed in freeze-fracture replicas.

Animals↗
PubMed1978

[Cryoprotective ability of glycerin and DMSO in the deep freezing of dog sperm].

The sperm-bearing fractions of 163 ejaculates collected from 6 Beagles aged two years were used to test the cryoprotective effect of DMSO and glycerol in deep freezing investigations. It could be shown that the cryoprotective capacity of glycerol was better than the one of DMSO. Glycerol should rather be added to the sperms at + 5 degrees C than at + 25 degrees C. The best preservation results were achieved by a combined addition of DMSO and glycerol under the following conditions: time of cooling down to +5 degrees C of the extended sperm fractions 50 minutes, final concentration of DMSO 1% (max.), final concentration of glycerol 8% (max.), glycerol equilibration time 30 minutes.

Animals↗
PubMedDate not supplied

[Effect of cryoprotectants on protein-synthesizing activity of cell-free system from rat liver].

The protein-synthetizing activity in postmitochondrial supernatant (S15) was studied as affected by ethylene glycol, PEG-400, PEG-4000, DMSO, ethanol, methanol, and glycerol. It is found that cryoprotectants inhibit 14C-leucine incorporation in the presence of physiological concentrations of Mg2+ (5 mM) in the cell-free system. All cryoprotectants, exept of glycerol, shift the maximum of 14C-leucine incorporation towards lower Mg2+ concentrations in the cell-free system.

Alcohols↗
PubMedDate not supplied

[Low temperature and cryoprotectant effect on oxygen uptake in rat kidney cortex homogenates].

Respiration and oxidative phosphorylation of the rat kidney cortex mitochondria are studied as affected by low temperatures and cryoprotectants. There is no change in respiration in state 2 during freezing and thawing of the rat kidney cortex slices both in case of using the succinate oxidation substrate and alpha-ketoglutarate. Respiration in state 3 is inhibited in the medium with alpha-ketoglutarate during freezing under protection of polyethylene oxide-100 (PEO-100) and glycerol, and does not significantly change in case of using dimethyl sulphoxide (DMSO). The effectiveness of cryoprotectants used in the experiments reduces in the following sequence: DMSO--glycerol--PEO-100. Preservation of the main mitochondria function, ATP synthesis, is shown to be possible for the kidney cortex slices exposed to freezing and thawing.

Adenosine Triphosphate↗
PubMed1977

Polymer cryoprotectants in the preservation of biological ultrastructure. I. Low temperature states of aqueous solutions of hydrophilic polymers.

The solid states formed by vitrified and frozen aqueous solutions of some hydrophilic polymers, able to act as biological cryoprotectants, have been studied by differential scanning calorimetry and freeze fracture electron microscopy. Glass transitions, devitrification, recrystallization and melting behaviour of aqueous solutions of polyvinylpyrrolidone, hydroxyethyl starch and dextran have been established. The vitrified polymer solutions exhibit a characteristic microspheral morphology which is not induced by the quench cooling process but is an inherent feature of the solutions themselves.

Cryoprotective Agents↗
PubMed1976

A copper block method for freezing non-cryoprotected tissue to produce ice-crystal-free regions for electron microscopy. I. Evaluation using freeze-substitution.

A wide variety of plant and animal tissues were prepared for electron microscopy by freeze-substitution, after rapid freezing on a liquid nitrogen cooled copper block by the van Harreveld method. Measurements were made of ice crystal size versus depth in tissues that had not been treated with any cryoprotectants. Ice crystal size increased exponentially with depth. It was confirmed that a narrow surface band increased exponentially with depth. It was confirmed that a narrow surface band (approximately 12 mum) was frozen sufficiently rapidly to prevent the formation of electron microscopically-visible ice crystals.

Animals↗
PubMed1976

Observations on the production of frozen-dried thin sections for electron microscopy using unfixed fresh liver, fast-frozen without cryoprotectants.

Thin sections of unfixed liver, fast-frozen without cryoprotectants, have been cut using conditions under which momentary thawing of the sections is unlikely to occur. A transfer stage which facilitates this procedure is described. Sections show hole damage probably due to ice-crystal formation during the freezing process and have well defined edges, but despite hole damage, some morphological features of the cell are discernible. Presumptive mitochondria appear smaller in frozen sections than in conventional Araldite sections. Sections devoid of hole damage have indistinct edges and are presumed to have undergone transient thawing. Carbon coating of freeze-dried sections to exclude atmospheric moisture during transference of sections to the electron microscope (EM) appear unnecessary as regards preservation of morphological structure. The results are discussed in relation to the limitations of the method and the potential value of the technique.

Animals↗