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At least 649 records · Page 36Linked to original sources
PubMed1998

Multiple testicular sampling in non-obstructive azoospermia--is it necessary?

Spermatogenesis may be focal in non-obstructive azoospermia. The present study was conducted to determine whether the performance of multiple, rather than a single testicular sample contributes to obtaining spermatozoa in amounts sufficient for fertilization and cryopreservation in non-obstructive, azoospermic patients. Furthermore, the aim was to clarify the significance of location for retrieval from the testis in such cases. Three biopsies were taken from identical locations in 55 testes of 29 men with non-obstructive azoospermia: (i) the rete testis region, ii) the midline, and (iii) the proximal region of the testis. When sperm cells were detected, they were used for intracytoplasmic sperm injection (ICSI), and the remainder were then cryopreserved in as many aliquots as possible (adjusted for ICSI procedure). Spermatozoa were found in 28 testes (50.9%) of 18 men (62.1%). In the testes from which spermatozoa were obtained, they were present in three, two or one locations in 15 (53.6%), five (17.9%) and eight (28.6%) cases respectively. The possibility of finding spermatozoa was not influenced by the location in the testis. Multiple testicular sperm extraction is recommended in cases of non-obstructive azoospermia, since it may enhance diagnostic accuracy of absolute testicular failure and increase the number of sperm cells retrieved.

Adult↗
PubMed1997

Characterization and application of an avian in vitro spermatozoa-egg interaction assay using the inner perivitelline layer from laid chicken eggs.

The inner perivitelline layer, separated from laid chicken eggs, was investigated as readily available material for studying the spermatozoa-egg interaction in vitro. This layer was found to have a similar response to hydrolysis by spermatozoa as the inner perivitelline layer from ovulated and follicular ova, in terms of the numbers of points of hydrolysis made by spermatozoa during a 5 min incubation at 40 degrees C. Initiation of hydrolysis of the inner perivitelline layer was found to occur within 2.5 min, after which the size, but not the number of holes, increased with time. The frequency of the points of hydrolysis per unit area of the inner perivitelline layer was positively correlated with the concentration of spermatozoa in the incubation medium. The perivitelline hydrolysis assay was able to detect more damaged spermatozoa in samples that had been either stored at 5 degrees C or cryopreserved in liquid N2 than did other tests of sperm quality, which are known to overestimate the fertilizing ability of stored avian semen.

Animals↗
PubMed2003

Human hepatocytes as a tool for studying toxicity and drug metabolism.

Drugs are usually biotransformed into new chemical species that may have either toxic or therapeutic effects. Drug metabolism studies are routinely performed in laboratory animals but, due to metabolic interspecies differences when compared to man, they are not accurate enough to anticipate the metabolic profile of a drug in humans. Human hepatocytes in primary culture provide the closest in vitro model to human liver and the only model that can produce a metabolic profile of a given drug that is very similar to that found in vivo. However their availability is limited due to the restricted access to suitable tissue samples. The scarcity of human liver has led to optimising the cryopreservation of adult hepatocytes for long-term storage and regular supply. Human hepatocytes in primary culture express typical hepatic functions and express drug metabolising enzymes. Moreover, qualitative and quantitative similarities between in vitro and in vivo metabolism of drugs were observed. Different strategies have been envisaged to prolong cell survival and delay the spontaneous decay of the differentiated phenotype during culture. Thus, hepatocytes represent the most appropriate model for the evaluation of integrated drug metabolism, toxicity/metabolism correlations, mechanisms of hepatotoxicity, and the interactions (inhibition and induction) of xenobiotics and drug-metabolising enzymes. However, in view of limitations of primary hepatocytes, efforts are made to develop alternative cellular models (i.e. metabolic competent CYP-engineered cells stably expressing individual CYPs and transient expression of CYPs by transduction of hepatoma cells with recombinant adenoviruses). In summary, several cellular tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment.

Cells, Cultured↗
PubMed2004

Semen cryopreservation from oncological patients: the Italian experience.

