Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “sample cryopreservation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,261 records · Page 70Linked to original sources
PubMedDate not supplied

Cryopreservation of in vitro-grown shoot tips of cassava by encapsulation-vitrification method.

Shoot tips of cassava (Manihot esculenta Crantz) in vitro plantlets were successfully cryopreserved using the encapsulation-vitrification technique. Nodal cuttings of 5 mm length with one leaf were cultured on modified MS medium in Petri dishes (90 mm x 20 mm) for about 28 days. Excised shoot tips were precultured on sucrose enriched (0.3 M) medium for 16 h, encapsulated and osmoprotected with a mixture of 2 M glycerol and 0.6 M sucrose for 90 min at 25 degree c before dehydration with PVS2 at 0 degree C for 4 h, then plunged in liquid nitrogen. Successfully vitrified shoot tips resumed growth within 3 days, without intermediary callus formation, and developed shoots. Shoot tips sampled from 21 day-old plantlets produced the highest survival of 80 %. The percentage survival of vitrified shoot tips differed from 38 to 80 % depending on the day of excision. The protocol was successfully applied to four cultivars of cassava with about 80 % average percentage of survival.

Cell Survival↗
PubMed1992

Freeze-substitution of chemically stabilized samples for biological field emission scanning electron microscopy.

The high resolution imaging capabilities of modern field emission scanning electron microscopes require adequately improved tissue preparation procedures to prevent the collapse of macromolecular structures and the extraction of molecules. A routine cryo-stabilization technique is described which utilizes chemical crosslinking and cryo-dehydration for mechanical and chemical stabilization of protein and lipid structures and increase of electrical conductivity of the sample. Thiocarbohydrazide (TCH) serves as a general mordant for osmium tetroxide crosslinking. However, extensive washing after all impregnation steps is necessary to dissolve unspecific osmium black precipitations at the sample surface. Collagen I aggregates showed increased stability against collapse after TCH osmification alone, whereas pulmonary surfactant liposomes require additional freeze-substitution in methanol and Freon 113 for stabilization during critical point drying. Environmental scanning electron microscopy (at water vapor pressures of 5-10 torr within the specimen chamber) was used to control, in the wet phase, the stabilization procedure at the level of chemical crosslinkage. It could be confirmed that tannic acid, often used to stabilize lipids, leads to artificial rearrangement of bilayered liposomes into compact presumable multilayered bodies, whereas the TCH osmification preserved liposome structures and their aggregates. The increase of electrical conductivity of sliced tissue was demonstrated on kidney. Support technologies for the cryo-stabilization procedures are described in detail, as well as simple routines for first stabilization trials with new samples. On pulmonary tissue, the excellent preservation of alveolar shape and fine structures of intermediate forms of surfactant are described.

Animals↗
PubMed2005

Changes over time in thymidine phosphorylase and dihydropyrimidine dehydrogenase when colon cancer samples are left at room temperature.

The changes over time in thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) were studied when colon cancer samples were left at room temperature. The TP levels and the TP/DPD ratio showed no changes over time in either the tumor or normal tissues. DPD levels increased over time in the normal tissues (P = 0.015), but showed no changes in the tumor tissues. When the DPD level in normal tissues is to be examined, the samples should be frozen as soon as possible. In all other cases, the ELISA results will not be affected if the samples are frozen within 6 h after collection.

Colon↗
PubMed2006

Use of ethanol for preserving steroid and indoleamine hormones in bird plasma.

Endocrinological research on wild animals inhabiting remote areas has been hampered by the need to store plasma samples at subzero temperatures. In an attempt to remedy this logistical issue, we here investigate the use of ethanol as an alternative to freezing for the preservation of steroid and indoleamine hormones in avian plasma. Known quantities of the steroids 5alpha-dihydrotestosterone (DHT), testosterone, 17beta-estradiol, corticosterone, and the indoleamine melatonin were added to a stripped pool of chicken plasma. Samples were either immediately frozen at -40 degrees C, or treated with pure ethanol. Ethanol-treated samples were either immediately frozen, or-to simulate storage conditions at various field locations-left sitting at room temperature for one to two months, or incubated at 36 degrees C for one month before all treatment groups were frozen at -40 degrees C. All samples were then analyzed by radioimmunoassay. For DHT and estradiol there were no differences among treatment groups suggesting that ethanol-treatment is as effective as immediate freezing in preserving plasma steroid concentrations. For testosterone, corticosterone and melatonin ethanol-treated samples differed significantly from immediately frozen samples suggesting that caution is needed when comparing absolute concentrations of hormones between samples preserved in different ways. However, differences among ethanol-treated samples in general were small, demonstrating the feasibility of this preservation method in the field at remote locations.

