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PubMed1982

[Critical role of rate previous freezing on the liberation of heat of fusion the preservation of hematopoietic cell lines].

71 bags of bone marrow cryopreserved and stored for periods up to 2 years, were thawed and CFUc recoveries were studied in relation to the rate of freezing following the heat of fusion. Excellent CFUc recoveries (greater than 50%) were obtained in all cases (11/11) for freezing rates smaller than 5 dgC/mn, whereas freezing rates greater than 10 dgC/mn produced poor recoveries in 78% of the cases (4/18). Intermediate freezing rates (5 less than 0 less than 10) resulted in intermediate yields of CFUc, with good recoveries in only 45% of the cases (19/42). These results indicate that CFUc recoveries and subsequently the kinetics of autologous engraftment following the infusion of cryopreserved marrow, which are already known to be related both to the freezing rate before the heat of fusion and the duration of the phase transition period, are also related to this new parameter, which should be carefully monitored during the freezing procedure.

Bone Marrow↗
PubMed1983

[Cryogenic preservation of erythrocytes by a droplet freezing technic].

The droplet freezing technique has two major advantages: firstly the good recovery of frozen erythrocytes with low concentration of cryoprotectants, and secondly the simplification of the post-thaw treatment. However, this technique has not been utilized for blood preservation for transfusion due to difficulties of the sterile freeze-thawing operation. In this study, the technique was modified to perform the freeze-thawing in a closed system under sterile conditions. An apparatus with a new type of nozzle was devised for this purpose. Fifty milliliters of packed red blood cells mixed with an equal volume of additive solutions were frozen in liquid nitrogen in droplet form (0.7 mm in diameter). The frozen droplets were collected into an aluminium container (18 X 18 X 0.5 cm) and thawed by immersing the container into a water bath at 47 degrees C. After thawing, the red cells were washed with either normal saline or hypertonic sugar solutions and resuspended in normal saline. Although the washing procedure involving centrifugation was necessary at least once to remove free hemoglobin and fragile cells produced during freeze-thawing, the post-thaw treatment of the modified droplet freezing was much more simple than that of ordinary bulk freezing technique. Approximately 85% of the frozen cells were recovered with cryoprotective additive solutions consisting of 0.4-0.5 M sugar (maltose or glucose) and 0.4-0.5M glycerol in 0.03 M sodium chloride. The osmotic fragility and ATP level of the post-thawed red cells were little changed in comparison with that of unfrozen cells. These results suggests a possibility of this technique for practical application in the future.

Blood Preservation↗
PubMed1980

The use of freeze-dried blood in the membrane feeding of tsetse flies (Glossina p. palpalis, Diptera: Glossinidae).

Rearing of tsetse flies using membrane feeding became a method of routine during recent years. HOwever, the use of blood does require donor animals. Now, for the first time, a technique has been developed using freeze-dried blood as nutrient for Glossina p. palpalis. This report contains an account of the experiments carried out in obtaining a freeze-dried blood product. Initially, fresh bovine blood was processed prior to freeze-drying. It was later reconstituted with distilled water and the osmolarity adjusted to that of fresh blood. ATP was necessary for optimal blood uptake and the addition of albumin increased the weights of puparia. A colony of flies was reared for more than two years by feeding the flies with this reconstituted blood formulation. Later, in a modified freeze-drying programme the processing of the blood prior to drying was eliminated. The addition of ATP was still required, but the quality of the product was not improved anymore by enrichment with albumin, plasma or serum. In comparisons between freeze-dried blood, fresh blood and in vivo feeding of tsetse flies, no differences were found, except the puparial weights were considerably higher in the colony fed on freeze-dried blood than those of the fresh blood colony. This membrane feeding technique eliminates the dependence on blood donor animals for rearing of tsetse flies and can be recommended for breeding colonies in Africa.

Adenosine Triphosphate↗
PubMed1981

The effects of cooling, freezing and thawing on vascular permeability and perfusion in experimental liver metastases.

