Freeze-drying of mononuclear cells and whole blood of human cord blood

The research on haematopoietic stem cells of human cord blood has become more important recently. People have concentrated on the preservation of cord blood stem cells. At present, cord blood can be preserved at ultra-low temperatures. In this study, we try to preserve cord blood and its constituents by freeze-drying. The experiments on both the mononuclear cell content and the whole blood of human cord blood were carried out respectively. The samples were frozen firstly by different cooling protocols in the presence of PVP, sucrose, and mannitol. Afterwards, they were vacuum-dried at a selected shelf temperature of -30 degree C for the main drying stage, and then vacuum-dried at 15 degree C for the second drying stage. The entire time of the freeze drying was 52 hours. Samples were stored at room temperature for 2 days prior to evaluation. Subsequently, the dried samples were suspended in an isotonic phosphate-buffered saline solution. The recovery of the cells were tested by a haemacytometer, and the highest cell numerical count recovery of MNC was 75.0 percent (SD = 4.1 percent) (P = 0.01), obtained in the protocol of 40 percent PVP + 20 percent sucrose + 10 percent Mannitol. The viability of the nucleated cells measured by PI staining and the ratio of the number of CD34+ to the number of lymphocytes (by the FITC anti-human CD34+ conjugated antibody method) were measured using a flow cytometer (FCM). The protocol of 40 percent PVP + 20 percent sucrose + 10 percent fetal bovine serum had the highest viability of 98.6 percent (SD = 0.7 percent) (P = 0.01). The highest ratio of CD34+ to lymphocytes was 1.2%, and the highest recovery of CD34+ was 68.4 percent (SD = 39.5 percent) (P = 0.05). Comparing the results of the lyophilized MNC subfraction with that of the whole blood, the lyophilization of the isolated MNC was more successful than that of whole blood.     

Evidence for neuroprotective properties of human umbilical cord blood cells after neuronal hypoxia <Emphasis Type="Italic">in vitro</Emphasis>

Background  

One of the most promising options for treatment of stroke using adult stem cells are human umbilical cord blood (HUCB) cells that were already approved for therapeutic efficacy in vivo. However, complexity of animal models has thus far limited the understanding of beneficial cellular mechanisms. To address the influence of HUCB cells on neuronal tissue after stroke we established and employed a human in vitro model of neuronal hypoxia using fully differentiated vulnerable SH-SY5Y cells. These cells were incubated under an oxygen-reduced atmosphere (O₂< 1%) for 48 hours. Subsequently, HUCB mononuclear cells (MNC) were added to post-hypoxic neuronal cultures. These cultures were characterized regarding to the development of apoptosis and necrosis over three days. Based on this we investigated the therapeutic influence of HUCB MNC on the progression of apoptotic cell death. The impact of HUCB cells and hypoxia on secretion of neuroprotective and inflammatory cytokines, chemokines and expression of adhesion molecules was proved.     

A novel dehydration technique for carrot slices implementing ultrasound and vacuum drying methods

A novel drying technique using a combination of ultrasound and vacuum dehydration was developed to shorten the drying time and improve the quality of carrot slices. Carrot slices were dried with ultrasonic vacuum (USV) drying and vacuum drying at 65 °C and 75 °C. The drying rate was significantly influenced by the drying techniques and temperatures. Compared with vacuum drying, USV drying resulted in a 41–53% decrease in the drying time. The drying time for the USV and vacuum drying techniques at 75 °C was determined to be 140 and 340 min for carrot slices, respectively. The rehydration potential, nutritional value (retention of β-carotene and ascorbic acid), color, and textural properties of USV-dried carrot slices are predominately better compared to vacuum-dried carrot slices. Moreover, lower energy consumption was used in the USV technique. The drying data (time versus moisture ratio) were successfully fitted to Wang and Singh model.     

Unusual freezing and melting of gallium encapsulated in carbon nanotubes

The freezing and melting behavior of gallium (Ga) encapsulated in carbon nanotubes was investigated through in situ observation in a transmission electron microscope. It is shown that Ga remains liquid up to -80 degrees C when encapsulated in carbon nanotubes. Results of detailed electron diffraction analysis show that the encapsulated Ga can crystallize in either beta phase or gamma phase rather than the common alpha phase upon freezing. Both beta-Ga and gamma-Ga melt at around -20 degrees C. While this is very close to the melting point of bulk beta-Ga (-16 degrees C), it is considerably higher than that of bulk gamma-Ga (-35.6 degrees C). It was observed that upon solidification, Ga has its unique crystallographic orientation relative to the host carbon nanotube.     

