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PubMed1988

Cryopreserved human fetal pancreas: a source of insulin-producing tissue?

Human fetal pancreata (HFP) were obtained from dilatation and extraction aborted fetuses of 11-18 weeks' gestation. The pancreas was excised under sterile conditions and kept in culture medium at 4 degrees C, prior to stepwise digestion into 50- to 150-micron fragments. The fragmented pieces were allowed to sediment by gravity, then transferred to tissue culture for 24-48 h, and cryopreserved. The freeze-thaw protocol used stepwise equilibration with dimethyl sulfoxide, nucleation of the sample at -10 degrees C, and a slow cooling rate of 0.25 degrees C/min to -40 degrees C, followed by submersion in liquid nitrogen (-196 degrees C). Rapid thawing at 300 degrees C/min from -196 degrees C was employed. Both fresh and frozen-thawed HFP fragments appeared viable as judged by light and electron microscopy, and secreted insulin in a perifusion system upon stimulation with glucose (28 mM) and theophylline (10 mM) or glucose (2.8 mM) and theophylline (10 mM). Six patients with Type I insulin-dependent diabetes mellitus, already requiring immunosuppression for a kidney transplant, had intraportal injection of 20 cryopreserved-thawed and pooled HFP fragments. Up to the 1-year post-transplant follow-up, there has been no evidence of in vivo insulin or C-peptide production. The usefulness of cryopreserved human fetal pancreata as a source of insulin-producing tissue for diabetic patients, therefore, remains to be demonstrated.

Abortion, Spontaneous↗
PubMedDate not supplied

[Examination of the fertility of patients with malignant testicular cancer at the time of orchiectomy. The question of the opinion on cryopreservation].

Semen analyses from 29 patients with testicular tumours were done, 19 were examined before and after orchidectomy but before any other treatment. There was no evident difference in both of these examinations. Therefore it is possible to obtain enough time for cryopreservation, because the stricken patients can be first treated by unilateral orchidectomy without loss in quality of the semen. Hendry et al. (1983) accepted for sperm freezing only samples with more than 10 mill/ml spermatozoa and more than 30% motility. 17 of our 29 patients (58.6%) fulfilled that condition. Thus, in more patients cryopreservation can be considered.

Fertility↗
PubMed1991

Successful cryopreservation of human bone marrow does not require a controlled-rate freezer.

We studied CFU-GM recovery from bone marrow samples frozen either in a machine for controlled-rate freezing or in a -70 degrees C freezer. We found no statistically significant difference between the two methods. Cooling-curve investigations also demonstrated that it was possible to obtain satisfactory cooling rates simply by manipulation of the shape and volume of the marrow and its container. Five patients received a total of six successful autografts for which their marrow cells were frozen without controlled-rate freezing. Thus it is not necessary to use a sophisticated machine in order to obtain satisfactory cryopreservation of human marrow stem cells.

Bone Marrow↗
PubMed2005

Evaluation of hematological reconstitution potential of autologous peripheral blood progenitor cells cryopreserved by a simple controlled-rate freezing method.

A novel and simple procedure for the controlled-rate cryopreservation of peripheral blood progenitor cells (PBPCs) was introduced. A freezing bag housed in a protective aluminum canister was placed on top of a styrene foam box in the -85 degrees C electric freezer. A second set of samples was kept in cryotubes placed in a double styrene foam box in the same electric freezer. Measurement of the freezing rate in the PB bags and cryotubes demonstrated that this simple method for PBPC cryopreservation provided optimal conditions for both large-scale and small-scale cryopreservation. Within several days after autologous peripheral blood stem cell transplantation, we thawed the cells in the small sample tubes and evaluated the cell viability, the cell recovery, and the recovery rates of hematopoietic progenitor cells (HPCs), such as CD34+ cells and colony-forming unit-granulocyte/macrophage (CFU-GM) colonies. The median duration of cryopreservation was 59 days (range, 14-365 days). According to our analysis, infusions of more than 2 x 10(6) CD34+ cells/kg body weight and 0.5 x 10(6) CFU-GM colonies/kg body weight after thawing had favorable influences on the neutrophil engraftment. We have therefore established a simple freezing method for cryopreservation of human PBPCs, which ensures the transplantability of hematopoietic progenitors even after thawing. In vitro HPC assay after thawing is important to evaluate the quality of cryopreservation procedures.

