Cold preservation of isolated hepatocytes in UW solution: experimental studies on the respiratory activity at 0 degrees C

To date, little attention has been paid to the role of the gas milieu in preservation solutions and its effect on cell viability. Dissolved O2 in the preservation media may be an important parameter to consider. In this study we polarographically measured the O2 concentration in air-equilibrated UW solution at 0 degrees C, as well as the respiratory activity of isolated hepatocytes cold-preserved in this solution up to 72 hours. To perform measurements at 0 degrees C, it was first necessary to characterize the sensor behavior at low temperatures. We verified that the sensor response is still linear at this temperature but the rate of response is significantly slower. The O2 solubility in UW-air solution at 0 degrees C was determined using a modified physical method and it was 410 microM O2, which, as expected, is lower than the solubility in water at the same temperature (453 microM O2). Isolated hepatocytes cold-stored in UW-air solution retained a measurable respiratory activity during a period of 72 hours. The O2 consumption rate was 0.48 +/- 0.13 nmol/O2/min/10(6) cells, which represents 1% of the control value at 36 degrees C (61.46 +/- 14.61 nmol/O2/min/10(6) cells). The respiratory activity and cell viability were well maintained during the preservation period. At present, preservation conditions need to be improved for cells to remain functionally active. Dissolved O2 may be required for energy re-synthesis but it also leads to an increment in reactive oxygen species. The O2 concentration in the preservation solution should be carefully controlled, reaching a compromise between cell requirement and toxicity.     

Interactive effect of pressure and temperature on the preservation of rat primary-cultured astrocytes and human glioblastoma cell line A172

Environmental factors such as temperature and pressure are important determinants of cell survival. Although the effect of temperature on cell preservation has been previously reported, the effects of pressure, an equally important thermodynamic parameter, have not been sufficiently investigated. In this study, we investigated the effect of temperature and pressure on cellular viability, morphology, adhesiveness, cell death, cell cycle and glucose metabolism in rat primary-cultured astrocytes and A172 human glioblastoma cell line subjected to 4-day preservation. It was revealed that under favorable preservation conditions (temperature: 15°C–20°C, pressure: 0.1–30?MPa) (1) cell morphology and adhesiveness of preserved cells were maintained similar to freshly isolated cells; (2) cell cycle was arrested; (3) glucose uptake and intra/extra-cellular pH decrease were suppressed. These results suggest that lowering temperature to 15°C–20°C or increasing pressure up to 30?MPa at temperatures of 20°C–25°C can reduce cellular metabolism and maintain cell-membrane fluidity, thus resulting in higher viability.     

High intensity focused ultrasound-induced gene activation in sublethally injured tumor cells in vitro

Cultured human cervical cancer (HeLa) and rat mammary carcinoma (R3230Ac) cells were transfected with vectors encoding green fluorescent protein (GFP) under the control of hsp70B promoter. Aliquots of 10-microl transfected cells (5 x 10(7) cells/ml) were placed in 0.2-ml thin-wall polymerase chain reaction tubes and exposed to 1.1-MHz high intensity focused ultrasound (HIFU) at a peak negative pressure P- = 2.68 MPa. By adjusting the duty cycle of the HIFU transducer, the cell suspensions were heated to a peak temperature from 50 to 70 degrees C in 1-10 s. Exposure dependent cell viability and gene activation were evaluated. For a 5-s HIFU exposure, cell viability dropped from 95% at 50 degrees C to 13% at 70 degrees C. Concomitantly, gene activation in sublethally injured tumor cells increased from 4% at 50 degrees C to 41% at 70 degrees C. A similar trend was observed at 60 degrees C peak temperature as the exposure time increased from 1 to 5 s. Further increase of exposure duration to 10 s led to significantly reduced cell viability and lower overall gene activation in exposed cells. Altogether, maximum HIFU-induced gene activation was achieved at 60 degrees C in 5 s. Under these experimental conditions, HIFU-induced gene activation was found to be produced primarily by thermal rather than mechanical stresses.     

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.     

Applications of chitosan-based thermo-sensitive copolymers for harvesting living cell sheet

A thermo-sensitive chitosan-based copolymer hydrogel was used for harvesting living cell sheets. The hydrogel was tested for harvesting 3T3 cells after carrying out cell culture at 37 °C and incubating the confluent cells at 20 °C for spontaneous detachment of cell sheets from hydrogel surface without enzyme treatment. Results from cell viability assay and microscopy observations demonstrated that cells could attach to the hydrogel surface and maintain high viability and proliferation ability. Cell detachment efficiency from the hydrogel was about 80%. The detached cell sheet retained high viability and could proliferate again after transferred to a new culture surface.     

