Abstract: Disclosed is a method for selective electrofusion of at least two fusion partners having cell-like membranes and cellular or subcellular dimensions, comprising the following steps: A) the fusion partners are brought into contact with each other and B) an electrical field of a strength sufficient to obtain fusion and highly focused on the fusion partners is applied. The fusion partners are independently selected from the group consisting of a single cell, a liposome, a proteoliposome, a synthetic vesicle, an egg cell, an enucleated egg cell, a sperm cell at any development stage and a plant protoplast.

Abstract: The present invention relates generally to mass spectrometry. The present invention relates more particularly to methods and systems for use in mass spectrometric identification of a variety of analytes, including high molecular weight species such as proteins. One embodiment of the invention is a method for analyzing an analyte. The method includes nebulizing a suspension of the analyte in a solvent with a surface acoustic wave transducer; and performing mass spectrometry on the nebulized suspension. The surface acoustic wave transducer can be used, for example, to transfer non-volatile peptides and proteins (as well as other analyztes, such as oligonucleotides and polymers) to the gas phase at atmospheric pressure. Nebulization using surface acoustic waves can be conducted in a discontinuous or pulsed mode, similar to that used in MALDI, or in a continuous mode, as in ESI.

Abstract: The present invention encompasses methods of preparing non-segmented negative-stranded RNA viruses from cells utilizing electroporation.

Abstract: A system and method are described for electroporating a sample that utilizes one or more sets of electrodes that are spaced apart in order to hold a surface tension constrained sample between the electrodes. The first electrode is connected to the lower body of the system while the second electrode is connected to the upper body. Both electrodes are connected to a pulse generator. Each electrode has a sample contact surface such that the first electrode and the second electrode may be positioned to hold a surface tension constrained sample between the two sample contact surfaces and the sample may receive a selected electric pulse.

Abstract: Disclosed is a DNA construct that is useful for efficient generation of recombinant CHO cells useful for the production of target proteins. The DNA construct is a construct comprising, from a 5? end toward a 3? end, a first homologous DNA fragment, a target protein gene, and a second homologous DNA fragment. The first and second homologous DNA fragments have homology allowing for homologous recombination with a part of a hypoxanthine-phosphoribosyltransferase enzyme (hprt) locus in a CHO cell genome and have a chain length of not less than 1 kbp.

Abstract: Transfecting biology cells with nucleic acid molecules (DNA, siRNA) is an essential prerequisite in elucidating how genes function in complex cellular context and how their activities could be modulated for therapeutic intervention. Traditionally studies are carried out on a low throughput gene-by-gene scale, which has created a huge bottleneck in functional genomic study and drug discovery. Development of high-throughput cell transfection technology will permit functional analysis of massive number of genes and how their activities could be modulated by chemical or biological entities inside cells. This invention describes design, construction of device and apparatus for high throughput effective cell transfection. Procedures and protocols for using the device and apparatus are also described in the application. Novel methods of using the device in cell-based assays are also disclosed.

Abstract: A manipulator system and a manipulation method of a micromanipulation target object, which are capable of automatically executing a variety of operations about a micromanipulation target object such as an ovum that have hitherto required a skilled technique, are disclosed.

Abstract: Disclosed is a method for parallel delivery of agents to and/or into a cell structure, wherein at least two electrolyte-filled tubes are provided together with a counter electrode, the tubes being connected to a voltage or current generator, said agents being introduced into the electrolyte solution contained in the tubes, which are placed close to the cell structure, whereupon the agents are transported through the tubes to said cell structure and into the said structure through pores which have been formed by application of an electric field focused on the cell structure, resulting in electroporation of the cell structure. Also different applications of the method is disclosed, e.g. use of the method in order to transfer cell-impermeant solutes, such as drugs or genes, into the cell structure or out of the cell structure.

Abstract: A method and apparatus are provided for delivering an agent into a cell through the application of nanosecond pulse electric fields (“nsPEF's”). The method includes circuitry for delivery of an agent into a cell via known methods followed by the application of nanosecond pulse electric fields to said cell in order to facilitate entry of the agent into the nucleus of the cell. In a preferred embodiment, the present invention is directed to a method of enhancing gene expression in a cell comprising the application of nanosecond pulse electric fields to said cell. An apparatus for generating long and short pulses according to the present invention is also provided. The apparatus includes a pulse generator capable of producing a first pulse having a long duration and low voltage amplitude and a second pulse having a short duration and high voltage amplitude.

