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PubMed2006

Feasibility of vitrification as a storage method for tissue-engineered blood vessels.

It is well established that, in multicellular systems, conventional cryopreservation results in damaging ice formation, both in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation, we performed a feasibility study using vitrification (ice-free cryopreservation) to cryopreserve tissue-engineered blood vessels. Fresh, frozen, and vitrified tissue-engineered blood vessels were compared using histological methods, cellular viability, and mechanical properties. Cryosubstitution methods were used to determine the location of ice in conventionally cryopreserved engineered vessels. Ice formation was negligible (0.0 +/- 0.0% of vessel area) in the vitrified specimens, and extensive (68.3 +/- 4.5% of vessel area) in the extracellular matrix of frozen specimens. The metabolic assay and TUNEL staining results indicated that vitrified tissue had similar viability to fresh controls. The contractility results for vitrified samples were >82.7% of fresh controls and, in marked contrast, the results for frozen samples were only 10.7% of fresh controls (p < 0.001). Passive mechanical testing revealed enhanced tissue strength after both freezing and vitrification. Vitrification is a feasible storage method for tissue-engineered blood vessel constructs, and their successful storage brings these constructs one step closer to clinical utility.

Animals↗
PubMed1995

Optimum abstinence time for cryopreservation of semen in cancer patients.

Although an abstinence period of 48 to 72 hours is the most commonly prescribed interval for diagnostic semen analysis, to our knowledge the association between the abstinence period and sperm quality after cryopreservation has not been identified. Patients with a malignant disease who are consulting for semen banking most often require urgent therapy. Consequently, defining the shortest abstinence period that allows for frequent semen collection within a limited interval and the best post-thaw sperm quality is necessary. We investigated the relationship between abstinence period, and the pre-freeze and post-thaw motility variables in semen specimens obtained from cancer patients for sperm banking. Samples collected from 95 patients were divided according to abstinence period: group 1-15 patients at 24 to less than 48 hours, group 2-53 at 48 to less than 72 hours and group 3-27 at more than 72 hours. Pre-freeze and post-thaw motile sperm count and motion variables (motility, velocity, linearity, amplitude of lateral head movement and motility index), and percentage decrease in sperm variables after cryopreservation were analyzed. Semen volume, the pre-freeze and post-thaw motile sperm count, motion parameters and the percentage decrease in semen variables did not differ significantly among the groups. We conclude that semen collection for cryopreservation after 24 to less than 48 hours of abstinence results in post-thaw quality comparable to that after an abstinence of 48 to less than 72 hours or longer. Thus, an abstinence period of 24 to less than 48 hours can be recommended for sperm banking in cancer patients.

Cryopreservation↗
PubMed1995

Ability of spermatozoa to bind to the zona pellucida during oligozoospermia induced with testosterone during a male contraceptive trial.

To determine the ability of spermatozoa to bind to the zona pellucida (ZP) in testosterone-induced oligozoospermia, previously fertile men participating in the World Heath Organization (WHO) male contraceptive trial in Melbourne were studied while oligozoospermic to various degrees. Semen analysis were performed according to WHO methods. One or two ejaculates from each subject were cryopreserved before commencing weekly intramuscular injections of 200 mg testosterone enanthate. The frozen spermatozoa were used as controls for ZP-binding tests of spermatozoa obtained during testosterone-induced oligozoospermia (< 10 x 10(6)/ml) in either the suppression or efficacy (n = 6) and recovery (n = 3) phases. Two other subjects in the recovery phase with normozoospermia were also tested. Human oocytes that failed to fertilized in vitro from infertile patients were used for the sperm-ZP binding test. Control (frozen) spermatozoa were labelled with fluorescein isothiocyanate and test (oligozoospermic semen) spermatozoa were labelled with tetramethylrhodamine B isothiocyanate. A mixture of equal numbers of labelled motile control and test spermatozoa were incubated with 4-6 ZP. There was a significantly (p < 0.01) lower number of spermatozoa bound per ZP in oligozoospermic samples (65 +/- 7, mean +/- SEM) than in controls (80 +/- 7). However, there were still large numbers of spermatozoa bound to the ZP for all the oligozoospermic samples. Five subjects had similar numbers of spermatozoa bound to the ZP for both control and oligozoospermic samples. Overall, the ZP-binding ratio of test and control spermatozoa averaged 0.82 (range 0.51-1.13).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2003

Sperm cryopreservation in adolescents with newly diagnosed cancer.