The aim of the study is to summarize official data on semen cryopreservation for oncological patients in Italy. The first private semen bank for oncological patients in Italy was founded by Colpi in 1983 in Milan. In 1987, the first public semen bank was established in "Macedonio Melloni" Hospital (Milan) by Ragni. Normal semen samples are usually present in only 40% of oncological patients. The rate of semen sample use for assisted reproduction techniques is similar to international data (5%). At present, semen cryopreservation is possible only in about 20 Italian centers.

Cryopreservation↗
PubMedDate not supplied

Cryopreservation of tea (Camellia sinensis L.) seeds and embryonic axes.

This study investigated the tolerance to desiccation and freezing of tea seeds, embryonic axes (EAs) and cotyledonary embryonic axes (CEAs, consisting of EAs with portions of cotyledons still attached). No seeds germinated after desiccation and cryopreservation. EAs extracted from seeds desiccated to 18.9% moisture content (fresh weight basis) and cryopreserved showed 20.7% survival but plantlet production from these EAs was impossible. When EAs and CEAs were extracted from seeds before being submitted to desiccation and freezing, survival of control and frozen samples was equivalent with both types of materials. However, plantlet production was significantly higher from control and cryopreserved CEAs than EAs. The maturity stage of the seeds from which CEAs were extracted had an important effect on their survival and plant production percentages, mature seeds providing better results than early mature and late mature seeds. The highest percentages of plantlet production from cryopreserved CEAs, which ranged between 75.1 and 80.4%, were achieved for EA moisture contents between 21.5 and 15.0%.

Camellia sinensis↗
PubMed2001

Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity.

OBJECTIVE: To investigate effects of cryopreservation on sperm motility and DNA integrity. DESIGN: Pre-cryopreservation and post-cryopreservation analysis of motility and DNA integrity of semen and prepared sperm samples. SETTING: A hospital andrology laboratory. PATIENT(S): Forty men attending the Regional Fertility Centre, Belfast, Northern Ireland. INTERVENTION(S): Each sample was divided, and an aliquot was frozen unprepared. Remaining aliquots were prepared by Percoll density centrifugation (95.0:47.5) or direct swim-up procedure and divided into aliquots to allow direct comparison of fresh and frozen semen and prepared sperm (frozen with or without the addition of seminal plasma) from the same ejaculate. Samples were frozen by static-phase vapor cooling and being plunged into liquid nitrogen. Thawing was carried out at room temperature. MAIN OUTCOME MEASURE(S): Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay, and motility was determined using computer-assisted semen analysis. RESULT(S): Sperm frozen unprepared in seminal fluid appeared more resistant to freezing damage than frozen prepared sperm. Further improvements can be achieved by selecting out the subpopulation of sperm with best motility and DNA integrity and freezing these sperm in seminal plasma, making this the optimal procedure. CONCLUSION(S): Freezing sperm in seminal plasma improves postthaw motility and DNA integrity.

Cryopreservation↗
PubMed1982

A comparison of freezing and thawing methods for the cryopreservation of human semen.

The purpose of this study was twofold. In the first part are compared cryosurvival rates for human semen following two different freezing and three different thawing techniques. In the second part a larger number of samples processed by the best of the six methods described were examined to ensure reproducibility. Eighteen human semen samples with initial motility greater than 60% and density greater than 20 X 10(6)/ml were mixed with a cryopreservative medium at 35 degree C and vacuumed into 0.5 ml straws. Six aliquots were prepared from as many specimens as possible. Three straws were frozen by a programmed method (P) and three by a rapid freezing technique (V). All six straws were stored in liquid nitrogen vapor (-196 degree C) for 1 week. The straws were thawed by 1 of 3 methods: (1) room temperature for 10 minutes and 37 degree C hot plate for 10 minutes, (2) ice water for 10 minutes and 37 degree C hot plate for 10 minutes, (3) 35 degree C water for 12 seconds and 37 degree C hot plate for 10 minutes. Postthaw motility was assessed for each aliquot. The freeze/thaw method P/1 was judged optimal. In the second part of the study a larger number of samples were processed by P/1. The mean +/- standard deviation postthaw motility o 57 semen specimens processed by this method was 61.4 +/- 12.1.

