Thawing Step Research Articles

Beef quality assessment of local and imported sources illustrating a contrary view that freezing is the best way of beef preservation using morphometric analysis

The histological analysis of local and imported beef samples throughout storage at various intervals in 4 °C, before and after being frozen at − 18 °C, to detect the changes happened in the microstructure of muscle fibers to evaluate the meat nutritive properties in a step toward rapid evaluation of meat quality. The obtained results illustrated that freezing–thawing step of beef leads to the loss of its muscle fiber structure due to the high moisture content failure, highlighted the idea that imported beef show significant shrinkage in their muscle fibers from the beginning of its purchase to consumers as they seem to be imported as frozen and thawed just before exposed and sold as fresh. Through consumption survey, however consumers prefer local meat, it was detected that 67% of population is eating imported beef with 39.4% more than twice per week. Therefore, consumers’ minds should be changed to depend on locally slaughtered beef on facing their needs of the recommended daily intake of protein.

Effect of swelling and deswelling on the elasticity of polyacrylonitrile gels prepared by freezing and thawing techniques

. The repeating cycle of freezing and thawing brings about a physical gelation for the solutions of polyacrylonitrile (PAN) in N, N-dimethylacetamide (DMA) for appropriate concentrations. It was revealed from wide-angle-X-ray diffraction that the PAN molecules were “cross-linked” by the crystallization to form a network through the freezing and thawing steps. The profiles of small-angle-X-ray scattering for the gels showed the excess scattering from the network structure formed through gelation, which could not be observed for the pure PAN solution. A reversible swelling and shrinking were observable for the PAN gel as a function of the relative content of DMA and benzene by varying the mixing ratio. The degree of swelling measured by weight, for the swollen and shrunk gels, was in a range from 0.7 to 8. A scaling relation appeared for the gel swelling between the PAN volume fraction (ϕ) and the shear moduli (G) which was measured by tensile and indentation tests, as G = G s ×(ϕ)s; G s= 1200kPa and s = 2.26 ± 0.23. A discussion is given on the value of the scaling exponent, s, on the basis of the conformation of flexible chain molecules in solution and the analogy in the microscopic and macroscopic properties between semidilute polymer solutions and gels.

Performance of nasopharyngeal swab and saliva in detecting Delta and Omicron SARS-CoV-2 variants.

A prospective cohort study was conducted during the Delta and Omicron severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) epidemic waves from paired nasopharyngeal swab (NPS or NP swab) and saliva samples taken from 624 participants. The study aimed to assess if any differences among participants from both waves could be observed and if any difference in molecular diagnostic performance could be observed among the two sample types. Samples were transported immediately to the laboratory to ensure the highest possible sample quality without any freezing and thawing steps before processing. Nucleic acids from saliva and NPS were prospectively extracted and SARS‐CoV‐2 was detected using a real‐time reverse‐transcription polymerase chain reaction. All observed results were statistically analyzed. Although the results obtained with NP and saliva agreed overall, higher viral loads were observed in NP swabs regardless of the day of specimen collection in both SARS‐CoV‐2 epidemic waves. No significant difference could be observed between the two epidemic waves characterized by Delta or Omicron SARS‐CoV‐2. To note, Delta infection resulted in higher viral loads both in NP and saliva and more symptoms, including rhinorrhea, cough, and dyspnea, whereas Omicron wave patients more frequently reported sore throat. An increase in the mean log RNA of SARS‐CoV‐2 was observed with the number of expressed symptoms in both waves, however, the difference was not significant. Data confirmed that results from saliva were concordant with those from NP swabs, although saliva proved to be a challenging sample with frequent inhibitions that required substantial retesting.