A simple GC method for determination of cryoprotector diols 1,4-butanediol or 2,3-butanediol in isolated rat hepatocytes

We devised a simple method for determining the cryoprotectant agents 1,4-butanediol or 2,3-butanediol in isolated rat hepatocytes. After extraction of of hepatocytes with water (containing internal standard - ethylene glycol 1.25 mg/mL) the diol content was analyzed by gas chromatography. The method shows a linear response in the range 0.125 to 2.50 mg/mL for 1,4-butanediol and 0.25 to 3.75 mg/mL for 2,3-butanediol. The accuracy and precision of the method were evaluated and the coefficients of variation were found to be within = 6.0 %. The recoveries from hepatocyte samples containing 0.50, 1.00 and 2.00 mg/mL were 91.0 to 108 % for 1,4-butanediol and 80.6 to 100.3 % for 2,3-butanediol, respectively. This method allowed the determination of the intracellular concentration of diols in hepatocytes preserved for up to 120 hours at - 4 (C in UW solution + 8 % w/v 1,4-butanediol (or 2,3-butanediol).     

Vitrification of encapsulated hepatocytes with reduced cooling and warming rates

We have used microencapsulated hepatocytes as model to develop a method of vitreous cryopreservation of large quantities of cell-containing constructs. The method included a pre-equilibration procedure in which the amount of penetrating cryoprotectant was gradually increased by 15% in each step. The optimal vitrification solution consists of 40% ethylene glycol and 0.6M sucrose. The concentration of 1M sucrose used for the first dilution solution with subsequent decrease of sucrose concentration to 0.7 M sucrose and by 0.2-0.15M for each subsequent step. This sucrose dilution procedure had no adverse effect on cell functions. Three cooling rates (400 degrees C/min and above) and three warming rates (650 degrees C/min and above), in combination with the proposed vitrification solution, were equally effective. The optimization of the procedure and solutions allow microencapsulated hepatocytes to be preserved with almost 100% retention of cell functions and no detectable damage to the fragile microcapsules. The de-linking of the cooling/warming rates with the effectiveness of vitrification potentially paves the way for large scale cryopreservation of complex tissue engineered constructs.     

Effect of surfactants on the degradation of perfluorooctanoic acid (PFOA) by ultrasonic (US) treatment

Perfluorooctanoic acid (C₇F₁₅COOH, PFOA) is an aqueous anionic surfactant and a persistent organic pollutant. It can be easily adsorbed onto the bubble-water interface and both mineralized and degraded by ultrasonic (US) cavitation at room temperature. The aim of this study is to investigate whether the effect of US on the degradation of PFOA in solution can be enhanced by the addition of surfactant. To achieve this aim, we first investigated the addition of a cationic (hexadecyl trimethyl ammonium bromide, CTAB), a nonionic (octyl phenol ethoxylate, TritonX-100), and an anionic (sodium dodecyl sulfate, SDS) surfactant. We found the addition of CTAB to have increased the degradation rate the most, followed by TritonX-100. SDS inhibited the degradation rate. We then conducted further experiments characterizing the removal efficiency of CTAB at varying surfactant concentrations and solution pHs. The removal efficiency of PFOA increased with CTAB concentration, with the efficiency reaching 79% after 120 min at 25 °C with a 0.12 mM CTAB dose.     

Rapana thomasiana hemocyanin (RtH): dissociation and reassociation behavior of two isoforms, RtH1 and RtH2

Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin isoforms, termed RtH1 and RtH2. The two subunit types, purified by ion exchange chromatography, were used for macromolecular reassociation studies. In vitro reassociation was achieved with Tris-saline stabilizing buffer at pH 7.4, containing 100mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation was monitored, and the different oligomeric forms of RtH1 and RtH2 were studied by transmission electron microscopy, using samples negatively stained with 1% (w/v) uranyl acetate or 5% (w/v) ammonium molybdate containing 1% (w/v) trehalose at pH 7.0. The two subunits reassociate to produce characteristic didecamers, oligomeric and polymeric forms depending on the dissociated material and the reassociation conditions (i.e. divalent ion concentration, duration). In contrast to the didecamers of the freshly isolated RtH preparations, RtH1 and RtH2 show after 2 weeks' reassociation a clear tendency to generate multidecameric structures. The behavior of the native RtH1 and RtH2 during reassociation in the presence of 100mM calcium and magnesium chloride corresponds to the reported common oligomerization characteristics of KLH1/HtH1 and KLH2/HtH2, respectively. It is important to note that during the reassociation of the RtH isoforms: (I) no smaller diameter tubular polymers (ca. 25-27nm) were formed from the subunits as well as from the decamers; (II) multidecamers with one or more 'nucleating' didecamers were detected in addition to the multidecamers, composed of didecamers with associated decamers at one or both ends.     

