Investigation of the influence of cell density of human fibroblasts cryopreserved inside collagen sponges at various cooling rates

The influence of cell density of cells cryopreserved inside a collagen matrix at various cooling rates was investigated. Human fibroblasts were three-dimensionally cultured for 2 days in a collagen sponge (20 mm in diameter and 1 mm in thickness) as an extracellular matrix to imitate biological tissue (artificial tissue). Different cell densities for the artificial tissue were used, from 10(5) to 10(7) cells/cm(3). Four artificial tissues were first stacked in a test chamber, frozen at a cooling rate of 0.3 to 50 degrees C/min in a solution of Dulbecco's Modified Eagle Medium, 20% fetal bovine serum and 10% dimethylsulfoxide, kept frozen below -185 degrees C for 2 hours, and then finally thawed. Membrane integrity of fibroblasts using a trypan blue exclusion assay was evaluated as an index for post-thaw cellular viability. Results show that with increasing cell density, the post-thaw membrane integrity decreased. Therefore, in the cryopreservation of biological tissue, it seems high cell density is one factor which causes a decline in viability.     

Probability of lethal damages of cryopreserved biological objects during storage

This study demonstrated that with a cyclic change of the temperature from -196 degree C up to -130 degree C or -100 degree C part of cryopreserved microorganisms, tumor and human embryo cells died. This should be taken into account when moving and transporting the cryopreserved objects. Storage of samples under the above-mentioned stable temperatures did not result in additional death of cells.     

Cryopreservation and metabolic profiling analysis of Arabidopsis T87 suspension-cultured cells

We established a simple cryopreservation protocol for Arabidopsis T87 cells using an encapsulation-dehydration method. T87 cells were encapsulated into alginate beads containing 2 M glycerol and 0.4 M sucrose. Alginate beads containing T87 cells were dehydrated with silica gel for 2 h (to c. 0.7 g water per g dry weight followed by immersed in LN. After rewarming at 35 degree C for 3 min and 1-d incubation under continuous illumination at 22 degree C, cryopreserved T87 cells exhibited considerable regrowth. Exponentially-grown 7-d-old T87 cells regrew more vigorously (86% of control) than 14-d-old cells after cryopreservation without preculture in medium containing 0.3 M sucrose. Genetic stability of cryopreserved T87 cells was demonstrated by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and principal component analysis (PCA). Transformed T87 cells were cryopreserved using established protocols, and GUS expression was maintained within a 2-fold variance. These results indicate that cryopreservation of T87 cells is useful for comprehensive metabolomics research and for the large scale collection of transformed cultured cell lines for functional genomics research.     

电子膨胀阀对电动汽车空调系统的调节特性研究

电子膨胀阀是电动汽车空调系统的主要调控部件,探究其调节特性对于制定系统控制策略具有重要意义.本文搭建了以R134a为制冷剂的电动汽车空调系统实验台,研究了电子膨胀阀调节过程中,空调系统内制冷剂流量和压力的动态变化规律,分析了不同压缩机转速下,阀开度对系统制冷量、空调箱出风温度、压缩机功耗和系统COP等性能参数的影响.结果表明：阀前制冷剂相态是影响电子膨胀阀调节时系统压力变化强弱的重要因素.在阀前制冷剂为过冷液态时,调节阀开度对系统压力影响更大,并且在阀前制冷剂具有较大过冷度(大于10℃)时,下调阀开度会导致短时间的过节流造成系统压力大幅波动;系统中制冷剂循环流量与阀开度呈线性变化趋势,不受阀前制冷剂相态的影响.在实验工况下,100%阀开度对应的制冷剂循环流量为97.2～115.5 kg/h,阀开度每下调10%,系统中制冷剂循环流量下降6%～9%.     