Use of natural deep eutectic systems as new cryoprotectant agents in the vitrification of mammalian cells

In this work we present the potential of Natural Deep Eutectic Systems (NADES) as new vitrification media for the cryopreservation of mammalian cells. Several NADES composed of natural metabolites were prepared and tested as CPAs in two cell lines, L929 and HacaT cells. After the harvesting, cells were mixed with the eutectic systems, and frozen directly into liquid nitrogen to achieve a vitreous state. Then, the cells were thawed and it was observed that NADES were able to exert a significant cryoprotective effect in L929 cells, when compared with DMSO or in the absence of a CPA. For HacaT cells, only a eutectic system showed a slightly improvement in cell survival, while DMSO caused complete cell death. Moreover, the thermal behaviour of the best systems was studied for further understanding the protective properties of NADES as CPAs, and have shown a significant difference in terms of Tm and Tc when compared with DMSO and water. Additionally, the results obtained showed that NADES can be maintained in the growth media after the thawing step, without compromising cell viability. In summary, we have shown the great potential of NADES to be used as CPAs for the cryopreservation of different cell types, using the vitrification method.

An intermittent outbreak of Burkholderia cepacia contaminating hematopoietic stem cells resulting in infusate-related blood stream infections.

Microbial contamination of hematopoietic stem cells (HSC), used for autologous and allogenic transplantations, is rare but could cause serious blood stream infection in transplanted patients. These infections occur immediately, or later following the formation of biofilm on the catheter lumen. The present study describes an intermittent B. cepacia HSC contamination associated with nosocomial bacteremia: from October 2011 to April 2015, 17B. cepacia strains were isolated in HSC bags (n = 14) and blood cultures (n = 3) in patients hospitalized in the National Bone Marrow Transplant Center. Two epidemiologic investigations in the National Blood Transfusion Center, allowing the isolation of three strains in hygiene samples, and four interventions in this institution were done. To identify the source of this contamination, a molecular investigation was done on 23B. cepacia strains isolated in our center from 2007 to 2015. PFGE analysis revealed five clusters. The major cluster included 18 strains isolated from HSC bags (n = 14), blood culture (n = 1), and water cans and bath (n = 3). The second cluster (B) including only two and the remaining clusters (C, D, and E) contained single strains isolated before the epidemic period. These findings confirmed that the origin of the outbreak was the contaminated water used in the water bath during the thawing step of HSC bags. Based on this result, new sterile water was used for every defrosting, but HSC bags contamination persisted. In May 2015, the water bath was replaced with a dry bath and no B. cepacia strain was isolated from that date to April 2020.

Potentialities of using cryoprotectants in gluten-free frozen dough and microwave baking as an emerging technology

Disorders related to human health involving proteins that form the gluten network are increasingly in society, justifying the need to offer bakery products for this market niche. This study aimed to gather important information about the challenges involving technological production in the gluten-free bakery market. The approach encompasses the development of frozen dough for gluten-free baking, the use of cryoprotectants to maintain cell viability during the freeze-thawing step, the application of emulsifiers to improve the quality of the bread and the baking by an unconventional method using the microwaves. The introduction of frozen dough in the gluten-free bakery sector represents an interesting tool. However, these bread formulations require a higher amount of water compared to a traditional bread formulation (70 to 120 %), which makes the freezing and thawing steps extremely challenging, especially when it comes to the biopreservation of yeast cell viability. Cryopreservation with isolated and/or combined application of penetrating (glycerol) and non-penetrating (sucrose, fructo-oligosaccharide, trehalose, proteins and hydrocolloids) cryoprotectants promotes changes in the viscosity of the medium and is an excellent alternative for the maintenance of the microorganism responsible for the fermentation process. The appropriate choices of cryoprotectants, as well as the control of the freezing and thawing and baking processes, are important steps in the production line, to guarantee products with adequate technological and sensory qualities.

Freeze-Thawing-Induced Macroporous Catechol Hydrogels with Shape Recovery and Sponge-like Properties.