BACKGROUND: Referring male patients (pts) for pretreatment sperm cryopreservation (SCP) is a routine practice in adult oncology. Our aim was to evaluate the semen quality and feasibility of sperm cryopreservation in male adolescents diagnosed with cancer prior to the commencement of treatment. METHODS: All consecutive adolescents from 14 to 19 years of age with newly diagnosed cancer were referred to this study. The following parameters of semen analysis were investigated: (1) volume of collected sample (N >or= 2.0 ml); (2) total sperm concentration (N >or= 20 x 10(6)/ml); (3) percentage of motile spermatozoa (N >or= 50%). The results were compared with normal values characteristic of healthy young men. RESULTS: Sixty-two attempts to collect sperm were made by the 27 adolescents. Of the 40/62 (64.5%) attempts, which resulted in a normal sperm count in each sample, only nine (22.5%) demonstrated normal sperm motility. Only 9/62 (14.5%) attempts resulted in normal sperm motility. Nineteen of 62 (30.6%) attempts produced a normal volume of ejaculate, while three pts were unable to produce any sperm. Only 4/62 (6.5%) attempts produced semen that could be considered normal in all the parameters. CONCLUSIONS: Only a minority of adolescents newly diagnosed with cancer is able to produce sperm that can be considered normal, compared with healthy young men. Despite this, SCP should be offered and is a technically feasible procedure for these patients in light of the recent advances in assisted reproductive technologies. Further studies are required to develop treatment protocols for this group of pts to lessen damage to fertility function.

Adolescent↗
PubMed2001

Short-term tumor cell lines from breast cancer for use as autologous tumor cell vaccines in the treatment of breast cancer.

OBJECTIVE: We tried to establish short-term cultures of autologous tumors from patients with breast carcinoma for potential use as active specific immunotherapy (i.e., autologous vaccine) after resection of primary breast cancer, and/or for the treatment of metastases. METHODS: Between 10/90 and 12/99 the cell biology laboratory of the Hoag Cancer Center attempted to establish short-term tumor cell lines from 115 breast cancer specimens from 56 primary breast lesions, 17 axillary nodes, 14 other lymph node/soft tissue sites, 10 chest wall recurrences, and 6 thoracentesis of malignant pleural effusions. Success was defined by growth of 5 x 10(7) viable cells whose malignant nature and breast cancer origin was confirmed by histology of the submitted tissue, cell morphology and antigenic phenotyping. Variables associated with successful growth of short-term cell lines were examined. RESULTS: Expansion to 5 x 10(7) cells was achieved for only 8/115 samples [7%] including two from chest wall recurrences, and one each from a supraclavicular node, an umbilical node, liver, omentum, and pleural fluid. Two of the successful cell lines were established from tissue that originally had been cryopreserved; the others were initiated from fresh tumor. The success rate was better from regional/distant metastases 7/55 (13%) compared to primary tumors 1/56 (1.8%) (p = 0.063). The success rate for tumors harvested at Hoag Hospital was 4/97 (4%) compared to 4/14 from (31%) distant sites, but all but one of the tumors from a distant geographic site was a metastatic lesion. Tumor cell lines were successfully established from metastatic lesions ranging in size from < 1.0 g to 19 g. Four patients were treated with their autologous vaccine in the setting of chemotherapy-refractory metastatic disease without any significant toxicity. CONCLUSIONS: We were unable to establish short-term cell lines for most patients with primary or metastatic breast cancer using this methodology. However, two long-term cell lines have been established and characterized. Treatment with the autologous irradiated cell product was not associated with acute toxicity.

