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Results for “Cryopreservation”

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At least 19 recordsLinked to original sources

PubMed1992

The contribution of the toxicity of a glycerol-egg yolk-citrate cryopreservative to the decline in human sperm motility during cryopreservation.

The contribution of the toxicity of glycerol-egg yolk-citrate (GEYC) cryopreservative medium to the loss of function of human spermatozoa during cryopreservation was determined by investigating the effect of mixing semen with the medium on sperm motility. The percentage of progressively motile spermatozoa, velocity (micron s-1) and lateral head displacement (micron) (mean +/- SEM, n = 28) were 55 +/- 4.1, 47 +/- 2.7, 4.4 +/- 0.2 and 32 +/- 3.8, 40 +/- 2.5, 3.6 +/- 0.25 and 15 +/- 2.5, 28 +/- 1.1, 2.8 +/- 0.15 in suspensions of washed spermatozoa prepared from fresh, GEYC-treated and frozen-thawed semen, respectively. The variables changed only slightly after incubation for 3 h. The toxicity of GEYC did not vary significantly between samples which survived the complete freeze-thaw cycle well or very poorly. The toxicity of GEYC is responsible for about 50% of the loss of progressively motile spermatozoa during the complete cryopreservation process, but has little effect on the quality of motility. Susceptibility to GEYC does not explain observed differences in the ability of semen samples to survive freezing.

Citrates↗

PubMedDate not supplied

Cryopreservation of human semen. Comparison of cryopreservatives, sources of variability, and prediction of post-thaw survival.

Human semen was cryopreserved using Human Sperm Preservation Medium, TEST-Yolk buffer, or glycerol alone. Sperm characteristics for each specimen were measured before and after freezing to determine which cryopreservative resulted in better cryosurvival and recovery of motile sperm. Sperm frozen in Human Sperm Preservation Medium had a significantly better recovery of all semen parameters (motility, velocity, and recovery) than either TEST-Yolk or glycerol alone. Statistical analyses also were done to examine the variability between and within donor semen specimens. Differences between donors, between specimens, and measurements within donors all contributed to variability of sperm characteristics. Specimen-to-specimen variability for a given donor represented 12% to 47% of the total variability, whereas processing and measurement variability represented 12% to 41%. Donors also varied in the ability of their sperm to tolerate freezing. There was a relationship between motile count after dilution with cryopreservative and post-thaw motile count. This relationship allows the prediction of poor-thaw survival before freezing a specimen.

Cell Survival↗

PubMed1991

[Development of an improved cryopreservation vial for cryopreservation of donor corneas].

To date, corneal cryopreservation has been performed in vials made of glass or plastic, where the cornea is placed in a large volume of culture medium. For improved cell survival, an attempt was made to define freezing curves, which are induced by variations in the temperature in the freezing chamber. Depending on the mass of the specimen to be frozen and on the material conducting the heat, the freezing vials used so far cannot be regarded as optimum. As glass and plastic do not conduct heat very well, we developed a new freezing vial that is especially suitable for corneal cyropreservation. In our experiments, the interaction of the chamber temperature and the temperature near the corneal endothelium were monitored. Additionally, endothelial cell survival was studied by postculturing the tissue and by vital staining. As a result, a cylindrical vial made of aluminum 2-4 mm thick was designed, which enables even heat transfer from the freezing chamber to the cornea. In this vial, the amount of freezing medium could be reduced to 400 microliters, so that the heat from the crystallization process was very low. In a small series the conditions that had been optimal with porcine tissue were tested on ten human donor corneas. The endothelial cell density before (2365 cells/mm2, range 1675-2800) and after cryopreservation (2199 cells/mm2, range 1600-2720) did not differ significantly.

Animals↗

PubMedDate not supplied

Functional changes in canine saphenous veins after cryopreservation.