Catechol-containing hydrogels have been exploited in biomedical fields due to their adhesive and cohesive properties, hemostatic abilities, and biocompatibility. Catechol moieties can be oxidized to o-catecholquinone, a chemically active intermediate, in the presence of oxygen to act as an electrophile to form catechol-catechol or catechol-amine/thiol adducts. To date, catechol cross-linking chemistry to fabricate hydrogels has been mostly performed at room temperature. Herein, we report large increases in catechol cross-linking reaction kinetics by the freeze-thawing process. The formation of ice crystals during freezing steps spatially condenses catechol-containing polymers into nearly frozen (yet unfrozen) regions, resulting in decreases in the polymeric chain distances. This environment allows great increases in catechol cross-linking kinetics, a phenomenon that can also occur during thawing steps. The increased cross-linking rate and spatial condensation in the cryogels provide unique wall and pore structures, which result in elastic, spongelike hydrogels. The moduli of the cryogels prepared by glycol-chitosan-catechol (g-chitosan-c) were improved by 3-6-fold compared to room temperature-cured conventional hydrogels, and the degree of improvement increased depending on the freezing time and the number of freeze-thawing cycles. Unlike typical cell encapsulations before cross-linking, which have often been a source of cytotoxicity, the macroporosity of cryogels allows nontoxic cell seeding with ease. This research offers a new way to utilize catechol cross-linking chemistry by freeze-thawing processes to simultaneously regulate mechanical strength and porous structures in catechol-containing hydrogels.

Application of HACCP system in catering system and microbiological quality of roasted chicken meals

This study aimed to work out the microbiological quality of roasted chicken ready to eat meals (n=240), swabs of labor surfaces (n=390), and the food handlers hands (n=90) in catering services within the university dormitory females to assure a secure supply of food for colleges students.The results obtained appeared no contamination with L. monocytogenes, Salmonella spp., Shigella spp., and Clostridium perfringens before the applying of the HACCP framework. While the fluctuation within the microbial numerous total viable bacteria, total molds and yeasts, B. cereus, Staph. aureus, spore-formers, and coliforms in served meals ascribed to inadequate handling or processing procedures, multiplication of microorganisms during thawing and cutting of chicken, poor hygiene of utensils, and equipment as well as the survival of microorganisms to the cooking process.The examined chicken samples from receiving to serving were 1.12×105, 7.4 X 103, 2.8 X 104, and 1.6 X 103 in washing chicken, thawing, and washing steps respectively. The lowest value was 3.98×103, 3.2 X 102, 1.2 x 103, and 6.1 X 102 in serving, dressing, washing, and receiving steps, respectively. Swabs samples analysis which taken before and after HACCP application from handlers, utensils, equipment and work surfaces observed different levels of significance in the reduction of microbial load in one or more of selected examination. Application of the HACCP framework shows a low rate of examined microorganism with a decreasing percentage reached 100% of 1 or more microbial groups in serving step and p < 0.05 which demonstrates a critical impact of HACCP application. HACCP framework can be moreover utilize to control the safety and quality of prepared ready to eat meals, based on specializing laboratory to do the necessary microbial analysis, whether by the ministry of Health, Universities or by the specifications to reduces the reliance on the end product inspection that ultimately resulted in improving food safety, reducing costs associated with food hazards.

The preparation of graphene foam by one-step reduction and air-drying for oil–water separation

The freeze-drying or supercritical CO2 drying methods are usually used to remove the solvent to keep the shape of material in traditional methods for preparing graphene foam. Porous frozen tofu is a kind of traditional Chinese food and prepared through a process of freezing casting and thawing. Inspired by it, a green and economic approach for graphene foam (GF) synthesis using one-step reduction and air-drying is reported in this article. By controlling the reduction time and precursor concentration, monolithic GF with a little or without volume shrinkage can be obtained after a simple pre-freezing, thawing, and air-drying step of the graphene hydrogel. The obtained GF shows low density (17.5 mg/cm3), superelastic, high recyclability, and excellent oil absorption ability. During the whole preparation process, the conditions were mild, and no special equipment such as freezer dryer was used, which is beneficial to large-scale production of porous GF with high oil adsorption and recyclability.