Freezing↗
PubMed1994

[Characterization of 24-hour survival rate and duration of survival of hydroxyethyl starch cryopreserved erythrocytes after autologous transfusion in the dog].

BACKGROUND: Cryopreservation of erythrocytes using hydroxyethyl starch (HES) as cryoprotecting additive could result in a nearly unlimited storage stability of preserved red cells. In addition, it would allow its immediate use for transfusion. In order to assess the therapeutic efficacy of erythrocytes cryopreserved with HES, their 24-hour post-transfusion survival and long-term survival was evaluated. MATERIALS AND METHODS: The experiments were carried out with dog erythrocytes as an animal model for human erythrocytes. To each of 6 German shepherd dogs a 15-ml sample of erythrocyte suspension, labeled with 51Cr (25 microCi) after thawing, was autologously injected. Caused by hemolysis 29% of the formerly cryopreserved erythrocytes have not been labeled. To each of 6 control animals 15 ml of a suspension of freshly drawn and 51Cr-labeled erythrocytes was injected. The 51Cr radioactivity in later taken blood samples was a measure for the number of injected erythrocytes having remained in the circulation until the moment of blood withdrawal. The effect of cryopreservation was assessed by comparison of the test group with the control group. RESULTS: In both groups 30% of the applied cells left the circulation within 30 min. This was effected by pharmacological enlargement of the dogs' spleen and not by hemolysis of the erythrocytes. After the first 24 h all of the cryopreserved labeled erythrocytes had survived to the same amount (> 95%) as the labeled fresh red cells. Between 12 h and 20 days after injection, in both groups the 51Cr activity decreased exponentially by 4.8 and 4.5%/d. This difference was not significant. The area under the curve amounted to 1253 and 1257% d, respectively. CONCLUSIONS: There exists a subpopulation of red cells that is destroyed by freezing stress. As a result the freed stroma would be a serious transfusion risk. All erythrocytes having survived the cryopreservation procedure resemble the fresh erythrocytes with regard to the in-vivo survival; their therapeutic efficacy is not impaired. In the context of in-vitro results with human erythrocytes it can be expected that at the present developmental state of the cryopreservation procedure at least 93% of the human erythrocytes cryopreserved with HES have a normal 24-hour and long-term post-transfusion survival.

Animals↗
PubMedDate not supplied

Relationships between computerized measurements of motion of frozen-thawed bull spermatozoa and fertility.

A computerized system (CellSoft, CRYO Resources, Ltd.) was validated using video tapes of frozen-thawed bull spermatozoa diluted in filtered (0.2 micron) egg yolk-citrate extender (8 X 10(6) spermatozoa/ml) and analyzed at 30 frames/sec for the percentage of motile spermatozoa (greater than or equal to 20 microns/sec) and linear velocity of motile spermatozoa. Virtually all motile spermatozoa were detected and debris rarely were classified as immotile spermatozoa if the extender had been filtered. Variation about the mean for percent motile cells was similar when only 12 rather than 20 or 30 frames/field were analyzed. Use of 20 frames/field was adequate to determine the percentage of motile bull spermatozoa. Five mixtures of live and killed spermatozoa were analyzed (four bulls) to evaluate accuracy. Percent motile spermatozoa was correlated (r = 0.97) with the ratio of live:killed spermatozoa. Mean linear velocity of motile spermatozoa was similar for each mixture (P greater than 0.05). To further evaluate accuracy, percent motile spermatozoa was determined by computer and by "track motility" (20 samples; 0 to 63% motile spermatozoa); values were correlated (r = 0.95). The system was precise (CV of 6% based on triplicate analyses of the same samples) and reasonably accurate for evaluating bull sperm motility if the extender had been filtered and 20 to 25 fields (greater than or equal to 200 spermatozoa) were evaluated. Correlations between measurements of sperm motion and fertility were studied using cryopreserved semen from two fertility trials. For the first, 75-day nonreturn rate data for 20 samples of bull semen (10 bulls) were not significantly correlated with evaluations made by CellSoft. For the second fertility trial, the competitive fertility index (a measure of relative fertility) for nine bulls was correlated (r greater than or equal to 0.68; P less than 0.05) with percent motile spermatozoa, linear velocity and straight-line velocity. Multiple correlations based on six characteristics evaluated by CellSoft, at 0 or 1.5 hours, and the competitive fertility index were greater than or equal to 0.94. Based on the latter data, the system may facilitate prediction of the relative fertility of bull spermatozoa.

