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PubMedDate not supplied

Semen of spinal cord injured men freezes reliably.

The objectives of the present study were to: 1) determine the effect of cryopreservation on the percent and the grade of motility of sperm from spinal cord injured (SCI) men and 2) determine which method of freezing yields the best post-thaw motility in sperm from SCI men. Antegrade semen samples were obtained from 9 SCI subjects and 10 age-matched healthy control subjects. Motility in fresh samples was determined and cryopreservative medium was added to each sample. Aliquots of each sample were frozen according to three methods: 1) liquid nitrogen vapor only (V); 2) vapor for 12 minutes followed by submersion into liquid nitrogen (V+N2); and 3) direct submersion into liquid nitrogen (N2). Samples were frozen for 1 week, then thawed. The post-thaw percent and grade of motility was determined. The mean percent motility of fresh samples for SCI subjects (21.0%) was significantly lower than for control subjects (55.7%). After thawing, the mean percent drop in motility for V, V+N2, and N2 for controls was 65.2%, 73.5%, and 79.4%, respectively, and for SCI subjects, it was 64.7%, 74.5%, and 81.6%, respectively. There was no statistically significant difference between control and SCI subjects by method of freezing. Vapor only as a freezing method was superior to all other methods for retention of sperm motility in both control and SCI subjects. We conclude that the semen of SCI men may be frozen reliably and that their sperm retain motility similar to that of normal men. Vapor only, being the most gentle method used, gives the best recovery of sperm motility in either group.

Adult↗
PubMed2000

Comparison of three polymerase chain reaction methods for routine detection of bovine herpesvirus 1 DNA in fresh bull semen.

Five bulls were inoculated intrapreputially with Bovineherpes virus 1 (BHV 1), in order to compare the relative sensitivity of three polymerase chain reaction (PCR) assays for routine diagnosis of fresh bovine semen for the presence of BHV 1 Semen was collected twice a week up to 107 days post-infection (dpi). To reactivate latent virus, the bulls were treated with dexamethasone from 44 until 48 dpi. All samples were examined before and after cryopreservation treatment using a standard virus isolation (VI) method and three PCR assays: PCR A, PCR B and PCR C. PCR A and PCR C used an internal control plasmid DNA template and PCR B used the split sample method in order to control for false negative results. Of the 149 fresh semen samples that were tested, PCR A detected 45 positive, PCR B detected 39 positive and PCR C detected 66 positive, while virus was isolated from 22 samples. Of the 149 samples treated by cryopreservation, the virus was isolated from 13 samples and PCR C was positive in 21 samples. The results demonstrate that all three PCR assays are more sensitive than virus isolation, particularly during the later phases of infection.

Animals↗
PubMed2005

Isolation, cryopreservation, and mitogenesis of peripheral blood lymphocytes from chickens (Gallus domesticus) and wild herring gulls (Larus argentatus).

Monitoring the toxicity of environmental contaminants on the physiologic function of wild birds often includes measures of immune function. The purpose of this study was to apply methods of isolation, cryopreservation, and cell culture of chicken lymphocytes to blood samples from herring gulls, which are a sentinel species for biomonitoring studies in the Great Lakes and northern North America. Slow-spin centrifugation and density gradient isolation of lymphocytes were compared using chicken blood. Significant thrombocyte contamination of density gradient isolated samples (40% to 77% thrombocytes) led to the use of slow-spin centrifugation (2% thrombocytes) for blood from herring gulls. Cryopreserved blood samples were collected from adult and prefledgling herring gulls at sites of low environmental contamination around the Great Lakes and the Bay of Fundy between 1999 and 2002. Cryopreservation decreased the viability of lymphocytes from wild birds, but a high proportion of samples yielded enough live lymphocytes to assess function in culture. Cryopreserved lymphocytes from herring gulls proliferated in response to phytohemagglutinin (PHA), concanavalin-A, phorbol myristate acetate (PMA), PHA plus PMA, and lipopolysaccharide stimulation. Weber and Roswell Park Memorial Institute medium (RPMI) 1640 media were compared for culture of lymphocytes. Weber medium better supported chicken B-lymphocyte proliferation than RPMI 1640 and supported chicken T-lymphocyte proliferation of a similar magnitude as RPMI. Proliferation responses for lymphocytes from prefledgling herring gulls were stronger in Weber medium than RPMI medium, especially for PHA, for which there was no stimulation in RPMI. Proliferation responses of lymphocytes from adult herring gulls were up to twofold greater than that for prefledgling herring gulls. The magnitudes of proliferation responses were similar to that for chicken lymphocytes. These methods have subsequently proven useful in ecotoxicology studies that involve species in remote locations.

