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Cryopreservation of coronary endothelium and endothelial-mediated responses.

Limited availability of functionally viable biological tissues for graft transplantation and scientific research necessitates the development of an effective storage method for preserving existing tissue samples. In this report we briefly reviewed recent attempts in the cryopreservation of coronary endothelium and endothelial-mediated vasoregulatory function. In contrast to partial preservation of vascular smooth muscle cell function, cryostorage of coronary arteries in dimethyl sulfoxide and fetal calf serum resulted in a near complete preservation of the endothelium and its vasoregulatory function. This protection was observed in both animal and human epicardial conduit coronary arteries, as well as in dog intramyocardial resistance arteries. The apparent better preservation of the latter resistance arteries, with a luminal diameter less than 50 microns, is consistent with earlier findings that cryopreservation efficacy improves with fewer and less packing of cells. To further investigate the cryopreservation of endothelial function, we cryostored canine cardiac valves and evaluated the vasorelaxant effects of valvular endothelium-derived vasoactive factors using a bioassay system. As was observed with intact coronary arteries, a near complete preservation of valvular endothelium and its production of vasoactive factors was noted following the freeze and thaw procedures. Thus, these results demonstrate that cryopreservation may provide a useful and efficient storage method for the conservation of limited supplies of biological vascular tissues for graft transplantation and scientific research.

Acetylcholine↗

The provision of a semen cryopreservation service for male cancer sufferers in the Republic of Ireland.

BACKGROUND: Chemotherapy, radiotherapy and surgery for cancer all carry risks of sterility. Semen cryopreservation would allow procreational ability to be preserved. METHODS: A human-assisted reproduction unit was set up in the Rotunda Hospital where semen was cryopreserved and stored for use. RESULTS: Semen was frozen for procreational potential during 1998 from 58 males about to have oncology therapy likely to render them sterile. The planning for this service and the modus operandi now in place are described. While cryopreservation was not unsuccessful in any case, substandard samples were obtained from 14 men. Pre-freeze viral tests tested positive for CMV in six patients. One pregnancy is already on-going utilising IVF with thawed semen. One patient's death has been notified and the frozen semen disposed of. CONCLUSION: The service is, at present, outside public purse provision. It is hoped that in the near future this will change, as the deficit between charges and costs cannot be sustained in-house forever.

Cryopreservation↗

CD38 expression on cryopreserved CD8+ T cells predicts HIV disease progression.

Previous studies have revealed that the expression of CD38 on CD8+ T cells is a strong predictor of disease progression in human immunodeficiency virus (HIV)-infected individuals. Those studies were performed using fresh patient samples over an extended trial period. After demonstrating the validity of assay results on cryopreserved cells, we performed a retrospective study using frozen cell samples to determine the predictive value of CD38 expression in patients with CD4 counts above 400 cells/microl. The CD38 expression as measured by antibody binding capacity and the CD38 median channel were shown to be associated with time to new opportunistic infection or death (both P < 0.001). These results suggest that CD38 expression on CD8+ T cells, whether fresh or frozen, provides a useful predictor of HIV disease progression.

ADP-ribosyl Cyclase↗

In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices.

With the exception of cytochrome P450 (CYP) 1A1 and its mRNA, in vitro induction of other CYP forms has not been demonstrated in cryopreserved liver slices until now. Therefore precision-cut rat liver slices were cultured after cryopreservation and thawing in William's medium E for up to 24 h in the presence of inducers to demonstrate CYP2B1- and CYP3A1-mRNA induction. CYP-mRNA expression was determined by competitive RT-PCR. Exposure to 100 microM phenobarbital caused a more than 20-fold increase in CYP2B1-mRNA expression within 24 h, reaching concentrations comparable with those of PB-exposed fresh rat liver slices. Exposure to 1 microM pregnenolone 16 alpha-carbonitrile enhanced CYP3A1-mRNA expression by more than 30-fold within 24 h. This is in the same range, although with higher variability, as detected with fresh liver slices. In spite of considerable variability among the thawed slices, the induction factors are high enough for a sensitive detection of an induction at mRNA level. Additionally, immunostaining of respective CYP-forms was performed in sections of few samples, indicating CYP increase in viable cells of cryopreserved slices.