Adolescent↗
PubMed2003

The effect of cooling rate on the survival of cryopreserved bull, ram, and boar spermatozoa: a comparison of two controlled-rate cooling machines.

Spermatozoa from three species, bovine, ovine, and porcine, were frozen using standard techniques in two controlled-rate cooling machines, a commercial instrument and a custom-built device. Ice crystallisation was induced mechanically by touching the straws with a pre-cooled rod. The sperm samples were stored 24h, and then thawed rapidly and evaluated for motility, viability, and acrosomal integrity in the membrane-intact population. The custom-built controlled-rate cooling machine proved significantly better at all cooling rates for all species. This was particularly evident for the ram and the boar spermatozoa. In general, -30 or -50 degrees C/min were better than -1 degrees C/min, with a slight advantage being evident for -30 degrees C/min. However, this became very apparent for boar spermatozoa. It is clear that the higher cooling rates are necessary for successful freezing of spermatozoa from these species, and that careful control of the cooling rate is essential for maximal recovery of viable and functional cells. This is best achieved when the cooling profile is controlled from within a dummy sample.

Animals↗
PubMed1998

Cryopreservation of single-donor platelets with a reduced dimethyl sulfoxide concentration by the addition of second-messenger effectors: enhanced retention of in vitro functional activity.

BACKGROUND: The potential for bacterial contamination limits the storage of platelets at 22 degrees C to 5 days. This creates an inventory problem, which could be overcome by the use of cryopreservation to allow long-term storage of platelets. It has been demonstrated that the addition to platelets of a mixture of second-messenger effectors (platelet storage solution), allows these cells to retain significant in vitro functional activity following cold storage. Analysis is needed of the ability of this second messenger effector mixture both to protect platelets during cryopreservation and to reduce the need for a cryoprotectant. STUDY DESIGN AND METHODS: Fresh single-donor platelet units (n = 8) were divided into three samples and treated with 6-percent dimethyl sulfoxide (DMSO), 2-percent DMSO or the platelet storage solution and 2-percent DMSO. The samples were placed directly into a -80 degrees C freezer and stored for 1 week, after which they were thawed and analyzed for in vitro functional activity. RESULTS: Platelets cryopreserved with the platelet storage solution and 2-percent DMSO displayed statistically higher retention of functional activity and viability--including cell number, percent of discoid cells, extent of shape change, and hypotonic shock response--than did platelets stored by the method using 6-percent DMSO. In addition, the treated platelets displayed statistically lower expression of p-selectin. The treated platelets showed no loss of cell number, > 88-percent retention of discoid morphology, and > 75-percent retention of ristocetin-induced aggregation as compared to values for these measures in fresh platelets. CONCLUSION: The use of this platelet storage solution in the cryopreservation of platelets yields a significant improvement in their postthaw in vitro recovery and allows for a reduction of the DMSO concentration from 6 to 2 percent, with superior maintenance of in vitro viability and function.

Blood Platelets↗
PubMed2000

The mechanical properties of fresh and cryopreserved arterial homografts.

OBJECTIVES: To assess the effect of cryopreservation on the elasticity and compliance of arterial allografts. MATERIALS AND METHODS: Iliofemoral segments of arteries and veins harvested from multiorgan donors were divided into two groups: fresh-control, tested for 24 hours after harvesting, and cryopreserved in liquid nitrogen after pretreatment with 20% dimethylsulphoxide and stored for an average time of 22 days. Vessel wall elastic properties were evaluated from the stress-strain relationship in a specially designed test cell fixed to the Instron Universal Testing Machine. RESULTS: The elastic modulus of the artery control group (1.54+/-0.33 MPa, n=20) was not significantly different from the cryopreserved group (1.69+/-0.61 MPa, n=15). Similarly, values for unfrozen veins (3.11+/-0.65 MPa, n=47) were not significantly different from those of frozen samples (2.71+/-0.85 MPa, n=38). Control compliance (6. 86+/-1.79x10(-5)%/Pa, for arteries; 3.84+/-0.81x10(-5)%/Pa, for veins) was similar to that of the cryopreserved group (6.66+/-1. 80x10(-5)%/Pa, for arteries; 4.16+/-1.21x10(-5)%/Pa, for veins). CONCLUSIONS: Cryopreservation maintains the important elastic properties of arterial and venous allografts during average storage time of 22 days.

Biomechanical Phenomena↗
PubMed1993

Effects of four different processing techniques on the microstructure of potatoes: comparison with fresh samples in the ESEM.

