On the role of intermembrane contact in cell electrofusion

The question: is cell electrofusion mediated by a long-lived fusogenic state or does the electric field fuse the membranes directly, was investigated by a new centrifugal approach. Mouse L-cells were brought into contact by a special centrifuge device allowing high voltage pulses to be applied upon the cell pellet during centrifugation. Both stages, membrane contact and electrical breakdown of cell membranes, were controlled. The degree of cell-to-cell compression and corresponding intermembrane contact area were estimated by measuring the low-voltage resistance R of the cell pellet which grows sharply with the increase of centripetal acceleration G. The extent of electrical membrane poration and critical pulse parameters were detected by recording the breakdown current. Supercritical pulse delivery to a cell pellet compressed by intensive centrifugation (400–600 g) leads to polycaryon mass formation. The pulse amplitude required for efficient fusion (2–3 kV/cm, 20–50 μs: fusion index F ∼ 25–35%) was found to be several times higher than the amplitude sufficient to induce noticeable breakdown (300 V/cm). The shapes of the F(G) and R(G) dependences were similar, which revealed a correlation between the area of intermembrane contact and fusion probability. Fusion was negligible if the moment of pulsation and the period of intensive centrifugation were separated in time. The data obtained allow us to conclude that in the case of fusion of L-cells the action of the electric field is not mediated by any long-lived fusogenic state. The process of the common membrane surface formation driven directly by the electric field is discussed.     

Factors controlling the electrofusion of murine embryonic cells

A wide variety of physical and biological factors are involved in determining the success of electrofusion procedures. The optimal conditions for the fusion and survival of mouse two-cell embryos have been determined by manipulating the electric field parameters, medium composition, degree of cell-cell contact and the relationship between current flow and membrane orientation. The experiments demonstrate that the events which initiate embryonic cell fusion are dependent upon a closely defined electric field strength and associated pulse duration. We show further that high cell fusion rates are the product of an inverse relationship between dc field strength and pulse duration and the initiation of pore formation by electric field application is insufficient to induce successful fusion unless accompanied by appropriate post-pulse medium and adequate membrane contact. Manipulation of the direction of current flow, membrane orientation and degree of cell-cell contact have shown that the initiation of pore formation occurs across the entire surface of the cell membrane.     

Highly controlled electrofusion of individually selected cells in dielectrophoretic field cages

The prospect of novel therapeutic approaches has renewed the current interest in the fusion of rare cells, like stem cells or primary immune cells. While conventional techniques are only capable of mass fusion, lab-on-a-chip systems often still lack an acceptable method for making the cells available after processing. Here, we present a microfluidic approach for electrofusion on the single-cell level that offers high control over the cells both before and after fusion. For cell pairing and fusion, we employed dielectrophoresis and AC voltage pulses, respectively. Each cell has been characterized and selected before they were paired, fused and released from the fluidic system for subsequent analysis and cultivation. The successful experimental evaluation of our system was further corroborated by numerical simulations. We obtained fusion efficiencies of more than 30% for individual pairs of mouse myeloma and B cell blasts and showed the proliferating ability of the hybrid cells 3 d after fusion. Since aggregates of more than two cells can be fused, the technique could also be developed further for generating giant cells for low-noise electrophysiology in the context of semi-automated pharmaceutical screening procedures.     

On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation

This article describes the development and full characterization of a microfluidic chip for electrofusion of human peripheral blood B-cells and mouse myeloma (NS-1) cells to generate hybridomas. The chip consists of an array of 783 traps, with dimensions that were optimized to obtain a final cell pairing efficiency of 33±6%. B cells were stained with a cytoplasmic stain CFDA to assess the different stages of cell fusion, i.e. dye transfer to NS-1 cells (initiating fusion) and membrane reorganization (advanced fusion). Six DC pulses of 100 μs (2.5 kV/cm) combined with an AC field (30 s, 2 MHz, 500 V/cm) and pronase treatment resulted in the highest electrofusion efficiency of paired cells (51±11%). Hybridoma formation, with a yield of 0.33 and 1.2%, was observed after culturing the fused cells for 14 days in conditioned medium. This work provides valuable leads to improve the current electrofusion protocols for the production of human antibodies for diagnostic and therapeutic applications.     