Autoantigens↗
PubMed2003

Quantitation of blood lymphocyte mitochondrial DNA for the monitoring of antiretroviral drug-induced mitochondrial DNA depletion.

OBJECTIVE: To investigate the impact of antiretroviral treatment on the mitochondrial DNA (mtDNA) content of peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients. DESIGN: As absolute mtDNA copy numbers widely differ between individuals, we performed a longitudinal analysis where the patient's first historical specimen was obtained as a baseline reference for relative comparison with subsequent samples from that patient. METHODS: mtDNA and nuclear DNA quantitation per cell (beta-globin gene copies) were both measured by real-time polymerase chain reaction analysis of whole DNA extracts of 361 serial live-cryopreserved PBMCs collected in former trials and clinical follow-ups from 60 individuals with established or recently acquired HIV-1 infections before and during administration of various antiviral combination therapies. RESULTS: mtDNA amounts were stable or increasing over years of natural HIV-1 infection in untreated patients (n = 7), consistent with our finding of a lack of differences in mtDNA copy numbers in patients with either a long established or recent lentivirus infection. Our quantitation system revealed significant changes in mtDNA copy number depending on the designated triple, quadruple, or quintuple anti-HIV drug combinations. Zidovudine + zalcitabine + ritonavir and zidovudine + lamivudine + didanosine regularly lead to mtDNA depletion in each of the treated patients, whereas none of 7 patients (and 35 cell specimens) receiving a stavudine + lamivudine + indinavir combination had any significant mtDNA content variations. In 7 patients, mtDNA copy numbers returned to pretreatment levels and/or higher levels without any interruption of the previously mtDNA-depleting antiretroviral drug combination. CONCLUSION: Our assay system allowed the detection of significant changes in the mtDNA content of PBMCs from HIV-1-infected patients taking antiretroviral drugs, as has been reported in the literature with other detection systems. Yet, mtDNA copy numbers regularly diminished during administration of some but not all nucleoside analog-containing combinations. This, plus the occasional finding that depleted mtDNA contents spontaneously increased to baseline levels and/or higher levels during uninterrupted treatment, should raise a note of caution about resorting to the PBMC mtDNA marker for monitoring of antiretroviral drug-related mitochondrial toxicities.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Cryopreservation of mitochondria and mitochondrial function in cardiac and skeletal muscle fibers.

Long-term preservation of muscle mitochondria for consequent functional analysis is an important and still unresolved challenge in the clinical study of metabolic diseases and in the basic research of mitochondrial physiology. We here present a method for cryopreservation of mitochondria in various muscle types including human biopsies. Mitochondrial function was analyzed after freeze-thawing permeabilized muscle fibers using glycerol and dimethyl sulfoxide as cryoprotectant. Using optimal freeze-thawing conditions, high rates of adenosine 5(')-diphosphate-stimulated respiration and high respiratory control were observed, showing intactness of mitochondrial respiratory function after cryopreservation. Measurement of adenosine 5(')-triphosphate (ATP) formation showed normal rates of ATP synthesis and ATP/O ratios. Intactness of the outer mitochondrial membrane and functional coupling between mitochondrial creatine kinase and oxidative phosphorylation were verified by respiratory cytochrome c and creatine tests. Simultaneous confocal imaging of mitochondrial flavoproteins and nicotinamide adenine dinucleotide revealed normal intracellular arrangement and metabolic responses of mitochondria after freeze-thawing. The method therefore permits, after freezing and long-term storage of muscle samples, mitochondrial function to be estimated and energy metabolism to be monitored in situ. This will significantly expand the scope for screening and exchange of human biopsy samples between research centers, thus providing a new basis for functional analysis of mitochondrial defects in various diseases.

Adenosine Triphosphate↗
PubMed1983

Cryopreservation of human bone marrow cells by a modification of the two-step cooling technique.