Four common scanning electron microscope (SEM) processing techniques involving freeze-substitution and chemical fixation were compared with fresh unprocessed samples imaged in an environmental scanning electron microscope (ESEM) using small pieces of potato tubers as test specimens. Potato tubers were chosen for this investigation because of their high moisture content and, consequently, the common need for extensive processing for conventional, high vacuum SEM imaging. ESEM results showed that the fresh unprocessed specimens were essentially unaltered, showing clear potato cell structure, morphology, and cell content. However, processed samples showed strong differences to the fresh samples: freeze-substituted specimens showed fine networks of material stretching across the surface of cells. These structures may represent fibrillar material or may be artifact caused during processing. Chemical fixation almost entirely destroyed the microstructure of these potato samples.

Cryopreservation↗
PubMed2000

Circulating donor-specific cytotoxic T lymphocytes with high avidity for donor human leukocyte antigens in pediatric and adult cardiac allograft valved conduit recipients.

OBJECTIVE: Specific immunological responses may be involved in the process of cryopreserved allograft valved conduit (AVC) degeneration, which is more frequently seen in young recipients. Rejection of heart and corneal allografts is preceded by an increase in the fraction of cytotoxic T lymphocytes (CTL) with high avidity for donor human leukocyte antigens (HLA) circulating in both peripheral blood and the affected graft. These donor-specific high-avidity CTLs are regarded as the destructive cells capable of causing graft damage. To monitor the precursors of these cells (CTLp) in young and adult AVC recipients, in vitro quantitative tests were performed on sequentially taken blood samples to quantitate CTLp frequencies and their avidity for donor antigens. METHOD: Six children and nine adults who received a cryopreserved AVC in the period between 1994 and 1997 were included in the study. From these patients, two to six blood samples were obtained up to 3 years after valve implantation. The number of circulating CTLp present within the peripheral blood mononuclear cell (PBMC) population was determined by limiting dilution analysis (LDA). The fraction of CTLp with high avidity for donor HLA class I was determined by addition of CD8 monoclonal antibodies (mAb) during the cytotoxic phase of the assay. Third-party stimulator cells were used to verify the donor-specificity of the response. RESULTS: The number of donor-specific CTLp increased significantly in the period 6-12 months after AVC implantation, while third-party-specific CTLp frequencies were not affected. Additionally, we found a significant increase of the high-avidity fraction of CTLp directed against donor antigens as early as during the first 6 months after AVC implantation. The fraction of high-avidity CTLp remained significantly higher post- compared with pre-implantation, even after 12 months. We observed no significant difference in the kinetics of CTLp frequencies between pediatric and adult AVC recipients. CONCLUSION: Implantation of cryopreserved human AVC induces an increase in the total number of circulating CTLp directed against donor HLA class I in both adults and children. The shift towards more destructive high-avidity CTLp in the peripheral blood indicates their potential damaging effect towards the heart valve allograft.

Adolescent↗
PubMedDate not supplied

[Hemoperfusion via cryopreserved tissue fragments of xenogeneic spleen in suppurative-septic states in urology].

Hemoperfusion through cryopreserved fragments of porcine xenospleen tissue was used in combined treatment of 42 patients with pyoseptic urologic diseases: urosepsis, purulent intoxication due to acute pyelonephritis, pyoinflammatory scrotal and prostatic lesions (in 15, 19, 8 patients, respectively). Indications and contraindications are specified. Altogether 57 sessions were performed. Seventeen patients underwent the perfusion pre- and postoperatively, twenty-five in combination with chemotherapy. After the removal porcine spleen was dissected, treated with cryoprotective mixture (-196 degrees C) and placed into liquid nitrogen. One-year viability of the tissue retained in 82-93% of the samples. Duration of the hemoperfusion was 40-80 min, the rate--30-40 ml/min. Two-four columns with cryopreserved tissue were utilized which were alternately connected with the circuit. Heparinization reached 12,000-15,000 units. All the patients survived. Positive clinical and laboratory findings were obtained for 38 patients (90%). Urine and blood samples appeared sterile on day 7-12. The improvement of the severe condition of the patients is attributed to detoxication, immunostimulation and immunocorrective effects.

Acute Disease↗
PubMed2004

Fertility after cancer: a prospective review of assisted reproductive outcome with banked semen specimens.

