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PubMed2004

Comparison of fertility between intracytoplasmic sperm injection and in vitro fertilization with a partial zona pellucida incision by using a piezo-micromanipulator in cryopreserved inbred mouse spermatozoa.

Cryopreservation of mouse spermatozoa has been widely applied for maintenance of transgenic and knockout lines. However, the fertility of cryopreserved spermatozoa from some inbred strains such as C57BL/6 and BALB/c is extremely poor. We have recently reported that a partial zona-pellucida incision by piezo-micromanipulator (ZIP) significantly improves the fertilization rate and subsequent embryonic development after in vitro fertilization (IVF) using cryopreserved C57BL/6 transgenic mouse spermatozoa and that inbred C57BL/6 mice could be produced by intracytoplasmic sperm injection (ICSI). These findings prompted us to compare the efficiency of fertilization and subsequent embryonic development between ICSI and IVF with ZIP (ZIP/IVF) using cryopreserved spermatozoa. In conventional IVF, BALB/cA, C57BL/6J, and B6C3F1 cryopreserved spermatozoa fertilized 19%, 0%, and 51% of oocytes, respectively. The fertilization rates of manipulated oocytes by ICSI versus ZIP/IVF using cryopreserved BALB/cA spermatozoa were 52% versus 68%, cryopreserved C57BL/6J spermatozoa were 43% versus 63%, and cryopreserved B6C3F1 spermatozoa were 58% versus 82%, respectively. In these strains, fertilization rates for ZIP/IVF were significantly higher (P < 0.05) than for other techniques. However, embryonic development to term for oocytes fertilized by cryopreserved spermatozoa was not significantly different between ZIP/IVF and ICSI in C57BL/6J and B6C3F1. The overall efficiency of mouse production in ZIP/IVF was higher than for ICSI and conventional IVF in C57BL/6J and B6C3F1. Furthermore, ZIP/IVF required approximately 3.3 times less manipulation time than did ICSI. Our results indicate that ZIP is a useful assisted reproductive technique for IVF of ova by cryopreserved spermatozoa and improves production in some mouse strains.

Animals↗
PubMed1990

Functional analysis of cryopreserved veins. Preliminary report.

Functional comparisons of cryopreserved and fresh canine vein endothelium, smooth muscle, and connective tissue were performed. Morphometric analysis of saphenous vein endothelium revealed no significant loss of endothelial integrity as a result of cryopreservation. Endothelial cell culture revealed similar numbers of clonogenic intimal cells from cryopreserved and fresh saphenous, cephalic, and jugular veins. Smooth muscle function was assessed by measurement of the isometric force generated by vein rings in response to norepinephrine, serotonin, and potassium chloride. There was no significant difference in the dose responses of cryopreserved and fresh saphenous veins to the reagents tested. Similar results were obtained for the cephalic and jugular vein experiments with norepinephrine. The maximum tensions generated in response to norepinephrine were 52% of fresh control segments. Connective tissue function was assessed by quantitation of 3H-proline incorporation. The results indicate that cryopreserved veins retained approximately 43.5% of values of fresh vein collagen synthesis. Finally, eight cryopreserved cephalic vein autografts were placed as femoral artery grafts and were removed electively after 1 to 8 weeks. All grafts were patent. Both light and electron microscopy demonstrated that the cryopreserved veins remained intact in vivo and that arteriolization occurred as described for fresh autografts in the literature. In conclusion, cryopreserved veins retain much of their cellular and tissue functions on thawing. Transplantation of cryopreserved veins suggests that cryopreservation does not change the sequence of histologic events associated with the use of autologous fresh vein as an arterial substitute.

Animals↗
PubMed1995

Establishment of epithelial lines from cryopreserved lenses and capsule-epithelial preparations.

PURPOSE: To determine if lens epithelial lines can be established from cryopreserved whole rabbit lenses and from cryopreserved capsule-epithelial preparations (CEPs). METHODS: Lenses or freshly isolated CEPs were cryopreserved and subsequently thawed. Thawed whole lenses were cultured for 48 hours in growth medium and fixed, and whole mounts were examined for mitosis. In addition, CEPs were peeled from cryopreserved lenses and placed in tissue culture. Viability of cryopreserved cells was assessed measuring attachment efficiency and growth. RESULTS: Whole mounts from cryopreserved lenses that were thawed and placed in organ culture in a serum-containing medium exhibited numerous mitotic figures. Freshly isolated CEPs that were cryopreserved and CEPs from cryopreserved lenses generated cell lines. Attachment efficiency was 90% within 3 hours of plating. When 50,000 cells from cryopreserved CEPs were cultured in growth medium, 10(6) cells were noted after 7 days of culture. The cells completed 27 population doublings and showed no sign of senescence. CONCLUSIONS: Rabbit lens epithelial cell lines can be initiated from cryopreserved lenses or CEPs.