Beneficial effect of post -thawing osmoconditioning on the recovery of cryopreserved coffee (Coffea arabica L.) seeds

Osmoconditioning-controlled rehydration of seeds in a solution with low osmotic potential -has been shown to reinvigorate aged seeds. The present work aimed at investigating the effect of osmoconditioning on the germination of cryopreserved seeds of Coffea arabica, whose viability and vigour are drastically affected by cryopreservation. For cryopreservation, seeds were desiccated to 0.21 g H2O/g dw, cooled at 1 degree C/min to -50 degree C, then immersed rapidly in liquid nitrogen. After rapid rewarming, seeds were osmoconditioned for 1 to 6 weeks using solutions with osmotic potentials between -1 and -4 MPa. The time to produce half of the final percentage of normal seedlings, T50, was about three fold lower with osmoconditioned seeds than with non-osmoconditioned seeds (12-14 d vs 36 d). Moreover, after a 6-week osmoconditioning treatment with solutions with osmotic potential of -1 and -1.25 MPa, the percentage of seedlings recovered from cryopreserved seeds was 64-74%, against 13-16% only for cryopreserved seeds which were not osmoconditioned.     

Oxygen-sensitive 19F NMR imaging of the vascular system in vivo

The fluorine nuclear magnetic resonance spin-lattice relaxation rate (1/T1) of the perfluorochemical blood substitute perfluorotripropylamine (FTPA) is very sensitive to oxygen tension. This presents the possibility of measuring blood oxygen tension by 19F MR imaging. We obtained oxygen-sensitive 19F NMR images of the circulatory system of rats infused with emulsified FTPA. Blood oxygenation was assessed under conditions of both air- and 100% O2-breathing. T1 relaxation times were derived from MR images using a partial saturation pulse sequence. The T1 times were compared with a phantom calibration curve to calculate average blood pO2 values in the lung, liver, and spleen. The results showed marked, organ-specific increases in blood oxygen tension when the rat breathed 100% O2 instead of air.     

RGDS-fuctionalized alginates improve the survival rate of encapsulated embryonic stem cells during cryopreservation

Cryopreservation of stem cells, especially embryonic stem cells, is problematic because of low post-thaw cell survival rates and spontaneous differentiation following recovery. In this investigation, mouse embryonic stem cells (mESCs) were encapsulated in arginine-glycine-aspartic acid-serine (RGDS)-coupled calcium alginates (1.2 percent, w/v), allowed to attach to the substratum and then cryopreserved in 10 percent (v/v) dimethyl sulfoxide (DMSO) solution at a slow cooling rate of 1 C per min. RGDS coupling to alginate was confirmed by Transmission Fourier Transform Infra-Red spectroscopy (T-FTIR) and quantified by using ninhydrin-Ultraviolet/Visible light (ninhydrin-UV/VIS) assay. Flow cytometry data showed that mESCs cryopreserved in RGDS-alginate beads had a higher expression of stem cell markers compared with cells cryopreserved in suspension or cells cryopreserved in unmodified alginates. Cell viability after thawing was assessed using trypan blue exclusion assay and monitored using Alamar blue assay for 6 hours. It was shown that post-thaw cell survival rate was significantly higher for cells encapsulated in RGDS-modified alginate (93 ± 2 percent, mean and standard error) than those in suspension (52 ± 2 percent) or in unmodified alginates (62 ± 3 percent). These results showed that cells encapsulated and attached to a substratum have better survival rate and stem cell marker expression 24 hours after cryopreservation than those in suspension. Encapsulation in RGDS-alginate was optimized for peptide concentration, cryoprotective agent loading time and cooling rate. The best result was obtained when using 12.5 mg peptide per g alginate, 30 minutes loading time and 1 C per min cooling rate.     