Concentration Effects in Frozen Polyacrylonitrile Solutions During the Gelation Process as Studied by Viscoelastic Techniques

Dynamic viscoelasticity measurements were carried out for N, N-dimethylacetamide solutions of polyacrylonitrile (PAN) frozen at −40 °C to characterize the effect of concentration in the frozen state in relation to the freezing and thawing method of the PAN gel. The storage (G′) and loss moduli (G″) were measured for samples obtained by separating the frozen and unfrozen regions during the thawing step. The G′ were also acquired for solutions without the separation (referred to as post-thawing solution), written as and for frozen region, unfrozen region, and post-thawing solution, respectively. The relationship, < < was observed, showing that regions of higher and lower concentrations were developed in the thawing step. (/) was considered a relative index of the concentration increase, to be compared for those of the initial and later stages within the thawing step. (/) was much greater than 1 in the initial stage, and then decreased and approached 1 in the later stage. The behavior of (/) was discussed with reference to the effects of freezing time and concentration on the gelation time. The microscopic separation between the dilute and concentrated phases induced at the freezing step was proposed as the physical process to interpret the viscoelastic data.

Synthetic Polyampholytes as Macromolecular Cryoprotective Agents.

A series of polyampholytes based on different molar ratios on N, N-dimethylaminopropyl methacrylamide (DMAPMA), acrylic acid (AA), and optionally, N- tert-butylacrylamide ( t-BuAAm), were prepared by free radical copolymerization, and tested as DMSO-free cryoprotective agents for 3T3 fibroblast cells by using a standard freeze-rethaw protocol. Polybetaines prepared by reaction of DMAPMA homo and copolymers with 1,3-propane sultone were used as additional controls. Results showed strong effects of copolymer composition, molecular weight, polymer and NaCl concentrations, on post-thaw cell viability. Binary (DMAPMA/AA) copolymers showed best post-thaw cell viability of 70% at a 30/70 mol % ratio of DMAPMA/AA, which increased to 90% upon introduction of 9 mol % t-BuAAm while maintaining the 30/70 mol % cation/anion ratio. The use of acrylamide linkages in DMAPMA ensures absence of hydrolytic loss of cationic side chains. These polyampholytes were found to decrease ice crystal size and to form a polymer-rich, ice-free layer around cells, reducing damage from intercellular ice crystals during both freezing and thawing steps. These polyampholytes also dehydrate cells during freezing, which helps protect cells from intracellular ice damage. While cell viability immediately after thawing was high, subsequent culturing revealed poor attachment and long-term viability, which is attributed to residual cell damage from intracellular ice formation. Addition of 2 wt % DMSO or 1% BSA to the polymer-based freeze medium was found to mitigate this damage and result in post-thaw viabilities matching those achieved with 10 wt % DMSO.

A validated cellular biobank for β-thalassemia.

BackgroundCellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on β-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning.MethodsErythroid precursor cells were obtained from 72 patients, mostly β-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC.ResultsIn this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from β-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers.ConclusionThe use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1016-4) contains supplementary material, which is available to authorized users.

Cord Blood: Spotlight on the Processing Step

Cord blood (CB) can be stored processed or un-processed. The advantage of applying a processing step to the cord blood units (CBUs) for cord blood banks (CBBs) is that the volume can be reduced by depletion of plasma and sometimes also by reduction of red blood cell (RBC) number. This decreases the associated costs for storage. Another advantage is the reduction of the quantity of cryopreservation solution in the final product. As the RBCs tend to burst during the freezing and thawing step the decrease in RBC numbers reduces the possibly harmful cellular debris and free hemoglobin released by burst RBCs into the CBUs. However processing can potentially cause also a loss of the wanted cells, like the stem cell compartment or cause stress to these cells. Thus, the optimal processing system reduces volume and RBCs while at the same time leaving the wanted cells like the stem cell compartment with its hematopoietic as well as non-hematopoietic stem cells unaffected. Hematopoietic stem cells are mainly used as a source for transplants to reconstitute the blood forming cells while non-hematopoietic stem cells can support the hematopoietic stem cells in the transplant setting but are potentially useful also for other regenerative medicine or immune modulatory purposes. Many CBBs today use a processing step instead of storing the CB un-processed just by the addition of cryopreservation solution. Different processing systems exist which often employ a centrifugation step for processing of the CBUs. In addition, we described recently a manual system, CellEffic CB, which employs a filtration step eliminating the need for centrifugation and thus any machines and its associated costs. We will highlight here recent development in the cord blood industry and how the new filtration system can contribute to this industry.