This study was designed to determine the functional characteristics of the endothelium, smooth muscle and nerve terminals of cryopreserved veins. Freshly harvested and cryopreserved canine saphenous veins were cut into rings. In some rings, the endothelium was removed. Cryopreserved veins were stored at -196 degrees C for at least 3 weeks prior to use. All rings contracted in a concentration-dependent manner to depolarization with KCl and to alpha-adrenergic stimulation; the maximal tensions were significantly less in cryopreserved than in freshly harvested veins. Calcium ionophore A23187 caused greater relaxations in rings with than without endothelium in freshly harvested and cryopreserved veins. These relaxations were reduced significantly by methylene blue and NG-monomethyl-L-arginine (L-NMMA) only in fresh veins. Cocaine-sensitive uptake of H3-norepinephrine was reduced following cryopreservation. Immediately after cryopreservation, the production of prostacyclin was elevated. The calcium ionophore A23187 stimulated production of prostacyclin only in freshly harvested veins. Tissue content of endothelin did not change following cryopreservation. These results suggest that cryopreservation of canine saphenous veins alters nerve terminals and decreases the ability of the smooth muscle to contract. The endothelium releases an endothelium-derived relaxing factor and prostanoids following cryopreservation but the ability to synthesize nitric oxide is probably reduced. These changes following cryopreservation may affect patency of the veins when used as arterial grafts.

Animals↗

PubMed1990

The effects of cryopreservation on membrane integrity, membrane transport, and protein synthesis in rat hepatocytes.

The cryopreservation of hepatocytes is of particular interest as a step in the possible treatment of some inborn disorders of metabolism. This study examines the metabolic damage that occurs as a result of the freeze-thaw procedures and during subsequent incubation periods of isolated rat hepatocytes. Even for freshly prepared hepatocytes, the presence of 1.8 M of Me2SO during incubation led to a rapid decline in viability. Optimal recovery after cryopreservation was obtained when incubation was started after the progressive removal of Me2SO. A buffer medium characterized by an intracellular electrolyte composition (Euro-Collins) proved particularly beneficial to the membrane integrity, probably by protecting the (Na+,K+)ATPase pump activity. The interpretation of viability using the trypan blue exclusion test was generally confirmed by the metabolic analysis of protein synthesizing activity and membrane transport function which are regarded as more rigorous tests of functional viability. The incorporation of L-[U-14C]isoleucine into the proteins of fresh hepatocytes during the first hour of incubation progressively leveled off over the next 2 hr. The cryopreserved hepatocytes showed a similar pattern although at a lower level of activity. Even after 3 hr of preincubation, the subsequent addition of labeled isoleucine still indicated a residual protein synthesizing activity. The active transport of alpha-amino[1-14C]isobutyric acid through the cell membranes reached a peak value after 60 min of incubation of fresh hepatocytes, and after 40 min of incubation of cryopreserved cells, followed by a steep decline as expression of rapid membrane deterioration. Again, the membrane transport pattern for the cryopreserved samples occurred at a lower level of activity. After preincubation of fresh and cryopreserved hepatocytes for 180 min, subsequent addition of labeled alpha-aminoisobutyric acid did not show any further significant metabolic activity. Initially the amino acid availability appeared to control protein synthesizing activity while, as membrane transport became seriously damaged, incorporation leveled off with only a low metabolic activity remaining. Although cryopreserved hepatocytes were susceptible to faster deterioration during subsequent incubation, considerable metabolic activity was retained. However, fresh and cryopreserved hepatocytes expressed metabolic functions at significantly different activities. Moreover, the differences between fresh and cryopreserved cells varied with the particular cellular function being examined.

Amino Acids↗

PubMed1991

Cryopreservation of pig and human liver slices.

The ability to cryopreserve human liver slices would greatly enhance the opportunities to test potentially hepatotoxic drugs and environmental contaminants as well as the metabolism of these compounds. This study focused on trying to cryopreserve pig and human liver slices. Since the acquisition of human liver tissue is unpredictable and scarce, an animal model was sought to predict problems associated with cryopreservation of human tissue. The pig liver was chosen because of its anatomical and physiological resemblance to human liver. The human liver tissues that did become available were obtained through the Arizona Organ Bank and the National Disease Research Interchange and from surgical liver resections. An in vitro culture system that employed precision-cut liver slices was used in this study. Different types and concentrations of cryoprotectants, cooling rates, and culture media were all tried in an attempt to cryopreserve pig and human liver slices. The viabilities of fresh and cryopreserved liver slices were evaluated using slice K+ retention and protein synthesis. Pig liver slices following cryopreservation retained between 80 and 85% of intracellular K+ content and protein synthesis as compared to controls using 1.4 M Me2SO, a 12 degrees C/min cooling rate, and a rapid rewarming rate of direct submersion of the slice into 37 degrees C fetal calf serum. Human liver slices following cryopreservation retained between 54 and 89% of intracellular K+ content and protein synthesis as compared to controls using the same protocol as for pigs, except that lower cooling rates were giving better results. The large variation seen in cryopreserved human liver slices was due to the length of warm and cold ischemia to which the tissue was exposed before arriving at the laboratory. This study indicated that pig and human liver slices can be cryopreserved and used for future toxicological and metabolic studies.

