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PubMed2002

Extensive early apoptosis in frozen-thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation.

Stem cell doses necessary for engraftment after myelo-ablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34+ cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed. After freeze-thawing large numbers of non-viable, early apoptotic cells appeared, leading to only 42% viability compared to 72% using 7-AAD only. Based on this Syto16 staining in the frozen-thawed grafts, threshold numbers for adequate haematological recovery of 2.8-3.0 x 10(6) CD34+ cells/kg body weight determined for fresh grafts, now decreased to 1.2-1.3 x 10(6) CD34+ cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low doses of CD34+ cells were sufficient for recovery (0.3-0.4 x 10(6)CD34+ cells/kg). In contrast to freeze-thawing of leucapheresis material, a high viability of CD34+ cells was preserved during storage for 3 days at 4 degrees C, leaving threshold doses for recovery unchanged. In conclusion, the Syto16 assay reveals the presence of many more non-functional stem cells in frozen-thawed transplants than presumed thus far. This led to a factor 2.3-fold adjustment downward of viable CD34+ threshold doses for haematological recovery.

Antigens, CD34↗
PubMed2005

Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer.

We have compared two methods for salvaging epididymal sperm from post-mortem samples from Iberian red deer. Of each pair of testicles (29 samples), one cauda epididymis was processed by means of cuts (sperm was immediately diluted with extender) and the other was detached from the corpus and flushed from the vas deferens with 1 mL of extender. Sperm was processed for cryopreservation, and analyzed just after recovery, pre-freezing and post-thawing. Total spermatozoa recovered, contamination (concentration of epididymal cells and red blood cells (RBCs)) and quality (motility by CASA, and acrosomal status, viability and mitochondrial status by flow cytometry) were used to compare both methods. The number of recovered spermatozoa was similar for both methods. Contamination was higher for the cuts method, but when considering the final dilution before freezing, only RBCs concentration was significantly higher. Motility was similar just after extraction, but higher for both pre-frozen and post-thawed flushed sperm. Pre-freezing acrosomal status (P < 0.05) and viability (P < 0.1) were better for flushing; however post-thawing results were similar for the two methods. A clustering analysis using CASA data showed that the subpopulation pattern of motile sperm was different depending on the method, being better for flushing. With regard to yield, lower contamination (especially RBCs) and, in general, better quality results, flushing seems to be a more recommendable method for post-mortem sperm recovery. The cuts method may be more practical on certain occasions, but care must be taken in order to achieve rapid extension of the sample and to avoid contamination in order to improve sample condition.

Acrosome↗
PubMed2006

Morphometric changes in goat sperm heads induced by cryopreservation.

Two experiments were designed to evaluate the effect of cryopreservation on morphometric characteristics of the goat sperm head. To address this question, we evaluated the size of the sperm head in fresh control cells, post-cooling cells after equilibration with the glycerol preservation solution, and post-thawing cells. Assessment was by automated morphometric sperm head analysis (ASMA) using phase-contrast microscopy without staining. In the first experiment, ASMA was performed on heterospermic pooled samples (fresh, post-cooling after equilibration with the glycerol preservation solution and post-thawing): length, width, area and perimeter were measured. In the second experiment, sperm viability was assessed by Hoechst staining and head morphometry was carried out as before, simultaneously during the cryopreservation process, and the head size was identified for both live and dead spermatozoa. The data were analysed by principal component analysis (PCA). The purpose of PCA is to derive a small number of linear combinations (principal components) from a set of variables (length, width, area and perimeter), that retain as much of the information in the original variables as possible. The main findings that have emerged from this study are that (i) a simple procedure has been developed for measuring spermatozoa heads without staining, which minimises the possibility that sperm head dimensions were influenced by procedural artefacts; (ii) the dimensions of goat sperm heads after cryopreservation in skimmed milk-glucose medium were smaller than in fresh sperm, but this was due to the equilibration phase with the cryoprotectant and not to the cryopreservation process itself; and (iii) dead spermatozoa showed smaller heads than live sperm, consequent upon the loss of membrane function. No differences were observed between post-cooling cells after equilibration with the glycerol preservation solution and post-thawing spermatozoa and only minor osmotic differences between them and fresh sperm were observed.

Animals↗
PubMed2004

Utility of bag segment and cryovial samples for quality control and confirmatory HLA typing in umbilical cord blood banking.