Animals↗
PubMed1987

CFU-GEMM correlate with neutrophil and platelet recovery in patients receiving autologous marrow transplantation after high-dose melphalan chemotherapy.

CFU-GEMM, multipotent progenitor cells, give rise to erythroid, granulocyte-macrophage and megakaryocytic cells. We have examined the utility of this assay for predicting hematologic recovery in patients treated with high-dose chemotherapy and autologous marrow rescue with cryopreserved marrow. In marrow samples from 50 patients CFU-E, BFU-E and CFU-GEMM significantly decreased following cryopreservation. The median value for CFU-E declined from 132,659 to 10,648, BFU-E decreased from 36,112 to 3345 and CFU-GEMM decreased from 3242 to 260 colonies per ml of marrow frozen. Once cryopreserved, the number of CFU-E, BFU-E and CFU-GEMM remained stable with prolonged storage in liquid nitrogen. In 48 patients uniformly treated with high-dose melphalan (180 mg/m2) and rescued with cryopreserved autologous marrow, univariate analyses showed that the time to platelet recovery (greater than 50 x 10(9)/l) was correlated with the number of CFU-E, BFU-E and CFU-GEMM infused (p less than 0.05). The time to neutrophil recovery was only correlated with the number of BFU-E and CFU-GEMM infused (p less than 0.01). However, by multivariate analyses, only the number of CFU-GEMM, and not CFU-E and BFU-E, infused correlated both with the time to neutrophil and platelet recovery. These data indicate that the CFU-GEMM assay may be useful for determining the repopulating ability of cryopreserved bone marrow.

Bone Marrow↗
PubMed1997

HIV-1 detection by nested PCR and viral culture in fresh or cryopreserved postmortem skin: potential implications for skin handling and allografting.

AIMS: To date, the risk relating to the handling or allografting of human immunodeficiency virus type 1 (HIV-1) infected postmortem skin remains hypothetical. While blood screening for HIV antibodies is still the key safety procedure to detect HIV infected cadavers, false negative results are a concern. Conversely, false positive results may hamper the collection of skin allografts. Accordingly, viral culture was used to clarify skin infectivity and the nested polymerase chain reaction (PCR) was used to assess the reliability of skin PCR testing. METHODS: Viral culture and nested PCR performed with gag and pol specific primers were investigated in cadaveric skin and blood from 12 HIV-1 infected patients. Samples were collected repeatedly between one and five days in seven patients. In most cases, analyses were performed on triplicate skin samples: fresh (n = 26); cryopreserved in 5% dimethylsulphoxide (n = 21), or cryopreserved in 30% glycerol (n = 26). RESULTS: HIV was isolated in two of 26 cultures of fresh skin specimens (8%), seven of 47 cryopreserved skin specimens (15%), and eight of 26 blood specimens (31%). The nested PCR detected HIV-1 in all skin samples (n = 73), regardless of the postmortem interval or cryopreservation. In blood, a positive signal was found in eight of 12 patients but two of them had discordant results on successive samples. CONCLUSIONS: These data suggest that nested PCR on postmortem skin samples can detect HIV more reliably than on blood. They also demonstrate the potential viral infectivity of fresh or stored skin postmortem samples in HIV infected patients. They underscore the need for caution during the handling of skin tissue from HIV infected cadavers and confirm the potential risk related to accidental allografting of HIV contaminated skin.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Cryopreservation of semen from pubertal boys with cancer.