Neuronal hypoxia <Emphasis Type="Italic">in vitro</Emphasis>: Investigation of therapeutic principles of HUCB-MNC and CD133<Superscript>+</Superscript>stem cells

Background  

The therapeutic capacity of human umbilical cord blood mononuclear cells (HsUCB-MNC) and stem cells derived thereof is documented in animal models of focal cerebral ischemia, while mechanisms behind the reduction of lesion size and the observed improvement of behavioral skills still remain poorly understood.     

Micro-computed tomography observation of sublimation interface and image analysis on sublimation process during freeze-drying

The freeze-drying process is complicated with complex heat and mass transfer during sublimation. The sublimation interface of freeze-drying has become more attractive and meaningful recently. In this study, apple slices undergoing sublimation were scanned by a Micro-CT scanner. The cross-sectional images were reconstructed with those scanning images respectively. The technique of grey value analysis was used to recognize the procedure. The results showed that, from direct scanning images and 2-D reconstructed images, a 3-D moving mode of sublimation interface which contracted to the centre of the sample could be seen, sublimation process proceeded from edge to center gradually. The grey value of ice crystals was determined to be 154 through gauss calculation. By comparing frozen sample with freeze-dried one, the ice crystals regions in the beginning became the porous regions after drying, grey values increased correspondingly. Samples shrunk slightly after drying for 3 to 7 hours, which could be distinguished by the change in grey values.     

柠檬酸溶胶凝胶法合成LiCoO2的晶体结构研究

用柠檬酸为络合剂,采用溶胶凝胶法按照Li ∶Co2 =1∶1合成锂钴氧化物。利用热分析TG-DSC、X射线衍射、红外光谱、共焦显微Raman光谱和透射电子显微技术分析了锂钴氧化物凝胶体晶化过程及在不同煅烧温度下合成的LiCoO2晶体结构的变化。结果表明:煅烧温度由400℃升高到700℃时,晶体颗粒尺寸增大,其尺寸大小约20~40 nm,但在800℃时有所减小。光谱分析和电子衍射结果表明,随着煅烧温度的升高,LiCoO2晶体结构由改性尖晶石结构转变为层状岩盐结构。在改性尖晶石结构中,二价钴离子占据氧四面体间隙,三价钴离子占据氧八面体间隙;层状结构中,只有三价钴离子占据氧八面体间隙。     

Chondrocyte recovery in cryopreserved porcine articular cartilage after bone carrier alteration

In order to investigate the consequences on the distribution of cell recovery through a cross-section of articular cartilage, the pathway for ice nucleation and diffusion of water and solutes in porcine osteochondral tissue was altered by drilling a 2mm diameter hole through the subchondral bone to the base of the cartilage. Samples equilibrated with 1M dimethyl sulfoxide were cooled at 1 C/min to -30 degrees C then stored in liquid nitrogen. A significant increase in chondrocyte recovery was documented when compared to samples cryopreserved without holes (48.3 percent vs 28.6 percent, P=0.003). The most significant change due to bone base modification was an increase in recovery in the middle section of the cartilage. These results provide insight into mechanisms of cryoinjury in tissue systems.     

Ultrastructural localization of the RNA of immunodeficiency viruses using electron microscopy in situ hybridization and in vitroinfected lymphocytes

Cells infected in vitro with immunodeficiency viruses have been examined by electron microscopy in situ hybridization (EM ISH) methods for localization of viral RNA. Techniques used for preparation of specimens and probes are described. Unambiguous positive results were obtained using a mixture of two or three single negative strand DNA oligonucleotides complementary to regions of the gag, env and nef genes, each 200-300 bases and labelled with dig-11-UTP. Positive strand probes were used as a negative control. Cells were fixed with a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol with progressive lowering of temperature and embedded in Lowicryl K4M or HM20 at -35 degrees C. Permeabilization or pre-treatment of sections with proteinase K was not essential. The hybridization mixture was applied for 3-4h at 37 degrees C and probe was visualized by direct immuno-staining with sheep anti-digoxigenin antibodies conjugated to 10nm gold. This method would be suitable for future studies of the pathogenesis of retroviral infections and as a basis for further development of the EM ISH technique. EM ISH of in vitro infections of immunodeficiency viruses has shown the location of viral RNA in immature and mature viruses and its relationship to multimerized Gag protein during viral budding. The label for RNA has also been found in the cytoplasm of infected cells; it was mainly located adjacent to the plasma membrane and unassociated with visible Gag proteins. This may indicate that viral RNA migrates to the plasma membrane independently of the Gag protein and may, in some instances, arrive at the plasma membrane prior to the Gag protein. Viral RNA has also been found in the nucleus of peripheral blood mononuclear cells (PBMC) that were showing no morphological evidence of infection. The RNA was typically located in the nucleolus and in peripheral dense chromatin. These cells, which displayed morphological features of macrophage lineage, may have been the initial cell type to be infected in the PBMC.     