Animals↗

Membrane changes during different stages of a freeze-thaw protocol for equine semen cryopreservation.

Many theories have been postulated concerning the possible effects of cryopreservation on spermatozoa, including suggestions the freeze-thawing process produces membranes that have greater fluidity and are more fusogenic, thus inducing changes similar to those of capacitation. The main objectives of this study were to determine at what stage of the freeze-thaw process membrane changes occur and whether evaluation with chlortetracycline (CTC) stain could predict the freezability of stallion sperm. Sperm viability and state of capacitation were simultaneously evaluated using CTC and Hoechst 33258 (H258) techniques. Membrane function was evaluated using the hypo-osmotic swelling test (HOS) and progressive motility (PM) was evaluated under light microscopy at each stage of a freeze-thaw protocol. Evaluated were raw semen; after dilution and centrifugation; after redilution and equilibration at room temperature; after cooling to 5 degrees C; after super cooling to -15 degrees C; and after thawing. The most pronounced functional damage to membranes and the greatest decrease in PM occurred in samples of all stallions after thawing (P<0.05). Cryopreservation, as evaluated by CTC/H258 staining, significantly (P<0.05) affected sperm membrane integrity after centrifugation, after redilution and equilibration at room temperature and after cooling to 5 degrees C. The HOS and H258 tests gave similar results (R values of approximately 0.75) and correlated inversely with the number of live noncapacitated sperm cells (R values of approximately -0.75). Remarkably, the subpopulation of capacitated live cells was unaffected in all freeze-thawing steps and the number of live acrosome reacted cells increased by a factor of 4. However, it was not possible to determine whether the changing CTC patterns reflect a true capacitation phenomenon or an intermediate destabilized state of the sperm cell membrane. This increase may indicate that the subpopulation of functional sperm cells capable of binding to the zona pellucida increases after freeze-thawing despite the deteriorative effect of this procedure for the entire live sperm population.

Acrosome↗

The effect of highly purified eicosapentaenoic and docosahexaenoic acids on monocyte phagocytosis in man.

The n-3 fatty acids (FA) from marine sources are known to exert antiinflammatory effects on monocyte function. There is still controversy whether n-3 FA may increase the susceptibility to infections. The present study was designed to assess the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on monocyte phagocytosis and respiratory burst activity. Fifty-eight healthy men were randomized to take a daily supplement of 3.8 g highly purified EPA (n = 20), 3.6 g DHA (n = 19), or corn oil (n = 19) for 7 wk. Mononuclear leukocytes were collected, isolated, and cryopreserved prior to and after dietary supplementation. Paired samples were analyzed in the presence of autologous serum in a crossover design. Monocyte phagocytosis and respiratory burst activity were measured by flow cytometry after ingestion of Escherichia coli. Monocytes retained their phagocytic ability and respiratory burst activity after supplementation. No reduction in internalization of bacteria was registered. Dietary n-3 FA and particularly EPA improved bacterial adherence to the monocyte surface. In the crossover experiments, there was an adverse effect of serum enriched with n-3 FA on bacterial adherence. We conclude that monocytes retain their phagocytic potential after supplementation with purified EPA and DHA.

Adult↗

Characterization of ultrastructural damage of valves cryopreserved under standard conditions.

Cryopreserved allograft valves are increasingly being used as valvular replacements. This study was conducted to characterize the ultrastructural damage on the allograft valves obtained by a current standard protocol of valve procurement, antibiotic exposure, and cryopreservation, as a basis for future studies on allograft valve preservation. Materials used were seven aortic and seven pulmonary fresh porcine valves, which were cryopreserved according to the requirements of the American and European Associations of Tissue Banks. The samples were randomly assigned into four groups: (1) fresh, untreated; (2) fresh, treated with antibiotics for 24 h.; (3) treated with antibiotics and exposed to dimethyl sulfoxide (without freezing); and (4) treated with antibiotics, exposed to dimethyl sulfoxide, and then cryopreserved and stored until the study. All tissue samples were processed simultaneously for routine light microscopy and transmission electron microscopy. Fresh-untreated, antibiotic-treated, and dimethyl sulfoxide-exposed valves showed adequate preservation of cellular components. However, after cryopreservation significant damage was observed in fibroblasts with signs of apoptotic cellular injury. Our observation suggests that apoptosis occurs during valve processing. This apoptotic process may be related to various factors, including chemical injury or hypoxia.