Animals↗
PubMed1984

Antibody-dependent cell-mediated cytotoxicity of cryopreserved human monocytes.

Human peripheral blood monocytes (PBM) modulate and participate in a variety of host defences. Cryopreservation of PBM has facilitated studies of their function. Peripheral blood samples cleared of red cells and granulocytes by centrifugation over Ficoll-Hypaque were cryopreserved at 1 degree C/min in 10% Me2SO and stored at -150 degrees C. Cryopreserved cells were thawed rapidly, diluted at a constant rate over 10 min with 9 vol of media, and washed twice prior to study. Antibody-dependent cell-mediated cytotoxicity (ADCC) activity against anti-D-coated Rh-positive erythrocytes of both fresh and cryopreserved PBM was tested and found to be equal (52.5 vs 51%). The myeloperoxidase positive, EA-rosette-positive population in cryopreserved cells was 39% compared with 17% for fresh cells (P less than 0.0001). This difference is due to preferential recovery of cryopreserved monocytes among mononuclear cells. The proportion of cells expressing Fc receptors among the myeloperoxidase-positive mononuclear cell population increased after freezing, suggesting an alteration in membrane structure induced by cryopreservation. It is concluded that PBM can be cryopreserved in Me2SO and that ADCC function is fully retained in the cryopreserved cells. This study along with a previous study (R.S. Weiner and S.J. Norman, J. Natl. Cancer Inst. 66, 255-260, 1981) demonstrate the feasibility of using cryopreserved human PBM for functional studies.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1999

A survey of oncologists regarding sperm cryopreservation and assisted reproductive techniques for male cancer patients.

BACKGROUND: The authors surveyed the current knowledge, opinions, and clinical practices of oncologists regarding pretherapy cryopreservation of semen from male cancer patients since the introduction of intracytoplasmic sperm injection (ICSI). METHODS: A survey was sent to all members of the American Society of Clinical Oncology in Minnesota. RESULTS: Forty-six of 165 oncologists (28%) responded. Factors considered important in how strongly to recommend cryopreservation were patient age at the time of diagnosis (94%), type of treatment (83%), type of cancer (65%), urgency to initiate treatment (63%), and preexisting infertility (57%). Oncologists perceived patients to be significantly more concerned about cryopreservation than they were themselves during pretherapy counseling (P = 0.0005). Oncologists estimated that 27% of their patients chose to cryopreserve sperm. However, only 26% of the oncologists knew about ICSI. The cancers perceived to warrant cryopreservation the most were lymphomas, leukemias, and testicular carcinomas. The treatment modalities perceived to warrant cryopreservation the most were distributed among various chemotherapy and radiation regimens. A majority of respondents to the survey knew where patients could go to cryopreserve sperm (89%), but less than half of the respondents gave accurate information about the cost. CONCLUSIONS: Most of the oncologists surveyed were unaware of recent advances in reproductive technology in which only a few sperm are needed for successful in vitro fertilization with ICSI. This lack of awareness may be contributing to underutilization of sperm cryopreservation by male cancer patients. Currently, all male cancer patients of reproductive age who will have treatment that may affect testicular function and who may desire children in the future should cryopreserve sperm before the initiation of therapy.

Cryopreservation↗
PubMed1994

Differential sensitivity to cryopreservation of clonogenic progenitor cells and stromal precursors from leukemic and normal bone marrow.

The survival of human leukemic and normal progenitor cells was determined after cryopreservation. Thirteen marrows from patients with acute myeloid leukemia (AML) were studied as fresh and eight as cryopreserved samples. Marrows from five normal donors were studied as both fresh and cryopreserved samples. Although the number of bone marrow mononuclear cells (BMMC) recovered after cryopreservation was always lower than that originally stored, no significant difference was observed between the clonogenic potential of fresh and cryopreserved BMMC from either the leukemic or the normal samples. When grown in long-term bone marrow culture (LTBMC), the cultures initiated with cryopreserved BMMC failed to form a confluent stroma, and the duration of nonadherent and progenitor cell production was significantly lower than that from fresh samples. However, when these cryopreserved samples were recharged onto preformed irradiated stroma, the duration of the cultures improved significantly. We conclude that it is the bone marrow stromal cells rather than the clonogenic progenitors which are sensitive to the effects of cryopreservation. Thus cryopreservation does not appear to influence the activity of AML progenitor cells. Our results also indicate that frozen marrow can be used for LTBMC experiments if cultured on a preformed stromal layer.