OBJECTIVE: To examine the outcome of assisted reproduction techniques (ART) using cryopreserved semen from patients with cancer. DESIGN: Prospective. SETTING: Therapeutic semen banking program at a tertiary healthcare center. PATIENT(S): Twenty-nine men with cancer who cryopreserved their sperm before treatment at our facility from 1982 to 2001 and withdrew their samples for assisted reproduction (IUI, IVF, or intracytoplasmic sperm injection [ICSI]). INTERVENTION(S): Sperm bank records were used to identify the patients. Information on fertility potential indices was obtained from medical records and through interviews. Of the 29 patients, 9 had testicular cancer, 12 had Hodgkin's disease, and 8 had other types of cancer. MAIN OUTCOME MEASURE(S): Pregnancy and live births. RESULT(S): A total of 87 ART cycles (42 IUI, 26 IVF, and 19 ICSI) was performed. Of those cycles, 18.3% resulted in pregnancy (7% IUI, 23% IVF, and 37% ICSI), and 75% of the pregnancies resulted in a live birth (100% IUI, 83% IVF, and 57% ICSI). There was no significant difference in the outcomes when the results were stratified by type of ART and malignancy. None of the 11 infants who were born had congenital anomalies. CONCLUSION(S): Our findings emphasize the need for physicians to discuss the issue of semen cryopreservation with all men of reproductive age who have cancer before antineoplastic therapy is started.

Antineoplastic Agents↗
PubMed2002

Freezing and thawing of murine bone marrow-derived dendritic cells does not alter their immunophenotype and antigen presentation characteristics.

The aim of this paper was to assess whether the BMDC after freezing and thawing are capable to retain the immunophenotype and antigen-presenting capacity. BMDC were generated from bone marrow precursor cells as described previously by culturing the cells in medium containing GM-CSF and IL-4. Afterwards, the cells were harvested, counted and used for phenotyping and priming of syngeneic spleen cells. For cryopreservation, the BMDC were frozen in the presence of 10% of dimethylsulphoxide (DMSO) and 90% foetal calf serum. Forty to fifty percent of both samples, frozen/thawed as well as fresh BMDC, exhibited characteristic DC morphology, and the DC obtained from the frozen/thawed samples expressed a similar level of MHC class I-, MHC class II-, CD80-, CD86-, CD11c-, CD11b-, CD54- and CD205-molecule as fresh DC. To examine the in vitro priming effect of cryopreserved BMDC on syngeneic non-adherent murine C57BL/6 (B6) spleen cells, the BMDC were thawed, pulsed with the lysate prepared from HPV 16-associated tumour MK16 and used for 3H-thymidine assay. The findings of the experiments indicate that fresh as well as cryopreserved murine BMDC preparations pulsed with tumour lysate were efficient to prime the mitogenic activity of syngeneic non-adherent splenocytes. Taken together, the results suggest that frozen/thawed BMDC are morphologically, phenotypically and functionally comparable with fresh BMDC and can be used for construction of dendritic cell-based tumour vaccines.

Animals↗
PubMed2006

Induction and cryopreservation of embryogenic cultures from nucelli and immature cotyledon cuts of mango (Mangifera indica L. var Zihua).

In this paper, we described the direct somatic embryogenesis from both immature cotyledon cuts and nucelli in the same mango cultivar (Mangifera indica L. var Zihua), studied the effect of growth conditions of embryogenic cultures (EMs) on cryopreservation and compared the cryopreservation response of EMs induced from these two different explants. Histological studies demonstrated that EMs derived from nucelli could be induced directly from epidermal cells of both sides of nucelli, whereas EMs derived from cotyledon cuts were induced only from epidermal cells of the adaxial side of the cotyledons. EMs from either nucelli or cotyledon cuts could be maintained in liquid medium or on solid medium and cryopreserved using a vitrification procedure. Success of cryopreservation of EMs depended on the dehydration treatment and the defined growth conditions during culture but not on their origins. When EMs were sampled during their exponential growth phase in liquid medium and dehydrated with PVS(3) solution for 5 min, survival of the EMs induced from cotyledon cuts and nucelli reached 77.7 and 80%, respectively, after cryopreservation in liquid nitrogen for 24 h. Furthermore, when dehydrated with PVS(3) solution for 30 min, all EMs induced from cotyledon cuts and 96.7% of EMs induced from nucelli could survive after cryopreservation. Cryopreservation did not affect the plant regeneration potential of EMs through somatic embryogenesis. The protocols of somatic embryogenesis and cryopreservation of mango EMs established in this study may offer potential ways to improve mango germplasm conservation and genetic improvement.

