Abstract: A chamber for treating cells contained in a suspension in an electric field, the chamber having a vessel for holding the suspension and at least two electrodes with electrode surfaces that face each other. The suspension may be placed between the electrode surfaces and the electrodes are connectable to different poles of a source of voltage to generate an electric field between the electrode surfaces. The electrodes are laminate and continuous and are configured to generate a non-uniform field. Moreover, the electrodes have electrode surfaces made of electrically conductive material, wherein the electrode surface of at least one of the two electrodes is shaped such that a non-uniform field with several maxima distributed over the electrode surface may be generated between the two electrodes.

Abstract: The invention comprises an apparatus and a method of effecting localized electroporation in a relatively small target area and for introducing foreign matter into cellular material in which the target area is located. The cellular material may be in vitro, in ovo or in vivo.

Abstract: Methods and apparatus for the reproducible, consistent and efficacious delivery of a therapeutic agent to a patient. The invention comprises means for the controlled administration of the therapeutic agent through an orifice to the patient, a plurality of penetrating electrodes arranged with a predetermined spatial relationship relative to the orifice, and means for generating an electrical signal operatively connected to the electrodes.

Abstract: DNA is efficiently transformed into a host by electroporation in the presence of a methylation package, which greatly improves the efficiency of the transformation. The methylation package comprises a source of cysteine, such as cysteine, homocysteine, or glutathione, with optional iron and magnesium ions.

Abstract: Intracellular material is released from bacterial, yeast, plant, animal, insect or human cells by the application of a low voltage such as 1 to 10 V to a suspension containing the cells. The conditions may be selected such that DNA released from the cells is electrochemically denatured so as to be available for use in an amplification procedure.

Abstract: The invention relates to a method for selectively releasing an agent loaded into a red blood cell, comprising electrosensitizing the red blood cell by application of an electric field and subsequently disrupting the cell selectively using ultrasound.

Abstract: An object of the invention is to provide a method for delivery of macromolecules into biological cells in the tissues of a patient and includes the steps of: (a) providing electrodes (16) in an electrode assembly (12), wherein the electrodes have fixed electrode surfaces (42) which are coated with at least one static layer of electrode releasable molecules (44) to be delivered; (b) providing a waveform generator (15) for generating electric fields; (c) establishing electrically conductive pathways between the electrodes (16) and the waveform generator (15); (d) locating the electrodes (16) such that the biological cells are situated therebetween, and (g) providing electric fields in the form of pulse waveforms from the waveform generator (15) to the electrodes (16), such that molecules in the at least one static layer of the electrode releasable molecules (44) on the electrodes (16) are delivered into the biological cells.

Abstract: The present invention relates to methods and apparatus for the encapsulation of biologically-active substances in various cell populations. More particularly, the present invention relates to a method and apparatus for the encapsulation of biologically-active substances in various cell populations in blood by electroporation to achieve therapeutically desirable changes in the physical characteristics of the various cell populations in blood.

Abstract: Methods are proved for introducing a biologically active agent into cells of a subject by introducing the agent in a form suitable for electrotransport into a region of tissue of the subject using one or more needle-free injectors, and applying a pulsed electric field to the region of tissue, thereby causing electroporation of the region of tissue. The combination of needle-free injection and electroporation is sufficient to introduce the agent into cells in skin, muscle or mucosa. For example, the region of tissue can be contacted with two oppositely charged injectors, one acting as the donor electrode and one acting as the counter electrode, or a single injector and one or more electrodes can be used. In addition, needle-free injection may be used in combination with suitable non-invasive electrode configurations. The active agents delivered into cells using the invention method can be small molecules, polynucleotides, polypeptides, and the like.

Abstract: The present invention relates to a method and apparatus for the encapsulation of substances and drugs into cells and platelets. The present invention is also related to the incorporation of thrombus dissolving drugs, such as tissue plasminogen activator and streptokinase into platelets using the apparatus described herein. The treated platelets can then be used to treat patients suffering from a thrombus blocking a blood vessel. The present invention is also related to a preparation of red blood cells that has a stable right shift of the oxygen dissociation curve.

Abstract: The invention relates to a measuring device which permits a very simple positioning of cells and vesicles respective of cell membranes on planar carriers. The invention also relates to a corresponding highly efficient method for the positioning and electric characterization of such membranes with a consistently high signal-to-noise ratio. In addition, statements concerning interactions of substances with lipid membranes respective of materials bonded thereon or therein respective of signal transduction mechanisms connected thereto are possible.