Adolescent↗
PubMed1996

Actions of selected hepatotoxicants on freshly isolated and cryopreserved rat hepatocytes.

The present study compares the actions of the hepatotoxic agents allyl alcohol, acetaminophen, and carbon tetrachloride on energy metabolism in freshly isolated and cryopreserved rat hepatocytes. After 30 min incubation of freshly isolated hepatocytes at 37 degrees C to allow metabolic equilibration, hepatocytes were supplemented with cryoprotectants and cooled in a stepwise manner to liquid nitrogen temperature. Hepatocytes stored in liquid nitrogen for 2 weeks to 6 months were thawed and centrifuged through Percoll to remove damaged cells. Despite similarities in energy status as indexed by ATP content and the rate of urea synthesis in freshly isolated and cryopreserved hepatocytes, cryopreserved hepatocytes were more sensitive to hepatotoxicants. All three hepatotoxicants caused ATP and rates of urea synthesis to decline to a greater extent in cryopreserved than in freshly isolated hepatocytes. Rates of oxygen uptake were higher in cryopreserved cells than in freshly isolated hepatocytes and declined in cryopreserved cells but not in freshly isolated cells during the initial period of incubation. Rates of mitochondrial respiration stimulated with site-specific substrates were comparable in freshly isolated and cryopreserved cells permeabilized with digitonin. Allyl alcohol and acetaminophen inhibited site-specific respiration to the same extent in both groups of cells. Collectively, these results suggest that increased sensitivity to hepatotoxic agents and elevated oxygen consumption in cryopreserved hepatocytes recovered after storage in liquid nitrogen are related to higher demand for energy in these cells rather than to permanent injury caused by cryopreservation and irreversible uncoupling of oxidative phosphorylation.

1-Propanol↗
PubMed2000

Assessment of some critical factors in the freezing technique for the cryopreservation of precision-cut rat liver slices.

A number of studies on the cryopreservation of precision-cut liver slices using various techniques have been reported. However, the identification of important factors that determine cell viability following cryopreservation is difficult because of large differences between the various methods published. The aim of this study was to evaluate some important factors in the freezing process in an effort to find an optimized approach to the cryopreservation of precision-cut liver slices. A comparative study of a slow and a fast freezing technique was carried out to establish any differences in tissue viability for a number of endpoints. Both freezing techniques aim at the prevention of intracellular ice formation, which is thought to be the main cause of cell death after cryopreservation. Subsequently, critical variables in the freezing process were studied more closely in order to explain the differences in viability found in the two methods in the first study. For this purpose, a full factorial experimental design was used with 16 experimental groups, allowing a number of variables to be studied at different levels in one single experiment. It is demonstrated that ATP and K(+) content and histomorphology are sensitive parameters for evaluating slice viability after cryopreservation. Subsequently, it is shown that freezing rate and the cryopreservation medium largely determine the residual viability of liver slices after cryopreservation and subsequent culturing. It is concluded that a cryopreservation protocol with a fast freezing step and using William's Medium E as cryopreservation medium was the most promising approach to successful freezing of rat liver slices of those tested in this study.

Adenosine Triphosphate↗
PubMed1996

Endothelium and vascular smooth muscle function in internal mammary artery after cryopreservation.