Cotyledon↗
PubMed2005

Non-transmission of bacterial and viral microbes to embryos and semen stored in the vapour phase of liquid nitrogen in dry shippers.

Cryopreservation and storage of germplasm is an important factor in the prevention of disease transmission by embryo transfer and artificial insemination. Here we report the results of an investigation on transmission of selected bacterial and viral pathogens by the vapour phase of liquid nitrogen (VPLN) to embryos and semen in dewars designed for short-term storage and transportation of biological specimens. In this study transmission of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, BVDV, and BHV-1 was examined from: (1) contaminated dry shippers to germplasm; (2) between contaminated and non-contaminated cryopreserved germplasm; and (3) between stock culture of pathogenic agents and germplasm. Contaminated and non-contaminated samples of embryos and semen were stored in proximity in the vapour phase LN in open containers for 7 days prior to testing for the presence of microbes. The results showed that there was no cross-contamination from either the contaminated dewars to germplasm or between contaminated and non-contaminated samples of embryos and semen during 7 days storage under LN vapours. The outcome of our investigation indicates that VPLN is a safe means for short-term storage of embryos and semen in dry shipper dewars commonly used for transportation of bovine germplasm.

Animals↗
PubMed1994

Methodologies for the preservation of proliferation associated antigens PCNA, p120, and p105 in tumor cell lines for use in flow cytometry.

The retention of antigen expression of PCNA, p120, and p105 in two tumor cell lines (MOLT-4 and MDA-MD-175-VII) under various conditions of fixation was investigated using flow cytometric analysis. Four currently utilized procedures for fixation/permeabilization of intracellular antigens were compared for their ability to stain the nuclear antigens. A procedure using a brief incubation in a solution of lysolecithin in paraformaldehyde followed by fixation in ice-cold methanol prior to antibody staining was selected to evaluate reagent protocols aimed at preserving antigen expression. Holding samples overnight at 4 degrees C in 2.5% fetal bovine serum after the lysolecithin/paraformaldehyde and methanol fixation steps prior to staining with monoclonal antibodies resulted in no decline in the percentage of cells positively stained for all three markers with little decrease in intensity of fluorescence and no increase in DNA coefficient of variation (c.v.). Fixed/permeabilized MOLT-4 cells held longer than 24 h before staining were lower in PCNA fluorescence than freshly stained cells; holding samples of either cell line longer than 48 h resulted in decreased PCNA and p120 staining. Prefixing and holding cells in 50% methanol or 50% ethanol overnight before processing and staining severely depressed PCNA and p120 fluorescence. Prefixing either cell line in a range of concentrations (0.25-1.0%) of paraformaldehyde also resulted in reduced intensity of PCNA and p120 fluorescence along with increased DNA c.v. P105 staining appeared to be relatively unaffected by all prefixation/storage conditions tested, except for a decline of fluorescence when MDA cells were prefixed in 50% ethanol. Cells cryopreserved in liquid nitrogen for 1 week before processing showed < 5% loss of PCNA and p120 fluorescence compared to freshly processed cells, but p105 fluorescence dropped 29% in cryopreserved MDA cells. These results underscore the fact that specific protocols for the fixation and storage of biological samples prior to staining and analysis must be determined for the specific nuclear antigen marker under investigation.

Cell Cycle↗
PubMedDate not supplied

Stimulation of ejaculated domestic cat sperm motility with caffeine, pentoxifylline, and 2'-deoxyadenosine.

Sperm motility patterns of cryopreserved domestic cat ejaculates treated at 37 degrees C with 1 mM caffeine, pentoxifylline, or 2'-deoxyadenosine were analyzed using computer-assisted semen analysis (CASA). The percent motility (MOT), curvilinear velocity (VLC), straight-line velocity (VSL), linearity (LIN), and amplitude of lateral head displacement (ALH) were measured for each group following 15 min of treatment. Motility indices were examined during a 6-h treatment period to determine the effect of each chemical on sperm longevity. Caffeine, pentoxifylline, and 2'-deoxyadenosine each increased (p > .05) the MOT and VCL of the ejaculates compared to the controls. The longevity of the treated and control samples were not significantly different throughout the incubation period. These results, similar to previous findings with cryopreserved epididymal cat sperm, demonstrate that motility stimulants can significantly elevate the MOT and VCL of cryopreserved ejaculated cat sperm without having deleterious effects on longevity.

Animals↗