Many cord blood (CB) banks have been established worldwide as a response to the increasing number of CB transplantations. In this study, we describe a quality control program in which the utility of an integral bag segment and cryovial containing aliquots of cryopreserved product as haematopoietic content control and HLA typing confirmation for CB units has been evaluated. For this purpose, every month one stored CB unit and its satellite cryovials were thawed and washed. Nucleated cell counts, viability and clonogenic assays were performed from the bag and cryovial before washing. After washing, total nucleated cell, CD34+ counts, viability, and clonogenic assays were performed from the bag. In order to assure the ability of bag segments to confirm hematopoietic potential of CB units, clonogenic assays and viability were performed from attached segments of 10 CB units and the results were compared with those from bags and cryovials. When comparing all variables between thawed bag and cryovial samples, they showed similar results. Mean colony-forming unit (CFU) content of segment samples was 118.8 +/- 93.72 x 10(4) that resulted similar to bags and cryovials haematopoietic content. In conclusion, the quality control system described in this paper demonstrates that CB units are processed preserving the quantity and quality of the progenitor cells. The contiguous segment haematopoietic content is representative of the final product.

Blood Banks↗
PubMed1986

The cryoprotective effects of dimethyl sulfoxide on human bone marrow as studied by 31P nuclear magnetic resonance spectroscopy.

31P nuclear magnetic resonance spectroscopy has been used to study freshly aspirated normal human bone marrow samples. The pH within the intact cells of the samples was determined from the chemical-shift position of the resonance for inorganic phosphate within the cell; the intracellular pH was found to be 7.35 for fresh bone marrow. The chemical-shift positions of the alpha and beta phosphate resonances of adenosine 5'-triphosphate were used to assess the fraction of this metabolite complexed with Mg2+. It was found that 84% of the total intracellular adenosine 5'-triphosphate was in the Mg2+-complexed form. The concentration of Mg2+ uncomplexed to any ligand was 0.4 mM. The areas of the resonances for the major phosphorus-containing metabolites were used to determine intracellular concentrations. For fresh human bone marrow, the intracellular concentrations determined were phosphate monoesters less than 0.3 mM, 2,3-diphosphoglycerate 3.9 +/- 1.0 mM, inorganic phosphate 1.2 +/- 0.6 mM, phosphodiesters 2.8 +/- 1.0 mM, adenosine 5'-triphosphate 1.6 +/- 0.4 mM, adenosine 5-diphosphate less than 0.2 mM, and nicotinamide adenine dinucleotide less than 0.2 mM. These metabolite concentrations within the intact cell samples did not change over 2.0 h and changed only gradually over a 24-h period. 31P nuclear magnetic resonance spectroscopy was then used to study the cryopreservation of normal human bone marrow in the presence of increasing concentrations of the penetrating cryopreservative dimethyl sulfoxide. Dimethyl sulfoxide alone without freezing was found to cause some gradual hydrolysis of 2,3-diphosphoglycerate, presumably by stimulating diphosphoglycerate phosphatase. The effect of freezing human bone marrow to liquid nitrogen temperatures, storage, and rapid thawing was a dramatic fall in intracellular adenosine 5'-triphosphate levels. The half-life of the metabolite, after thawing, was about 0.3 h. If the bone marrow was frozen in the presence of 2.5, 5.0, and 7.5% dimethyl sulfoxide, the post-thaw half-life was prolonged to 0.4, 0.5, and 0.6 h, respectively. 15% dimethyl sulfoxide afforded complete cryoprotection, with adenosine 5'-triphosphate levels constant for 15 h after thawing the human bone marrow.

2,3-Diphosphoglycerate↗
PubMed1998

Vascular rejection post heart transplantation is associated with positive flow cytometric cross-matching.