Animals↗
PubMed2001

Is cryopreservation a homogeneous process? Ultrastructure and motility of untreated, prefreezing, and postthawed spermatozoa of Diplodus puntazzo (Cetti).

This study subdivides the cryopreservation procedure for Diplodus puntazzo spermatozoa into three key phases, fresh, prefreezing (samples equilibrated in cryosolutions), and postthawed stages, and examines the ultrastructural anomalies and motility profiles of spermatozoa in each stage, with different cryodiluents. Two simple cryosolutions were evaluated: 0.17 M sodium chloride containing a final concentration of 15% dimethyl sulfoxide (Me(2)SO) (cryosolution A) and 0.1 M sodium citrate containing a final concentration of 10% Me(2)SO (cryosolution B). Ultrastructural anomalies of the plasmatic and nuclear membranes of the sperm head were common and the severity of the cryoinjury differed significantly between the pre- and the postfreezing phases and between the two cryosolutions. In spermatozoa diluted with cryosolution A, during the prefreezing phase, the plasmalemma of 61% of the cells was absent or damaged compared with 24% in the fresh sample (P < 0.001). In spermatozoa diluted with cryosolution B, there was a pronounced increase in the number of cells lacking the head plasmatic membrane from the prefreezing to the postthawed stages (from 32 to 52%, P < 0.01). In both cryosolutions, damages to nuclear membrane were significantly higher after freezing (cryosolution A: 8 to 23%, P < 0.01; cryosolution B: 5 to 38%, P < 0.001). With cryosolution A, the after-activation motility profile confirmed a consistent drop from fresh at the prefreezing stage, whereas freezing and thawing did not affect the motility much further and 50% of the cells were immotile by 60-90 s after activation. With cryosolution B, only the postthawing stage showed a sharp drop of motility profile. This study suggests that the different phases of the cryoprocess should be investigated to better understand the process of sperm damage.

Animals↗
PubMed2003

Risk of non-Hodgkin's lymphoma and prediagnostic serum organochlorines: beta-hexachlorocyclohexane, chlordane/heptachlor-related compounds, dieldrin, and hexachlorobenzene.

Increases in non-Hodgkin's lymphoma (NHL) incidence and mortality rates during the past few decades remain largely unexplained. Studies suggest that organochlorine pesticides may contribute to an increased risk of NHL. In 1974, serum samples were obtained from 25,802 participants in the Campaign Against Cancer and Stroke in Washington County, Maryland (USA), and cryopreserved for future study. We measured prediagnostic levels of chlordane, lindane (gamma-hexachlorocyclohexane), beta-hexachlorocyclohexane, transnonachlor, heptachlor, heptachlor epoxide, oxychlordane, dieldrin, and hexachlorobenzene in serum samples of 74 cases of NHL and 147 matched controls. Previously, we found an association between NHL and serum levels of total PCBs (polychlorinated biphenyls), but not DDT (dichlorodiphenyltrichloroethane) and related compounds. In this instance, there was no evidence of an association between NHL risk and serum levels of any of the individual lipid- and recovery-corrected organochlorines that we evaluated, nor of the summed chlordane-related compounds (transnonachlor, heptachlor, heptachlor epoxide, oxychlordane). These findings do not support the hypothesis that the organochlorine compounds included in this study are strongly linked to the development of NHL. The possibility of a weak association cannot be excluded by these data.