An optimal cryopreserved arterial conduit should have anatomic and physiologic characteristics similar to those of the fresh artery. We have cryopreserved canine internal mammary artery (IMA) with intact, prostacyclin (PGI2)-producing endothelial cells, but the underlying vascular smooth muscle appeared nonfunctional. Thus the aim of this study was to evaluate which steps of the cryopreservation method compromise vascular smooth muscle function in cryopreserved IMA. Isometric tension recording responses to vasoconstrictor agonists were used to evaluate the level of vascular muscle integrity, while endothelial function was assessed by relaxation responses to acetylcholine and by PGI2 production. These variables were measured in vessels rewarmed to 37 degrees C, following initial exposure to different stages of an eight-step computerized cryopreservation protocol (n = 10). In this protocol, tissue temperature was lowered to -10 degrees C (steps 1-2), rapidly reduced to -12 degrees C (steps 3-4), reduced further to -40 degrees C at a rate of -0.5 degrees C/min (steps 5-6), lowered to -70 degrees C (step 7), and finally equilibrated at - 196 degrees C by immersion in liquid nitrogen (step 8). Compared to fresh IMA, the amplitude of dose-dependent contractions to norepinephrine (NE 10(-9) to 10(-4) M) was step-wise depressed after cooling to < or = -12 degrees C (steps 3-4), showing only 75 and 19% of maximal contraction after steps 3-4 and step 7, respectively. Similarly, depolarization-induced contractions to KCl (10 to 60 mM) also were progressively depressed after step-wise cryopreservation, showing reduced contractile amplitudes even after steps 1-2 (-10 degrees C). Vascular muscle agonist sensitivity was unchanged (KCl) or only mildly influenced (NE) by these same temperature reductions. In contrast, endothelial-dependent relaxation to acetylcholine and PGI2 production were maintained after all steps in the cryopreservation process, and baseline PGI2 production was higher in cryopreserved IMA. These data indicate that IMA smooth muscle cell viability is poorly preserved after cooling to below -10 to -12 degrees C of the cryopreservation process, whereas endothelial cell function appears intact after profound cooling to -196 degrees C. The loss of vascular smooth muscle responsiveness coupled with the protection afforded by an intact endothelium may provide an arterial conduit less susceptible to vasospasm. Such a graft, however, would lack the dynamic properties of flow regulation in response to the metabolic needs of the myocardium.

Acetylcholine↗
PubMed1993

Cryopreserved arterial homografts: preliminary study.

The use of arterial or venous allografts for vascular reconstruction was first reported in 1951, but long-term results have been disappointing. Rejection and inappropriate methods of preservation are the main reasons for failure. A successful solution to this problem could be achieved by programmed cryopreservation with cryoprotectant. Our study had two aims: to define the biomechanical properties of cryopreserved arterial allografts and to study their histologic appearance. Arteries were removed as part of a protocol for multiorgan harvesting for transplantation. Cryopreservation was performed within the first 24 hours after harvesting. Programmed cryopreservation with 15% dimethyl sulfoxide (cryoprotectant) was used. Mechanical testing was done immediately after thawing. Two groups were tested: a control group of fresh aortas and a group of cryopreserved aortas. Axial and circumferential strips were tested. High strain modulus and stress and strain characteristics were calculated for each strip. There was no statistically significant difference between the mechanical properties of fresh and cryopreserved human descending thoracic aortas. Biochemical tests were performed in the preservative solution at 1 and 7 days in both groups. There was no statistically significant difference between the two groups at day 1 or day 7 (p > 0.05). Histologic studies before and after arterial cryopreservation included standard and electron microscopy and showed that arteries had normal structure after cryopreservation. These results confirm that programmed cryopreservation with cryoprotectant does not alter the molecular or geometric configuration of collagen or elastic fibers. Endothelial cells were still present, however, their viability and function were not assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aorta↗
PubMed1996

Simultaneous program of natural-cycle in vitro fertilization and cryopreserved-thawed embryo transfer.

PURPOSE: This clinical study was designed to identify and compare the pregnancy rates of simultaneous program of natural-cycle IVF and cryopreserved-thawed ET (NICE) with those of natural-cycle IVF and cryopreserved-thawed ET. METHODS: All three groups comprised spontaneously ovulating infertile women under the age of 40 and without any male factor present. The NICE program was performed in 36 patients (47 cycles) who had previously undergone IVF-ET resulting in cryopreserved embryos. As control groups, the natural-cycle IVF was performed in 45 patients (80 cycles), and the cryopreserved-thawed ET alone in 29 patients (40 cycles). RESULTS: The cancellation rate of the initiated cycles prior to ET was 19.1% (9/47) in the NICE group, 23.8% (19/80) in the natural-cycle IVF group, and 2.5% (1/40) in the cryopreserved-thawed ET group. The mean number of embryos transferred was 4.0 +/- 1.1 (2-5) in the NICE group, 1.0 in the natural-cycle IVF group, and 4.2 +/- 1.8 (1-5) in the cryopreserved-thawed ET group. The clinical pregnancy rates per aspiration cycle (32.5%) and per ET cycle (34.2%) in the NICE group were significantly higher than those (14.9, 16.4%) in the natural-cycle IVF group. The clinical and delivered pregnancy rates per ET (34.2, 26.3%) in the NICE group were higher than those (20.5, 15.4%) in the cryopreserved-thawed ET group, without statistical significance. CONCLUSIONS: Since the NICE program results in saving the fresh oocyte for patients participating in cryopreserved-thawed ET, more favorable pregnancy rates may be obtained from NICE cycles in women ovulating normally who had previously undergone IVF-ET with embryo cryopreservation.