Abstract: Techniques for streaming electroporation. A representative but non-limiting method includes: generating a spatially inhomogeneous electric field with a pair of electrodes and displacing the pair of electrodes and a sample relative to one other while the electric field is substantially constant in terms of magnitude so that the sample is displaced across electric field lines for a time sufficient to effect electroporation.

Abstract: A method and apparatus for introducing a preselected molecule into a living cell by contacting the cell with the preselected molecule and applying a multiple series of electrical pulses to the cell. The method can be utilized ex vivo. The multiple electrical pulses generate rotating electric fields which introduce transient pores in the living cell without killing the cell. The rotating electric fields are provided in a flow through chamber apparatus having more than two electrodes. A three-step pulse process, e.g. collection, electroporation, electrophoresis, is used to introduce the preselected molecule into the cell. A mechanical means of repositioning cells between successive pulses is also provided. The apparatus can also provide a means to pulse cells at different temperatures and then after pulsing, let the cells recover for a specified residence time at another temperature.

Abstract: Cuvette inserts adapted and configured to fit within an electroporation cuvette. The inserts each include a support structure that holds a porous membrane. When positioned within the cuvette, the membrane is positioned proximal the cuvette electrodes to facilitate membrane-based fusion of cells. In certain aspects, a tube extends through the support structure to allow for application of negative pressure in a convenient location away from electrode contacts and other components of the cuvette or cuvette holder.

Abstract: An object of the invention is to provide a method for delivery of macromolecules into biological cells in the tissues of a patient and includes the steps of: (a) providing electrodes (16) in an electrode assembly (12), wherein the electrodes have fixed electrode surfaces (42) which are coated with at least one static layer of electrode releasable molecules (44) to be delivered; (b) providing a waveform generator (15) for generating electric fields; (c) establishing electrically conductive pathways between the electrodes (16) and the waveform generator (15); (d) locating the electrodes (16) such that the biological cells are situated therebetween, and (g) providing electric fields in the form of pulse waveforms from the waveform generator (15) to the electrodes (16), such that molecules in the at least one static layer of the electrode releasable molecules (44) on the electrodes (16) are delivered into the biological cells.

Abstract: The present invention relates to a method and an apparatus for separating substances from or in biological materials.

Abstract: The present invention relates to ionic electrodes, particularly microelectrodes and electrode arrays, and also relates to fabrication methods for such electrodes. In particular, the present invention relates to planar polymer electrodes for making patch clamp measurements of ionic currents through biological membranes, such as the plasma membranes of living cells. The electrodes of the present invention are useful for measuring individual and multisite cell membrane currents and voltages, as well as in high-throughput screening procedures.

Abstract: A housing has a chamber containing a low speed, e.g., 6 rpm, rotating shaft of highly polished stainless steel to avoid bubbles and arcing. A fluid containing cells to be electroporated and molecules associated with the electroporation are introduced into the chamber formed by the gap between the shaft and housing in either batch or continuous flow modes. A capacitor network applies alternating positive and negative pulses of high voltage across the electrodes formed by the housing and shaft to electroporate the cells. After a few pulses, a resistive load is placed across the network to reduce the value of the pulses applied to the electrodes for a relatively longer period than the high voltage pulses to a relatively low harmless level.

Abstract: The object of the invention is to provide a method and apparatus for treating membrane-containing material with electrical fields, in vitro, and with an added treating substance. With the method, a plurality of electrodes are arrayed around the material to be treated and are connected to outputs of an electrode selection apparatus. Inputs of the electrode selection apparatus are connected to outputs of a pulse sequence generator. A treating substance is added to the membrane-containing material. Electrical pulses are applied to the electrode selection apparatus and are routed through the electrode selection apparatus in a predetermined, computer-controlled sequence to selected electrodes in the array of electrodes, whereby the membrane-containing material is treated with the added treating substance and with electrical fields. The routing of applied pulses through the electrode selection apparatus to selected electrodes can be done in an enormous number of ways.

Abstract: Delivery of metal particles to living tissue, then applying external energy that interacts with the metal particles, is found to selectively increase the energy deposition and interaction surrounding the metal particles. The method is useful to improve treatment of various conditions, since targeted cells may be selectively altered or killed. Metal particles are also loaded into cells or membrane vesicles by placing metal seed particles into the cells or vesicles, then chemically depositing additional metal on the metal seed particles. The metal particles are useful to improve imaging and therapies by their interaction with externally applied energy.