Cryopreservation of human embryonic stem cells: a protocol by programmed cooling

Human embryonic stem (ES) cells have far-reaching applications in the areas of tissue engineering, regenerative medicine, pharmacology and basic scientific research. Although the culture conditions can maintain the human ES cells in an undifferentiated state for a transient period, spontaneous differentiation has also been observed during the routine culturing of ES cells. However, the maintenance of ES cells in the undifferentiated, pluripotent state for extended periods of time will be required in many areas of scientific research. Cryopreservation is a technology with potentially far reaching implication for the development and widespread use of such cell lines. This study was undertaken to develop and optimize a protocol for cryopreservation of human ES cells through programmed cooling. The effects of the seeding temperature, the cooling rate and the sub-zero temperature to which the samples were cooled before plunging into liquid nitrogen(the terminal temperature), all significantly affected the recovery of cryopreserved ES cells. After studying these factors, an improved protocol was obtained: the sample was cooled from 0 degree C to -35 degree C at a cooling rate of 0.5 degree per min, with seeding was set at -10 degree C, before being plunged immediately into the liquid nitrogen. Using this protocol, 9 of 11 colony fragments survived freezing and thawing and could be cultured for prolonged periods. They retained the properties of pluripotent cells, had a normal karyotype and showed histochemical staining for alkaline phosphatase.     

Effects of adsorbed carbon and oxygen on the chemisorption of H2 and Co on Mo(100)

The deposit of carbon and oxygen adatoms on Mo(100) was characterized by AES and LEED. Carbon was introduced by the thermal cracking of ethylene; several ordered structures were observed as a function of coverage with carbon atoms residing on four-fold sites. The Mo(100)—O system exhibited two different sequences of LEED patterns depending on the adsorption temperature of oxygen. The effects of adsorbed carbon and oxygen on the chemisorption properties of Mo(100) was investigated by FDS. The presence of either carbon or oxygen severely hindered the ability of Mo(100) to dissociatively adsorb hydrogen or carbon monoxide. The amount of CO dissociated was directly related to the available four-fold sites on the carbide surfaces. The molecular adsorption of CO was not significantly affected by the adlayers. It was found that one monolayer of adsorbed oxygen reduced the binding energy of molecular CO considerably more than the same amount of adsorbed carbon. A continuous shift in the binding energy of CO with the C/O ratio on the surface was observed.     

Development of a vitrification-based cryopreservation protocol for the storage of saltcedar (Tamarix boveana Bunge)

We cryopreserved in vitro shoot tips of saltcedar (Tamarix boveana Bunge) using the vitrification technique. The success of the cryopreservation protocol was strongly affected by preculture, loading duration, dehydration duration in plant vitrification solution 2 (PVS2), and medium composition during post-warming regrowth. The highest explant regrowth (50 percent) occurred when the following conditions were employed: preculture in 0.4 M glycerol; treatment with a loading solution (LS) consisting of 2 M glycerol + 0.4 M sucrose in culture medium for 40 min at room temperature; and dehydration in PVS2 at 0 degree C for 45 min before rapid immersion in liquid nitrogen (LN). Rewarming was performed in a water-bath at 40 degree C for 2 min. Explants were then immersed in unloading solution for 10 min before plating on recovery medium supplemented with 0.01 mg per liter thidiazuron (TDZ). TDZ was progressively eliminated from the medium over a period of 6 weeks. Plantlets were transferred to a double-layer medium to enhance rooting. This protocol was successfully applied to three individuals of T. boveana harvested from the wild.     

Oxygen consumption and chemisorption in low-temperature oxidation of sub-bituminous pulverized coal