Animals↗

Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium.

BACKGROUND: Oocyte cryopreservation may have significant potential for assisted reproductive technology. However, to date, successful results have been limited. We report a preliminary series of IVF outcomes after fertilization of oocytes that were frozen in a low-sodium medium. METHODS: In this retrospective analysis, 12 patients (21-41 years old), who underwent IVF in a fertility clinic affiliated to the University of Buenos Aires, had oocytes cryopreserved in a modified phosphate buffered saline medium, in which sodium chloride was replaced by choline chloride. A slow-freezing, rapid-thawing protocol was used and oocytes were inseminated by ICSI. Outcome measures included oocyte survival, fertilization, implantation and pregnancy rates. RESULTS: Median oocyte survival was 63%. Median fertilization rate was 59%. Overall implantation rate was 25%. Six clinical pregnancies were achieved; two of these pregnancies went to term resulting in the birth of two babies. CONCLUSIONS: To the best of our knowledge, these are the first pregnancies and normal births using oocytes that were cryopreserved in a choline-based medium. The small sample size prevents us from concluding that freezing in a low-sodium medium is superior to using a conventional one.

Adult↗

Cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa in a chemically defined extender.

AIM: To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender. METHODS: Semen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris mTTE synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1 %, 3 %, 5 %, 10 % and 15 % [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity. RESULTS: The post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P0.05) for 5 % glycerol (42.95 +/- 2.55 and 50.39+/- 2.42, respectively) than those of the other groups (1%: 19.19 +/- 3.22 and 24.84 +/- 3.64; 3%: 34.23 +/- 3.43 and 41.37 +/- 3.42; 10%:15.68 +/- 2.36 and 21.39 +/- 3.14; 15%: 7.47 +/- 1.44 and 12.90 +/- 2.18). The parameters for 30 min equilibration(42.95 +/- 2.55 and 50.39 +/- 2.42) were better (P0.05) than those of the other groups (10 min: 31.33 +/- 3.06 and 38.98 +/- 3.31; 60 min: 32.49 +/- 3.86 and 40.01 +/- 4.18; 90 min: 31.16 +/- 3.66 and 38.30 +/- 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity. CONCLUSION: Cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.

Animals↗

Freeze-drying of red blood cells: how useful are freeze/thaw experiments for optimization of the cooling rate?

A red blood cell suspension, prepared according to a high-yield HES cryopreservation protocol, was frozen at selected cooling rates of 50, 220, 1250, 4200, and 13,500 K/min. After either thawing or vacuum-drying, the cell recovery was determined using a modified saline stability test. As expected, the recovery of thawed samples followed the theory of Mazur's two-factor hypothesis. The best result was found at a cooling rate of 220 K/min. In contrast, the recovery of freeze-dried and rehydrated samples was very poor at that rate, but maximal at 4200 K/min where thawing caused almost complete hemolysis. This discrepancy is attributed to different damaging mechanisms involved with the respective sample processing subsequent to freezing. While thawing leads to increased devitrification and recrystallization at supraoptimal cooling rates for cryopreservation, the resultant almost vitreous sample structure seems to be advantageous for vacuum-drying. It can be concluded that freeze/thaw experiments are not sufficient for optimization of the cooling rate for freeze-drying.

Adult↗

[Relationship between exposure time and cryopreservation of articular cartilage].