BACKGROUND: The possibility of cryopreservation of semen from adolescents has until now received only little attention. Therefore, we have investigated the possibility of cryopreservation of semen in adolescent boys with cancer. PROCEDURE: Forty-five boys, aged 13-18 years, admitted because of cancer during the period January 1, 1995 to July 31, 1998 were eligible. Semen was obtained after masturbation in the majority of the cases. In three boys, semen was preserved after penile vibration or electroejaculation in general anaesthesia. The semen samples were analysed for concentration, motility, and morphology according to the WHO guidelines. The sample was transferred into straws prior to cryopreservation at 196 degrees C in liquid nitrogen. RESULTS: Twenty-one boys delivered a semen sample for cryopreservation. Four boys were offered and accepted sperm banking but were not able to produce a sample. In 20 cases time did not allow an attempt of sperm banking, the boy was not assessed to be mature enough to deliver a semen sample, or the procedure was not accepted. The boys delivered 1-3 samples, and the total number of spermatozoa ranged from 0-210 millions. Median percentage of motile sperm was 50% (range 9-86%). Semen quality improved with age; however, a 13- year- old boy produced 75 million spermatozoa with 38% motile cells. CONCLUSIONS: Pubertal maturation should be assessed in all boys admitted for cancer, and the possibility of sperm banking should be discussed with the patient and his parents.

Adolescent↗
PubMed2000

The use of buck and ram extenders and two packaging systems to cryopreserve aoudad (Ammotragus lervia) spermatozoa.

Because the aoudad has been hunted to near extinction, cryopreservation of their semen would be useful for DNA conservation and for the possible re-establishment of captive bred animals to their former ranges. This study was conducted to investigate the effectiveness of cryopreserving aoudad spermatozoa. Semen samples from four post-pubertal animals were collected using electro-ejaculation. Microscopic analysis was performed to assess the percentages of progressively and non-progressively motile spermatozoa as well as intact acrosomes in samples prior to freezing and post-thaw. Extended samples (0.2 mL) were frozen using 2 different extenders and packaging systems and stored in LN2 Post-thaw data were arcsine-transformed and analyzed using ANOVA, 2 x 2 factorial. Samples that were processed using the ram/straw method had a significantly higher percentage (P < 0.05) of spermatozoa with intact acrosomes than did any other system. In addition, samples that were processed with the buck/pellet system had significantly greater percentages (P < 0.05) of progressive and non-progressively motile spermatozoa than the samples processed using either extender and packaged in straws. This study illustrates that some aoudad spermatozoa may be cryopreserved using the extender/processing systems developed for the domestic buck and ram.

Animals↗
PubMed2005

Bovine embryo development after IVF with spermatozoa having abnormal morphology.

The study was conducted to evaluate the effects of scrotal insulation on semen samples collected from bulls on embryonic development after IVF. Semen samples were obtained and cryopreserved from four Holstein bulls before and after a scrotal insulation period of 48 h (Day 0). Three types of samples were used for IVF: (1) semen from the test bulls collected 5 d prior to scrotal insulation (pre-insult); (2) semen from Day 13 (2-week post-insult; 2-week PI); and (3) semen from Day 20 (3-week PI). After 18 h of sperm-oocyte co-incubation, the zygotes were cultured for 8 d when a developmental score (0=degenerate, 1=2-cell embryo through 5=blastocyst) was assigned to each embryo. The post-thaw morphological evaluation of sperm samples revealed a decrease (P<0.01) in the percentages of normal spermatozoa in the 3-week PI samples in comparison with the pre-insult samples for Bulls I and III (74-22.3% and 67.7-0.5 %, respectively). The percentage of vacuolated spermatozoa increased significantly for Bull II. The cleavage and blastocyst formation rates and embryo development scores were affected (P<0.01) by the interaction of bull by sample collection time. For Bulls I and III (severe responders) the scrotal insulation effects persisted from the time of cleavage through blastocyst formation. In contrast, the cleavage and blastocyst formation rates for Bulls II and IV were unaffected, despite high percentages of vacuolated spermatozoa present in the post-insult samples for Bull II. In conclusion, the use of scrotal insulation to elevate scrotal temperature was an effective method to obtain semen samples with high percentages of abnormal spermatozoa. The decrease in embryonic development after IVF when using spermatozoa with morphological abnormalities seemed to be multifaceted and related to changes in head morphology.

