Ultra-rapid cooling and rewarming rate is a critical technical approach to achieve ice-free cells during the freezing and melting process. A set of ultra-rapid solid surface freeze-thaw visualization system was developed based on a sapphire flim, and experiments on droplet freeze-thaw were carried out under different cryoprotectant components, volumes and laser energies. The results showed that the cooling rate of 1 μL mixed cryoprotectant [1.5 mol/L propylene glycol (PG) + 1.5 mol/L ethylene glycol (EG) + 0.5 mol/L trehalose (TRE)] could be 9.2×10 ³ °C/min. The volume range of 1-8 μL droplets could be vitrified. After comparing the proportions of multiple cryoprotectants, the combination of equal proportion mixed permeability protectant and trehalose had the best vitrification freezing effect and more uniform crystallization characteristics. During the rewarming operation, the heating curve of glassy droplets containing gold nanoparticles was measured for the first time under the action of 400-1 200 W laser power, and the rewarming rate was up to the order of 10 ⁶ °C/min. According to the droplet images of different power rewarming processes, the laser power range for ice-free rewarming with micron-level resolution was clarified to be 1 400-1 600 W. The work of this paper simultaneously realizes the ultra-high-speed temperature ramp-up, transient visual observation and temperature measurement of droplets, providing technical means for judging the ice free droplets during the freeze-thaw process. It is conducive to promoting the development of ultra-rapid freeze-thaw technology for biological cells and tissues.
Freezing ; Vitrification ; Cryopreservation/methods* ; Trehalose ; Gold ; Rewarming ; Metal Nanoparticles ; Cryoprotective Agents ; Lasers

Intrinsically disordered proteins (IDPs) are proteins or protein regions that fail to get folded into definite three-dimensional structures but participate in various biological processes and perform specific functions. Defying the traditional protein "sequence-structure-function" paradigm, they enrich the protein "structure-function" diversity. Ubiquitous in organisms, they show extreme hydrophilicity, charged amino acids, and highly repetitive amino acid sequences, with simple arrangement. As a result, they feature highly variable binding affinities and high coordination, which facilitate their functions. IDPs play an important role in cell stress response, which can improve the tolerance to a variety of stresses, such as freezing, high salt, heat shock, and desiccation. In this study, we briefed the characteristics, classifications, and identification of IDPs, summarized the molecular mechanism in improving cell stress resistance, and described the potential applications.
Freezing ; Intrinsically Disordered Proteins/metabolism* ; Protein Conformation

Objective To investigate the effects of self-made carriers on the cryopreservation of ovarian tissue of sheep. Methods Thirty-two ovaries were randomly assigned to fresh group,programmed freezing group,self-made carrier I vitrification group,and self-made carrier Ⅱ vitrification group.The morphology,proliferation,apoptosis,and estrogen level of the ovarian tissue in each group were observed. Results After cryopreservation,the morphology normal rate of the primordial follicles in programmed freezing group,self-made carrier I vitrification group,and self-made carrier Ⅱ vitrification group were 74.2%,72.8%,and 72.3%,respectively,lower than that(83.7%)in the fresh group(χ
Animals ; Cryopreservation ; Female ; Freezing ; Ovarian Follicle ; Ovary ; Sheep ; Vitrification

Objective To investigate the stability of IgE in postmortem plasma and hemolyzed samples under different storage conditions and freezing-thawing. Methods Thirty nine cardiac blood samples were collected from non-frozen corpses with the postmortem interval of less than 48 hours, including 20 plasma samples and 19 hemolyzed samples taken from whole blood. The samples were stored at -20 ℃, 4 ℃ and 25 ℃ for 28 d and at -80 ℃ for 1 year to evaluate the stability of IgE under different storage conditions. Repeated freezing-thawing treatment was conducted for 5 times to explore the stability of IgE in postmortem plasma and hemolyzed samples. IgE concentration in plasma and hemolyzed samples was detected by electroluminescence before and after treatment. Results The degradation rates of IgE in plasma samples under the three storage conditions, -20 ℃, 4 ℃ and 25 ℃ were close. After 28 d, the mean value was about 15%, the degradation speed of IgE in hemolyzed samples was faster than that of plasma under the same condition （P<0.05） and the degradation rate was faster than other two conditions under 25 ℃ （P<0.05）. The differences in the concentration of plasma samples after freezing at -80 ℃ for 1 year and that before freezing had no statistical significance （ P>0.05）, while the concentration of hemolyzed samples was degraded after freezing at -80 ℃ for 1 year （P<0.05）. The differences between the detection results of plasma and hemolyzed samples after repeated freezing-thawing for 5 times and that before freezing-thawing showed no statistical significance （ P>0.05）. Conclusion IgE has good freezing-thawing stability in postmortem plasma and hemolyzed samples. Stability of IgE is better in postmortem plasma samples than hemolyzed samples, thus it is recommended to separate plasma from postmortem blood samples as soon as possible in forensic practice.
Autopsy ; Forensic Medicine ; Freezing ; Immunoglobulin E ; Plasma