The first attempt to freeze human bone marrow cells with a two-step cooling method is reported. A simple and reliable way of obtaining stable first-step subzero freezing baths is described. One-milliliter samples each containing 20 X 10(6) bone marrow cells and 10% Me2SO were frozen in polypropylene cryotubes. Using these experimental conditions, the optimal freezing temperature was found to be in the range of -36 to 37.5 degrees C for BM progenitor cell (GM-CFC, CFUE, and BFUE) survival. Such temperatures were easily obtained in stable sludges of anisole or K2CO3 eutectic solution in water. The optimal holding time was 20 min before plunging tubes into liquid nitrogen. Similar or improved progenitor cell recoveries were observed compared with the conventional cooling technique. Adaptation of this two-step technique for the freezing of large volumes of BM cells for autografting is under investigation.

Anisoles↗
PubMed2004

Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001.

The number of human embryonic stem cell (hESC) lines available to federally funded U.S. researchers is currently limited. Thus, determining their basic characteristics and disseminating these lines is important. In this report, we recovered and expanded the earliest available cryopreserved stocks of the BG01, BG02, and BG03 hESC lines. These cultures exhibited multiple definitive characteristics of undifferentiated cells, including long-term self-renewal, expression of markers of pluripotency, maintenance of a normal karyotype, and differentiation to mesoderm, endoderm, and ectoderm. Each cell line exhibited a unique genotype and human leukocyte antigen (HLA) isotype, confirming that they were isolated independently. BG01, BG02, and BG03 maintained in feederfree conditions demonstrated self-renewal, maintenance of normal karyotype, and gene expression indicative of undifferentiated pluripotent stem cells. A survey of gene expression in BG02 cells using massively parallel signature sequencing generated a digital read-out of transcript abundance and showed that this line was similar to other hESC lines. BG01, BG02, and BG03 hESCs are therefore independent, undifferentiated, and pluripotent lines that can be maintained without accumulation of karyotypic abnormalities.

Alkaline Phosphatase↗
PubMedDate not supplied

Relationship between the changes in cellular volume of fish spermatozoa and their cryoresistance.

We have investigated the hypothesis that the spermatozoa of marine fish are more resistant than freshwater species to the dynamic changes in osmotic pressure that occur during the process of cryopreservation. We show that while the spermatozoa of marine fish can be successfully activated across a wide range of osmotic pressures (0-2000 mOsmol/l), those of the freshwater species only survive activation within a more restricted range (0-300 mOsm/l). After freeze-thawing, up to 30 percent of motile cells were found in silver carp samples, while up to 90 percent of motile cells were observed in samples from the haarder (Mugil soiuy B). Haarder spermatozoa showed no change of cell volume after dilution in activating or cryoprotective media, while the silver carp spermatozoa responded by swelling and eventual cell disruption. We propose that the differences in cryoresistance of silver carp (Hypophthalmichthys molitrix V.) and haarder spermatozoa may be determined by the ability to preserve cellular volume under non-isotonic conditions.

Animals↗
PubMed1997

Donor-specific anti-human leukocyte antigen class I antibodies after implantation of cardiac valve allografts.

The allospecific humoral immune response was examined in 31 patients in the first year after implantation of cryopreserved human cardiac valves. We determined the percentage antibodies against human leucocyte antigens (HLA) class I in a complement-dependent microlymphocytotoxicity test against a panel of 50 selected donors carrying most of the defined HLA-A and HLA-B specificities (panel-reactive antibodies). In blood samples taken immediately before implantation, no antibodies could be detected. Thereafter, antibodies were present in 23 of 31 (74%) patients (median panel-reactive antibodies: 57%, range 9% to 91%). In 21 patients the HLA-type of the valve donor was available. In four patients no blood samples taken after 4 weeks were available. In 14 of 17 patients (82%) with a follow-up of more than 1 month antibodies were present, and in 12 of these 14 (86%) antibodies were specifically directed against HLA class I of the donor. In conclusion, the formation of donor-specific antibodies is frequently observed after human cardiac valve replacement. It could be one of the factors leading to valve destruction and dysfunction.