The effects of cooling and freezing temperatures on vascular perfusion in solitary Walker carcinosarcomas implanted in the liver were studied in Sprague-Dawley rats. With cooling to above freezing temperatures, perfusion with Microfil decreased significantly in both the encircling tumor plexus as well as in the internal tumor circulation. Freezing and thawing produced acute, significant increases of arterial perfusion in encircling and internal tumor circulations. Tumor vascular permeability, as measured by an Evans blue extraction method, increased tremendously as a result of freezing and thawing. Significant rises in permeability were observed at five minutes to 24 hours, with peak activity at six hours. The tumor circulation appeared to respond to the insult of freezing and thawing in a manner similar to that of normal blood vessels, whereas results of previous studies have shown different reactions to chemical vasoactive agents. The acute increases of perfusion and permeability in the tumors as a result of freezing and thawing could be useful as a means of improving the concentration of antitumor agents.

Animals↗
PubMedDate not supplied

The effect of hetastarch on the stability of L-asparaginase during freeze-thaw cycling.

L-asparaginase, a therapeutic agent for the treatment of acute lymphoblastic leukemia, was evaluated for its susceptibility to cold denaturation. It was found that the enzyme derived from Erwinia chrysanthemi loses its activity when exposed to freeze-thaw cycling. When it was frozen at -40 degrees C and thawed, the enzyme lost 67.3% of its activity; whereas, when frozen in liquid nitrogen (-190 degrees C), it lost almost all of its activity. Rheological studies of hetastarch showed that its viscosity dramatically increases with decreasing temperature, suggesting that at sub-zero temperatures it will create a highly viscous environment around the enzyme. It is proposed that this highly viscous environment retards the rate of conformational changes leading to losses in activity. Hetastarch solutions of various concentrations and degrees of hydroxyethylation were evaluated for their protective ability against the freeze-thaw denaturation of L-asparaginase. It was found that the cryoprotective effect of hetastarch with 0.8 degree of substitution at a concentration of 0.2% was sustained over many freeze-thaw cycles while that of the lesser substituted starch was not. The cryoprotective effect of hetastarch was compared to that of other commonly used additives such as glucose and lactose, which failed to protect the enzyme from freeze-thaw denaturation. In addition, the protective effect of a monomer of hetastarch was evaluated in order to distinguish whether the protective effect of hetastarch was due to physicochemical interactions with the individual monomer units or to its polymeric nature. The monomer showed significant cryoprotection through the first freeze-thaw cycle which was not sustained over additional freeze-thaw cycles.(ABSTRACT TRUNCATED AT 250 WORDS)

Asparaginase↗
PubMed1995

Effect of freezing on the relative escape force of sperm as measured by a laser optical trap.

OBJECTIVE: To determine the possible effect of freezing on sperm escape force as measured by laser trap. DESIGN: Controlled clinical study. SETTING: Normal volunteers, academic setting. PATIENTS: Normal, healthy volunteers. INTERVENTION: Sperm selection, freezing and thawing. Measurement of relative (sperm) escape force before and after freezing. MAIN OUTCOME MEASURE: Comparison of escape force as a measurement of freezing effects. RESULTS: Wide individual variations noted, with some individuals showing significant increases or decreases. The relative escape force in fresh (76.1 +/- 31.1 mW) and in frozen-thawed samples (75.6 +/- 40.0 mW) were similar. CONCLUSION: Freezing does not affect the relative escape force of normal sperm. Any possible detrimental effect of freezing on sperm may be related to other sperm functions.

Freezing↗
PubMed1976

Historical recollections of freeze-drying.

The history of freeze-drying is surprisingly recent. Although Altmann used freeze-drying for the preparation of histological sections as early as 1890, his technique went unnoticed for over 40 years. Shackell independently rediscovered the technique in 1909 for the preservation of biologicals. The industrial applications of freeze-drying do not appear to have been appreciated prior to the patients of Tival in 1927 and Elser in 1934, rapidly followed by the important contributions of Flosdorf in the United States and Greaves in England who were largely responsible for making large scale applications of freeze-drying possible. Stimulated particularly by a series of symposia in England and the United States and the renowned courses on freeze-drying organized by Rey in France, the atmosphere in the 1950s and early 60s was one of optimism for the future of freeze-drying, particularly in its application to food stuffs. Many of the dreams of that time remain unfulfilled, largely because of the higher costs of quality processing. Nevertheless, although the optimism may have somewhat dimmed, the promise remains, and economic changes in the future may well stimulate another surge in development.

Freeze Drying↗
PubMed1993

[LDH, SDH assay for the effect of freezing on MEC-1, Ca9-22, Tca8113 cell lines in vitro].