Animals↗
PubMedDate not supplied

Immunoquantitation of cytochromes P450 1A and P450 2B and comparison with chlorinated hydrocarbon levels in archived polar bear liver samples.

The present study examined the utility of an immunoblot method for quantitation of cytochrome P450 isozymes in archived liver samples as a bioassay of exposure to halogenated hydrocarbons. Hepatic microsomes were prepared from 44 archived polar bear (Ursus maritimus) liver homogenates that had been stored at approximately -40 degrees C for 9-10 years and analyzed on blots probed with antibodies to rat cytochromes P450 1A1 and P450 2B1. The results revealed a positive correlation between cytochrome P450 1A and total polychlorinated biphenyl (PCB) levels in the archived liver samples, suggesting that cytochrome P450 1A was induced in polar bears by environmental exposure to PCBs.

Animals↗
PubMed2003

Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem.

Post-mortem spermatozoa recovery is an important technique for obtaining germplasm reserves from genetically valuable animals or endangered species. However, there are many factors that influence the outcome of this technique. We have studied the effect of the interval between animal's death and sperm recovery (0, 24 or 48 h) on the quality and freezability of ram spermatozoa from cauda epididymidis. Storage temperature of epididymis (room temperature or 5 degrees C) was also analysed. Spermatozoa were diluted with Tes-Tris-Fructose solution supplemented with egg yolk (10%) and glycerol (4%), and frozen using a programmable biofreezer (-20 degrees C/min). Pre-freeze and post-thaw sperm samples showed viable spermatozoa up to 48 h after the animal's death, although their quality declined significantly as post-mortem storage time increased. Epididymis sperm stored at 5 degrees C showed better motility and a lower percentage of abnormal forms than epididymis stored at room temperature after 24 and 48 h. The fertilizing ability of cauda epididymis ram spermatozoa obtained at 0 and 24h after the animal's death is similar to that of ejaculated spermatozoa. Therefore, a good protocol for post-mortem semen collection in rams when epididymal spermatozoa cannot be collected immediately, is to preserve the epididymis at 5 degrees C and process the samples in the first 24h after the animal's death.

Acrosome↗
PubMed1994

Viability of Bordetella pertussis in four suspending solutions at three temperatures.

We studied the survival of Bordetella pertussis in four suspending solutions (Casamino Acids broth, deionized water, phosphate-buffered saline, and serum inositol), subjected to three storage temperatures (4, -20, and -70 degrees C) and two freezing methods (direct freezing and fast-freezing in an ethanol-dry-ice bath). Recovery rates were higher for longer periods for suspensions stored at -70 degrees C than those stored at -20 or 4 degrees C. Serum inositol showed the highest recovery rates for all experimental conditions, followed by Casamino Acids, deionized water, and phosphate-buffered saline. Cell viability was significantly reduced in phosphate-buffered saline suspensions fast-frozen before storage. These results identify optimal conditions for storing B. pertussis cells and are applicable to the collection, transport, and storage of aspirated nasopharyngeal samples for use in the laboratory diagnosis of pertussis.

Amino Acids↗
PubMed1995

Modification of standard proteinase K/phenol method for DNA isolation to improve yield and purity from frozen blood.

Research in medical genetics may frequently involve freezing of large numbers of peripheral blood samples. This is a convenient method for storing blood for subsequent DNA isolation and analysis. An area of potential concern is the low yield of DNA from blood samples that have been frozen. Here we report a modification of the widely used standard proteinase K/phenol DNA isolation method for improving the yield and purity of DNA from frozen blood samples, by an initial trypsinisation of whole blood before cell lysis to obtain lymphocytic nuclei and subsequent DNA purification. We report an increased total yield of DNA with pretrypsinised blood as well as improved purity. These results indicate that trypsinisation of thawed whole blood helps the deproteinisation process, reducing the amount of protein associated with the nuclear pellet. This modification to improve yield and purity of DNA from frozen blood samples should be useful to laboratories performing DNA based diagnostic work or studying molecular genetic mechanisms of disease.

Blood Preservation↗
PubMed2001

Automated constant denaturant capillary electrophoresis applied for detection of KRAS exon 1 mutations.