Adolescent↗
PubMed1995

DSC measurement of cell suspensions during successive freezing runs: implications for the mechanisms of intracellular ice formation.

The formation of intracellular ice in biological cells during freezing is considered almost universally lethal and is the major contributor to cell damage at high cooling rates. Despite its importance, our understanding of the mechanisms of intracellular ice formation (IIF) is still incomplete. In this paper differential scanning calorimetry is used to study IIF in human lymphocytes in the presence of dimethyl sulfoxide (Me2SO). Under conditions where damage due to IIF on the initial cooling run is 40-60%, the samples are studied as a function of multiple successive cooling runs. This enables the study not only of the cell fraction which undergoes IIF, but also of the fraction which survives. The temperature at which IIF occurs and the fraction of cell volume which undergoes IIF are analyzed as functions of successive cooling runs. Taking advantage of the large number of cells present in the samples (ca. 10(6)), the effect of successive cooling runs on susceptibility to IIF is examined.

Calorimetry, Differential Scanning↗
PubMed1993

DNA isolation by a rapid method from human blood samples: effects of MgCl2, EDTA, storage time, and temperature on DNA yield and quality.

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl2 led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37 degrees C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at -70 degrees C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 microliters of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA.

Blood Preservation↗
PubMed2004

Assessment of in vitro sperm characteristics after flow cytometric sorting of frozen-thawed bull spermatozoa.

The effect of processing prior to sex-sorting, re-freezing and thawing of frozen-thawed bull spermatozoa on in vitro sperm characteristics was investigated. Frozen-thawed bull spermatozoa (three bulls; three ejaculates per bull) were prepared for sorting by washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility and forward progressive motility (FPM) after processing, staining, sorting and incubation (3 h; 37 degrees C). After frozen-thawed samples were processed and analyzed using a high-speed cell sorter, aliquots were removed and re-frozen and thawed (FTF-WASH; FTF-GRADIENT). Non-sorted frozen-thawed spermatozoa (FT-CONTROL) were also re-frozen and thawed (FTF-CONTROL). Spermatozoa from all treatments were assessed for penetration of an artificial cervical mucus at 0 h after sorting or thawing, and for motility, FPM and acrosomal status after 3-h incubation (37 degrees C). Frozen-thawed spermatozoa prepared by gradient centrifugation before sorting were sorted more efficiently than washed samples (P < 0.05). However, after sorting (FT) or thawing (FTF) and incubation, the percentage of motile spermatozoa and FPM rating was lower for GRADIENT than WASH (21.5 +/- 3.39%; 1.4 +/- 0.16 FPM versus 48.6 +/- 4.02%, 2.6 +/- 0.16 FPM; P < 0.01). Frozen-thawed sorted spermatozoa (FT) penetrated in greater numbers (151.0 +/- 19.50 spermatozoa) and distance (56.3 +/- 5.11 mm) in the artificial cervical mucus and had a higher proportion of motile spermatozoa (65.5 +/- 2.77%) and FPM rating (2.8 +/- 0.12) after incubation than spermatozoa that had been re-frozen and thawed after sorting (FTF: 14.0 +/- 3.67 spermatozoa, 21.6 +/- 3.05 mm, 12.2 +/- 1.31% and 1.2 +/- 0.10 FPM, respectively; P < 0.001). Regardless of processing prior to sorting, frozen-thawed sorted and non-sorted spermatozoa migrated similar distances in the artificial cervical mucus (FT-WASH: 60.0 +/- 1.2 mm; FT-GRADIENT: 57.2 +/- 0.76 mm; FT-CONTROL: 51.7 +/- 0.69 mm). The results of this preliminary study suggested that frozen-thawed bull spermatozoa can be efficiently sorted into high purity X- and Y-chromosome enriched samples with retained functional capacity.

Animals↗
PubMed2003

Frozen mitochondria as rapid water quality bioassay.