Animals↗

PubMed1991

Cryopreservation affects endothelial and smooth muscle function of canine autogenous saphenous vein grafts.

Cryopreserved veins used as arterial grafts may be affected by both rejection and the cryopreservation process. Experiments were designed to study changes in endothelial and smooth muscle function after cryopreservation but independent of rejection. One saphenous vein from each of eight dogs was cryopreserved for subsequent use as autografts. After 3 weeks one cryopreserved and one freshly harvested autogenous saphenous vein were implanted as bilateral femoral arterial interposition grafts. Platelet deposition was studied in vivo with indium 111-labeled platelets. At 4 weeks the autografts were removed, and the functional characteristics of the grafts were studied in organ chambers; and the ability of nerve terminals to uptake transmitter was studied with 3H-norepinephrine. Neither patency rates, blood flows, nor platelet deposition were significantly different between freshly harvested and cryopreserved grafts. Uptake of 3H-norepinephrine was significantly reduced in both grafts as compared to unoperated veins. The smooth muscle of the cryopreserved and fresh grafts contracted comparably to alpha-adrenergic agonists and endothelin. In cryopreserved grafts, the maximal tensions that developed to KCl, prostaglandin F2 alpha, and endothelin were greater when the endothelium was present compared to that developed by the smooth muscle alone. Calcium ionophore A23187 caused relaxations only in rings with endothelium; these were not significantly different between graft types. However, relaxations of the smooth muscle to nitric oxide were decreased in the cryopreserved grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

The effect of cryopreservation on cell viability and hormone secretion in human parathyroid tissue.

BACKGROUND: To explain the decreased success of cryopreserved versus fresh parathyroid autotransplants, we investigated the effect of cryopreservation on parathyroid function. METHODS: Parathyroid cryopreserved tissue from surgical specimens of 18 patients was analyzed in a comparative fashion with 10 fresh abnormal glands. Cell viability was evaluated by a double-fluorescence technique, and parathyroid hormone secretion was detected from individually dispersed parathyroid cells with the reverse hemolytic plaque assay. The plaque area reflected the amount of parathyroid hormone secreted by each cell, and the percentage of plaque-forming cells represented the number of secreting cells. Feedback regulatory mechanisms remained intact for both fresh and cryopreserved tissue as shown over a wide range of calcium concentrations. RESULTS: The percentage of viable cells from fresh and cryopreserved tissue was always in excess of 88%. No significant difference was noted in the percentage of plaque-forming cells nor the amount of hormone released per individual cell comparing fresh and cryopreserved glands (6 months to 2 years). Specifically, at physiologic calcium concentrations no significant differences existed in the secretory behavior between fresh and cryopreserved tissue. CONCLUSIONS: Without showing decreased viability or parathyroid hormone secretion after cryopreservation, the cause of graft failure in cryopreserved tissue still remains unknown.

Calcium↗

PubMed1991

Infectivity of cryopreserved Giardia cysts for Mongolian gerbils (Meriones unguiculatus).

Although Giardia species trophozoites have been cryopreserved successfully, no report on the successful cryopreservation of cysts could be found. Using infectivity to Mongolian gerbils (Meriones unguiculatus) as a measure of cyst viability, we tested the viability of 4 strains of Giardia cysts that had been cryopreserved for 1-67 wk. Cysts were frozen in either Keister's medium or physiological saline, both containing 5% or 7.5% dimethylsulfoxide as the cryoprotectant. Viability of cryopreserved cysts was dependent upon the number of cysts inoculated, the length of time cysts were held at 4 C before cryopreservation, and the cryopreserving medium. Infection was established in gerbils by inoculating them with cysts that had been cryopreserved for up to 67 wk, cysts that had been held for less than 30 days before cryopreservation, and cysts frozen in Keister's medium. Saline appears to be unsuitable as a freezing medium for the cryopreservation of Giardia cysts.