Three human oral cancer cell lines were tested with the purpose of investigating the effect of freezing temperature and freeze-thaw cycle on the cells by LDH-release, SDH assay. The results showed that freezing can kill the cancer cells. The effective freezing temperature for cryonecrosis of the cells was -40 degrees C-(-)50 degrees C. Two freeze-thaw cycles were potent enough to obtain the most effectual cryonecrosis of the cancer cell lines. Amount of LDH-release was freezing-temperature dependent. At -40 degrees C, more then 90% LDH of cells were released. The results showed that the membranes of 90% survival cells were injured after cryotreat.

Carcinoma, Mucoepidermoid↗
PubMed1997

Effects of multiple freezing-thawing cycles on ultimate indentation load and stiffness of bovine cancellous bone.

OBJECTIVE: To assess the effects of multiple freezing-thawing cycles on ultimate indentation load and stiffness of bovine tibial cancellous bone with regard to freezing conditions (saline solution or air) or methods of thawing (saline solution or air). SAMPLES: 4 tibias from 4 adult cows. PROCEDURE: The proximal portions of the tibias were sectioned to produce 20-mm-thick bone slices. Slices were subjected to 5 freezing-thawing cycles under 4 conditions: freezing with and without saline solution, then thawing in saline solution or exposed to the air. The mechanical properties of the bone slices before and after the treatments were measured, using an indentation test for comparison. Indentation testing was performed before and after freezing-thawing cycles to measure differences in mechanical parameters. RESULTS: Significant differences in mechanical parameters were not found for bone specimens frozen in saline solution and thawed in saline solution; frozen without saline solution and thawed in air; frozen in saline solution and thawed in air; or frozen without saline solution and thawed in saline solution. CONCLUSIONS: Multiple thawing-freezing cycles do not significantly affect the ultimate indentation load and stiffness of bovine tibial cancellous bone.

Animals↗
PubMed1998

Freeze duration influences postfreeze survival in the frog Rana sylvatica.

Survival, cryoprotection, and the time course for recovery of vital functions were studied in autumn-collected juvenile wood frogs (Rana sylvatica) following freezing at -1.5 degrees C for various periods. Frogs readily tolerated freezing for 3 or 9 d, but only 50% survived a 28-d freezing trial. Generally, the postfreeze return of vital functions (vascular circulation, pulmonary breathing, righting reflex, jumping reflex) occurred later in frogs frozen for longer periods. Augmenting endogenous levels of the cryoprotectant glucose (via injections) prior to freezing substantially increased freeze endurance, as these frogs had excellent survival after remaining frozen for as long as 49 d. The improved freeze endurance of glucose-loaded frogs apparently was not associated with a reduction in ice content but rather may reflect the greater availability of energy substrate needed to support metabolism of frozen ischemic tissues.

Animals↗
PubMed1998

[The stability of freeze-drying lysosomal enzymes].

OBJECTIVE: Enzyme assays for the diagnosis of lysosomal storage diseases have been underway in China only in a few laboratories, and the specimens for enzyme assay must not be inactivated by environmental factors. To solve the problem, the stability of freeze-drying lysosomal enzymes was tested. METHODS: Three pools for samples were set up: control leucocytes pool and plasma pool(40 individuals in each pool), control fibroblasts pool( 3 cell lines). 4 groups of different dispositions of samples were designed for the pools:(1)pellets or plasma directly stored at -80 degrees C as routine(non-freeze-drying group or N);(2)after freeze-drying, pellets or plasma immediately stored at -80 degrees C (0 week or 0W); (3) after freeze-drying, pellets or plasma stayed at room temperature for 1 week(simulated mail) then stored at -80 degrees C (1 week or 1W);(4) the same with 1W but 3 weeks at room temperature(3 weeks or 3W). Microassay for enzyme activity was employed: 20 enzymes in leucocytes pool, 14 enzymes in fibroblasts pool and 7 in plasma pool. RESULTS: Enzyme activities among 4 groups in each sample pool were compared. Single factor analysis of variance (F test) showed no significance of differences (P>0.05) among 4 groups in leucocytes and fibroblasts pool, but highly significant differences (P<0.01) among 4 groups in plasma pool. "t test" showed no significance of differences (P> 0.05) between N and 3W in leucocytes and also fibroblasts pool, but very significant differences (P<0.01) between N and 3W in plasma pool. CONCLUSION: The results suggest that freeze-drying samples from leucocytes and fibro- blasts can provide a stable resource for enzyme assay and be simple for transportation of the diagnostic samples. Though freeze-drying is not appropriate for the plasma samples, it is still applicable for wide use since only a few kinds of lysosomal enzyme being indicated to use plasma for enzyme assay.