OBJECTIVE: Use of flow cytometry cross-matching for measurement of donor-specific alloreactivity and monitoring anti-donor antibodies is well established. This study was performed to determine (1) its accuracy as a marker of vascular rejection, (2) its correlation with post-transplant outcome and (3) its ability to monitor highly sensitized patients requiring antibody removal with plasma exchange. METHODS: Serial serum samples from 99 heart transplant recipients were examined for the presence of anti-donor antibodies of the IgG class that were reactive with T and/or B cryopreserved donor lymphocytes. A sub-group of 20 HLA sensitized patients required plasma exchange to remove the anti-HLA antibodies and were monitored with flow cytometry cross-matching to assess the degree of antibody removal. RESULTS: Positive T-cell reactions were observed in 26 patients and positive B-cell reactions in 54. Twenty patients had vascular rejection. A significantly larger number of patients with a positive flow cytometry cross-match had vascular rejection (42% versus 12% for T-cell reactions, and 32% versus 7% for B-cell reactions; P = 0.002 each). Of the patients who had vascular rejection, 11 had a positive T-cell reaction (flow cytometry cross-match sensitivity of 55%), and 17 had a positive B-cell reaction (sensitivity of 85%). Of the 79 patients who did not develop vascular rejection, 64 had a negative T-cell reaction (specificity of 81%), and 42 had a negative B-cell reaction (specificity of 53%). The actuarial 2-year survival estimates were significantly higher in patients with negative T-cell reactions (90% versus 75%; P = 0.04), and B-cell reactions (95% versus 78%; P = 0.02). In the highly sensitized subgroup (n = 20) the effectiveness of plasma exchange to decrease anti-HLA antibody reactivity was a strong predictor of outcome. For patients in whom plasma exchange (PE) reduced anti-donor reactivity, 1-year survival was 87% compared to 25% in those whom PE did not reduce the level of antibody binding as assessed with flow cytometry cross-matching (P < 0.0001). CONCLUSIONS: Flow cytometry cross-matching provides a valuable marker for the detection of vascular rejection after cardiac transplantation. Quantitative measurements may allow evaluation of the efficacy of treatment modalities employed in the management of vascular rejection in an attempt to improve outcome.

Actuarial Analysis↗
PubMed2000

Paper-based archiving of mammalian and plant samples for RNA analysis.

The ability to archive biological samples for subsequent nucleic acid analysis is essential for tissue specimens and forensic samples. FTA Card is a chemically treated filter paper designed for the collection and room temperature storage of biological samples for subsequent DNA analysis. Its usefulness for the preservation of biological samples for subsequent RNA analysis was tested. Here, we demonstrate that RNA in biological samples stored on FTA Cards is stable and can be used successfully for RT-PCR and northern blot analysis. RNA stability depends on the storage temperature and the type of biological specimen. RNA in mammalian cells stored on FTA Cards is stable for over one year at temperatures at or below -20 degrees C and for two to three months in samples stored at room temperature. For plant leaf, longer storage times (> 5 days) require temperatures at or below -70 degrees C following sample application. FTA Cards may constitute a method not only for convenient collection and storage of biological samples but also for rapid RT-PCR analysis of tissue and cell samples.

Animals↗
PubMed1988

Avoidance of contamination in element analysis of serum samples.

The selection of sampling needles and plastic material used for the storage of serum samples were studied. Sampling needles and plastic material were analyzed by neutron activation analysis. Leaching of elements from containers by acids and EDTA-solution was also followed. Also the long-term storage of serum samples for element analysis was investigated. Zn, Cu and Se concentration of serum was followed over the course of four years. The selection of sampling needles, as well as all plasticware which comes into contact with the serum, should be based on the knowledge of possible removal of elements from materials into the sample. Storage of serum samples at -18 degrees C does not change the copper, zinc or selenium concentration over the course of 4 years despite repeated melting and refreezing of the samples.

Blood Chemical Analysis↗
PubMed1993

Progress in scanning electron microscopy of frozen-hydrated biological specimens.

Modern scanning electron microscopy yields structural information down to 2 to 5 nm from thin, beam transparent biological specimens. This paper examines the possibilities of garnering this level of structural information from bulk, frozen-hydrated samples. Freeze-fractured, frozen-hydrated yeast cells, frequently taken as a yardstick to monitor progress in low-temperature scanning electron microscopy, have been used to optimize both metal shadowing methods and observation parameters (e.g. accelerating voltage, electron beam irradiation of the specimen). Uncoated frozen-hydrated yeast cells do not change electrically at an accelerating voltage of 30 kV. Increasing charging effects are however observed with decreasing accelerating voltages. Very thin metal films are therefore used for specimen coating to localize and enhance the specific secondary electron signal. Planar-magnetron sputtering of a 1 nm metal layer provides high resolution secondary electron images, at 30 kV, of freeze-fractured, frozen-hydrated yeast cells in an in-lens field-emission scanning electron microscope. Structural information comparable to that of transmission electron microscopy of freeze-fractures is attained. Planar-magnetron sputtering of either chromium, tungsten or platinum results in essentially the same information density (smallest visible significant structural detail). Frozen-hydrated samples are very beam sensitive and have to be observed under minimum dose conditions.

Cryopreservation↗
PubMedDate not supplied

Cryopreservation of kiwi shoot tips.