Acrosome↗
PubMed1997

Clonogenic capacity and ex vivo expansion potential of umbilical cord blood progenitor cells are not impaired by cryopreservation.

Umbilical cord blood (UCB) progenitor cells have been demonstrated to possess significant advantages over bone marrow (BM), in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been recently considered an attractive potential alternative to BM as a source of hematopoietic progenitor cells for clinical applications. Since several programs throughout the world are currently evaluating the feasibility of large-scale UCB banking for unrelated transplants, it was the aim of this study to evaluate whether cryopreservation procedures might heavily impair the clonogenic capacity, the feasibility of CD34+ selection and the ex vivo expansion potential of UCB progenitor cells. UCB samples were collected and cryopreserved as unseparated (n = 21) or mononuclear (MNC) cells (n = 15) within 12 h from delivery, and evaluated for viability, immunophenotype, cell and progenitor numbers after a minimum stay in liquid nitrogen of 6 months (range 6-14 months). Viability was always > 97% and no statistically significant difference was detected by flow cytometric analysis. Clonogenic recovery from unseparated cells was 80-87% for HPP-CFC, CFU-GEMM, BFU-E and CFU-GM, and from MNC cells ranged from 82 to 91% for LTC-IC, CFU-GEMM, BFU-E and CFU-GM. CD34+ selection (n = 8) was performed on fresh and cryopreserved MNC cells using the MiniMACS immunomagnetic separation device, showing no difference in yield (68 +/- 7% vs 57 +/- 4%, P < or = 0.4) or in purity (89 +/- 2% vs 81 +/- 6%, < or = 0.4), for fresh in comparison to cryopreserved MNC cells. After 14 days of liquid culture in the presence of different combinations of SCF, IL-3, IL-6 and G-CSF no statistically significant difference was detected in CFC fold-expansion for fresh or cryopreserved MNC cells and for CD34+ cells, either selected and cultured from fresh or cryopreserved MNC cells. In conclusion we can state that UCB is a potential source of primitive progenitor cells that can be cryopreserved unmanipulated or after physical separation without major losses in clonogenic capacity and immunophenotypic composition. Moreover, CD34+ selection from cryopreserved MNC cells is feasible and ex vivo expansion is not impaired. These results have important implications in the large scale UCB banking, in view of the potential applications of ex vivo expanded hematopoietic progenitor cells for the engraftment of adult patients.

Antigens, CD34↗
PubMed2006

The "in situ" expression of human leukocyte antigen class I antigens is not altered by cryopreservation in human arterial allografts.

This study was aimed to establish whether the cryopreservation procedure we currently use in clinics can modify arterial homograft antigenicity. To this purpose, we performed an immunohistochemical study on fresh and cryopreserved human arterial homografts to visualize the expression of HLA class I heavy and light chains "in situ" by using the HC-10 and Namb-1 monoclonal antibodies. Human femoral arteries and thoracic aortas were harvested from 18 heart-beating donors and sampled before and after cryopreservation. Arterial segments were frozen in liquid nitrogen vapors in a controlled rate freezing system. After thawing, samples were processed for routine immunohistochemistry. To standardize immunostaining, flow-cytometry indirect immunofluorescence analysis was performed on HUVEC; immunohistochemistry of human ovarian cortical vessels was performed as an additional positive control. Negative controls were performed by omitting tissue incubation with primary antibodies. HLA-class I antigens were markedly expressed by endothelial cells lining surface intima and adventitial vasa vasorum; a moderate expression was found in medial smooth muscle cells. Except for the surface unreactivity caused by loss of endothelium, results from cryopreserved arterial allografts were strictly comparable to those observed in fresh, unfrozen tissues. These results support the view that cryopreserved arterial allografts are immunogenic as their fresh counterparts; apart from smooth muscle cells which retained a moderate expression of HLA class I antigens following cryopreservation, our study suggests that the highly HC-10 positive endothelial cells we found to line the rich adventitial network of vasa vasorum are expected to be one of the major targets of the serological response in the recipient.