Animals↗

PubMed1992

Characterization of cryopreserved rat liver parenchymal cells by metabolism of diagnostic substrates and activities of related enzymes.

The metabolism of testosterone and benzo(a)pyrene (BaP) which is mediated by diverse enzymes was determined in cryopreserved rat liver parenchymal cells and compared with that found in freshly isolated cells. In addition, the activities of single xenobiotic-metabolizing enzymes were measured by using specific substrates. The cytochrome P450 (P450)-mediated total metabolic conversion of testosterone was reduced to 55% in cryopreserved cells. The metabolite profile, i.e. the formation of single metabolites compared with total metabolic conversion, was however unchanged when compared with freshly isolated cells. A concomitant reduction in the activities of the involved P450 isoenzymes can therefore be postulated. The amount of detected phase I-metabolites of BaP was unaffected by the cryopreservation method. The formation of phase II-metabolites and total metabolic conversion of BaP in cryopreserved cells was however reduced to about 50-60%. The reduced glutathione S-transferase and more obviously phenol sulfotransferase activities measured in cryopreserved cells, may explain the impaired conjugation of BaP. The ratio between phase I- and phase II-metabolites was thus changed by cryopreservation. Density separation on Percoll yielded cryopreserved cells with a viability and metabolic capacity not measurably different from freshly isolated cells. To this extent, cryopreserved, Percoll-purified liver parenchymal cells are a useful in vitro system for drug metabolism studies. However due to the extensive loss in cell number during this procedure (recovery = 22% of freshly isolated cells) the application of this system is limited.

Animals↗

PubMed1991

Transplantation of cryopreserved canine venous allografts.

Local vascular reconstructions frequently require the use of vein grafts to bridge arterial or venous defects. Most previous studies on the use of cryopreserved veins have used relatively large caliber vessels. There have been few studies on the effectiveness of cryopreserved micro- or small-venous allografts. Here, we tested two types of cryopreserved venous allografts: (1) 1.5- to 1.9-mm diameter microvenous grafts (MVG); and (2) 4- to 5-mm diameter small venous grafts (SVG). Cryopreserved MVG allografts were placed into saphenous arteries of six experimental dogs and SVG cryopreserved allografts were placed into femoral arteries of six experimental dogs for 3 to 6 weeks. Two fresh MVG autografts were also transplanted into experimental dogs as controls and autografts were transferred to the contralateral side in SVG dogs as controls. None of the six cryopreserved MVG grafts retained patency but three/six cryopreserved SVG allografts were patent at harvest. Histological examination of grfts revealed control autografts were undergoing arterialization with an intact intima. Experimental cryopreserved allografts showed extensive medial fibrosis, significant lymphocytic infiltrates, and sporadic areas of intact intima for both patent and nonpatent grafts.

Animals↗

PubMed1992

Cryopreservation of islets of Langerhans does not affect angiogenesis and revascularization after free transplantation.

Cryopreservation of isolated islets of Langerhans will be a necessary procedure if pancreatic islet transplantation crosses the threshold for clinical treatment of diabetes mellitus. Although successful cryopreservation of rodent, canine, porcine and human islets has been documented in the past few years, little is known about the influence of the freeze-thaw procedure on the islet's potential to induce angiogenesis and revascularization, a process which is of crucial importance after free transplantation. We have analyzed the process of revascularization of 1- and 10-week-cryopreserved hamster islet isografts using intravital fluorescence microscopy. First signs of angiogenesis of cryopreserved islet grafts were observed on day 2 after transplantation, characterized by the protrusion of capillary sprouts. During the following days these sprouts formed a microvascular network, and revascularization was completed on day 10 after transplantation. Quantitative analysis of functional capillary density, capillary red blood cell velocity, capillary diameter and flow of individual capillaries did neither show differences between 1- and 10-week-cryopreserved islets, nor differences between cryopreserved islets and islets transplanted without cryopreservation were observed. From these results we conclude that cryopreservation of isolated pancreatic islet grafts is an adequate technique for long-term storage prior to transplantation.

Animals↗

PubMed1991

Production of transgenic mice from cryopreserved fertilized ova.