A rapid and relatively low cost bioassay, usable in routine screening water test has been developed modifying the beef heart mitochondria test. In our experiments, mitochondria (FM22) were frozen at -22 degrees C, instead of -80 degrees C (FM80), and their applicability and sensitivity was verified. The oxygen consumption was measured by a Clark electrode that was interfaced to a PC to collect test analysis data. Blank tests were carried out to verify the oxygen consumption linear fitting. Toxicity tests were performed using pure organic and inorganic compounds, such to verify the FM22 sensitivity. A piecewise regression, through an Excel Macro, identified the break-point in the oxygen consumption and calculated the toxicity. The IC50s of the tested compounds were calculated and ranged from 0.123 to 0.173 mg/l for heavy metals (Cd, Cr, Cu, Ni, Pb and Zn) and from 0.572 to 10.545 mg/l for organics (benzene, DMSO, DDE, endrin, dichloromethane, chlorobenzene, 1,2-dichlorobenzene and 1,3-dichlorobenzene). Water effluent samples were then tested. The FM22 gave different toxic reactions to them. Water samples were characterised for heavy metals. The FM22 bioassay had a higher sensitivity than the FM80 and a high reproducibility in the toxicity test with pure compounds. The FM22 test was a good predictor of toxicity for water samples; the bioassay is easy, low cost and rapid, then usable for routine tests.

Animals↗
PubMed1995

Validity of urinary biomarkers of exposure to tobacco smoke following prolonged storage.

BACKGROUND: The utility of using biomarkers of smoking in epidemiological studies depends not only on the validity and precision of the laboratory procedure but often on the long-term stability of the analytes of interest in stored biological samples. METHODS: We retrieved urine samples collected in 1976-1977 from women included in a cohort study in Utrecht and for whom information on smoking status was available. Creatinine and thiocyanate were measured in 1976-1977 on fresh samples. Cotinine and creatinine were analysed in 1988 on urine stored at -20 degrees C. RESULTS: Measurements of creatinine more than 10 years apart showed a correlation of 0.95 and equal means. Cotinine measurements made in 1988 allowed a clear separation of smokers and non-smokers (sensitivity 92%, specificity 100%), suggesting that concentrations retained their discriminant value even after 10 years of storage. CONCLUSION: These results emphasize the possibilities offered by long-term storage, under proper conditions, of biological samples for subsequent determination of analytes which may emerge as the study progresses.

Biomarkers↗
PubMedDate not supplied

[Application of SensiScript enzyme to a RT-PCR system for the obtention of RNA of HIV-1 starting serum samples stored at -20 degrees C during a decade].

The efficiency of SensiScript reverse transciptase to obtain useful genetic material in the sequencing of the nucleic acid from HIV-1, starting from sera collected between 1989 and 1998 and kept at suboptimal temperatures, was proved. On using the SensiScript enzyme it was obtained an amplification of the RNA of the HIV-1 in 86.5 % of the studied samples, compared with 20 % on using the AMV-RT enzyme . No amplification was obtained in 13.5 % of the studied samples with any of the 2 enzymes used.

Blood↗
PubMed1995

Effect of intramuscular polysulfated glycosaminoglycans on bovine semen production and quality of the cryopreserved semen.

Polysulfated glycosaminoglycans (PSGAG) are widely used to treat cartilage disease in horses. The drug's safety in breeding animals has not been documented. This study determined whether im PSGAG is detrimental to the bull's health and, in particular, the quantity of semen produced or the quality of the cryopreserved product. No clinically significant effect of treatment on hematologic or serum biochemistry parameters was detected, but a significant treatment x time interaction for mean corpuscular volume warrants further investigation. There was an unexplained treatment effect on total daily sperm numbers collected, even though the pre-treatment values were included as co-variates in the statistical analysis to control for any day 0 variability. There was no effect on the percent forward motile sperm in the fresh ejaculate. Although there was no significant effect of treatment on the post-thaw semen data, the power of each statistical analysis was low, ranging from 10% to 19%. Further studies using larger sample sizes are warranted. These preliminary studies suggest that im PSGAG is safe to use in bulls on an experimental basis.

Analysis of Variance↗