Adult↗
PubMed1997

Immunomagnetic selection of purified monocyte and lymphocyte populations from peripheral blood mononuclear cells following cryopreservation.

Cryopreservation is a method commonly used to store human blood samples. We sought to determine if cryopreserved peripheral blood mononuclear cells (PBMC) could be separated effectively into distinct populations by using monoclonal antibodies and immunomagnetic microspheres. PBMC obtained from healthy blood donors and from human immunodeficiency virus-infected subjects were cryopreserved for as long as 18 months. Recovered cells were separated into CD14+ monocytes and CD4+ T-cell subsets by immunomagnetic selection. Flow cytometry analysis indicated >95% depletion of monocytes from PBMC following immunomagnetic selection with anti-CD14. A highly enriched population of CD4+ T cells was obtained from the CD14-depleted cell fraction by using an anti-CD4 monoclonal antibody and detachable immunomagnetic beads. The CD4+ T cells were subsequently separated into CD4+ CD45RO and CD4+ CD45RA fractions. Each fraction contained >90% enrichment for the respective subpopulation and <5% of the reciprocal subpopulation. No significant differences in cell surface expression of leukocyte markers, in efficiency of selection of PBMC subpopulations, or in mitogen-induced proliferation were detected in freshly isolated or cryopreserved cells. Efficient recovery of cryopreserved specimens means that targeted assays can be performed on selected, prospectively stored samples once clinical endpoints have been achieved.

Antibodies, Monoclonal↗
PubMed1999

In vitro interactions of cryopreserved stallion spermatozoa and oviduct (uterine tube) epithelial cells or their secretory products.

Formation of a spermatozoa ('sperm') reservoir in the mare is thought to occur through lectin-mediated sperm attachment to the oviductal epithelium. Once attached, prefertilization sperm survival is supported by oviductal factors. Cryopreservation of stallion sperm decreases the number of sperm attaching to oviduct epithelial cells (OEC) and the length of time these sperm survive. Quantification of in vitro interactions between sperm and OEC in a co-culture system may provide an assay for functional integrity of cryopreserved or fresh sperm samples. Additionally, superior additives for in vitro handling of stallion sperm may be isolated from OEC secretory products. Experiment 1 compared first service conception (FSC) rates resulting from the use of cryopreserved sperm of seven stallions, with sperm function in co-culture such as attachment to OEC and subsequent survival time. Stallions were grouped by cumulative FSC rates observed over three seasons as having average (44 +/- 3%) or high (65 +/- 2%) fertility over a total of 217 first services (31 +/- 9 per stallion). Samples from stallions in the high fertility group had more (P = 0.04) sperm attached to OEC and longer subsequent sperm survival in co-culture (P = 0.05) as compared with those from the average fertility group. FSC rates correlated with numbers of sperm attaching to OEC and their survival time in co-culture (r > or = 0.71). In Experiment 2, the function of cryopreserved stallion sperm was evaluated in culture with OEC secretory products from three different sources. After 5 h of culture, sperm incubated with medium conditioned by bovine OEC which had been 'bioactivated' (e.g. previously exposed to sperm in culture) were found to be more (P < or = 0.05) motile and capacitated as compared to sperm in basal TALP medium alone. Sperm in this conditioned medium also survived longer (P = 0.05; 27 +/- 5 h vs. 17 +/- 4 h) than did those in control medium.

Animals↗
PubMed2006

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study.

BACKGROUND: Cryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and peripheral blood mononuclear cells. RESULTS: Paired samples of PBMC were processed freshly, or after cryopreservation in a Nalgene container (NC) or a controlled-rate freezer (CRF). Although there were some differences between the methods, cryopreservation inhibited PHA-induced IL-10 secretion and Der f 1-induced IL-2 secretion, and augmented PHA-induced IL-2 secretion and spontaneous secretion of TNF-alpha. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-gamma, TNF-alpha) by PHA-stimulated cord blood mononuclear cells. With the exception of PHA-induced IL-13, results from fresh and cryopreserved cord blood samples were not significantly correlated. Finally, in reproducibility studies involving processing of identical cell samples in up to 4 separate laboratories, variances in cytokine responses of fresh cells stimulated at separate sites did not exceed those in cryopreserved cells stimulated at a central site. CONCLUSION: Collectively, these studies indicate that cryopreservation can affect mononuclear cell cytokine response profiles, and that IL-10 secretion and antigen-induced responses may be especially vulnerable. These studies also demonstrate that mononuclear cell responses can be standardized for performance in a small number of laboratories for multicenter studies, and underscore the importance of measuring reproducibility and of testing whether cryopreservation techniques alter specific immunologic outcomes.