A novel approach to identifying physical markers of cryo-damage in bull spermatozoa.

Cryopreservation is an efficient way to store spermatozoa and plays a critical role in the livestock industry as well as in clinical practice. During cryopreservation, cryo-stress causes substantial damage to spermatozoa. In present study, the effects of cryo-stress at various cryopreservation steps, such as dilution / cooling, adding cryoprtectant, and freezing were studied in spermatozoa collected from 9 individual bull testes. The motility (%), motion kinematics, capacitation status, mitochondrial activity, and viability of bovine spermatozoa at each step of the cryopreservation process were assessed using computer-assisted sperm analysis, Hoechst 33258/chlortetracycline fluorescence, rhodamine 123 staining, and hypo-osmotic swelling test, respectively. The results demonstrate that the cryopreservation steps reduced motility (%), rapid speed (%), and mitochondrial activity, whereas medium/slow speed (%), and the acrosome reaction were increased (P < 0.05). Differences (Δ) of the acrosome reaction were higher in dilution/cooling step (P < 0.05), whereas differences (Δ) of motility, rapid speed, and non-progressive motility were higher in cryoprotectant and freezing as compared to dilution/cooling (P < 0.05). On the other hand, differences (Δ) of mitochondrial activity, viability, and progressive motility were higher in freezing step (P < 0.05) while the difference (Δ) of the acrosome reaction was higher in dilution/cooling (P < 0.05). Based on these results, we propose that freezing / thawing steps are the most critical in cryopreservation and may provide a logical ground of understanding on the cryo-damage. Moreover, these sperm parameters might be used as physical markers of sperm cryo-damage.

Challenges in the Development of an Immunochromatographic Interferon-Gamma Test for Diagnosis of Pleural Tuberculosis

Existing diagnostic tests for pleural tuberculosis (TB) have inadequate accuracy and/or turnaround time. Interferon-gamma (IFNg) has been identified in many studies as a biomarker for pleural TB. Our objective was to develop a lateral flow, immunochromatographic test (ICT) based on this biomarker and to evaluate the test in a clinical cohort. Because IFNg is commonly present in non-TB pleural effusions in low amounts, a diagnostic IFNg-threshold was first defined with an enzyme-linked immunosorbent assay (ELISA) for IFNg in samples from 38 patients with a confirmed clinical diagnosis (cut-off of 300pg/ml; 94% sensitivity and 93% specificity). The ICT was then designed; however, its achievable limit of detection (5000pg/ml) was over 10-fold higher than that of the ELISA. After several iterations in development, the prototype ICT assay for IFNg had a sensitivity of 69% (95% confidence interval (CI): 50-83) and a specificity of 94% (95% CI: 81-99%) compared to ELISA on frozen samples. Evaluation of the prototype in a prospective clinical cohort (72 patients) on fresh pleural fluid samples, in comparison to a composite reference standard (including histopathological and microbiologic test results), showed that the prototype had 65% sensitivity (95% CI: 44-83) and 89% specificity (95% CI: 74-97). Discordant results were observed in 15% of samples if testing was repeated after one freezing and thawing step. Inter-rater variability was limited (3%; 1out of 32). In conclusion, despite an iterative development and optimization process, the performance of the IFNg ICT remained lower than what could be expected from the published literature on IFNg as a biomarker in pleural fluid. Further improvements in the limit of detection of an ICT for IFNg, and possibly combination of IFNg with other biomarkers such as adenosine deaminase, are necessary for such a test to be of value in the evaluation of pleural tuberculosis.

