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 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

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

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

We explored the improved method to prepare decellularized kidney scaffold and provide experimental basis for kidney tissue engineering and renal pathology and toxicology in vitro research. We perfused rat kidneys with PBS (group control) and prepared the decellularized kidney scaffolds with sodium dodecyl sulfate (SDS) (group S), Triton X-100 combined with SDS (group TS), and Triton X-100 combined with SDS after repeated freezing and thawing (group FTS) in different flow velocity. Meanwhile we measured their fluid distributions and vascular resistance. We examined the degree of decellularization of acellular scaffolds by HE, DAPI staining and DNA quantification. We examined the retention of main composition and structural integrity of decellularized scaffolds by Masson, PAS and immunohistochemical staining. We also detected the ultrastructure, cytotoxicity and the level of growth factor of the scaffolds by scanning electron microscope, MTT and ELISA, respectively. The results showed that the time of decellularization in group FTS was less than that in group S and TS. The vascular resistance of scaffolds decellularized at 10 mL/min flow velocity was lower. The fluid distribution in groups S, TS and FTS was different from that in control group. No residual cell was detected by HE and DAPI staining. DNA content was less than 50 ng/mg. Masson, PAS and immunohistochemical staining results showed that there was extracellular collagen, polysaccharide, type I collagen, type IV collagen, fibronectin and laminin in the decellularized scaffolds, and the scanning electron microscope result showed the scaffolds had the honeycomb structure. The cytotoxicity level of decellularized scaffolds was between grade 0 to 1. The level of VEGF, EGF, IGF-1 and PDGF-BB in group FTS were significantly higher than those in group S and TS. In concluding, combining freeze-thawing with perfusion can produce more ideal and effective whole organ decellularized scaffold of rat kidney, and make a foundation for the study of kidney tissue engineering and in vitro pathology and toxicology of kidney.
Animals ; Collagen ; Extracellular Matrix ; Freezing ; Kidney ; Perfusion ; Rats ; Tissue Engineering ; Tissue Scaffolds

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

PURPOSE: The purpose of the present study was to compare the accuracy of four different metal copings fabricated by CAD/CAM technology and to evaluate clinical effectiveness. MATERIALS AND METHODS: Composite resin tooth of the maxillary central incisor was prepared for a metal ceramic crown and duplicated metal die was fabricated. Then scan the metal die for 12 times to obtain STL files using a confocal microscopy type oral scanner. Metal copings with a thickness of 0.5 mm and a cement space of 50 µm were designed on a CAD program. The Co-Cr metal copings were fabricated by the following four methods: Wax pattern milling & Casting (WM), Resin pattern 3D Printing & casting (RP), Milling & Sintering (MS), Selective laser melting (SLM). Silicone replica technique was used to measure marginal and internal discrepancies. The data was statistically analyzed with One-way analysis of variance and appropriate post hoc test (Scheffe test) (α=.05). RESULTS: Mean marginal discrepancy was significantly smaller in the Group WM (27.66 ± 9.85 µm) and Group MS (28.88 ± 10.13 µm) than in the Group RP (38.09 ± 11.14 µm). Mean cervical discrepancy was significantly smaller in the Group MS than in the Group RP. Mean axial discrepancy was significantly smaller in the Group WM and Group MS then in the Group RP and Group SLM. Mean incisal discrepancies was significantly smaller in the Group RP than in all other groups. CONCLUSION: The marginal and axial discrepancies of the Co-Cr coping fabricated by the Wax pattern milling and Milling/Sintering method were better than those of the other groups. The marginal, cervical and axial fit of Co-Cr copings in all groups are within a clinically acceptable range.
Ceramics ; Crowns ; Dental Marginal Adaptation ; Freezing ; Incisor ; Methods ; Microscopy, Confocal ; Printing, Three-Dimensional ; Replica Techniques ; Silicon ; Silicones ; Tooth ; Treatment Outcome