Abstract: A method is provided for measuring a state variable of a biological cell (3) located in a nutrient medium (2) and supported on and adhering to a support area (5). Within the support area (5) for the cell (3) and at a distance from the support area edge, an opening is made in the membrane of the cell (3). The edge of the cell membrane that surrounds the opening and adheres to the support area (5) seals off the liquid found inside the cell (3) from the nutrient medium (2). Through the opening the state variable (2) is measured. An apparatus for performing the method is also provided.

Abstract: The present invention involves methods and devices which enable discrete objects having a conducting inner core, surrounded by a dielectric membrane to be selectively inactivated by electric fields via irreversible breakdown of their dielectric membrane. One important application of the invention is in the selection, purification, and/or purging of desired or undesired biological cells from cell suspensions. According to the invention, electric fields can be utilized to selectively inactivate and render non-viable particular subpopulations of cells in a suspension, while not adversely affecting other desired subpopulations. According to the inventive methods, the cells can be selected on the basis of intrinsic or induced differences in a characteristic electroporation threshold, which can depend, for example, on a difference in cell size and/or critical dielectric membrane breakdown voltage.

Abstract: A system for applying energy to cells so as to elicit the formation of pores, to enhance transfection, and/or cell transformation, includes a computer, a plurality of acoustic probes for controllably applying acoustic energy to batches of cells, and a robot operatively for effecting relative movement between the probes and the batches of cells. Preferably, the acoustic energy comprises ultrasonic energy, which is applied in combination with optical or electrical energy to enhance the formation of pores in surface membranes of the cells.

Abstract: The invention concerns a method for treating an aqueous flow colonized by cells with a pulsed electric field applied to a flow, characterized in that the applied field is substantially parallel to the direction of flow and to its application to the transfer of nucleic acids (RNA, DNA, oligonucleotides) into cells, to the transfer of proteins to cells, to the extraction of cytoplasmic macromolecules and molecules contained in the cells, to cell fusion and the production of hybrids and/or to insertion of membrane proteins. It also concerns an electropulsing chamber, a method for destroying cells and a membrane permeabilization method.

Abstract: A method of sorting cells, particularly, but not exclusively, sperm cells, is described. The method comprises the use of a surface decontamination medium to strip the cells of any extra-cellular surface contamination and to then subject to stripped cells to a charge based separation.

Abstract: An improved electrode for use in generating an electrical field in a saline solution is provided. In particular, a continuous crystalline metal nitride coated electrode is provided for use in a variety of saline solution applications, such as in an electrophoresis device for separating proteins or nucleic acids or an electroporation apparatus for the encapsulation of biologically-active substances in various cell populations. A method and apparatus are provided for the encapsulation of biologically-active substances in red blood cells, characterized by an optionally automated, continuous-flow, self-contained electroporation system which allows withdrawal of blood from a patient, separation of red blood cells, encapsulation of a biologically-active substances in the cells, and optional recombination of blood plasma and the modified red blood cells thereby producing blood with modified biological characteristics.

Abstract: Systems, methods and algorithms for automatically performing optimization of an electroporation system. A system according to the present invention typically includes a cuvette holding assembly configured to hold a plurality of electroporation cuvettes, wherein each cuvette includes a first and second electrode, and a shocking chamber configured to hold the cuvette holding assembly, the chamber having a commutator assembly configured to provide an electrical contact to the first electrode of each of the plurality of cuvettes in turn. The system also typically includes a control system communicably coupled to the shocking chamber, wherein the control system controls the commutator to automatically contact the first electrode of each cuvette in an order and to provide a potential across the cuvette electrodes when contact is made.

Abstract: The present invention involves methods and devices which enable discrete objects having a conducting inner core, surrounded by a dielectric membrane to be selectively inactivated by electric fields via irreversible breakdown of their dielectric membrane. One important application of the invention is in the selection, purification, and/or purging of desired or undesired biological cells from cell suspensions. According to the invention, electric fields can be utilized to selectively inactivate and render non-viable particular subpopulations of cells in a suspension, while not adversely affecting other desired subpopulations. According to the inventive methods, the cells can be selected on the basis of intrinsic or induced differences in a characteristic electroporation threshold, which can depend, for example, on a difference in cell size and/or critical dielectric membrane breakdown voltage.