OBJECTIVE: To search an optimal method for improving viability of cryopreserved articular cartilage. METHODS: Articular cartilage which was sampled from the rabbits were randomly divided into 5 groups. Fresh cartilage was group I, other groups were frozen. Before frozen, other cartilage was exposured in 10% DMSO at 4 degrees C for 30 minutes(group II), 1 hour(group III), 2 hours (group IV), 4 hours(group V), then were stored in liquid nitrogen for 1 week. Viabilities of the chondrocytes were detected by Typan-blue staining, electron transmission microscope, and determination of incorporation 3H-TdR after the temperature returned to normal. RESULTS: 1. The cells were injuried at different extent after the cartilage was frozen. In group I, survival rate of cells was 96% and incorporation of 3H-TdR was (4,953.13 +/- 583.27)%, statistic difference was significant between group I and other groups(P < 0.01). The microstructure of group I was normal while other groups all had damage of the organella, 2. Structures and functions of cells in group IV were best among frozen groups. Organella were less damaged than group II, III, V, survival rate of cells was 56% and incorporation of 3H-TdR was (1,139.88 +/- 146.39)%, statistic difference was significant between group IV and group II, III, V(P < 0.01). CONCLUSION: If cartilage are exposured in 10% DMSO at 4 degrees C for 2 hours before frozen, optimal cryopreservation can be achieved.

Animals↗

Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts.

BACKGROUND: Successful cryopreservation of gonadal tissue is an important factor in guaranteeing the fertility preservation via germ cell or testicular tissue transplantation. The aim of this study was to evaluate the effects of cooling and cryopreservation on spermatogonial stem cell survival and function of immature non-human primate testicular tissue xenografted to nude mice. METHODS: Group 1 (control group) received subcutaneous grafts of fresh immature rhesus monkey testes. The treatment groups received grafts after 24 h cooling in ice-cold medium (Group 2), after 24 h of cryopreservation without cryoprotectant (Group 3), with ethylene glycol (Group 4: 1.4 M) or with dimethylsulphoxide (DMSO) (group 5: 1.4 M; group 6: 0.7 M), using cooling rates of 0.5 degrees C/min. The graft number, weight and histology were examined 3-5 months later. RESULTS: After xenografting, grafts from fresh and cooled tissue showed good survival and spermatogenic induction to spermatocytes. Cryopreservation in 1.4 M DMSO also allowed grafts to initiate spermatogenesis. In contrast, 0.7 M DMSO and ethylene glycol showed inferior protection. CONCLUSIONS: Our observations suggest that cryopreservation of immature primate testis is a feasible approach to maintain spermatogonial stem cells and may serve as a promising tool for fertility preservation of prepubertal boys. The possibility to delay the transplantation of cooled samples suggests an option for clinical centralization of testicular tissue cryopreservation.

Animals↗

Keratocyte injury in human corneas cryopreserved under standard conditions.

This study was conducted to characterize ultrastructural damage to human corneas cryopreserved by a standard protocol. The materials used were seven human corneas that were unsuitable for transplantation due to the presence of positive bacteriological cultures; they were cryopreserved according the standard procedure. After freezing and thawing, samples were obtained for scanning and transmission electron microscopy studies. Marked damage was observed in keratocytes with signs of apoptotic cellular injury. However our observations have shown that apoptosis contribute less significantly than necrosis to cellular death in keratocytes of human corneas and although the control of apoptosis is clearly desirable, in order to improve the success of cryopreserved corneas for transplant, we need to continue our investigation to reduce the effects of the necrotic process.

Cornea↗

Computerized sperm motility and application of sperm cryopreservation.