Cryopreserved fertilized mouse ova were used to generate transgenic mice via micromanipulation. Five-DNA constructions were injected into a total of 1,052 cryopreserved ova, of which 683 (65%) survived the injection and were transferred into recipients. Of 35 recipients, 66% became pregnant and littered a total of 88 pups. As controls, these DNA constructions were also injected into 1,123 fresh ova, of which 744 (66%) survived and were transferred. Of 42 recipients, 79% became pregnant and littered a total of 167 pups. That is, 22% of fresh ova that were transferred developed into live pups, whereas only 13% of cryopreserved ova did so. Of the pups born, 42 of the 167 (25%) produced from fresh ova were transgenic, and 28 of the 88 (32%) produced from cryopreserved ova were transgenic. In terms of the injected ova that had been transferred, 5.6% of the 744 fresh and 4.1% of the 683 frozen ova developed into transgenic mice. These data indicate that the efficiency of production of transgenic mice from cryopreserved ova is close to that from fresh ova. That observation and the fact that cryopreserved ova allow more efficient utilization of animals suggest that cryopreserved ova can be used instead of fresh ova to produce transgenic mice.

Animals↗

PubMed1991

Cryopreservation of hamster pancreatic islets using a rapid cooling rate.

To clarify the possibility of developing a rapid cooling rate for islet cryopreservation, we used a cooling rate of 25 degrees C/min for hamster pancreatic islet cryopreservation using 15 per cent dimethylsulfoxide as a cryoprotectant. After preservation, these islets were examined for their morphology and function by assaying the insulin release after glucose stimulation and the contents of the insulin and DNA in 10 islets. In addition, islet cell replicatory activity was investigated by an autoradiographic technique. The effects of transplantation of the islets upon isogeneic and xenogeneic transplantation were also examined. Freezing using a rapid cooling rate of 25 degrees C/min was found to be as effective as a slow cooling rate of 1 degree C/min for hamster islet cryopreservation. Morphologically, the cryopreserved islets appeared to be similar to the non-frozen cultured islets. The glucose-stimulated insulin release and cell replicatory activities in vitro also remained unchanged, whereas the number of cells per islet decreased slightly after cryopreservation. The grafting of cryopreserved islets normalized streptozotocin induced hyperglycemia following isogeneic transplantation. On the other hand, no prolongation of graft survivals in the case of the xenogeneic transplantation of hamster islets to rats was observed. The isogeneically transplanted islets exhibited the same cell replicatory activities in vivo, which was even higher compared that of normal hamster pancreatic islets in situ. In conclusion, the present findings indicate that hamster pancreatic islets can be successfully cryopreserved using a rapid cooling rate, however, it does not appear that this treatment reduces islet vulnerability to xenogeneic graft rejection.

Animals↗

PubMed1991

Cryopreservation and transplantation of immature rat retina into adult rat retina.

A bank of freeze-stored donor retinas would free transplantation research from dependence on availability of fresh donor tissue. Donor retinas from E13, E16, E19 and E22 (P1) rat embryos were cryoprotected and stored in liquid nitrogen for up to 8 months. Cryopreserved and fresh donor retinas were grafted to adult rat retina. After 4 weeks survival, transplants were evaluated according to a scoring protocol for the criteria of size, viability, lamination and integration. All donor ages of fresh and cryopreserved retina resulted in successful transplants, with the exception of cryopreserved E13. Cryopreserved grafts were significantly less laminated than grafts of fresh tissue. The best lamination scores of cryopreserved transplants were achieved with donor age E16. Surviving transplants were found in the epiretinal and/or subretinal space. Subretinal transplants had higher viability scores than did epiretinal grafts; the difference was more pronounced with transplants of cryopreserved than with fresh tissue. Fresh subretinal transplants were also significantly better laminated than fresh epiretinal transplants. This study shows that (1) cryopreserved retinal donor tissue can successfully be transplanted to rat retina; and (2) the subretinal space appears to be more favorable than the epiretinal space for retinal transplants.

Animals↗

PubMed2026

Cryopreservation-induced proteomic alterations in Pêga donkey (Equus asinus) spermatozoa.