Abstract: Disclosed is a method for electromanipulation of at least one cell or cell-like structure having cell-like membranes, comprising the following consecutive steps: (a) at least one cell or cell-like structure is transported from one or more sample containers located on a chip through at least one microchannel located on said chip into a chamber located on said chip, wherein said chamber contains at least one electrode connected to a voltage generator, and wherein said microchannel provides a fluid contact between the sample containers, (b) either said at least one cell or cell-like structure is placed or aligned close to said at least one electrode, or said at least one electrode is placed or aligned close to said at least one cell or cell-like structure in said chamber, and (c) an electrical field is applied and focused on said at least one cell or cell-like structures, said electrical field being of a strength sufficient to obtain pore-formation in said at least one cell or cell-like structure or sufficient t

Abstract: A molecule introducing apparatus is provided with a power supply circuit 20 for supplying power, a step-up transformer 30 which is supplied with power from the power supply circuit 20 and which outputs a high voltage, a switching transistor 22 that blocks off and allows the supply of power from the power supply circuit 20 to the step-up transformer 30 and which generates an instantaneous high voltage, and a pair of electrode probes 50 for applying the instantaneous high voltage generated at the step-up transformer 30 to a predetermined region in a body. The molecule introducing apparatus oscillates DNA arranged outside a cell by the instantaneous high voltage so as to introduce the DNA into the cell.

Abstract: Electroporation is performed in a controlled manner in either individual or multiple biological cells or biological tissue by monitoring the electrical impedance, defined herein as the ratio of current to voltage in the electroporation cell. The impedance detects the onset of electroporation in the biological cell(s), and this information is used to control the intensity and duration of the voltage to assure that electroporation has occurred without destroying the cell(s). This is applicable to electroporation in general. In addition, a particular method and apparatus are disclosed in which electroporation and/or mass transfer across a cell membrane are accomplished by securing a cell across an opening in a barrier between two chambers such that the cell closes the opening. The barrier is either electrically insulating, impermeable to the solute, or both, depending on whether pore formation, diffusive transport of the solute across the membrane, or both are sought.

Abstract: The present invention relates to methods and apparatus for the encapsulation of biologically-active substances in various cell populations. More particularly, the present invention relates to a method and apparatus for the encapsulation of biologically-active substances in various cell populations in blood by electroporation to achieve therapeutically desirable changes in the physical characteristics of the various cell populations in blood.

Abstract: The invention relates to a method for permeabilization of a cell structure consisting of a single cell, an intracellular structure or an organelle comprising the following steps: (a) microelectrodes, preferably two carbon fibre electrodes or hollow fibre electrodes, are provided, (b) the microelectrodes are connected to a power supply, (c) the electrodes, individually controlled by high-graduation micromanipulators, are placed close to the cell structure at an appropriate inter-electrode distance, and (d) a highly focused electric field of a strength sufficient to obtain electroporation is applied between the electrodes. The method may be used in order to transfer cell impermeant solutes, such as drugs or genes, into the cell structure or out of the cell structure, in biosensors, in the treatment of tumours and neurodegenerative diseases and in the study of biophysical processes.

Abstract: A loading system for providing a cell suitable for delivery of an agent to a vertebrate, the system comprise a loading module for loading a cell with an agent; and a sensitisation module in fluid communication with the loading module, the sensitisation module for sensitising a cell to an energy field, such that said cell is induced to release the agent upon exposure to said energy field. The system can be used to transform a cell, such as a red blood cell, into a delivery vehicle for delivering a therapeutic agent and/or an imaging agent to a vertebrate, and in particular, to a mammal, such as a human being.

Abstract: The present invention is discloses an improved method of producing infectious papillomavirus in vitro, with a method comprising (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and (b) providing conditions that produce papillomavirus, wherein the conditions comprise not contacting the epithelial cell a fibroblast and does not comprise an organotypic raft culture or a dermal equivalent. The present invention also discloses a papillomavirus infected non-keratinocyte epithelial cell produced by the methods of the present invention. Further, uses of the disclosed method includes detection methods, methods for screening anti-papillomavirus drugs, methods of making recombinant papillomavirus for vaccines and studying the life cycle. Additionally, a method of reducing and assessing the risk of spontaneous abortion is disclosed.

Abstract: An improved electrode for use in generating an electrical field in a saline solution is provided. In particular, a continuous crystalline metal nitride coated electrode is provided for use in a variety of saline solution applications, such as in an electrophoresis device for separating proteins or nucleic acids or an electroporation apparatus for the encapsulation of biologically-active substances in various cell populations. A method and apparatus are provided for the encapsulation of biologically-active substances in red blood cells, characterized by an optionally automated, continuous-flow, self-contained electroporation system which allows withdrawal of blood from a patient, separation of red blood cells, encapsulation of a biologically-active substances in the cells, and optional recombination of blood plasma and the modified red blood cells thereby producing blood with modified biological characteristics.