Clinical Enzyme Tests↗
PubMed1988

Application of spray-freezing to the study of rapid platelet reactions by a quenched-flow approach.

A spray-freezing approach coupled to quenched-flow techniques has been developed to study rapid (0.3-5.0 s) biochemical and morphological changes during platelet activation. Platelet-rich plasma (PRP) pumped through the quenched-flow system was atomized and quenched within 50 ms in liquid propane at -196 degrees C. Analysis of platelet size and morphology by scanning electron microscopy (SEM) indicated that platelets can be sprayed without morphological damage. The spray-frozen platelets can be subjected to freeze-fracture, freeze-substitution, or freeze-drying procedures for later analysis by electron microscopy. Quenched-flow techniques have permitted investigation of rapid increases in cytosolic calcium protein phosphorylation and morphological changes which occur within 0.5 s after exposure to an activator. The combination of quenched-flow and spray-freezing methodologies should prove useful for studying equally rapid changes in protein distribution in activated platelets by means of electron microscopy coupled with immunocytochemical approaches.

Blood Platelets↗
PubMed1981

Patterns in quench-frozen, freeze-dried, blood proteins.

Scanning electron micrographs of whole blood prepared with cryotechnics demonstrated patterns assumed to be artifacts of the freezing process. A SEM investigation of the effects of freezing and freeze-drying on columns of purified blood proteins has been carried out using quench-freezing and cryo-fracturing followed by freeze-drying. Correlative studies with a light microscope and a transmission electron microscope are included. The resulting patterns depended on the type and concentration of protein as well as on the position within the sample and the orientation of the fracture plane relative to the column axis.

Blood Proteins↗
PubMed1988

Alarm call responsivity of mallard ducklings: VII. Auditory experience maintains freezing.

Mallard ducklings (Anas platyrhynchos) freeze upon hearing the maternal alarm call, which the hen issues when there are disturbances (e.g., potential predators) near the nest. Our previous work indicates that ducklings that have been devocalized embryonically and reared in auditory isolation exhibit a significant reduction in the incidence of freezing, as compared to vocal ducklings or to devocal ducklings that have been exposed to duckling sounds throughout the perinatal period up to the time of testing at 12 hr posthatch. The main purpose of this paper is to assess whether there is a prenatal or postnatal sensitive period for such auditory experience. Two groups of 30 devocalized ducklings were either (a) stimulated with duckling sounds prenatally and tested to the alarm call at 12 hr posthatch (Expt. 1), or (b) stimulated from the time of hatching to the time of testing at 12 hr (Expt. 2). These experiments rendered no conclusive evidence of either a prenatal or postnatal sensitive period, but they suggested that auditory experience might be important in maintaining freezing. To test this hypothesis, a third group of 30 ducklings was stimulated from hatching to 12 hr and tested at 24 hr (Expt. 3). Only those ducklings receiving auditory experience contiguous with the time of testing (Expt. 2) showed high levels of freezing. Whenever a gap occurred between the offset of stimulation and the onset of testing (Expts. 1 and 3), the incidence of freezing dropped. Thus, auditory experience is important in maintaining the freezing response. This effect contrasts markedly with traditional conceptions of sensitive periods.

Acoustic Stimulation↗
PubMed2006

Striatal dopa and glucose metabolism in PD patients with freezing of gait.