In this study, we have applied automated constant denaturant capillary electrophoresis (ACDCE) for the detection of KRAS exon 1 mutations. Samples from 191 sporadic colon carcinomas previously analyzed for KRAS mutations with allele-specific PCR (ASPCR), temporal temperature gradient electrophoresis (TTGE), and constant denaturant capillary electrophoresis (CDCE) were analyzed. In ACDCE, an unmodified ABI Prism 310 genetic analyzer with constant denaturant conditions separated fluorescein-labeled PCR products. Temperature in combination with a chemical denaturant was used for separation. The optimal separation conditions for PCR-amplified KRAS exon 1 fragments were determined by adjusting the temperature before electrophoresis. In the ACDCE analysis, the sequence of a mutant was determined by comparing the electropherogram of the fragment to that of known mutations followed by mixing the sample with control mutations before reanalysis. In a titration experiment mixing mutant and wild-type alleles, the sensitivity for mutation detection was shown to be 0.6% in this automated CDCE technique. The automation of CDCE allowed rapid analysis of a large number of test samples over as short period of time and with a commercially available apparatus.

Autoanalysis↗
PubMed2006

Bioactivity of human chorionic gonadotropin produced in frozen-thawed embryo transfer cycles with exogenous hormone replacement.

Human chorionic gonadotropin (HCG) is the glycoprotein hormone in pregnancy. It is known that hCG molecule has a variety of isoforms showing different potency in bioactivity. Taking advantage of rat Leydig cell assay to evaluate hCG bioactivity, we first tried to predict the prognosis of pregnancy in hormone replacement (HR)-cryopreserved embryo transfer cycles. There was no significant difference in serum estradiol (E2) level, immuno-hCG, or bioactive to immunoreactive hCG ratio (b/i) between normal pregnancies and miscarriages at 4 weeks of gestation. Linear regression did not show a significant correlation in E2 or b/i between normal pregnancies and miscarriages. The same analysis was performed on serum samples collected from spontaneously pregnant patients. In normal pregnancies b/i was significantly lower than that in miscarriages while serum E2 was significantly higher. The linear regression analysis was also performed to clarify the correlation between E2 and b/i, and no significant correlation was observed. To confirm the effect of E2 on hCG production, we treated trophoblastic cells with E2. E2 increased the immunoreactivity of hCG to a non-physiologically high concentration; however, it did not change its bioactivity, suggesting that E2 did not change hCG bioactivity by affecting trophoblasts directly. In this study, we presumed it possible to speculate the prognosis of the pregnancy in spontaneous cycles from b/i of hCG, but not in HR cycles. It is suggested that process of hCG secretion could undergo different endocrine regulations between the artificial sex steroid replacement cycles and spontaneous cycles with corpus luteum function.

Animals↗
PubMedDate not supplied

The motility of epididymal or testicular spermatozoa does not directly affect IVF/ICSI pregnancy outcomes.

Our objective was to determine whether the presence of motility in surgically obtained sperm samples improves fertilization and pregnancy rates for patients undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). This was a retrospective study in a hospital-based infertility center. Sixty-seven couples with a diagnosis of azoospermia or severe oligozoospermia who had undergone a sperm retrieval procedure in conjunction with 100 IVF/ICSI cycles from 1995 to 2004 were evaluated. The impact of sperm motility on fertilization and clinical pregnancy rates was determined. The motile and nonmotile sperm groups differed in the number of mature oocytes retrieved (10.7 +/- 5.8 vs 13.4 +/- 6.0), but fertilization (56.7% vs 59.1%) and embryo cryopreservation rates (35.9% vs 39.3%) were statistically similar. Clinical pregnancy rates did not differ between the motile (38.5%) and nonmotile (31.2%) groups, nor did they differ between obstructive and nonobstructive patients (35.3% vs 26.7%). There was also no statistical difference in pregnancy rates between testicular and epididymal aspiration (35.3% vs 26.7%), although epididymal sperm were significantly more likely to be motile than testicular sperm (100% vs 39.3%, P < .0001). Epididymal aspiration is more likely to produce motile sperm than testicular sperm retrieval. The use of motile sperm from epididymal or testicular samples, however, does not appear to enhance fertilization or clinical pregnancy rates.

Adult↗
PubMed1992

A comparative analysis of cytoplasmic mu (C mu) expression in acute lymphoblastic leukemia by molecular and immunologic techniques. Identification of leukemia cases expressing C mu mRNA transcripts in the absence of detectable C mu proteins.