Adult↗
PubMed1994

Coculture of mouse embryos with cryopreserved human oviduct epithelial cells.

PURPOSE: The effect of coculturing mouse embryos with cryopreserved human oviduct epithelial cells was investigated. The cryopreserved cells in Cellbanker were thawed and cultured in Richard D. Goldsby culture medium to establish monolayers. Two-cell-stage mouse embryos were cultured alone (control group) or cocultured with monolayers established from cryopreserved cells (cryopreserved coculture group) or from fresh cells (fresh coculture group). The rates of embryo development and the qualities of the blastocysts in the three groups were compared. RESULTS: The two coculture groups had significantly higher blastocyst development rates (cryopreserved coculture group, 81.6%; fresh coculture group, 82.2%) than the control group (63.1%). The two coculture groups had significantly more blastomeres (cryopreserved coculture group, 108.3 +/- 25.9; fresh coculture group, 108.4 +/- 25.1) than the control group (87.7 +/- 31.9). CONCLUSION: The method of cryopreservation of human oviduct epithelial cells using Cellbanker is simpler than conventional cryopreservation methods. These cryopreserved human oviduct epithelial cells may provide a constant supply of cells for coculture for in vitro fertilization and embryo transfer.

Adult↗
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↗
PubMed1995

In vitro development and implantation rates of fresh and cryopreserved sibling zygotes.

OBJECTIVE: To compare the developmental potential of cryopreserved human zygotes after thawing to sibling zygotes that were transferred without cryopreservation. METHODS: Retrospective analysis of embryo data and pregnancy outcome for all in vitro fertilization (IVF) patients who had sufficient zygotes to allow fresh embryo transfer and cryopreservation of additional sibling zygotes for later use. RESULTS: Zygotes survived cryopreservation at a high rate (87%). After thawing, cryopreserved zygotes developed at rates similar to those of fresh zygotes. Pregnancy occurred at similar rates after replacement of fresh embryos (27.9%) or replacement of cryopreserved-thawed zygote-derived embryos (24.3%). CONCLUSIONS: Human embryos can be cryopreserved at the pronuclear zygote stage with little loss of developmental potential. Cryopreservation allowed a reduction in the number of embryos transferred during an IVF retrieval cycle, thereby reducing the occurrence of multiple pregnancy. The total cumulative pregnancy rate per retrieval cycle doubled when pregnancies from cryopreserved-thawed zygotes were added to those originating from fresh zygotes.

Cryopreservation↗
PubMed2004

Restoring the endothelium of cryopreserved arterial grafts: co-culture of venous and arterial endothelial cells.