Abstract: A micromachined patch-clamp apparatus is disclosed for holding one or more cells and providing electrical, chemical, or mechanical stimulation to the cells during analysis with the patch-clamp technique for studying ion channels in cell membranes. The apparatus formed on a silicon substrate utilizes a lower chamber formed from silicon nitride using surface micromachining and an upper chamber formed from a molded polymer material. An opening in a common wall between the chambers is used to trap and hold a cell for analysis using the patch-clamp technique with sensing electrodes on each side of the cell. Some embodiments of the present invention utilize one or more electrostatic actuators formed on the substrate to provide mechanical stimulation to the cell being analyzed, or to provide information about mechanical movement of the cell in response to electrical or chemical stimulation.

Abstract: The present invention provides a method and apparatus for transferring an agent into a cell. The method includes the steps of providing an agent outside of a cell and generating a vapor bubble and a plasma discharge between an avalanche electrode and a conductive fluid surrounding the cell. The vapor bubble and plasma discharge generate a mechanical stress wave and an electric field, respectively. The combination of this mechanical stress wave and electric field results in permeabilization of the cell, which in turn results in transfer of the agent into the cell.

Abstract: The present disclosure relates to antigen specific tolerogenic antigen presenting cells presenting antigenic portions of an autoantigen and to related compositions, methods and systems.

Abstract: Disclosed is a method for sequential delivery of agents to and/or into a cell structure, wherein an electrolyte-filled tube is provided together with a counter electrode, said tube is connected to a voltage or current generator, at least two agents are introduced in a discrete mode into the electrolyte solution contained in the tube, which is placed close to the cell structure, one agent at the time being transported through the tube to and/or into said cell structure in which a pore has been formed by application of an electric field focused on the cell structure, resulting in electroporation of the cell structure. Also different applications of the method is disclosed, e.g. us of the method in order to transfer cell-impermeant solutes, such as drugs or genes, into the cell structure or out of the cell structure.

Abstract: An electroporation apparatus provides for the electroporation of adherent cells attached to an electrode surface or suspended cells in close proximity to an electrode surface. In one embodiment, the electrodes are transparent to allow cell viewing using a microscope or an automated image analysis machine. The geometry of the electrodes and associated electrically non-conductive structures may provide for well-defined regions of electroporated and non-electroporated adherent cells with a clearly defined interface between these regions, facilitating comparison of electroporated cells and non-electroporated cells, and evaluation of transfer of material from cell to cell via intercellular gap junctions.

Abstract: The present invention includes devices and methods for transfecting a cell or cell population and dynamic monitoring of cellular events. A variety of microelectronic devices are provide that incorporate functions such as electroporation, modulation of a transmembrane potential and dynamic monitoring of cellular functions and mechanisms.

Abstract: The present invention relates to genetic engineering and especially to the use of DNA transposition complex of bacteriophage Mu. In particular, the invention provides a gene transfer system for eukaryotic cells, wherein in vitro assembled Mu transposition complexes are introduced into a target cell and subsequently transposition into a cellular nucleic acid occurs. The invention further provides a kit for producing insertional mutations into the genomes of eukaryotic cells. The kit can be used, e.g., to generate insertional mutant libraries.

Abstract: A method for introducing biologically active molecules into animal or human cells using an electric current includes suspending the cells and dissolving the biologically active molecules in a buffer solution including HEPES and at least 10 mmol×1?1 magnesium ions (Mg2+), the buffer solution having a buffer capacity of at least 20 mmol×1?1 ×pH?1 at a change in the pH from pH 7 to pH 8 and at a temperature of 25° C., and an ionic strength of at least 200 mmol×1?1. An electric voltage is applied to the suspension.

Abstract: One aspect of the current invention is a method of preventing and/or treating arthritis and/or preventing or treating lameness in a subject. Additionally, subject quality of life and welfare, and body condition scores are improved by utilizing methodology that administers the nucleic acid expression construct encoding a GHRH or functional biological equivalent to a subject through a parenteral route of administration. Following a single dose of nucleic acid expression vector, subjects are healthier and effects are demonstrated long term without additional administration(s) of the expression construct.

Abstract: The invention relates to a novel circuit arrangement for electrotransfection or electrofusion, which enables the transportation of DNA and/or other biologically active molecules to the nucleus of higher eukaryotic cells or the fusion of cells, independent of cell division and with reduced cell mortality.