029 Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein organization of rooster sperm during cooling and cryopreservation using the fluorescent stains Merocyanine 540 (phospholipid order) and Rhodamine 640 (protein order). Rooster semen samples (3 roosters and breeds per pool; 4 pools total) were diluted in Lakes Low Temperature (LLT) and Sasaki diluent and cooled to 5 °C over 30 min (Step 1). The samples were then diluted with the penetrating cryoprotectants (CPA) glycerol (LLT) or methylacetamide (Sasaki), equilibrated for 30 min (Step 2), loaded into straws and cryopreserved. The cryopreservation and thawing (Step 3) was performed using the optimized procedures for each diluent. Computer automated semen analysis (CASA) was used to analyze motility characteristics and flow cytometry was used to analyze plasma membrane phospholipid, protein organization and plasma membrane integrity (PMI) at each step. Percent values from CASA and flow cytometry fluorescent medians (FM) were transformed using arcsine and Box–Cox, respectively. The GLM ANOVA was used to detect differences in motility characteristics and membrane organization and included the effects of cryopreservation step and rooster semen pool. In the total motility analyses cryopreservation diluent/CPA was a significant ( P P P P P Source of funding: USDA-ARS-NCGRP National Animal Germplasm Program. Conflict of interest: None declared. phil.purdy@ars.usda.gov

Effects of freezing and thawing on the microstructure of latex paints

Freeze–thaw (FT) cycles can aggregate particles in aqueous paint suspensions. To understand the mechanism of particle aggregation, cryogenic scanning electron microscopy (cryoSEM) was used to visualize the microstructure after the freezing and thawing steps of the FT cycle. After the freezing step, cryoSEM images show that the microstructure contains ice crystals and particle-rich regions. Adding propylene glycol, a FT stabilizing additive, leads to formation of larger ice crystals. After thawing, the dispersion structure revealed by cryoSEM shows that the particles redisperse only in the paint with the highest amount of propylene glycol. The other paints contain clusters that are different from the particle-rich regions found after the freezing step. Increasing the thawing rate leads to a more dispersed microstructure even in the absence of propylene glycol. Analysis of the cryoSEM results shows that particle aggregation into these clusters occurs during the thawing stage, and slow thawing conditions lead to more aggregation. The cryoSEM results reported here are used to propose a mechanism of aggregation of particles in the paint.

Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

Freeze-thawing as a path to concentrate aqueous solution

Phenol-dye aqueous ternary solutions were employed as model eutectic solution, and concentration phenomena during freezing and thawing were investigated. The freeze-concentration level during freezing step and the concentration profiles of the melt fractions during thawing step were examined. It was found that the present freezing conditions did not effectively separate the solutes from frozen zone. Phenol component was slightly concentrated in the top frozen layer though the dye was almost equivalently distributed in the frozen domain. However, the concentrations of melt fractions obtained during thawing step were clearly higher than the concentration level of the frozen domains. Approximately 50% of the both solutes could be recovered from the initial 30% of the melt fraction. It was suggested that concentration behaviour during thawing would be controlled by thawing condition as explained by the kinetics of mass transfer of solutes in the frozen domain and at the frozen interface.

Vitrification is a highly efficient method to cryopreserve human embryos in in vitro fertilization patients at high risk of developing ovarian hyperstimulation syndrome

This study aimed to evaluate the efficacy of embryo vitrification as an emergency procedure for patients at high risk of developing ovarian hyperstimulation syndrome (OHSS). A total of 69 embryos, derived from 24 patients for whom embryo transfer could not be performed because of the risk of developing OHSS, were vitrified and warmed for deferred embryo transfer. Surviving embryos were transferred, resulting in 10 clinical pregnancies, of which 4 were successfully delivered and the remaining 6 are still ongoing.