Sonochemical preparation of high surface area MgAl2O4 spinel

High surface area MgAl(2)O(4) has been synthesised by a sonochemical method. Two kinds of precursors were used, alkoxides and nitrates/acetates and in both cases nanostructured MgAl(2)O(4) was obtained. The effect of the addition of a surfactant during the sonication, cetyl trimethyl ammonium bromide, was also investigated. In the case of alkoxides precursors the as-made product is a mixture of hydroxides of aluminium and magnesium, while with nitrates/acetates a gel is obtained after sonication, containing the metal hydroxides and ammonium nitrate. Heating at 500 degrees C transforms the as-made products into MgAl(2)O(4) spinel phase. The surface area is up to 267m(2)/g after treatment at 500 degrees C and 138m(2)/g at 800 degrees C.     

The assessment of viability in isolated rat hepatocytes subjected to cold or subzero non-freezing preservation protocols using a propidium iodide modified test

A rapid and simple assay (6 min, two steps) is described for determination of cell viability of hepatocytes subjected to cold preservation protocols. In this method, cells are incubated with the fluorescent marker propidium iodide (PI) and the fluorescence intensity is measured before (direct fluorescence--Fd) and after (total fluorescence--Ft) addition of digitonin, which allows the dye to enter the hepatocytes. The Fd originated from non-viable cells that have membrane damage and taken up PI. The Ft originated from all cells in the sample. The ratio between the two fluorescence values is used as an indicator of cell viability. The assay was challenged versus two classical viability tests: LDH retention and Trypan Blue exclusion. Our assay shows good correlation only with Trypan Blue test. In addition, a fluorescence confocal microscopy protocol was used to evaluate the possible toxicity of PI in hepatocyte suspensions.     

磷酸-氨水蒸沉γ晶型聚酰胺6的热行为研究

尼龙6是一种多晶型的半结晶高聚物。实验采用蒸沉法制备出尼龙6,首先通过XRD和FTIR的手段进行表征,与采用沸水处理的尼龙6样品表征结果对比,确定其为γ晶型,然后在130～211℃的温度范围内进行热处理,通过DSC研究其在低于熔点热处理时的整个热行为变化过程,并运用FTIR观察其在不同条件下热处理发生的晶型变化。发现蒸沉法制备的γ晶型尼龙6随着热处理温度的升高,整体结晶完善度以及晶片厚度随着处理温度的升高而增加。而且在这个过程中,样品厚度不同的晶片在不同温度下发生了γ晶型向α晶型的转化,最终在接近熔点(211℃)热处理时,样品变为以α晶型为主。     

Evaluation of ammonia–nitrogen removal efficiency from aqueous solutions by ultrasonic irradiation in short sonication periods

In this study, mechanisms and efficiency of ammonia–nitrogen removal from aqueous solutions by ultrasonic irradiation were investigated. Depending on the factors affecting the sonication (initial concentration, initial pH, ultrasonic power density and sonication period), sonication tests were carried out and ammonium–nitrogen removal efficiency by ultrasonic irradiation was determined. In these experiments, ammonia–nitrogen removal efficiency was achieved in the range of 8–64%. In short sonication periods, the best ammonia–nitrogen removal efficiency was achieved at pH 8.2–11. Lower ammonia–nitrogen removal efficiency was observed in high initial ammonia–nitrogen concentration of solutions. It was observed that high initial ammonia–nitrogen concentrations may led to decreased ammonia–nitrogen removal efficiency however quantity of ammonia–nitrogen removal was higher. Because high initial concentration had a negative impact on the sonochemical reactions the heat of cavitation bubbles was reduced. Ammonia–nitrogen removal efficiency was increased with ultrasonic density and sonication period. This study showed that effective ammonia–nitrogen removal could be achieved by the ultrasonic irradiation in short sonication periods (as 60–600 s). Specific cost of ammonia–nitrogen removal by the ultrasonic irradiation from simulated ground water, surface water, wastewater and landfill leachate was also calculated. The specific removal cost was varied between 0.01 and 0.25 $/g ammonia–nitrogen.     