Abstract: Methods for expansion of antigen-specific T cells are provided. Said methods include following steps: generating antigen-specific T cells by stimulation of T cells with antigen A; introducing genes encoding immune recognition molecule specific to major histocompatibility complex (MHC) molecule bound with a peptide derived from antigen B into the antigen A specific T cell to produce bi-specific T cells recognizing both target cells expressing antigen A peptide associated MHC and target cells expressing antigen B peptide associated MHC; stimulating the bi-specific T cells by antigen A for expansion of the bi-specific T cells in vitro or in vivo. Methods of the present invention can be applied to expand various of T cells with specific to cancer cells with tumor antigen peptide loaded MHC molecules for adoptive therapy against unmet medical need such as tumors etc.

Abstract: The present invention is directed to adoptive immunotherapy using a lymphocyte in which an antigen-specific receptor and a bioactive material gene such as an IL-2 gene or a water-soluble TGF-beta receptor gene are transferred. The bioactive material is intensively secreted to, for example, a local site of a tumor, thereby reducing systemic side effects as much as possible, and the survival time of the lymphocyte is increased, thereby further improving the effect of the adoptive immunotherapy.

Abstract: The present invention relates to automated methods of introducing multiple nucleic acid sequences into one or more target cells.

Abstract: The present invention relates to a technology and method of priming of an immune response using invariant chain linked antigen, when these are used to prime a subsequent booster immunization using any suitable vacci.

Abstract: Disclosed is a circuit arrangement comprising at least one storage device for electrical charges to generate at least one voltage pulse by selectively discharging the storage device, and at least one control unit for controlling the discharge. A controller for monitoring the chronological progression of the voltage pulse is provided which controls at least one continuation of discharge after termination. Biomaterial is treated by using at least one electrical field generated by a first voltage pulse which is terminated once the value for an electrical parameter has exceeded or dropped below a preset limit. After the first voltage pulse has been terminated, it is continued by an additional voltage pulse.

Abstract: Compositions and methods for cancer treatment are discloses herein. More specifically the present invention describes an autologous cancer vaccine genetically modified for Furin knockdown and GM-CSF expression. The vaccine described herein attenuates the immunosuppressive activity of TGF-? through the use of bi-functional shRNAs to knock down the expression of furin in cancer cells, and to augment tumor antigen expression, presentation, and processing through expression of the GM-CSF transgene.

Abstract: An electroporation cuvette is constructed with electroporation electrodes arranged in non-parallel relation to form a gap whose width varies with the location within the cuvette, plus a pair of positioning electrodes that are arranged to cause electrophoretic migration of biological cells within the cuvette according to cell size. Once the cells, suspended in a solution of the impregnant, are distributed in the cuvette by the positioning electrodes, electric field pulses are generated by the non-parallel electroporation electrodes. Because of their distribution in the cuvette, the various cells will experience voltage differentials across their widths that approach uniformity regardless of cell diameter, since the larger cells will be positioned at locations where the gap between the electrodes is greater and the smaller cells at locations where the gap is relatively small while the voltage drop across the entire gap is uniform along the length of the cell.

Abstract: A buffer solution for suspending animal or human cells and for dissolving biologically active molecules in order to introduce the biologically active molecules into the cells using electric current. The buffer solution includes at least one of sodium succinate, mannitol and sodium lactobionate. The buffer solution has a buffer capacity of at least 20 mmol*l?1*pH?1 at a change in the from pH 7 to pH 8 and at a temperature of 25° C., and an ionic strength of at least 200 mmol * l?1.

Abstract: The present invention relates, generally, to improved methods of delivering a biologically active agent, in particular a therapeutic or prophylactic nucleic acid, to the ocular sphere of a subject comprising administering said agent to the ciliary body tissue(s) or cells and/or to the extra-ocular muscle tissue or cells. More particularly, the invention relates to devices, their uses, notably in gene therapy, and to methods for treating pathologies of the ocular sphere by specific ciliary body tissue(s) or cells and/or extra-ocular muscle or cells administration of a therapeutic product and transfer thereof into the ocular tissue to be treated. This invention also relates to pharmaceutical compositions comprising the product in a form suitable for ciliary body tissue(s) or cells and/or extra-ocular muscle or cells administration, their preparation and uses.

Abstract: A method and apparatus are provided for delivering an agent into a cell through the application of nanosecond pulse electric fields (“nsPEF's”). The method includes circuitry for delivery of an agent into a cell via known methods followed by the application of nanosecond pulse electric fields to said cell in order to facilitate entry of the agent into the nucleus of the cell. In a preferred embodiment, the present invention is directed to a method of enhancing gene expression in a cell comprising the application of nanosecond pulse electric fields to said cell. An apparatus for generating long and short pulses according to the present invention is also provided. The apparatus includes a pulse generator capable of producing a first pulse having a long duration and low voltage amplitude and a second pulse having a short duration and high voltage amplitude.