In Parkinson's disease (PD), freezing suggests sudden and transient blocks of motor behavior during initiating or continuous repetitive movements. Its underlying pathophysiology remains unclear. The objective of this study is to compare striatal dopamine metabolism and cerebral glucose metabolism between PD patients with and without freezing of gait (FOG). A total of 10 PD patients with FOG at off and 7 PD patients without FOG underwent brain positron emission tomography with (18)[F]-6-fluoro-levodopa (FDOPA) and (18)[F]-fluordesoxyglucose (FDG). Striatum decarboxylase activity was expressed by metabolic influx constants of the striatum related to the occipital lobe (Kocc). FDG uptake in caudate and putamen was normalized to global FDG uptake. Region of interest (ROI) analysis of striatal regions was used, as well as voxel-based analysis by statistical parametric mapping (SPM). ROI analysis did not reveal differences in striatal FDOPA and FDG uptake between the groups. SPM showed lower putaminal FDOPA uptake (P = 0.05 uncorrected) with increased FDG uptake (P = 0.01 uncorrected) in freezing PD, whereas caudate uptake of the two tracers was reduced. Freezing-related cortical FDG decrease was found in (right) parietal regions. In conclusion, in freezing PD, caudate uptake of FDG and FDOPA was reduced, whereas putamen FDOPA decrease was associated with FDG increase. Right hemisphere circuitry seemed to be more affected in freezing patients.

Blood Glucose↗
PubMed1993

Primary progressive freezing gait.

Freezing gait is an incapacitating symptom often observed in patients with Parkinson's disease. It has been less frequently described in association with multi-infarct state, multisystem atrophies, and normotensive hydrocephalus. In our movement disorder clinic, we have diagnosed (and followed up to 3 years; median, 16 months), 18 patients in whom progressive freezing gait was the sole neurological dysfunction. These 15 men and 3 women (aged 60-82 years; 74 +/- 6) were subjected to an extensive neurological workup that included clinical evaluation, videotaping for grading of gait disability, comprehensive blood and cerebrospinal fluid (CSF) analysis, and brain computed tomography (CT) and magnetic resonance imaging (MRI). Mean disease duration was 2.5 +/- 1.9 years (range, 0.5-6). Neurological examination disclosed freezing gait, often associated with varying degrees of postural instability. The degree of freezing gait ranged from sudden motor blocks only when confronted with obstacles to severe disability with total inability to start walking requiring a walker, massive assistance, or a wheelchair. However, patients could mimic gait movements with absolutely no freezing when seated or lying prone, and most of them could overcome arrests by the "walking-over-lines" maneuver. Otherwise, neurological examination was normal with no signs of bradykinesia, rigidity, or tremor. Blood chemistry and CSF analysis were normal. Brain CT and MRI were normal or showed mild cortical atrophy in 12 and putative lacunes in 6 patients. Therapy with levodopa or dopamine agonists was ineffective. During the follow-up period, a gradual progression of the freezing gait was observed. However, it remained unaccompanied by any other neurological findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1998

Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium.

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stage in vitro. Specifically, the effects of cooling to subzero temperatures, thawing rate, LN2 plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or -7.0 degreesC in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN2 from -10, -20, or -33 degreesC at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, -0.3 C/min, plunging at -33 degreesC) rapid thawing by direct submersion in 30 degreesC water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.

Animals↗
PubMed1999

Effect of heat shock or cold shock treatment on the resistance of lactococcus lactis to freezing and lyophilization

This study investigated the effect of heat shock or cold shock treatment on the resistance of commercial Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris cheese starter bacteria to freezing and freeze-drying. The ability to withstand freezing at -60 degrees C for 24 h was variable among lactococci, but could be significantly (P < 0.05) improved in most strains by a 25-min heat shock at 42 degrees C (L. lactis ssp. lactis) or 39 degrees C (L. lactis ssp. cremoris) or by a 2-h cold shock at 10 degrees C. In addition, stress treatments that improved lactococcal cryotolerance significantly (P < 0.05) enhanced the resistance of most strains to lyophilization. Greater resistance to freezing or lyophilization was not detected when stress treatments were performed in broth that contained erythromycin, which indicated that stress-induced proteins were required for cell protection. Membrane fatty acid analysis of stress-treated cells suggested that enhanced resistance to freezing and lyophilization may be related to changes in cell membrane lipid composition. Heat-shocked cells had a higher 19:0 cyclopropane fatty acid content than control cells, and cold-shocked cells contained a lower ratio of saturated to unsaturated fatty acids. Other factors must also be involved in cell protection, however, because similar changes in membrane lipid composition were detected in strains whose resistance to freezing and lyophilization was not improved by heat or cold shock treatment. Copyright 1999 Academic Press.

Journal Article↗