In this study, the effect of various factors, including the physiological state of mother-plants and parameters of the encapsulation-dehydration protocol (cryoprotective treatment, freezing protocol) on the regrowth of shoot tips of kiwi in vitro plantlets was studied. The optimal protocol established was the following: shoot tips were sampled on mother-plants after a one month cold-acclimation period at 5 degrees C. For preculture, which was performed at 5 degrees C, encapsulated shoot tips were transferred at 24-hour intervals on solid media with increasing sucrose concentrations, from 0.5 to 1.0M. Beads were then desiccated to 26% moisture content (fresh weight basis), prefrozen from 0 degrees C to -40 degrees C at 0.2 degrees C/min and immersed rapidly in liquid nitrogen. No differences were noted between in vitro plantlets produced from cryopreserved and control shoot tips as regards leaf color, average height, multiplication rate and peroxidase zymogram pattern. Apices of 3 kiwi accessions were frozen using the above protocol with regrowth percentages ranging between 22 and 56%.

Actinidia↗
PubMedDate not supplied

Continuous assessment of human spermatozoa viability during cryopreservation.

Cryomicroscopy has enabled direct observation of freezing and thawing of human spermatozoa. When used with a fluorescent viability kit, sperm membrane damage was not apparent down to temperatures of -5 degrees C, but significant damage occurred after thawing (55% of spermatozoa had damaged membranes). Semen samples were cooled or frozen to temperatures (at decrements of 10 degrees C) from 0 degree C to -110 degrees C. At all these temperatures the proportion of live to membrane-damaged cells remained constant. Samples held at temperatures above -30 degrees C were not adversely affected. Below -30 degrees C there was a gradual increase in the proportion of membrane-damaged cells on thaw and a decrease in the number of live cells recovering motility. At temperatures between -50 degrees C and -60 degrees C there was an equal proportion of live motile, immotile, and membrane-damaged cells. It is concluded that some irreversible damage to spermatozoa was a result of freezing processes in cells frozen to -30 degrees C or less, but most of the cryodamage was incurred during thawing, possibly due to recrystallization.

Cell Survival↗
PubMed1994

Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing.

The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoal motility significantly better throughout storage at 5 degrees C. Addition of 5 or 25% seminal plasma to perfused epididymal spermatozoa (0% seminal plasma) resulted in a significant stimulation of spermatozoal motility by 25% seminal plasma at 0 h (P<0.05) and to a lesser extent at 24 and 48 h. Post-thaw motility of ejaculated as well as epididymal spermatozoa was not influenced by slow cooling to 15 degrees or 5 degrees C with or without glycerol prior to rapid freezing in liquid nitrogen vapor. During cooled storage, seminal plasma had a stimulatory effect on epididymal spermatozoa and depressed motility in ejaculated spermatozoa. Results on cryopreservation indicate that freezability of equine spermatozoa is already determined when spermatozoa leave the tail of the epididymis.

Journal Article↗
PubMed2006

Effects of freezing and cryopreservation on the mechanical properties of arteries.

Cryoplasty, a freezing therapy, is being used for the treatment of restenosis in peripheral arteries. In addition, cryo-preserved arteries are increasingly used in vascular grafts. While studies are being performed to establish the efficacy of such treatments, very little is known about the postcryosurgical or postcryo-preservation changes in mechanical properties of the arteries. Few studies have examined the effect of freezing in the absence of cryoprotective agents (CPAs), and the several studies done in the presence of CPAs have given mixed results. To examine this issue further, we froze pig femoral arteries in a controlled rate freezer, using an aluminum probe, both in the presence at (-80 degrees C to 1 degrees C/min) and absence (at -20 degrees C for 2 or 5 mins) of CPA and Fetal bovine serum (FBS). Following freezing, artery samples were subjected to uniaxial tensile testing. The weights of the tissue were measured before and after freezing. Our results suggest that freezing does have an effect on stress-strain properties, particularly in the low stress region corresponding to physiological conditions. The mechanisms of this change in mechanical properties may include the loss of smooth muscle cell viability, damage to extra cellular matrix (ECM), bulk redistribution of water, or changes in alignment caused by ice crystal growth. In the case of samples frozen in the absence of CPA or FBS, the results indicated a drastic reduction in weight of the tissue suggesting the importance of bulk water redistribution as one underlying mechanism. To further examine potential mechanisms, we subjected cryopreserved vessels to the same uniaxial tests. The extent of changes in mechanical properties and bulk water redistribution was greatly attenuated; reinforcing that water movement might play a role in the changes observed with freezing.