BACKGROUND: It is currently unknown whether patients with a fever after controlled ovulation during egg retrieval could increase the risk of pelvic infection or not, and fever itself may affect endometrial receptivity or embryo quality with poor pregnancy outcomes. The aim of this study was to analyze the outcomes of patients with fever during oocyte retrieval after the first frozen-thawed embryo transfer (FET) cycle. METHODS: This was a 1:3 retrospective paired study matched for age. In this study, 58 infertility patients (Group 1) had a fever during the control ovulation, and the time of the oocyte retrieval was within 72 hours, they underwent ovum pick up and whole embryo freezing ("freeze-all" strategy). The control subjects (Group 2) are 174 patients matched for age who underwent whole embryo freezing for other reasons. The baseline characteristics, clinical data of ovarian stimulation, and outcomes, such as the clinical pregnancy rate, ongoing clinical pregnancy rate were compared between the two groups in the subsequent FET cycle. RESULTS: All patients had no pelvic inflammatory disease after oocyte retrieval. Anti-Mullerian hormone (AMH) levels (4.2 vs. 2.2, P <0.001) were higher in group 2, and the number of oocytes retrieved, and fertilization rate were lower in group 1 (P < 0.001), but the endometrial thickness, the number of embryo transfers, and the type of luteal support supplementation were similar between the two groups. Regarding pregnancy outcomes in the subsequent FET cycle, the implantation rate, clinical pregnancy rate, early spontaneous rate, ectopic pregnancy rate, and ongoing pregnancy rate were all not significantly different. Further regression analyses showed that the clinical pregnancy rate and ongoing pregnancy rate were also not significantly different. CONCLUSIONS Transvaginal ultrasound-guided follicular puncture for oocyte retrieval is a safe and minimally invasive method for patients with fever. Moreover, the fever had almost no effect on embryo quality.
Cryopreservation ; Female ; Fertilization in Vitro ; Freezing ; Humans ; Infertility ; Oocyte Retrieval ; Oocytes ; Ovulation Induction ; Pregnancy ; Pregnancy Outcome ; Pregnancy Rate ; Retrospective Studies

PURPOSE: To report a case of corneal collagen cross-linking for corneal ulcer caused by the Moraxella group.CASE SUMMARY: A 77-year-old male had decreased visual acuity for several days in his right eye. The patient showed severe stromal ring infiltrates with a corneal epithelial defect measuring (5.0 × 7.0 mm), a corneal endothelial plaque, and a hypopyon measuring less than 1.0 mm in height in the anterior chamber of the right eye. There was no abnormal finding in the right eye using B-scan ultrasonography. Before starting treatment, a corneal culture was conducted. The culture tests showed the presence of the Moraxella group. Because the patient was diagnosed with a corneal ulcer caused by the Moraxella group, corneal collagen cross-linking (CXL) was performed. The antimicrobial susceptibility test confirmed that this Moraxella group was sensitive to ceftazidime, so the patient was treated with 5% ceftazidime eye drops and 0.5% moxifloxacin eye drops every 2 hours for 9 months after corneal collagen CXL. The uncorrected visual acuity was 0.1 in the right eye, and there was almost no corneal stromal melting on anterior segment optical coherence tomography.CONCLUSIONS: This is the first known case of a corneal ulcer, in the Republic of Korea, caused by the Moraxella group and treated with corneal collagen CXL. Corneal collagen CXL should be considered as a surgical treatment for patients who have an impending corneal perforation due to a corneal ulcer because it is a simple procedure and causes fewer serious complications than other treatments.
Aged ; Anterior Chamber ; Ceftazidime ; Collagen ; Cornea ; Corneal Perforation ; Corneal Ulcer ; Freezing ; Humans ; Male ; Moraxella ; Ophthalmic Solutions ; Republic of Korea ; Tomography, Optical Coherence ; Ultrasonography ; Visual Acuity