The use of arterial homografts in clinical practice is becoming increasingly common, yet there is an urgent need to address one of the most well-established problems associated with their use: the loss of integrity of the endothelium following cryopreservation. The partial lack of endothelium causes contact between the extracellular matrix and blood flow, which, in turn, often gives rise to thrombosis and/or restenosis. Our objective was first to attempt to replace the arterial endothelial cells lost during the cryopreservation process by seeding autologous venous endothelial cells, and to evaluate the behaviour of venous and arterial endothelial cells in co-culture. The idea was to establish whether venous endothelial cells would be accepted by arterial endothelial cells and could therefore be used to restore the endothelial lining for the subsequent use of these vessels in in vivo grafting procedures. For the co-culture experiments, endothelial cells were obtained from the jugular vein and both iliac arteries of the minipig by treatment with 0.1% type I collagenase. The venous endothelial cells were fluorescently labelled with the membrane intercalating dye PKH26. Equal numbers of venous and arterial endothelial cells were mixed and co-cultured for 24h, 48h or 4 days. Cell viability, determined by 2% trypan blue staining and the TUNEL method, was established before and after fluorescence labelling. Cellular activity was determined by estimating PGI2 levels in the cultures. The proliferation index was established by [H(3)]thymidine (1muCi/ml) in the cell culture medium. For the in vivo tests, 5 cm length segments of minipig iliac artery were used to establish the groups: control (n = 6), fresh arterial segments; group I (n = 16), cryopreserved arterial segments and group II (n = 16), cryopreserved arterial segments seeded with autologous venous endothelial cells. The cryopreserved vessels in group II were seeded by flooding with a labelled venous endothelial cell suspension. Once seeded, the arterial segments were included in an in vitro flow circuit. All the specimens were processed for fluorescence and light microscopy, and scanning electron microscopy. The denuded endothelial surface was determined in each group. Cell death was evaluated by the TUNEL method. We confirmed the existence of intercellular PECAM1-type junctions between venous (PKH26+) and arterial cells in co-culture and the functional activity of the cells. The cryopreserved arterial segments showed a well-preserved wall structure. However, different size areas of marked endothelial denudation were detected. After seeding with labelled cells (PKH26+), these denuded areas of the cryopreserved artery were entirely covered by fluorescent cells. After seeding, a drop in the proportion of damaged endothelial cells was recorded. Despite some loss of seeded cells after inclusion in the in vitro flow circuit, the endothelial cell count was not significantly different to those recorded for control, non-cryopreserved specimens. In conclusion arterial and venous endothelial cells growing in co-culture modify their behaviour to form multilayers. The two cell populations form normal PECAM1 junctions and preserve their functional properties. Seeding autologous venous endothelial cells on the luminal surface of cryopreserved arterial segments serves to restore the integrity of the endothelial layer.

Animals↗
PubMed2005

Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm.

Cryopreservation causes several types of damage to spermatozoa, such as loss of plasma membrane integrity and functionality, loss of motility, and ATP content, resulting in decrease of fertility rates. This spermatozoal damage has been widely investigated for several marine and freshwater fish species. However, not much attention has been paid to the nuclear DNA. The objective of this study was to determine the degree to which cryopreservation induces spermatozoal DNA damage in two commercially cultured species, rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), both of which could benefit from the development of cryopreservation strategies on a large scale. We have used the single-cell gel electrophoresis, commonly known as Comet assay to detect strand breaks in DNA. This technique was performed on fresh and cryopreserved sperm from both species. In rainbow trout there was a significant increase in the averages of fragmented DNA and Olive tail moment after cryopreservation (11.19-30.29% tail DNA and 13.4-53.48% Olive tail moment in fresh and cryopreserved sperm, respectively), as well as in the proportion of cells with a high percentage of DNA fragmentation. For gilthead sea bream there were no significant differences in the percentage of tail DNA between the control samples and sperm diluted 1:6 and cryopreserved (28.23 and 31.3% DNA(t), respectively). However, an increase in the sperm dilution rate produced an increase in the percentage of DNA fragmentation (41.4%). Our study demonstrates that cryopreservation can induce DNA damage in these species, and that this fact should be taken into account in the evaluation of freezing/thawing protocols, especially when sperm cryopreservation will be used for gene bank purposes.

Animals↗
PubMed2006

Cryopreserved embryo transfers suggest that endometrial receptivity may contribute to reduced success rates of later developing embryos.

OBJECTIVE: To evaluate viability and implantation potential of cryopreserved blastocysts according to the day of blastocyst expansion and cryopreservation. DESIGN: Retrospective study. SETTING: Private ART center. PATIENT(S): Three hundred and seventy-five patients undergoing embryo transfer with cryopreserved blastocysts. INTERVENTION(S): Blastocyst cryopreservation on day 5, 6, or 7 after oocyte retrieval according to the day of blastocyst expansion and subsequent embryo transfer. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (PR) per embryo transfer. RESULT(S): Clinical PRs were similar between blastocysts cryopreserved on day 5 and blastocysts cryopreserved on day 6 (32% vs. 28%). The clinical PR was lower for blastocysts cryopreserved on day 7 (15%), but this difference was not statistically significant after accounting for the number of embryos per transfer (P=.15). CONCLUSION(S): Viability and implantation potential are similar for day 5 and day 6 blastocyst cryopreservation. Viability may be reduced for blastocysts cryopreserved on day 7, but not to the extent suggested by reports of fresh transfers. These results suggest that reduced success rates associated with fresh transfers of later developing blastocysts may be the result of asynchrony with endometrial receptivity instead of poorer embryo quality.

Adult↗