Synthesis and characterization of biocompatible magnetic glyconanoparticles

Magnetic glyconanoparticles were synthesized via the co-precipitation method. Iron (II) and iron (III) chloride were co-precipitated out of solution by the addition of ammonium hydroxide in an aqueous solution containing carbohydrate stabilizers such as d-gluconic acid, lactobionic acid and Ficoll^® at 75-80 °C. Stable magnetic glyconanoparticles were formed in a simple and direct process. Dynamic light scattering and transmission electron microscopy were used to characterize the surface-coated magnetic nanoparticles. In vitro cell viability studies of the glyconanoparticles were conducted with the mouse fibroblast cell lines. The magnetic glyconanoparticles revealed to be non-toxic at a concentration as high as 0.1 mg/mL.     

Cryopreservation of in vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant: effect of cryogenic procedure and storage duration

In vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant, were successfully cryopreserved using the vitrification and the encapsulation-dehydration techniques with subsequent high frequency plant regeneration. Using vitrification, post-liquid nitrogen (LN) shoot regeneration up to 83% was recorded when excised shoot tips were pretreated overnight on MS medium containing 0.3 M sucrose followed by loading with MS containing 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degree C, dehydration with PVS2 for 90 min at 0 degree C and quenching in LN. After 1 h of storage in LN, the shoot tips were rewarmed in a water-bath at 40 degrees C, unloaded with 1.2 M sucrose solution for 20 min and cultured on recovery growth medium. While using encapsulation-dehydration, the highest regeneration frequency recorded was 76% when sucrose-pretreated shoot tips were encapsulated with 3% calcium alginate, precultured in 0.75 M sucrose for 3 days, dehydrated to 25% moisture content (FW basis) under the laminar air flow, stored in LN for 1h and rewarmed at 40 degree C. The cryopreserved shoot tips maintained their viability and an unaltered level of regeneration capability after up to one year of storage in LN.     

Mammalian hibernation: lessons for organ preparation?

The adaptations to low environmental temperatures exhibited in mammalian hibernation are many and varied, and involve molecular and cellular mechanisms as well as the systematic physiology of the whole organism. Natural torpidity is characterised by a profound reduction in body temperature and other functions lasting from a few hours to several weeks. Controlled reduction of heart rate, respiration and oxygen consumption is followed by the fall in body temperature. However, thermoregulation persists such that a decrease in ambient temperature below dangerous levels typically triggers arousal, and shivering and non-shivering thermogenesis from brown fat provide the heat to restore body temperature to normal levels. Many of the cellular mechanisms for survival are similar to those brought into play during medium-term storage of organs destined for transplantation. For example maintenance of ionic regulation and membrane fluxes is fundamental to cell survival and function at low body temperatures. Differences between hibernating and non-hibernating species are marked by differences in Na+/K+ transport and Ca++pumps. These in turn are probably associated with alterations in the lipoproteins of the plasma membrane and inner mitochondrial membrane. We have accordingly conducted a series of pilot studies in captured Richardson's ground squirrels kept in laboratory conditions as a model for hypothermic organ preservation. Tissue function was compared during the summer (non-hibernating season) with that in the winter when the animals could be: (i) in deep hibernation in a cold chamber at 4 degree C; (ii) maintained in an ambient temperature of 4 degree C but active and awake; or (iii) active at an ambient temperature of 22 degree C. The studies involved: whole animal monitoring of standard physiological parameters; whole organ (kidney) storage and transplantation for viability assessment; storage and functional assessment on an ex vivo test circuit with capacity for perfusion at normothermic and hypothermic temperatures; measurement of thyroid function; measurements of total nucleotides (ATP, ADP and AMP)and ratios by standard techniques after freeze-clamping of organs; similar nucleotide and pH measurements using31P-NMR as a non-invasive whole animal technique; and measurement of O2 uptake and gluconeogenesis using isolated renal tubules and isolated hepatocytes. Marked differences in cold tolerance were demonstrated between organs taken from hibernating versus non-hibernating individuals. In particular kidneys transplanted from animals in deep hibernation were capable of withstanding up to 72 hours of cold storage as compared with up to 24 hours in non-hibernating squirrels or in comparable sized rats. Adaptations which might provide valuable clues in our attempts to better preserve human organs for transplantation are explored in some depth in this report.     