Among acute lymphoblastic leukemias derived from the B-cell lineage, the subset of cases expressing cytoplasmic mu heavy chain proteins (C mu) in the absence of surface immunoglobulin has been designated pre-B-cell acute lymphoblastic leukemia. This group, traditionally identified using immunologic smear techniques, has been associated with a poor prognosis in some series. In a comparative study, 25 cases of B-lineage acute lymphoblastic leukemia were analyzed for C mu expression using molecular and immunologic techniques. RNA derived from cryopreserved blast cells was hybridized in both Northern and slot-blot analyses using a probe (pBZ311) containing four exons of the human immunoglobulin heavy chain mu constant region gene. Expression of C mu proteins was assessed simultaneously by slide immunofluorescence and flow cytometric techniques in all samples. These studies were correlated with immunoglobulin heavy and light chain gene rearrangements, cell-surface immunophenotype, cytogenetics, and other clinicopathologic features. C mu mRNA transcripts were detected in 14 of 25 cases, whereas C mu proteins were detected in only 9 of these cases using flow cytometric techniques. Only four of these nine cases were positive by slide immunofluorescence techniques. These studies imply that molecular and flow cytometric techniques may be a more sensitive means to assess C mu expression. The identification of five cases that expressed C mu mRNA transcripts in the absence of detectable C mu proteins also suggests that molecular techniques may be valuable in identifying a unique subgroup of pre-B-cell acute lymphoblastic leukemia cases that contain C mu mRNA transcripts, but lack C mu proteins.

Adolescent↗
PubMed2003

Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility.

This study investigated the use of annexin-V/PI assay to assess sub lethal changes in bull spermatozoa post-thawing, and to further relate these changes to results obtained by fluorometric assessment of sperm viability and sperm chromatin structure assay (SCSA), as well as field fertility (as 56-day non-return rates, 56-day NRR) after AI. Frozen-thawed semen samples were obtained from 18 Swedish Red and White bulls (one to three semen batches/bull) and fertility data was based on 6900 inseminations. The annexin-V/PI assay revealed that post-thaw semen samples contained on average 41.8+/-7.5% annexin-V-positive cells. Most of the annexin-V-positive cells were dying cells, i.e. also PI-positive. The incidence of annexin-V-positive cells was negatively related (r=-0.59, P<0.01) to the percentage of viable cells, as detected by fluorometry. The incidence of annexin-V-positive spermatozoa significantly correlated to the SCSA variable xalphat (r=0.53, P<0.05). The incidence of annexin-V-negative, dead cells was the only annexin-V/PI assay variable that correlated significantly with fertility both at batch (r=-0.40, P<0.05), and bull (r=-0.56, P<0.05) levels. Among sperm viability variables, subjectively assessed sperm motility (r=0.52-0.59, P<0.01), CASA-assessed sperm motility (r=0.43-0.61, P<0.05), and the incidence of live spermatozoa, expressed as total numbers (r=0.39-0.54, P<0.05), or percentage values (r=0.68-0.68, P<0.01), correlated significantly with field fertility both at batch, and bull levels. Among the SCSA variables, only the COMP alphat correlated significantly (r=0.33-0.51, P<0.05) with fertility results. The results indicate a certain proportion of bull spermatozoa express PS on their surface after thawing, e.g. they have altered membrane function, and that the incidence of such cells is inversely correlated to sperm viability, and positively correlated to abnormal sperm chromatin condensation since they eventually undergo necrosis.

Animals↗
PubMed1997

Satisfactory recovery and viability of stem cells cryopreserved at high cell concentration.

Peripheral Blood Stem Cells (PBSC) are being extensively used in both oncology-haematology and in solid tumor therapy schedules. The latest generation of cell separators allow the collection of greater numbers of nucleated cells (NC) than is usually obtained in bone marrow transplantation (BMT) settings. Thus, larger volumes of components would be stored if the same NC concentration employed in BMT is to be used. In order to reduce large volume DMSO-infusion related side effects and to avoid storage problems, we froze components from 23 PBSC collections at higher NC concentrations, from 40-200 x 10(6)/mL. After 2-12 months of storage, we thawed the samples and determined if the high NC concentration had a detrimental effect on NC viability and on stem cell clonogenicity. No statistically significant differences emerged in regard to CFU-GM, CFU-GEMM and BFU-E percent recovery even at 200 x 10(6)/mL NC concentration.

Analysis of Variance↗
PubMed1996

In vitro penetration into bovine oocytes and zona-free hamster oocytes by bull spermatozoa treated with liposomes.