Studying the effect of oxygen in coal oxidation is very important for understanding and controlling coal spontaneous combustion. However, the oxygen effect is not very easy to determine clearly due to the large effect of heat source on coal oxidation in temperature rising experiments. Here, focused on sub-bituminous coal, the oxygen effect was separated from coal oxidation by continuously measuring FTIR spectra of coal with respect to varying temperatures and under oxygen and nitrogen. The active groups’ real-time changes of coal oxidation, thermal treatment and oxygen effect were measured. The carboxylic ester and carboxyl units are the main functional groups that increase with temperatures increasing under oxygen and nitrogen, while the other functional groups decrease in quantity. The oxygen effect promoted the consumption of aliphatic hydrocarbons and hydroxyl groups and also promoted the formation of oxygen-containing groups (except hydroxyl). Four characteristic temperature stages involved in the oxygen effect and their key functional groups were identified. Simultaneously, the relationship of oxygen consumption and chemisorption in oxygen effect was analyzed. The starting temperature of oxygen chemisorption is between 50 and 60°C. The maximum contribution of oxygen effect was observed in methyl and methylene groups. These results are important for chemical control of coal spontaneous combustion. The oxidation of aliphatic hydrocarbon should be controlled before oxygen chemisorption. The value of oxygen consumption between 70 and 80°C can be measured accurately due to the constant chemisorption rate, which help to identify the tendency for spontaneous combustion. These results will help in better understanding of the reaction mechanism of coal oxidation, especially the oxygen effect.     

Cryopreservation of Vanda coerulea protocorms by encapsulation-dehydration

Protocorms of Vanda coerulea were successfully cryopreserved by encapsulation-dehydration in combination with a loading solution. Protocorms were selected 70 days after sowing seeds harvested from 7-month-old fruits. After encapsulation in an alginate matrix composed of 2 percent Na-alginate, 2 M glycerol plus 0.4 M sucrose (loading solution), the protocorms were precultured in modified Vacin and Went (1949) (VW) liquid medium supplemented with 0.7 M sucrose on a shaker (110 rpm) at 25 +/- 3 degree C for 20 h. Encapsulated protocorms were then dehydrated in a sterile air-flow in a laminar air-flow cabinet at 25 +/- 3 degree C for 0-10 h and then directly plunged into liquid nitrogen for 1 d. After thawing at 40 degree C for 2 min, cryopreserved beads were cultured on modified VW agar medium for regrowth. The highest regrowth of 40 percent was observed with cryopreserved beads with 35 percent water content after 8 h dehydration. No morphological variation was detected between non-cryopreserved and cryopreserved plantlets, and ploidy level was unchanged as a result of cryopreservation.     

Preparation of cholesterol oxidase nanoparticles and their application in amperometric determination of cholesterol

Highly dispersed platinum nanoparticles were deposited on gram quantities of non-functionalized multiwalled carbon nanotubes (MWCNTs) by atomic layer deposition (ALD) in a fluidized bed reactor at 300 °C. (Methylcyclopentadienyl) trimethylplatinum and oxygen were used as precursors. The results of TEM analysis showed that ~1.3 nm Pt nanoparticles were highly dispersed on non-functionalized MWCNTs. The porous structures of MWCNTs did not change with the deposition of Pt nanoparticles. For comparison, the commercial 3 wt% Pt/C catalyst was also characterized. The ALD-prepared Pt/MWCNT was used for the hydrogenation of xylose to xylitol. The ALD-prepared Pt/MWCNT showed the best catalytic performance with 100 % conversion of xylose and 99.3 % selectivity to xylitol, compared to commercially available Pt/C, Ru/C, and Raney Ni catalysts. The stability of ALD produced Pt/MWCNT catalyst was higher than that of the commercial Pt/C, due to the presence of surface defects on the MWCNTs and the strong metal–support interaction for the ALD-prepared Pt/MWCNT catalyst.     