Abstract: In accordance with the present invention is provided a method of treating a subject having a cancerous tumor. The treatment protocol methodology includes injecting the cancerous tumor with an effective dose of plasmid coding for a therapeutic protein followed by administering electroporation therapy to the tumor, the electroporation therapy includes the administration of at least one high voltage, short duration pulse to the tumor.

Abstract: Methods for material transfer into a cell are provided according to the present invention which include electroporation of the cell in the presence of the material, such as nucleic acids, and 2,3-butanedione monoxime; and lipofection with a complex of a lipid-based carrier, such as liposomes, and the material, in the presence of 2,3-butanedione monoxime.

Abstract: A system and method are described for electroporating a sample that utilizes one or more sets of electrodes that are spaced apart in order to hold a surface tension constrained sample between the electrodes. The first electrode is connected to the lower body of the system while the second electrode is connected to the upper body. Both electrodes are connected to a pulse generator. Each electrode has a sample contact surface such that the first electrode and the second electrode may be positioned to hold a surface tension constrained sample between the two sample contact surfaces and the sample may receive a selected electric pulse.

Abstract: The introduction of genetic material or molecules of biological interest into cells is a procedure with an increasing interest both for experimental and application purposes, so that electroporation is a widely used technique, but the electroporation of single adhering cells is still impaired. The present application describes an apparatus for the electroporation of any kind of cell adhering to a substrate at any stage of development, where an electrical signal can be driven and applied to a single adhering cell in culture in order to obtain its electroporation. The method to electroporate a single adhering cell with the apparatus of the invention is also described.

Abstract: Electroporation is performed on a population of cells, liposomes, vesicles, or other membrane-encased structures with uniform results regardless of size variations within the population, by drawing the membrane-encased structures into micron-sized openings that contain paired electrodes. An electric potential is then imposed between the paired electrodes to permeabilize only that portion of each cell that extends into the openings and resides within the electric field focused in the area between the electrodes.

Abstract: A method for introducing biologically active molecules into animal or human cells using electric current includes suspending the cells and dissolving the biologically active molecules in a buffer solution which has a buffer capacity of at least 20 mmol×l?1×pH?1 and an ionic strength of at least 200 mmol×l?1 at a change in the pH from pH 7 to pH 8 and at a temperature of 25° C. to form a suspension. The method further includes applying an electric voltage to the suspension so as to introduce the biologically active molecules into animal or human cells.

Abstract: The present invention relates to a method for the generation of immortalized transduced cell lines susceptible to senescence whereby the senescence is exogenously inducible and said cells can switch severalfold between the senescent stage and the immortalized stage. In particular, the present invention relates to a method for the generation of immortalized transduced cell lines susceptible to senescence wherein two or more different immortalizing gene sequences have been incorporated whereby said immortalizing gene sequences are regulated by regulators controlled via exogenous means. In a further aspect, the present invention relates to immortalized transduced cell lines obtainable with said method. In addition, methods for screening molecules influencing senescence of cells are provided as well as kits for conducting the same.

Abstract: A method for introducing biologically active molecules into animal or human cells using an electric current includes suspending the cells and dissolving the biologically active molecules in a buffer solution including HEPES and at least 10 mmol×1?1 magnesium ions (Mg2+), the buffer solution having a buffer capacity of at least 20 mmol×1?1×pH?1 at a change in the pH from pH 7 to pH 8 and at a temperature of 25° C., and an ionic strength of at least 200 mmol×1?1. An electric voltage is applied to the suspension.

Abstract: A conductive substrate for introducing a nucleic acid into a cell, which comprises a carbon nanotube with a carboxyl group, and a nucleic acid, the carbon nanotube and the nucleic acid being loaded on an electrode substrate with a cationic surface.

Abstract: There is provided a method by which multiple types of substances desired to be transferred into cells can be continuously transferred into multiple types of cells by a convenient procedure, a cell in which the substance desired to be transferred into cells has been taken up by this method, and an apparatus for transferring a substance into cells by this method. The foregoing objects can be achieved by electrospraying cells with a liquid free from the substance to be transferred into cells while the cells are kept in contact with the substance to be transferred into cells, or first electrospraying cells with a liquid free from the substance to be transferred into cells and then bringing the cells into contact with the substance to be transferred into cells.