Case-Control Studies↗
PubMedDate not supplied

Use of a cocktail of probe substrates for drug-metabolizing enzymes for the assessment of the metabolic capacity of hepatocyte preparations.

A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation). The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CLint) were calculated from the disappearance of the compounds from the incubations. Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin. The highest CLint estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CLint was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CLint was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively. When the cocktail was incubated together with human hepatocytes and 1 microM ketoconazole, the CLint of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor. It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.

Animals↗
PubMed2002

The role of quality control in a skin bank: tissue viability determination.

New surgical procedures requiring viable skin have increased rapidly over the last few years. The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability. Various methods, both qualitative and quantitative, are currently used. Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting. Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability. Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing. Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes. The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength. It was seen that WST-1 is easier to handle, more stable, has a wider linear range, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension. However, after 72 hours of storage at 4 degrees C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks. Moreover, after thawing WST-1 tested samples were almost negative, whilst MTT samples continued to give strong signals. In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation. In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability. Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation.

Journal Article↗
PubMed1994

Post-thaw sperm motility, cAMP concentration and membrane lipid peroxidation after stimulation with pentoxifylline and platelet-activating factor.

Earlier studies have demonstrated that pentoxifylline (PTX) and platelet-activating factor (PAF) can significantly improve the motion parameters of post-thaw human spermatozoa. This study has investigated the effects of PAF, PTX and their combination on cyclic adenosine monophosphate (cAMP) concentrations and membrane lipid peroxidation (LPO) in post-thaw human spermatozoa. Washed spermatozoa from normal volunteers (n = 10) were cryopreserved in Test-yolk buffer using a standard protocol. After 2 weeks the sperm samples were thawed, washed and incubated with either 1 microM PAF, 3 mM PTX or 0.5 microM PAF plus 1.5 mM PTX. Video sequences were recorded at 0, 30, 60, and 120 min for analysis of sperm motion parameters using the Cell Track Sperm Analysis System. Concentrations of cAMP were assessed by radioimmunoassay, and LPO levels were measured by malondialdehyde-thiobarbituric acid reactivity. Our studies indicate a time-course stimulatory effect with overall maximal stimulation observed in samples treated with the combination of PAF and PTX. The maximal stimulation of percentage motility compared to control was observed at 60 min in samples treated with PAF, PTX, or PAF plus PTX. PAF plus PTX stimulated straight-line velocity (VSL), curvilinear velocity (VCL) and lateral head displacement (ALH) after 30 min incubation. The primary effect of PAF was observed on VSL, while the main effect of PTX was on VCL. cAMP concentrations were 3-fold higher than controls in samples treated with PTX or PAF plus PTX. cAMP concentrations in PAF-treated samples did not differ significantly from controls. No significant differences were observed between any groups for LPO.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2001

Cryopreservation of artificial cartilage: viability and functional examination after thawing.

In biomedical research and in reconstructive surgery, preservation of intact tissue has been an unsolved problem. In this study, we investigated the viability of cryopreserved artificial cartilage and its synthetic activity of cartilage-specific matrix proteins after thawing for in vitro use. A polymer fleece cylinder (diameter = 3 mm; height = 3 mm) was loaded with a suspension of bovine chondrocytes (25 x 10(6)/ml) and encapsulated with fibrin glue. After a culture period of 1 week, the artificial cartilage units were frozen in a cryoprotection solution containing 10% basal medium (RPMI 1640), 10% DMSO and 80% FCS. The freezing procedure consisted of three steps: a 30-min period at +4 degrees C followed by a 24-hour storage at -80 degrees C. After that, the tissue units were transferred into liquid nitrogen (-196 degrees C) for final storage. Using histochemical staining techniques of cryogenic slices, we investigated the ability of cryopreserved artificial cartilage to produce its specific matrix after thawing. A modified MTT assay was used to determine the viability of frozen tissue units in comparison with unpreserved samples at different moments after thawing. Depending on the chondrocytes used for the formation of artificial cartilage, the viability of cryopreserved tissue varied between 65 and 85%. Both the intensity of alcian blue staining for proteoglycans and the azan staining for collagens increased proportionally with incubation time after thawing. These findings indicate that cryopreservation of small artificial cartilage units is possible with a minor loss of cell viability. Secondly, its synthetic activity of cartilage-specific matrix did not decline after the freezing process.