Semen cryopreservation is a key tool for assisted reproduction and genetic conservation, but its efficiency remains limited in donkeys, compromising post-thaw sperm quality. This study aimed to characterize the proteomic profile of Pêga donkey spermatozoa and to investigate molecular mechanisms associated with cryopreservation-induced impairment of sperm function. Semen samples were collected from Pêga jacks and evaluated for sperm motility and vigor before and after cryopreservation. Quantitative proteomic analysis was performed by LC-MS/MS, followed by bioinformatic characterization of differentially abundant proteins. Cryopreservation markedly reduced sperm motility in all animals, whereas sperm vigor showed only a non-significant tendency toward reduction, suggesting sublethal cryoinjury primarily affecting flagellar efficiency. Proteomic profiling identified 554 proteins, of which 98 were differentially abundant between in natura and cryopreserved spermatozoa. Functional enrichment analyses showed that these proteins were mainly associated with energy metabolism, mitochondrial oxidative phosphorylation, glycolysis, cytoskeletal organization, signal transduction, proteostasis, and oxidative stress response. Notably, proteins involved in ATP production, mitochondrial function, and axonemal organization were significantly altered, supporting a mechanistic link between metabolic dysfunction, flagellar structural disorganization, and reduced post-thaw motility. Overall, cryopreservation induced coordinated and compartment-specific remodeling of the donkey sperm proteome, particularly affecting pathways essential for motility and functional competence. These findings provide new molecular insights into the cryobiological vulnerability of donkey spermatozoa and establish a mechanistic basis for the development of more effective, biology-driven cryopreservation strategies for this species.

Animals↗

PubMed2026

Research note: Efficient preservation of genome-edited chicken germplasm via N-methylacetamide-based semen cryopreservation.

The rapid development of genome-edited chicken lines for agricultural and biomedical use requires effective methods for long-term preservation. In birds, cryopreservation of oocytes and embryos is challenging due to the structure of the egg. This makes semen cryopreservation one of the most practical alternatives, however, this method has not been validated in genome-edited chickens. This study evaluated the feasibility of cryopreserving semen from genome-edited chickens using an N-methylacetamide (N-MA)-based protocol. Two genome-edited chicken lines were used in this study which are a DAZL-GFP germ cell reporter line and a RAG1 knockout line. Semen was stored for either one week or one month prior to assessing fertility and hatchability. The results demonstrated that cryopreserved semen from both genome-edited chicken lines maintained their fertilizing capacity, with no significant differences in fertility or hatchability compared to cryopreserved wild-type groups at either storage duration. Hatchability remained above 80% across all groups, indicating that semen cryopreservation did not significantly affect embryo development after successful fertilization. Furthermore, SYBR-14/PI staining revealed no significant differences in sperm viability between genome-edited and control groups, although both showed a significant decrease in viability compared to fresh semen. These findings demonstrate that N-MA-based semen cryopreservation provides a reliable and practical method for the preservation of genome-edited chicken lines.

Chicken↗

PubMedDate not supplied

Adverse reactions in patients transfused with cryopreserved marrow.

Marrow is cryopreserved for use in autologous bone marrow transplants, but little is known of the incidence of reactions in patients transfused with these cryopreserved marrows. Reactions in patients transfused during a 4-year period with 134 autologous marrows cryopreserved in dimethyl sulfoxide (DMSO) were compared with those in patients transfused with marrow that had been collected from HLA-compatible donors and that had not been cryopreserved. Patients transfused with cryopreserved marrow had significantly more nausea (44.8 vs. 14.1%; p less than 0.0005), vomiting (23.9 vs. 8.5%; p less than 0.01), chills (31.3 vs. 1.4%; p less than 0.0005), and fever (17.9 vs. 0%; p less than 0.005) than patients transfused with fresh allogeneic marrow. The incidence of emesis correlated with the dose of DMSO received, but that of nausea did not. All cryopreserved marrows were cultured for bacteria at the time of transfusion and 17 (12.7%) were found to be positive. Only 1 of the 17 patients transfused with culture-positive marrow developed sepsis during the transplant course with the same organism that was present in the transfused marrow. Although the reactions in donors transfused with cryopreserved marrow were readily treated, this study suggests that the incidence of some reactions might be decreased by reducing the dose of DMSO transfused. Bacterial contamination of transfused marrow was a worrisome complication, and efforts should be made to improve marrow collection and processing techniques to minimize that risk.

Bacteria↗