Algorithms↗
PubMed1996

Long-term storage of tissues by cryopreservation: critical issues.

The technique of cryopreservation (maintenance of biological samples in a state of 'suspended animation' at cryogenic temperatures), its potential use in tissue engineering applications and current obstacles to the development of effective cryopreservation methods for tissues are reviewed. A didactic overview of the principles of cryobiology and the methodology of cryopreservation is given, with emphasis on the processes of injury to cells during freezing and thawing, and how these are related to the physicochemical and biophysical changes occurring during cryopreservation. Critical issues relevant to the application of cryopreservation methods to tissues are then addressed, including heat and mass transfer limitations in these bulk systems, intrinsic differences between isolated and cultured cells, and mechanisms of freezing injury unique to tissue systems.

Animals↗
PubMed1997

Influence of human tooth cryopreservation on dentin bond strength.

OBJECTIVES: The goal of this study was to evaluate the effects of cryopreservation of teeth on dentin bond strength as a function of remaining dentin thickness. METHODS: Flat occlusal surfaces of human dentin were prepared in 54 freshly extracted teeth and 54 thawed, cryopreserved teeth. In each group, 18 bonds were performed in superficial dentin, 18 in mid-coronal, and 18 in deep dentin. A resin composite cylinder, 3 mm in diameter and in height, was bonded orthogonally to the surface. After storage in distilled water at room temperature for 1 wk, the bonded cylinders underwent shear testing at a crosshead speed of 0.5 mm min-1. The mean remaining dentin thickness was calculated after longitudinally sectioning the debonded samples through the center of the bonded area. Non-parametric statistical analyses were used to correlate the shear bond strength with the remaining dentin thickness among the storage modes and within the different dentin regions. RESULTS: The lowest shear bond strength values were found in the deep dentin of both fresh and cryopreserved dentin, while the values in deep and mid-coronal dentin were not significantly different in fresh and cryopreserved dentin. In the superficial and mid-coronal dentin of cryopreserved samples, the shear bond strength values were identical. There was a significant difference between the shear bond strength values in the superficial dentin of fresh teeth compared to the values for cryopreserved teeth. SIGNIFICANCE: According to the experimental conditions, tooth cryopreservation shows some promise as a substitute for freshly extracted teeth, provided that the experiments are performed in midcoronal and deep dentin.

Composite Resins↗
PubMed1999

Modified cryopreservation and xenotransplantation of human parathyroid tissue.