Melting temperature shift (T(m)-shift) is a new detection method that analyze the melting curve on real-time PCR thermocycler using SYBR Green I fluorescent dye. To establish a T(m)-shift method for the detection of Ancylostoma ceylanicum and A. tubaeforme in cats, specific primers, with GC tail of unequal length attached to their 5′ end, were designed based on 2 SNP loci (ITS101 and ITS296) of the internal transcribed spacer 1 (ITS1) sequences. The standard curve of T(m)-shift was established using the standard plasmids of A. ceylanicum (AceP) and A. tubaeforme (AtuP). The T(m)-shift method stability, sensitivity, and accuracy were tested with reference to the standard curve, and clinical fecal samples were also examined. The results demonstrated that the 2 sets of primers based on the 2 SNPs could accurately distinguish between A. ceylanicum and A. tubaeforme. The coefficient of variation (CV) of T(m)-values of AceP and AtuP was 0.07% and 0.06% in ITS101 and was 0.06% and 0.08% in ITS296, respectively. The minimum detectable DNA concentration was 5.22×10⁻⁶ and 5.28×10⁻⁶ ng/μl samples of AceP and AtuP, respectively. The accuracy of T(m)-shift method reached 100% based on examination of 10 hookworm DNA samples with known species. In the clinical detection of hookworm in 69 stray cat fecal sample, the T(m)-shift detection results were consistent with the microscopic examination and successfully differentiated between the 2-hookworm species. In conclusion, the developed method is a rapid, sensitive and accurate technique and can provide a promising tool for clinical detection and epidemiological investigation of cat-derived hookworms.
Ancylostoma ; Ancylostomatoidea ; Animals ; Cats ; DNA ; Freezing ; Methods ; Plasmids ; Polymorphism, Single Nucleotide ; Real-Time Polymerase Chain Reaction ; Tail

In the present study, the performance of the liquid nitrogen frozen and thinned bovine pericardium was studied and compared with the porcine pericardium. The microstructure and mechanical properties of the bovine pericardium were observed and tested by hematoxylin-eosin (HE) staining and tensile test respectively. In all conditions, porcine pericardium was selected as a control group. The results showed that there was little difference in the performance of bovine pericardium after being frozen by liquid nitrogen. The secant modulus and ultimate strength of the thinned bovine pericardium were similar to those of porcine pericardium, however, the elastic modulus was a little higher than porcine pericardium. The study suggested that the performance of the thinned bovine pericardium was similar to those of porcine pericardium. It was easy for the thinned bovine pericardium to obtain a relatively ideal thickness and expected performance, therefore, the thinned bovine pericardium can be used as the materials of transcatheter aortic valve leaflets.
Animals ; Aortic Valve ; Bioprosthesis ; Cattle ; Elastic Modulus ; Freezing ; Heart Valve Prosthesis ; Nitrogen ; Pericardium ; physiology ; Swine

Cell freeze-drying can be divided into the freezing and drying processes. Mechanical damage caused by ice crystals and damage from solute during freezing shall not be ignored and lyoprotectants are commonly used to reduce those damages on cells. In order to study the mechanism of lyoprotectants to protect cells and determine an optimal lyoprotectant formula, the thermophysical properties and percentage of unfrozen water of different lyoprotectants in freezing were investigated with differential scanning calorimeter (DSC). The survival rate indicated by trypan blue exclusion test and cell-attachment rate after 24 h using different lyoprotectants to freeze hepatoma Hep-G cells were measured after cell cryopreservation. The results show that 40% (W/V) PVP + 10% (V/V) glycerol + 15% (V/V) fetal bovine serum + 20% (W/V) trehalose formula of lyoprotectant demonstrate the best effect in protecting cells during freezing, for cell-attachment rate after 24 h is 44.56% ± 2.73%. In conclusion, the formula of lyoprotectant mentioned above can effectively protect cells.
Calorimetry, Differential Scanning ; Cryopreservation ; Cryoprotective Agents ; chemistry ; Freeze Drying ; Freezing ; Hep G2 Cells ; Humans ; Trehalose ; chemistry

To establish appropriate conditions for a disinfectant efficacy test at subzero temperatures, this study examined mixtures of frozen foot-and-mouth disease virus or avian influenza virus solutions and disinfectant diluents at −5℃ and monitored temperature and freezing status of an anti-freezing diluent (AFD, 15% ethanol + 30% propylene glycol + 55% distilled water) over time at various subzero temperatures. Viral solutions and disinfectant diluents froze before the mixtures reached −5℃, whereas the AFD was not frozen at −30℃. The times taken for the AFD to reach −10, −20, −30, and −40℃ from room temperature were 36, 39, 45, and 48 min, respectively.
Animals ; Ethanol ; Foot-and-Mouth Disease Virus ; Freezing ; Influenza in Birds ; Propylene Glycol