The objectives of this study were 1) to determine whether in vitro penetration of bovine and zona-free hamster oocytes, using spermatozoa treated with the lipid dilauroylphosphatidylcholine, was correlated with the fertility of bulls and 2) to determine whether utilizing results from several laboratory assays could effectively evaluate fertility. Cryopreserved semen was used from 12 bulls having lifetime nonreturn rates ranging from 66 to 81%. The lipid concentration that maximized penetration of hamster oocytes was determined as well as the number of spermatozoa entering all hamster oocytes, number of spermatozoa in each penetrated hamster oocyte, fertilization of bovine oocytes, blastocyst development of bovine oocytes, and the percentage of motile spermatozoa of each sample. Correlations were low between the lipid concentration that maximized each individual parameter and nonreturn rate (r < or = 0.34). The six-parameter regression equation accounted for 71% of the variation in bull fertility, and a four-parameter equation accounted for 61% of the variation in fertility. Pooling data for pairs of bulls into six fertility groups to increase information per data point still resulted in poor predictions of fertility for individual parameters. However, a four-variable model, including the penetration rates for hamster and bovine oocytes, the number of spermatozoa that penetrated hamster oocytes, and the percentage of motile spermatozoa, accounted for 98% of the variability in bull fertility.

Animals↗
PubMed2003

Live-birth rates and multiple-birth risk of assisted reproductive technology pregnancies conceived using thawed embryos, USA 1999-2000.

BACKGROUND: Increasing use of assisted reproductive technology treatments has been associated with the current rise in multiple births in the USA. Embryo cryopreservation and subsequent thawed embryo transfer may favourably impact the multiple-birth risk by relieving some pressure that patients and providers may feel to transfer several embryos in a single cycle. The study objective was to examine both live-birth rates and multiple-birth risk in thawed cycles. METHODS: The authors used a population-based sample of 21 555 assisted reproductive technology procedures performed in US clinics in 1999 and 2000 that used thawed embryos derived from the patient's oocytes. RESULTS: Both patient age and the number of embryos transferred were independent predictors of live birth. Even among women aged 20-29 years, the transfer of three embryos resulted in an increase in the live-birth rate compared with cycles in which one or two embryos were transferred. This increase in success was accompanied by an increased multiple-birth risk. In all age groups up to 40 years, the transfer of just two embryos resulted in a multiple-birth risk of 16-17%. The multiple-birth risk increased with the number of embryos transferred. CONCLUSIONS: Patient age and the number of embryos transferred significantly affect live-birth and multiple-birth rates among women who use thawed embryos.

Adult↗
PubMed1997

Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture.

Ovarian cortical tissue was obtained during gynaecological operations by biopsy or after oophorectomy from 20 women aged 25-42 years. It was placed in organ culture, either fresh or following thawing after cryopreservation, for 1-4 months. The tissue was cut in slices 0.1-0.3 mm in diameter and transferred to 12 mm inserts in 24-well culture plates. These slices were cultured for 4-21 days in either alpha minimum essential medium (alpha-MEM) or Earle's balanced salt solution with added pyruvate. Both media were supplemented with 10% human serum, insulin, gonadotrophins and antibiotics. Half of the inserts were precoated with extracellular matrix (Matrigel). Histological samples revealed that there were viable, non-atretic, primordial, primary and secondary follicles in all the cultures. Mitoses were seen in the granulosa cells of the secondary follicles. Although the proportion of atretic follicles increased during culture, non-atretic follicles were still present after 21 days. After 4-11 days the proportion of viable follicles was significantly higher when cultured in Earle's solution supplemented with pyruvate, than when cultured in MEM (77 versus 38%, P < 0.001). In cultures with extracellular matrix the proportion of viable follicles was significantly higher after 10-15 days than it was without matrix (85 versus 19%, P < 0.001). Culture after thawing frozen ovarian tissue did not affect the density or the proportion of the viable follicles. Two-thirds of follicles in cryopreserved tissue were viable after 10-15 days in culture. The results indicate that it is possible to culture human primary and primordial follicles in vitro, and follicles in cryopreserved tissue are viable.

Adult↗
PubMed2007

Microindentation test for assessing the mechanical properties of cartilaginous tissues.

Mechanical properties of the fresh control, frozen, and vitrified cartilaginous (cartilage and meniscus) samples were measured by microindentation. Indentation depth, elastic modulus, and indentation yield strength were obtained from the microindentation loading curves. Indentation deformation behavior was studied using Hertz contact model. The stress distribution of cartilaginous tissues under indentation loading was analyzed by finite element technique. It was found that fresh tissue shows the lowest indentation depth and the highest elastic modulus and indentation yield strength, followed by vitrified and frozen tissues. The vitrified tissue shows slightly lower but comparable mechanical properties with control tissue. The vitrification technique used in this study can preserve live cells with superior mechanical properties that make it an ideal technique for use in orthopedic and other biomedical applications. The microindentation tests and corresponding analysis methods used in this study offer a simple way to evaluate the mechanical properties of cartilaginous tissues. It suits small sample sizes and it may be used for other biological tissues.

Animals↗