INTRODUCTION: A modified cryopreservation technique for human parathyroid tissue was compared with the standard method using a programmed freezer. METHODS: Total parathyroidectomy was performed in three groups of 6-week-old Rowett nude rats. Group I (control) underwent no transplantation of parathyroid tissue (n=9). After 10 days, the rats of groups II (n=15) and III (n=15) underwent xenotransplantation of 20 mg cryopreserved human parathyroid tissue, which had been stored in liquid nitrogen at -196 degrees C for 1-22 months prior to xenotransplantation. The parathyroid tissue was derived from 15 parathyroidectomized patients with renal hyperparathyroidism. Two tissue samples were obtained from every patient. One sample from every patient was cryopreserved by means of the technique of programmed cryopreservation: programmed freezing of human parathyroid tissue at a controlled rate of -1 degrees C/min to -80 degrees C prior to transfer to liquid nitrogen (group II). The second sample from every patient was cryopreserved by means of a modified cryopreservation technique: immediate placement of human parathyroid tissue in a freezer at -20 degrees C and transfer to liquid nitrogen after 2 h (group III). Calcium and intact parathyroid hormone concentrations in serum were determined over a period of 60 days. Furthermore, individual differences in the calcium concentrations were assessed for each rat on the basis of the calcium levels recorded preoperatively and at day 60. RESULTS: All animals in the control group developed hypocalcemia. Serum calcium concentrations returned to normal levels 60 days after xenotransplantation of parathyroid tissue, which had been cryopreserved using the modified technique (group III) in 12 of 15 rats (80%). At day 60, serum calcium had normalized in 10 of 15 rats (67%) after xenotransplantation of parathyroid tissue cryopreserved using programmed freezing (group II). After modified cryopreservation (group III), the median individual difference in the calcium concentrations was -0.16 mmol/l after programmed freezing (group II). At the end of the study, a median level of human intact parathyroid hormone of 3.5 pg/ml and 2.4 pg/ml was estimated for groups II and III, respectively. There was no statistical significant difference of the individual differences in the calcium concentrations or of the levels of human intact parathyroid hormone between groups II and III. CONCLUSION: Human parathyroid tissue was successfully xenografted in the present experimental study. The results obtained after cryopreservation using the described modified technique were equivalent to those recorded after controlled freezing in a programmed freezer. The simplified cryopreservation technique therefore appears to be suitable for human parathyroid tissue.

Animals↗
PubMed2003

Effects of cryopreservation on lymphocyte immunophenotype and function.

Cryopreservation of isolated peripheral blood mononuclear cells (PBMCs) for phenotypic and functional analyses is considered a standard procedure in order to minimize operator-dependent inter-assay variability and to optimize the use of available resources. However, only few and somewhat conflicting data are presently available on the effects of cryopreservation on PBMCs, especially in samples from HIV-infected patients in which assessment of lymphocyte phenotype and function is of the outmost importance. In this study, we compared fresh versus frozen/thawed (F/T) samples isolated from 19 healthy individuals and 21 HIV-infected patients, showing that cryopreservation induces: (i) a profound decrease of CD62L expression, with a consequent significant decline of the calculated proportions of "naïve" (CD45RA+CD62L+) and "central memory" (CD45RO+CD62L+) T cells; (ii) an increase of the calculated proportions of "effector" CD8+ T cells (CD45RA+CD62L- and CD45RO+CD62L-) in the healthy subjects, while no changes were observed in the HIV-infected group; (iii) a significant decline of CC chemokine receptor 5 (CCR5) expression; (iv) a loss of proliferative responses to some HIV antigens (i.e. p24) and recall antigens [cytomegalovirus (CMV) and Influenza] in HIV-infected patients. We thus conclude that cryopreservation induces a consistent set of changes in PBMCs from both healthy and HIV-infected individuals, and that certain immunological studies of HIV-infected patients (i.e. studies of immune reconstitution following antiretroviral therapy in HIV-infected patients or studies of HIV-infectivity in vitro using CCR5-tropic strains) should be performed on fresh samples.

Cryopreservation↗
PubMedDate not supplied

Cryopreservation of mango (Mangifera indica L.) embryogenic cultures.

Three techniques were compared for cryopreserving embryogenic masses (EMs) sampled from mango (Mangifera indica L.) cv. Zihua embryogenic cultures: (i) encapsulation/dehydration; (ii) pregrowth/dehydration; and (iii) vitrification. In all experiments, EMs were sampled from embryogenic cultures during their exponential growth phase and pretreated for 24 h on solid medium containing 0.5 M sucrose before freezing. No recovery was achieved after cryopreservation using the encapsulation/dehydration technique, whatever the moisture content (fresh weight basis) of EMs, which ranged from 78.3% without dehydration to 40.8% after 6 h dehydration. With the pregrowth plus dehydration technique, limited recovery (8.3%) was achieved after desiccation of EMs for 1 h, to 58.5% MC. Using the vitrification technique, recovery ranged from 94.3% after treatment of EMs with the PVS3 vitrification solution for 20 min (EM moisture content of 34.7%) to 10.9% after a 120 min treatment with the vitrification solution (EM moisture content of 26.0%).

Cryopreservation↗