Abstract: The present invention refers to the construction of cloning vectors containing the max gene. Especially, the present invention refers to the introduction of cloning vectors containing the max gene in cells using transport vectors. In addition, the presence of cloning vectors containing the max gene in cells allows the differential expression of the max gene in the same cells. In addition, the present invention refers to a method of gene therapy in which the differential expression of the max gene has cytoprotective activity, especially neuroprotective activity, and is capable of application to medical and veterinary therapeutics of neurodegenerative conditions.

Abstract: The present invention provides a method and apparatus for transferring an agent into a cell. The method includes the steps of providing an agent outside of a cell and generating a vapor bubble and a plasma discharge between an avalanche electrode and a conductive fluid surrounding the cell. The vapor bubble and plasma discharge generate a mechanical stress wave and an electric field, respectively. The combination of this mechanical stress wave and electric field results in permeabilization of the cell, which in turn results in transfer of the agent into the cell.

Abstract: The present disclosure describes pathogenic bacteria that have been modified to be deficient in NiFe hydrogenase activity; compositions comprising such modified bacteria, and the use of such bacteria to protect animals from pathogenic enteric bacterial infections.

Abstract: The present invention relates to an electrofusion microelectrode made of a tube having a first proximal end and a second distal end. The tube has an electrically conductive coating on its exterior surface that extends continually from the first proximal end of the tube toward the second distal end of the tube. Also disclosed is an electrofusion microelectrode unit having an electrofusion microelectrode and a holding tool capable of receiving the electrofusion microelectrode at the second distal end of the tube. The present invention also relates to a system having two or more electrofusion microelectrodes of the present invention and to methods of manipulating cells and/or cellular components using the electrofusion microelectrodes, units, and systems of the present invention.

Abstract: A pulse generator circuit may include a diode configured to operate as an opening switch, a tank circuit in series with the diode having an admittance that is switchable from a first value to a second value that is different from the first value, and a switching system configured to cause the tank circuit to switch between the first value and the second value. The diode may saturate in less than 100 nanoseconds. A saturable core transformer may operate as a switch that controls the opening of the diode. The pulse generator may generate a plurality of pulses, each having a length of no more than 3 nanoseconds and an amplitude of at least 1 kilovolt. Electrodes may be connected to the pulse generator to deliver the plurality of pulses to biological cells.

Abstract: A pulse generator circuit may include a diode configured to operate as an opening switch, a tank circuit in series with the diode having an admittance that is switchable from a first value to a second value that is different from the first value, and a switching system configured to cause the tank circuit to switch between the first value and the second value. The diode may saturate in less than 100 nanoseconds. A saturable core transformer may operate as a switch that controls the opening of the diode. The pulse generator may generate a plurality of pulses, each having a length of no more than 3 nanoseconds and an amplitude of at least 1 kilovolt. Electrodes may be connected to the pulse generator to deliver the plurality of pulses to biological cells.

Abstract: The innate immune system of eukaryotes is able to recognise foreign genetic material by means of Toll-like receptors and to initiate signal transduction cascades that trigger an antiviral state of cell populations by way of an interferon response. That antiviral state is also a barrier for non-viral gene delivery systems. If the signal transduction cascade is interrupted intracellularly or intercellularly, transfection efficiencies of non-viral gene delivery systems can be increased and undesirable changes in the expression profile can be avoided. Since RNA-interference is to be attributed to the antiviral state, the RNAi machinery is likewise activated after activation of the innate immune system. In that way, knock-down efficiencies on transfection with siRNA can be increased.

Abstract: A method for facilitating a delivery of a molecule into an interior space of a cell includes the steps of introducing a molecule into a target tissue comprising a cell and applying a substantially continuous low-level electric field to the target tissue. The field is applied for a duration sufficient to effect a change in porosity the cell of the target tissue sufficient to facilitate an entry of a desired molecule into an interior of the cell.

Abstract: The present invention is directed to LAK-T cells, which have been transformed by a transgenic T cell receptor (tg-TCR). The invention is further directed to a method of generating those transgenic T cells, a pharmaceutical composition comprising said cells and the use of the LAK-T cells or of the pharmaceutical composition in the adoptive cell therapy and for treating hematological malignancies or solid tumors or acute or chronic infections or autoimmune diseases.

Abstract: The present invention provides methods for increasing the transfer of nucleic acids into cells. In particular, the present invention provides for the use of inhibitors of HDAC6, a cytoplasmic histone deacetylase present in mammalian cells by, for example, small molecules or siRNA treatment, in increasing gene transfer and/or expression in cells in vitro and in vivo for research and gene therapy applications.

Abstract: A method to increase self-renewal of an undifferentiaded human stem cell culture or cell line, by reducing or eliminating the presence of the protein “p18”.