Microstructure characterization of Al matrix composite reinforced with Ti-6Al-4V meshes after compression by scanning electron microscope and transmission electron microscope

Compressive properties of Al matrix composite reinforced with Ti-6Al-4V meshes (TC4(m)/5A06 Al composite) under the strain rates of 10(-3)S(-1) and 1S(-1) at different temperature were measured and microstructure of composites after compression was characterized by scanning electron microscope (SEM) and transmission electron microscope (TEM). Compressive strength decreased with the test temperature increased and the strain-rate sensitivity (R) of composite increased with the increasing temperature. SEM observations showed that grains of Al matrix were elongated severely along 45° direction (angle between axis direction and fracture surface) and TC4 fibres were sheared into several parts in composite compressed under the strain rate of 10(-3)S(-1) at 25°C and 250°C. Besides, amounts of cracks were produced at the interfacial layer between TC4 fibre and Al matrix and in (Fe, Mn)Al(6) phases. With the compressive temperature increasing to 400°C, there was no damage at the interfacial layer between TC4 fibre and Al matrix and in (Fe, Mn)Al(6) phases, while equiaxed recrystal grains with sizes about 10 μm at the original grain boundaries of Al matrix were observed. However, interface separation of TC4 fibres and Al matrix occurred in composite compressed under the strain rate of 1S(-1) at 250°C and 400°C. With the compressive temperature increasing from 25°C to 100°C under the strain rate of 10(-3) S(-1), TEM microstructure in Al matrix exhibited high density dislocations and slipping bands (25°C), polygonized dislocations and dynamic recovery (100°C), equiaxed recrystals with sizes below 500 μm (250°C) and growth of equiaxed recrystals (400°C), respectively.     

Cryopreservation of goldfish caudal fin explants using glycerol as a cryoprotecant

The objective of this study was to investigate the effect of glycerol on the cryopreservation fin explants of goldfish, Carassius auratus. Four different concentrations, 5, 10, 15, and 20% (v/v) of glycerol and a control were tested. These were prepared in Dulbecco's modified Eagle's medium with 20% (v/v) Fetal Bovine Serum. Attachment and outgrowing rates were monitored from day 3 to day 14. Results showed that fin explants cryopreserved in 20% concentration of glycerol was significantly higher (P < 0.05) with a 100% attachment rate compared to 5, 10, and 15% concentrations with 36.67, 84.19 and 86.51% attachment rate, respectively. Fin explants cryopreserved in 20% glycerol concentration also had significantly higher (P < 0.05) outgrowth of cells (73%) than the other three concentrations on day 3. Moreover, a 100% outgrowth of cells in all concentrations was achieved after 14 days of culture. No attachment and out growth of cells were observed in control group. Goldfish caudal fin explants cryopreserved in glycerol can produce live cells efficiently, regardless of concentration.     

Oxygen-induced changes in longitudinal relaxation times in skeletal muscle

The objective was to measure the effect of 100% oxygen inhalation on T1 relaxation times in skeletal muscle. Healthy volunteers were scanned using three different MRI protocols while breathing medical air and 100% oxygen. Measurements of T1 were made from regions of interest (ROIs) within various skeletal muscle groups. Dynamic data of subjects breathing a sequence of air-oxygen-air allowed the calculation of characteristic wash-in and -out times for dissolved oxygen in muscle. Contrary to previous findings, a statistically significant decrease in T1 in skeletal muscle was observed due to oxygen inhalation. We report approximate baseline characteristic values for the response of skeletal muscle to oxygen inhalation. This measurement may provide new biomarkers for evaluation of oxygen delivery and consumption in normal and diseased skeletal muscle.     

Heat Transfer Characteristics for Condensation of R134a in a Vertical Smooth Tube

In this study, condensation of pure refrigerant R134a vapor inside a smooth vertical tube was experimentally investigated. The test section was made of a copper tube with inside diameter of 7.52 mm and length of 1 m. Experimental tests were conducted for mass fluxes in the range of 20–175 kg/m²s with saturation pressure ranging between 5.8 and 7 bar. The effects of mass flux, saturation pressure, and temperature difference between the refrigerant and tube inner wall (ΔT) on the heat transfer performance were analyzed through experimental data. Obtained results showed that average condensation heat transfer coefficient decreases with increasing saturation pressure or temperature difference (ΔT). In addition, for the same temperature difference (ΔT), heat can be removed from the refrigerant at a higher rate at relatively low pressure values. Under the same operating conditions, it was shown that average condensation heat transfer coefficient increases as mass flux increases. Finally, the most widely used heat transfer coefficient correlations for condensation inside smooth tubes were analyzed through the experimental data. The best fit was obtained with Akers et al.'s (1959) correlation with an absolute mean deviation of 22.6%.     