Methodology for evaluation of melanin content and production of pigment cells in vitro

Melanin has a photo-screening, a biophysical/biochemical and a cosmetic effect. Melanin content of cultured pigmented cells can be measured by spectrophotometry and expressed either as melanin content per cell or melanin content per culture (area). Melanin production can be calculated from melanin content and cell number at the beginning and at the end of a culture using various formulas and expressed as melanin production per cell per day or melanin production per culture per day. Melanin content or production per cell have been used widely to compare melanin content in various cell lines or to compare the melanin content during different stages in the culture (e.g. growing stage and senescent stage). For the evaluation of changes in melanin content and production in a given pigment cell line after treatment with a special chemical, physical or biological stimulator or inhibitor, different parameters used for the evaluation of experimental data can lead to conflicting results. Melanin content per area is determined by melanin content per cell and the number of cells in this area. The biological and cosmetic effects of melanin in vivo are determined mainly by melanin content per area, not melanin content per cell. For example, if melanin content per cell is the same, but the number of cells in a given area is increased after the treatment, then the melanin content per area is also increased. Under this circumstance, the color of skin turns darker and the total antioxidant activity provided by melanin in this area is increased even though the melanin content per cell measured remains the same; therefore, melanin content or production per culture is more important than melanin content or production per cell under this circumstance.     

Optimization of double chamber microbial fuel cell for domestic wastewater treatment and electricity production

Microbial fuel cells (MFCs) represent a new approach for treating waste water along with electricity production. The present study addressed electricity production from domestic wastewater using a mediator-less double chamber MFC. The electricity production was monitored under different operational conditions for both summer and winter samples. Optimization of the anodic and cathodic chambers resulted in a maximal current of 0.784 and 0.645 mA with the maximal power intensity of 209 and 117 mW/m² in power duration of 24 h for the summer and winter samples, respectively. Scanning electron microscopy showed that the bacterial biofilm formation on the anode was denser for the summer sample than that when the winter sample was used, so was the total bacterial count. Therefore, samples taken during summer were considered better in electricity production and waste water treatment than those taken during winter basically because of the high microbial load during the hot season. In parallel, there was a decrease in both biological oxygen demand (BOD₅) and chemical oxygen demand (COD) values which reached 71.8% and 72.85%, respectively at the end of the operation process for the summer sample, while there was no evident decrease for the winter sample. Optimizing the operating conditions not only increased the potential of using domestic waste water in microbial fuel cells to produce electricity, but also improved the quality of the domestic waste water.     

Naphthodithiophene-2,1,3-benzothiadiazole copolymers for bulk heterojunction solar cells

Two newly synthesized naphthodithiophene-based copolymers, PNB, exhibit a low optical bandgap of ～1.64 eV with which the solar cells fabricated from the blend of PNB and PC(71)BM afforded a power conversion efficiency of 5.3% with external quantum efficiency over 60% in a broad spectral range.     

Absorption tuning of monosubstituted triazatruxenes for bulk heterojunction solar cells

A series of triazatruxene (TAT)-functionalized Bodipy dyes were prepared by a sequence of reactions involving either cross-coupling reactions promoted by Pd complexes or a Knoevenagel reaction leading to a vinyl linker. The new dyes show large absorption coefficients and fluorescence quantum yields as well as interesting electrochemical properties. The blue dyes of this series exhibit interesting photovoltaic effects (V(OC) = 0.83 V, J(SC) = 3.6 mA/cm(2), efficiency 0.9%) in bulk heterojunction solar cells, due to the good hole mobility imported by the TAT entity.     

Synthesis of n-type conjugated polymers for bulk heterojunction solar cells

A new conjugated alternating donor-acceptor copolymer is synthesized from perylene diimide and the benzodithiophene derivative via the Suzuki reaction. Temperatures corresponding to the 10% weight loss of the copolymer in air and argon are 336 and 362°C. The copolymer is soluble in common organic solvents. The maxima of absorption spectra of the copolymer in chloroform solution and thin films are at 456 and 567 and at 444 and 567 nm, respectively. The optical band gap, as calculated from the onset of the absorption spectrum, is 1.74 eV, and the electrochemical band gap is 1.89 eV. The energies of HOMO and LUMO derived from the onset of the first oxidation and reduction potential of the cyclic voltamperogram are ?5.97 and ?4.08 eV. It is found that the solar cell with copolymer-to-poly(3-hexylthiophene) = 1: 1 features the best characteristics: The open circuit voltage is 0.54 V, the short circuit current is 0.976 mA/cm², the fill factor is 0.397, and the efficiency is 0.14%. The conjugated polymer based on perylene diimide belongs to the class of n-type polymers and shows promise as an acceptor material for all-polymer bulk heterojunction solar cells.     