Advanced Fenton processing of aqueous phenol solutions: a continuous system study including sonication effects

Our previous report based on a batch reactor system for the Advanced Fenton Process (AFP) showed that pH, hydrogen peroxide and the organic substances treated are among the most important factors affecting the oxidation efficiency. As an extended study towards its potential commercialisation, this paper reports the effects of the main process parameters including those relating to a new laboratory scale AFP flow-through system. In order to systemise and correlate the results, the Taguchi experimental design method was used. Total organic carbon (TOC) removal was utilised as the measure of the oxidation efficiency and it was found that the removal of phenol from aqueous solution at pH 2.0 and 2.5 was very similar but hydrogen peroxide supply significantly affected the TOC removal with the change of flow rate from 14.4 ml/h to 60 ml/h. Also, the initial concentration of phenol was a highly significant factor, with higher concentrations resulting in a lower TOC removal rate. The temperature effects in the range of 14-42 degrees C were investigated and it was found that there was accelerated oxidation of phenol in the early stages but after 90 min there was no significant difference between the results. Sonication with a bath type sonicator resulted in relatively small enhancements of TOC removal but further studies with cup-horn sonication showed that TOC removal increased with higher intensity of sonication.     

All-solid-state high-power conduction-cooled Nd:YLF rod laser

A high-average-power conduction-cooled diode-pumped Nd:YLF rod laser has been developed. A new conduction-cooled side-pumping scheme with a solid prismatic pump-light confinement cavity was employed. A transparent, high-thermal-conductivity MgF>(2) prism was used as a highly efficient pump cavity as well as a low-thermal-resistance heat spreader. The pumping efficiency and thermal resistance of the cavity were 85% and 0.20 degrees C degrees W, respectively. When this scheme was combined with heat pipes for heat removal, a maximum average output power of 72 W was demonstrated, with an optical slope efficiency as high as 49%.     

Experimental investigations on ultrasound mediated particle breakage

This paper investigates the effect of high-intensity ultrasound on the breakage characteristics of particles suspended in water. A continuous sonicated flow experimental apparatus is used involving a 24 kHz horn type transducer and continuous in-line particle chord length measurement. The effects of sonication power (150-350 W) and temperature (10-50 degrees C) on the breakage characteristics are investigated. Higher breakage is favored at higher sonication power. An optimum temperature in the range tested is observed to exist between 25 degrees C and 37 degrees C. The acoustic cavitation field is influenced by temperature through a complex interplay of vapor pressure, surface tension and viscosity leading to the optimum observed in particle breakage. The efficiency of ultrasound energy conversion to particle breakage is calculated using calorimetry and found along with the net breakage efficiency to initially increase with temperature followed by a decrease after the optimum. It is found to be independent of input ultrasonic power. The effects of contact time is also investigated.     

3D-EEMs和PARAFAC解析土壤渗滤处理污水厂二级出水研究

利用三维荧光光谱（3D-EEMs）和平行因子分析（PARAFAC）的方法研究了土壤渗滤处理污水厂二级出水中氮、磷及溶解性有机物（DOM）的垂直分布特征。试验在一个中试规模的土壤渗滤系统中进行,反应器自上而下每隔30 cm设置一个采样口,采集的样品通过PARAFAC识别出系统不同点位的DOM具有4个荧光组分,包括2个类腐殖质物质（C1,C2）、2个类蛋白物质（C3,C4）。对荧光组分浓度得分F_max分析得出,C4代表的类色氨酸比其他3类物质更易于降解,即类色氨酸最易降解,其次为类富里酸、类胡敏酸类物质、类蛋白物质。四种组分的F_max变化幅度都以在0~30 cm处最大,表明此处生化反应最为剧烈,DOM的迁移转化速率最大。运用PARAFAC、主成分分析（PCA）和聚类分析（CA）等手段,可以揭示土壤渗滤系统中DOM的来源和不同深度的变化规律。土壤渗滤系统在4 L·d-1的低负荷条件下处理污水处理厂二级出水,对TN和NO3-N的去除是不利的,后续可以耦合反硝化滤池等工艺强化反硝化脱氮,进一步提高氮素的去除率。土壤对磷的吸附尚未达到饱和的状态,保持了较高的TP去除效率。