Animals↗
PubMed2004

Use of chromatin stability assay, mitochondrial stain JC-1, and fluorometric assessment of plasma membrane to evaluate frozen-thawed ram semen.

Cryopreservation of semen imposes deleterious effects on spermatozoa, either killing a certain proportion of cells or causing subtle damages on sperm function in the surviving population, changes not easily revealed by conventional assays. We have tested three functional assessment techniques in frozen-thawed ram semen from six adult rams, cryopreserved following eight different protocols (four extenders, and glycerol being added at two temperatures). Semen samples were thawed and the following analyses were carried out: motility (CASA), membrane integrity (Hoescht 33258 and fluorometry), chromatin status (chromatin stability test and fluorescence-assisted cell sorting, FACS) and mitochondrial activity (JC-1 and FACS). Fluorometry outcome did not correlate with the other parameters and showed large variation, albeit discriminating among cryopreservation techniques (P < 0.01). Mitochondrial activity correlated, but with low values, with total and progressive motility. However, good sperm motility and high velocity values were associated to high mitochondrial membrane potential. The chromatin stability assay was also successfully carried out, and had a good relationship with male factor (%COMP alpha(t) and SD alpha(t) parameters). In conclusion, fluorometric assessment of membrane integrity albeit rendering poor results, merits improvement, being a low-cost and handy technique, especially for work in the field. On the other hand, both assessments of chromatin stability and mitochondrial status (JC-1 staining), combined with FACS, are reliable techniques that can be used for the functional assessment of frozen-thawed ram semen.

Animals↗
PubMed1996

[Morphologic and functional changes in thrombocytes after deep freezing with DMSO].

BACKGROUND: During the past decade much work has been carried out towards establishing the optimum method for cryopreservation of platelets. Among the various cryoprotectants dimethyl sulfoxide (DMSO) has been shown to be the most effective. This report describes ultrastructural and functional changes of platelets during the deep-freezing process with DMSO. MATERIALS AND METHODS: Single-donor platelet concentrates were cryopreserved in liquid nitrogen by use of DMSO. Before, during and after the freezing process samples were taken for analysis of ultrastructure and platelet function. RESULTS: While after isolation and addition of DMSO a normal ultrastructure of platelets could be observed, clear signs of beginning cell necrosis were detected after thawing and resuspension in autologous plasma. Fibrinogen-binding capacity and platelet aggregation were significantly diminished. CONCLUSIONS: Although cryopreserved platelets are characterized by hemostatic effects in vivo, it seems conceivable that these effects could be improved by further development of platelet-freezing techniques.

Blood Platelets↗
PubMed2006

Cryopreservation of murine embryos, human spermatozoa and embryonic stem cells using a liquid nitrogen-free, controlled rate freezer.

A Stirling Cycle Cryocooler has been developed as an alternative to conventional liquid nitrogen controlled rate freezers. Unlike liquid nitrogen systems, the Stirling Cycle freezer does not pose a contamination risk, can be used in sterile conditions and has no need for a constant supply of cryogen. Three types of samples from two species (murine embryos, human spermatozoa and embryonic stem cells), each requiring different cooling protocols, were cryopreserved in the Stirling Cycle freezer. For comparison, cells were also frozen in a conventional liquid nitrogen controlled rate freezer. Upon thawing, the rates of survival of viable cells were generally greater than 50% for mouse embryos and human embryonic stem cells, based on morphology (mouse embryos) and staining and colony formation (human embryonic stem cells). Survival rates of human spermatozoa frozen in the Stirling Cycle freezer, based on motility and dead cell staining, were similar to those of samples frozen in a conventional controlled rate freezer using liquid nitrogen.

Animals↗