Nanocrystalline RE2O3:Tm3+ (RE: Gd3+, Y3+) Blue Phosphors Synthesized via the Combustion Method

This work reports on the preparation of a luminescent blue-emitting rare earth (RE) Tm-doped oxide phosphor. Nanocrystalline RE2O3:Tm3+ particles were prepared via the combustion method using citric acid, glycine, or urea as fuels. Samples were doped with different percentages of the activator Tm3+. The post-annealing treatment was performed in air for all the samples, at temperatures ranging from 800 to 1100 degrees C, for 4 h. The samples were characterized by X-ray diffraction (XRD), photoluminescence spectroscopy (PL) and scanning electron and transmission microscopies (SEM and TEM) in order to determine the best synthetic procedure. The post-annealed powders showed blue emission with maximum at 452 nm characteristics for Tm3+ transition 1D2 --> 3H4 (under UV excitation at 360 nm). Samples, presented a tri-dimensional porous structure (50-200 nm) formed of spheroid particles with a diameter between 20 and 60 nm. The best luminescent material was obtained when urea was used to prepare nanoparticles of Gd2O3 doped with 0.5% Tm3+, and 1100 degrees C was used as the post-annealing temperature.     

Freeze-drying of human platelets: influence of saccharide, freezing rate and cell concentration

Freeze-drying of human platelets is one potentially ideal approach for long-term preservation of platelets. In this study, effects of concentration and type of saccharides, freezing rate and initial cell concentration on the recovery of freeze-dried platelets were investigated. Annexin V binding platelet activation assays, scanning electron microscopy and platelet aggregation upon thrombin (1 U/ml) addition were used to evaluate the effectiveness of platelet freeze-drying. The numerical recovery of freeze-dried platelets was reached as high as 93.0+/-5.2 percent and the recovery of nonactive platelets was reached up to 85.7 +/- 3.4 percent in the presence of 1% BSA and 20% trehalose. Frozen by shelf pre-cooling was the best way to freeze the sample in this study and the numerical recovery of freeze-dried platelets was reached 93.0 +/- 5.2 percent at about 10 degree C/min. When the platelet concentration was increased from 0.2 to 4x10(9) platelets/ml, recovery remained higher than 81.4 percent. The morphology of freeze-dried and rehydrated platelets was intact but a little rounder compared with fresh platelets. The maximum aggregation rate to thrombin (1 U/ml) of freeze-dried platelets was 83.9 percent of the fresh ones, but aggregation speed was 43.0 percent of the fresh ones. Further research on rehydration process and scale up are required.     

Low temperature survival of slender naiad (Najas flexilis) seeds

The response to drying and storage at -20 degrees C or in liquid nitrogen was studied in seeds of the freshwater aquatic plant Najas flexilis. The seeds of this species show some desiccation sensitivity, although post-harvest storage in water at 16 degrees C resulted in improvements in desiccation tolerance. There was 63% germination of seeds dried to 9.5% moisture content (30% RH) following this maturation period. Optimum moisture contents for seeds stored at -20 degrees C for 3 months and in liquid nitrogen for 1 week were ~11% and ~15%, respectively.     

Heat Stress Induced Redistribution of Fluorescent Quantum Dots in Breast Tumor Cells

The probing of living cells in different colors over extended periods of time can be used to see the complicated processes that take place during carcinogenesis or heat stress, for example. Since most therapeutic laser tissue interactions are based on thermal effects a detailed characterization of thermal tissue damages in the cellular and sub-cellular levels is important. In order to study such microdosimetry laser-induced fluorescences of Quantum dots provide a suitable approach. Streptavidin conjugated Qdot 605 (Quantum Dot Corp., USA) were used in combination with the concanavalin A-biotin labeling system (Molecular Probes, NL) to observe membrane associated thermal lesions. Fluorescent Qdot conjugates are a promising alternative to organic dyes. The extinction coefficient of Qdot 605 streptavidin conjugate is 650,000 M(-1) cm(-1) at 600 nm. Red fluorescent Qdots 605 were selected because autofluorescence of cells in the red spectral range is not relevant. Fluorescence detection was performed with a confocal laser scan microscope LSM410 (Carl Zeiss, Germany). Breast cancer cells were used in the thermal stressing experiments performed at 40 degrees C, 42 degrees C, 45 degrees C, 50 degrees C or 56 degrees C for 30 min, each. In this methodical approach Qdot mediated labeling of heat stressed cells were demonstrated to show alterations of plasma membrane organizations and integrities, respectively.     