Development of nanocrystalline Mn-Zn ferrites for high frequency transformer applications

Microwave-Hydrothermal (M-H) method has been successfully used for the synthesis of nanocrystalline Mn-Zn ferrites which are used for high-frequency applications. As synthesized powders were characterized using X-ray diffraction (XRD) and transmission electron microscopy (TEM). The nanopowders were annealed at 600 °C/20 min using the microwave sintering method. The frequency dependence of dielectric constant (ε′) was measured in the range of 10 Hz-1.3 GHz and initial permeability (μ_i) was measured in the range of 10 Hz-1 MHz. The total power loss (P_t) was measured on the annealed samples at 100 kHz and 200 mT condition. Conductor-embedded-ferrite transformers were fabricated and output power (P_o), efficiency (η) and temperature rise (ΔT) were measured at sinusoidal voltage of 25 V at 1 MHz. The transformer efficiency (η) was found to be high and surface rise of temperature (ΔT) is very low.     

V-cryo-plate procedure as an effective protocol for cryobanks: case study of mint cryopreservation

A vitrification procedure using aluminium cryo-plates (V-Cryo-plate procedure) was successfully developed and adjusted for in vitro-grown mint (Mentha spp.) shoot tips. Shoots were cultured at 25°C on MS medium containing 0.088 M sucrose for 7 to 14 days after the last subculture. Shoot tips with a basal part (1-1.5 mm × 1 mm) were dissected from the shoots and precultured at 25°C for 1 day on the same medium. Precultured shoot tips were placed on aluminium cryo-plates with 10 wells and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 degree C in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 0.8 M sucrose). For dehydration, the cryo-plates were transferred and immersed in 25 ml pipetting reservoirs filled with PVS2 for 20 min at 25 degree C. Then the cryo-plates were transferred in uncapped 2 ml cryotubes and directly plunged into liquid nitrogen. For rewarming, shoot tips attached to the cryo-plates were immersed in cryotubes containing 2 ml 1 M sucrose solution at room temperature. Using this procedure, regrowth of cryopreserved shoot tips of line 'Fukuyamajisei' reached over 90 percent. This protocol was successfully applied to 16 additional Mentha lines, with regrowth ranging from 73 percent to 100 percent. This V-Cryo-plate method will facilitate the cryostorage of mint germplasm in our genebank.     

片层硅酸盐用于热固性PU/PLS复合可能性探讨

高岭土(Kaolinite)和蒙脱土(Montromollite)等粘土,被广泛用作添加剂来改善聚合物的耐热性能。文章用各种先进技术对高岭土与蒙脱土的形态、组成和结构进行表征比较。电子透射电镜及电子探针(TEM/EDX)结果显示高岭土较蒙脱土有较大的粒径和较小的硅铝比,TEM还显示出这些粘土是由片层粒子堆积而成。热重分析(TGA)的结果表明蒙脱土由于失去吸附水从60 ℃升温至90 ℃失重明显(约7%),而高岭土在此温度下几乎不失水。高岭土脱除结构羟基官能团脱水失重的温度大约在510 ℃,低于蒙脱土的670 ℃。室温下光声傅里叶变换(PAS-FTIR)红外光谱显示蒙脱土在羟基吸收区有较强的吸附水的吸收,1 650 cm-1处孤立的弱吸收谱带可给予左证。变温红外和TGA的结果显示,片层硅酸盐表面的结构羟基从100～500 ℃非常稳定,它可以和异氰酸酯基团(NCO)封端的预聚物反应形成热固性PU/PLS复合聚合物。