甲醇水蒸气重整制氢反应条件的优化

对共沉淀法制备的CuO/ZnO/CeO₂-ZrO₂催化剂在甲醇水蒸气重整制氢反应体系中的性能进行了考察,并利用统计学实验设计方法对该反应的反应条件进行了优化。选择反应温度、水醇比和甲醇气体空速为独立要因,利用全因子实验设计方法,得到反应温度对两个响应值(甲醇转化率和重整气中CO物质的量分数)的影响最为显著,甲醇气体空速对重整气中CO物质的量分数的影响最小。固定甲醇气体空速为300 h^-1,利用中心旋转组合设计实验方法对反应温度和水醇比进行优化,得出当反应温度在249~258℃、水醇比在1.76~2.00时,甲醇能全部转化,重整气中CO物质的量分数小于0.5%。此模型的计算值与实验结果较为接近,表明采用统计学实验设计方法得出的结论对甲醇水蒸气重整制氢反应条件的优化具有指导意义。     

Processing additives for improved efficiency from bulk heterojunction solar cells

Two criteria for processing additives introduced to control the morphology of bulk heterojunction (BHJ) materials for use in solar cells have been identified: (i) selective (differential) solubility of the fullerene component and (ii) higher boiling point than the host solvent. Using these criteria, we have investigated the class of 1,8-di(R)octanes with various functional groups (R) as processing additives for BHJ solar cells. Control of the BHJ morphology by selective solubility of the fullerene component is demonstrated using these high boiling point processing additives. The best results are obtained with R = Iodine (I). Using 1,8-diiodooctane as the processing additive, the efficiency of the BHJ solar cells was improved from 3.4% (for the reference device) to 5.1%.     

Design and characterization of Bodipy derivatives for bulk heterojunction solar cells

Two electron rich Bodipy dyes with strong absorptivities in the visible region were designed and synthesized as potential electron donors in bulk heterojunction photovoltaic constructs. Overall efficiency is above 1%, with impressive responsiveness at both UV and near-IR ends of the visible spectrum. Computational studies reveal an unexpected effect of meso-substituents on the electron transfer efficiency.     

Significance of miscibility in multidonor bulk heterojunction solar cells

Ternary organic blends have potential in realizing efficient bulk heterojunction (BHJ) organic solar cells by harvesting a larger portion of the solar spectrum than binary blends. Several challenging requirements, based on the electronic structure of the components of the ternary blend and their nanoscale morphology, need to be met in order to achieve high power conversion efficiency in ternary BHJs. The properties of a model ternary system comprising two donor polymers, poly(3-hexylthiophene) (P3HT) and a furan-containing, diketopyrrolopyrrole-thiophene low-bandgap polymer (PDPP2FT), with a fullerene acceptor, PC₆₁BM, were examined. The relative miscibility of PC₆₁BM with P3HT and PDPP2FT was examined using diffusion with dynamic secondary ion mass spectrometry (dynamic SIMS) measurements. Grazing incidence small and wide angle X-ray scattering analysis (GISAXS and GIWAXS) were used to study the morphology of the ternary blends. These measurements, along with optoelectronic characterization of ternary blend solar cells, indicate that the miscibility of the fullerene acceptor and donor polymers is a critical factor in the performance in a ternary cell. A guideline that the miscibility of the fullerene in the two polymers should be matched is proposed and further substantiated by examination of known well-performing ternary blends. The ternary blending of semiconducting components can improve the power conversion efficiency of bulk heterojunction organic photovoltaics. The blending of P3HT and PDPP2FT with PC₆₁BM leads to good absorptive coverage of the incident solar spectrum and cascading transport energy levels. The performance of this ternary blend reveals the impact of the miscibility of PC₆₁BM in each polymer as a function of composition, highlighting an important factor for optimization of ternary BHJs. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016, 54, 237–246     

Membrane microextension: a possible mechanism for establishing molecular contact in electrofusion.