Loss of moisture from harvested rice seeds on MRI

The drying of rough rice seeds was visualized with the single point mapping imaging (SPI) technique by magnetic resonance imaging (MRI) at various temperatures, and the results were compared with those of the oven-drying method. Most of the water was present in the embryo and endosperm. The water reduction rate was larger on the outside than in the central position of the rice seeds at 50 degrees C, although this discrepancy was not obvious at 40 degrees C. Water reduction was brought about with time according to the kinetics of the multiple components, for both MR imaging and the ventilated-oven method. Images were continuously measured (10 min per image for 100 min). The reduction rate of water from rice kernels increased rapidly with temperature (up) to near 60 degrees C then rose slowly above 60 degrees C. Latent heat was calculated as 15 kcal/mol. deg from the changes of drying rate at temperatures below 60 degrees C.     

Acoustic impedance changes in cartilage and subchondral bone due to primary arthrosis

This study aimed at assessing elastic changes of cartilage and subchondral bone in sections from osteoarthritic human tibia plateaus using a 50-MHz scanning acoustic microscope (SAM). Samples were obtained from 28 human individuals during alloplastic implant surgery. Sagittal sections were explored using a time-resolved acoustic microscope in hyperosmolar (2.5 molar) saline solution at 25 degrees C. Cartilage and bone impedance distributions were evaluated as a function of the distance to the cartilage-bone interface. The degree of cartilage degeneration was derived from histological and immunohistochemical analyses. The mean impedance value in cartilage was 2.12+/-0.02 Mrayl. The layered cartilage structure was revealed by means of distinctly different impedance values in most samples. Generally, values were higher close to the bone interface and decreased continuously towards the cartilage surface. Higher grades of degeneration show a loss of the layered structure and remarkable cartilage surface undulations. The mean impedance value in subchondral bone was 6.28+/-0.54 Mrayl. A significant increase of the acoustic impedance within the first 150 microm relative to cartilage-bone interface was observed in 65.5% of the investigated sections. We hypothesize that the impedance increase close to the bone cartilage boundary is an indicator for subchondral sclerosis.     

Imprinted optical pattern of low-softening phosphate glass

Thermal imprinting of transparent tin phosphate glass was performed at 250 degrees C using a fine-patterned silica mold. The glass sample was prepared by a conventional melt-quenching method and polished with a roughness of < or =10 nm for imprinting experiments. The imprinting temperature is optimized based on experimental viscosity data. Scanning electron microscope and atomic force microscope observations revealed that a square grid pattern has a surface roughness of < or =10 nm and 5 microm x 5 microm squares with ~1 microm intervals and 90-100 nm depth. Diffraction spots due to the micropattern are demonstrated by illuminating He-Ne laser light.     

Collapse temperature of bacterial suspensions: the effect of cell type and concentration

The characterisation of the physical state of frozen and freeze dried biological products delivers powerful information for freeze-drying process optimisation. The influence of lactic acid bacterial cell size, shape and concentration on collapse temperature of concentrated bacterial suspensions was investigated. Lactobacillus bulgaricus (long rods), and Streptococcus thermophilus (small spherical cells) were used as cellular models for this study. Whatever the strain, when lactic acid bacterial cells were added to protective solutions, the collapse temperature increased, thus allowing the use of higher sublimation temperatures during primary drying than expected from the protective medium alone. Moreover, the higher the cell concentration, the greater the effect, linear relationships existing between the collapse temperatures and the total dried matter. Cells of both strains gave a kind of robustness to the freeze-dried product, but the increase observed in collapse temperature was considerably higher (3 - 5 degree C) for L. bulgaricus compared to S. thermophilus. This result was ascribed to the different size and shape of the strains.     

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.     

Co9S8纳米晶聚集体的水热合成及其光谱研究

以七水合硫酸亚钴(CoSO4·7H2O)和无水亚硫酸钠(Na2SO3)为主要原料,以水合肼(N2H4·H2O)为还原剂,利用水热法合成了Co9S8纳米晶聚集体.运用X射线衍射(XRD)、场发射扫描电镜(FESEM)和透射电镜(TEM)对产物进行了表征,并利用傅里叶变换红外光谱仪(FTIR)对产物的红外光谱进行了测定和初步分析.实验结果表明,产物主要为Co9S8纳米晶聚集体,其中含有平均粒度约为2.5 nm Co9S8晶粒.聚集体的形貌呈六角片状,其平均直径约为2.1μm,厚度约为200 nm.