Adaptation, Physiological↗
PubMedDate not supplied

No evidence for altered cellular immune functions in personnel deployed in the Persian Gulf during and after the Gulf War--The Danish Gulf War study.

Veterans who have participated in the Gulf War suffer from a number of symptoms, collectively referred to as the Gulf War Syndrome. It has been hypothesized that a change in the systemic cytokine balance or other changes in immunological parameters could be responsible for some of the symptoms. We analyzed the peripheral blood natural killer (NK) cell activity of 686 Gulf War personnel who had been present in the Persian Gulf area during and immediately after the Gulf War as well as 231 gender and age-matched controls. The test material included individual samples of frozen peripheral blood mononuclear cells kept at -139 degrees C for a period of 50 to 380 days prior to NK cell analysis of freshly thawed cells. Significant differences in NK-cell activity were not observed by direct comparison of the levels of natural cytotoxic activity in the two groups. However, NK-cell cytotoxicity as such decreased due to cryopreservation. Surprisingly, the NK cells obtained from control donors were significantly (p<0.0001) more sensitive to freezing conditions than cells from the Gulf War personnel, leaving the marginal comparison between the two groups untrustworthy, in particular because of the marked difference between the -139 degrees C storage times used for the two groups. Freshly thawed samples of peripheral blood T lymphocytes (CD2+ cells) from 109 randomly selected Gulf War personnel and 68 gender- and age-matched controls were stimulated for 3 days with phytohemagglutinin followed by 4 h activation by phorbol ester and ionomycin, and were stained for intracellular content of interleukin-2, -5, -10 and interferon-gamma. As with natural cytotoxicity, the length of cell storage at -139 degrees C influenced the production of cytokines. No significant differences in the cytokine production between the two groups were observed when the influence of the storage period was taken into consideration. Together, these data suggest that no overall long-term effects on NK-cell function and T-cell cytokine production are present in the Danish Gulf War personnel. Moreover, cryopreservation is a major potential source of bias when studying the physiology of thawed NK and T cells.

Adult↗
PubMed1995

[Quality of cryopreserved ejaculate in Hodgkin disease].

Each year approximately 70 males are diagnosed with Hodgkin's disease (HD) in Denmark. Most of these are less than 50 years of age. Since chemotherapy and radiotherapy may induce infertility in about 80% of patients, it has become routine to offer cryopreservation of semen prior to initiation of treatment. In 41 men aged 17-42 years at diagnosis we evaluated the quality of semen (according to the WHO criteria) prior to cryopreservation and after thawing. The median sperm concentration at the time of diagnosis was 33 x 10(6)/ml (range: 0.3-458 x 10(6)/ml) and positively correlated to age (rs = 0.36 p = 0.02) and to the histopathological classification (highest for patients with nodular sclerosis (p = 0.04) but not to semen volume, stage or presence of B-symptoms. The percentage of motile spermatozoa (range: 0-90%, median value 72%) was correlated to the sperm concentration; but not to any of the other parameters studied. Prior to freezing, 19 of 41 patients had normal semen quality according to the WHO criteria, whereas following thawing this was the case for only 14 semen samples. Ten of 23 patients with nodular sclerosis HD had normal semen quality (according to the WHO criteria) after thawing whereas this was the case for only 2 of 15 patients with mixed cellularity HD (p = 0.05). Cryopreservation of semen may secure the possibility of fathering children for patients with HD following therapy, and is to be recommended.

Adolescent↗
PubMed1995

The application of in vitro isolation, cryopreservation and patch-clamp microelectrode recording methods to adult rat thymic nurse cells.

Characterisation of the ionic mechanisms possessed by immune cells has begun to reveal a range of transmembrane ion channel properties which may have immunological significance. Since thymic epithelial cells appear to influence selection of the T cell repertoire, understanding their membrane physiology may be of importance. A method is therefore outlined for the targeting of patch-clamp electrophysiological measurements to acutely isolated, fresh and cryopreserved adult rat thymic nurse cells. These cells represent a discrete and predominantly cortical population of epithelium. Cells were separated by enzymatic and mechanical dispersal of thymus, enriched by sedimentation and identified on the basis of their characteristic lympho-epithelial, multi-cellular morphologies. Phase-bright cells retaining this anatomical form survived freezing and the voltage-gated conductances in such cells are described. The merits of this approach include the preparation and storage of mature differentiated phenotypes of immune cells for in vitro studies. Their preservation permits temporal separation of experiments, correlative experimentation on identical samples, optimises cell preparation in terms of cost and labour, and reduces animal and tissue requirements.

Animals↗