True cell membrane contact is an essential condition for electro-pulsed cell fusion, but initial morphological perturbation leading to true contact is still not clear. Dielectrophoresis mediated compression and fusogenic pulse induced compaction of cells led to rapid merger of tight membranes, and deprived direct microscopic view of surface membrane perturbation. Freely suspending cells with large and different cell-cell gaps may proceed to electrofusion with perturbed membrane and initiates fusion events at different time. These pulsed exposed cells can be used for capturing changes in the membrane surface and early electrofusion events. Early stage of fusion of freely suspended intact human erythrocytes exposed to single exponential decay pulse was studied by scanning electron microscopy (SEM). Field pulse induces small membrane bumps. Interaction of bumps on adjacent membranes lead to true membrane contact and form bridges between the membranes as microextension, combining both membranes into a topologically single structure. Some fusion products showed expanded fusion zones, which suggest indication of open lumen at contact area.     

Material structure-composite morphology-photovoltaic performance relationship for organic bulk heterojunction solar cells

Conjugated PPV-PPE copolymer has been investigated in organic solar cells in combination with twelve different fullerene derivatives. It was shown that the length of solubilizing alkyl chains in the fullerene derivative structures correlates well with the performance of photovoltaic cells.     

Thiophene-free diphenyl-amino-stilbene-diketo-pyrrolo-pyrrole derivatives as donors for organic bulk heterojunction solar cells

An extended study on a group of four soluble diphenyl-amino-stilbene based diphenyl-diketopyrrolo- pyrrole molecules has been carried out. Using the materials in thin-film transistors it was shown that the above-mentioned compounds can be successfully used as donors in organic photovoltaic devices. Influence of the molecular symmetry and solubilizing chain on the morphology and solar cell performance are described. It was shown that a shorter and non-branched ethyl acetate chain leads to higher charge carrier mobility, short circuit current, and better fill factor. After the basic optimization, a power conversion efficiency of about 1.5 % was reached. This, to the best of our knowledge, is the highest reported efficiency of thiophene-free small-molecule diketo-pyrrolopyrroles.     

Optimization and validation of stability indicating RP-HPLC method for the quantification of gefitinib in bulk drug and nanoformulations: An application towards in vitro and ex vivo performance evaluation

Gefitinib (GFB) is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor used primarily to treat non-small cell lung cancer (NSCLC), but it also works by lowering AKT and MAPK phosphorylation, which makes it effective against the breast cancer cells. A high-performance liquid chromatography (HPLC) method was developed for detecting gefitinib by C18 analytical column with mobile phase containing acetonitrile and 1 % w/v ammonium acetate in water with ratio of 60:40. Box-Behnken design was used to optimize the chromatographic conditions, and method was validated for accuracy, precision, linearity, robustness, ruggedness, limit of detection, and limit of quantification. The developed method was found to be useful in estimating gefitinib in conventional marketed tablet dosage forms and nanoformulations such as SLNs and liposomes. With a calibration curve, linearity was attained in the drug concentration range of 8–56 µg/mL (r² = 0.9996), and sensitivity was found to be 1.3 and 3.9 µg/mLas LOD and LOQ, respectively. All the validation criteria for the method were within the acceptable limits. The drug content in the conventional tablet formulation was found to be 99.99 %. The HPLC analysis indicated that gefitinib was highly soluble in Precirol ATO5 as solid lipid and tween 80 as surfactant. Drug entrapment efficiency was found to be 83.1 % and 80.5 % for SLNs and liposomes respectively. In vitro release data revealed that 30 % of drug was released from plain suspension and more than 77 % released from both nanoformulations after 24 h at pH 7.4 respectively. By applying kinetic fit, data was most appropriately fitted into first order as compared to other. The apparent permeability of gefitinib from plain suspension, SLNs and Liposomes was found to be 3.98 × 10^?4 cm/min, 1.35 × 10^?4 cm/min and 1.79 × 10^?4 cm/min.     

Optimization of distilled monoglycerides production

Monoglycerides (MG) are emulsifiers widely used in food and pharmaceutical industries. Current industrial processes for MG production consist of the interesterification of triglycerides with glycerol (GL), in the presence of inorganic catalysts at high temperatures (>200 degrees C). This reaction is known as glycerolysis and produces a mixture of approx 50% of MG. This level of concentration is suitable for many applications, although, for some specific uses like margarine, shortening, icing, and cream filling, require distilled MGs, which are purified MG (min. 90%) obtained by the molecular distillation process. Therefore, in this work, a 2(3) factorial design was employed to evaluate the effects of reaction parameters in the MG content after the interesterification reaction of refined soybean oil with GL in the presence of sodium hydroxide as catalyst. After that, the MG content in the reaction product was enhanced through the molecular distillation process in order to obtain distilled MG.