An objective method for measuring sperm motion characteristics was developed on an Intellect 100 Quantel Image Analysis System suitable for various image cytometric applications. It provided overall analysis of percent motility (% MS) as well as individual and mean measurements of motion characteristics, including vigor characteristics such as curvilinear velocity (Vc), straight line velocity (Vsl), and trajectory pattern characteristics, that is, progressiveness ratio (PR) and amplitude of lateral head displacement (Alh). Evaluation of the method for reproducibility and accuracy showed reliable measurements of these parameters measured on a minimum of 70 motile sperm, sufficient to describe adequately the sperm population. A study was performed comparing motion characteristics of 30 semen samples falling in a normal range before and after cryopreservation in cryoprotector medium (CM). A mean motility rate of recovery (MRR) of 45% was obtained. Only sperm count and concentration in motile forms among initial semen variables correlated weakly with MRR. Velocity recovery rate (VRR) approached 1 with a marked variability among ejaculates. Distribution profile of Vc was highly modified by freezing in CM: spermatozoa that were initially fast and progressive were the most resistant to cryoaggression. PR and Alh values were little affected by freezing in CM. The tolerance of various samples from a given patient was highly variable for % MS and Alh and less variable for Vc and PR. This illustrates the difficulty in predicting the effect of freezing on motility characteristics and, therefore, of extrapolating from semen variables the ability of frozen-thawed samples to fertilize.

Freezing↗

Viability and functional activity of cryopreserved mononuclear cells.

Factors that influence viability and function of cryopreserved peripheral blood mononuclear cells (PBMC) were identified on 54 samples from 27 AIDS Clinical Trial Units. PBMC viability ranged from 1 to 96% with a median of 70%, was higher in laboratories with experienced staff, and was not significantly associated with CD4 cell number. Function of cryopreserved PBMC, measured by lymphocyte proliferation, was associated with viability. Preparations with viability greater than or equal to 70% had consistent proliferative responses and were suitable for functional analyses.

Cell Division↗

Diphtheria toxin resistance in human lymphocytes and lymphoblasts in the in vivo somatic cell mutation test.

It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6-thioguanine-resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus-specific differences in response to mutagens. Resistance to diphtheria toxin (Dipr) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinese hamster cells with no evidence for cell density or cross-feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein-Barr virus (EBV)-transformed lymphocytes were tested for continued DNA synthesis (3H-thymidine, autoradiography) or protein synthesis (35S-methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulated to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA-stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The findings were confirmed in cord blood lymphocytes, ruling out the possibility that diphtheria immunization could have led to a selection of Dipr lymphocytes. One lymphoblast line (EBV-transformed lymphocytes) showed a reduction in protein synthesis to 0.2% of controls only at 192 hr after exposure to 100 LF/ml. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dipr may not be a suitable marker for the somatic cell mutation test.

Cells, Cultured↗

ATAC-seq in Emerging Model Organisms: Challenges and Strategies.

The Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a versatile and widely utilized method for identifying potential regulatory regions, such as promoters and enhancers, within a genome. ATAC-seq has been successfully applied to a wide range of established and emerging model organisms. However, implementing this method in emerging model systems, such as arthropods, can be challenging due to several factors that influence data quality. These factors include the availability of a sufficient amount and quality of tissue or cells, the need for species- and tissue-specific protocol optimization, the completeness and accuracy of the reference genome, and the quality of the genome annotation. In this article, we emphasize the key steps in the ATAC-seq protocol that, based on our experience, have the greatest impact on data quality when adapting this method for emerging model organisms. Specifically, we discuss the importance of nuclei isolation, the incubation conditions of the Tn5 transposase, and PCR amplification of the library. Furthermore, we outline essential quality checkpoints during the bioinformatic analysis of ATAC-seq data to assist in assessing data integrity and consistency. Given that many emerging model organisms may not be readily available in laboratory cultures, we also emphasize the importance of evaluating how different preservation methods affect ATAC-seq data quality. Based on examples in one spider and one ant species, we demonstrate that replication and thorough quality controls at all steps of the protocol and data analysis are essential to assess the usability of ATAC-seq data. Our data highlights the importance of isolating the right number of intact nuclei, as well as ensuring optimal amplification conditions during library preparation to obtain good-quality sequence data for downstream analyses. We recommend using fresh tissue samples if possible because we show that direct cryopreservation of the tissue may affect chromatin integrity. This effect could be avoided or reduced by preserving the homogenate in cell culture medium. Overall, we explain the ATAC-seq protocol and downstream analyses in detail and give step-by-step advice to researchers who are new to the field and want to implement this method. With careful planning and validation, ATAC-seq can reveal the regulatory landscape of a genome and aid in identifying elements that govern gene expression.

Animals↗