Abstract: A process is provided for the intracellular manipulation of a biological cell (3) which is positioned adhering to a support area (5) in a culture medium (2). Inside the support area (5) for the cell (3) an opening into the membrane of the cell (3) is created spaced from its support edge. The edge of the cell membrane surrounding the opening, adhering to the support area (5), thus seals off the cell fluid situated in the interior of the cell (3) from the culture medium (2) and insulates the cell fluid against the culture medium (2). The interior of the cell (3) is manipulated through the opening. An apparatus for implementing the process is also provided, including an object carrier (4) with a support area (5) for adhering the cell and a poration tool (6) for creating the opening in the cell membrane. The poration tool (6) may be any of various chemical, mechanical and/or electrical devices.

Abstract: A method is provided for measuring a state variable of a biological cell (3) located in a nutrient medium (2) and supported on and adhering to a support area (5). Within the support area (5) for the cell (3) and at a distance from the support area edge, an opening is made in the membrane of the cell (3). The edge of the cell membrane that surrounds the opening and adheres to the support area (5) seals off the liquid found inside the cell (3) from the nutrient medium (2). Through the opening the state variable (2) is measured. An apparatus for performing the method is also provided.

Abstract: An electrodiffusion pump includes a housing having an inlet, drug reservoir, and outlet. A diffusion membrane is disposed between the reservoir and outlet for diffusing the drug outwardly therethrough. Internal and external electrodes are disposed on opposite sides of the membrane. The electrodes are porous, and the external electrode is additionally perforate to expose a portion of the membrane at the outlet. The electrodes are electrically powered for driving drug ions through the membrane under electrophoresis to complement diffusion thereof.

Abstract: An electroporation method and apparatus generating and applying an electric field according to a user-specified pulsing and temperature profile scheme. The apparatus includes a cuvette holder with a Peltier device forming part of the electrode structures that form part of the holder. Advantageously, one such pulse includes a low voltage pulse of a first duration, immediately followed by a high voltage of a second duration, immediately followed by a low voltage of a third duration. The low voltage electroporation field accumulates molecules at the surface of a cell, the appropriately high voltage field creates an opening in the cell, and the final low voltage field moves the molecule into the cell. The molecules may be DNA, portions of DNA, chemical agents, the receiving cells may be eggs, platelets, human cells, red blood cells, mammalian cells, plant protoplasts, plant pollen, liposomes, bacteria, fungi, yeast, sperm, or other suitable cells.

Abstract: The populating method for populating a substrate with living cells provides a population with at least one populating phase and at least one perfusion phase. During the populating phase the substrate can be held in contact with the cell suspension by continuous rotation in various spatial arrangements. The populating device of the invention (10) comprises a rollable container (10a) and a removable insert in it with a means (12) for inserting the substrate being populated, such as a plastic or collagen matrix. The insert is designed so that in interaction with the inserted substrate, two liquid-tight chambers are formed within the container on opposite sides of the inserted substrate, so as to allow separate populating of both sides of the substrate. The container have liquid inlets and outlets (16) and may also have gas inlets and outlets. The rollable populating reactor (10) is inserted into a roller cabinet for certain phases of the populating.

Abstract: Methods are disclosed for identifying an RNA fragment that mimics the structure of a defined or undefined target RNA molecule to which a compound binds inside of a cell resulting in retardation of cell growth or cell death. Methods using these RNA fragments for identifying unknown compounds of pharmaceutical interest, and for identifying unknown RNA targets for use in treating disease are disclosed. These methods and compositions are used in screening for novel antibiotics, bacteriostatics, or modifications thereof or for identifying compounds useful to alter expression levels of proteins encoded by mRNA. The methods involve providing random DNA fragments from DNA which encodes RNA target molecules, cloning such fragments to create a plasmid library of same; transfecting cells which contain the native RNA target molecule with the plasmid library and exposing the cells to one or more of test compounds.

Abstract: A new and useful apparatus for producing cell electrofusion is provided. The apparatus comprises: a. a chamber with a substrate disposed therein, b. means for directing the cells to be fused toward one side of the substrate; and c. a device for inducing fusion of the portion of the cells.

Abstract: Drug candidate screening methods are applied to discover compounds with activity against ion channel targets. The method may include modulating the transmembrane potential of host cells in a plurality of sample wells with a repetitive application of electric fields so as to set the transmembrane potential to a level corresponding to a pre-selected voltage dependent state of a target ion channel.

Abstract: A method of characterizing the biological activity of a candidate compound may include exposing cells to the candidate compound, and then exposing the cells to a repetitive application of electric fields so as to set the transmembrane potential to a level corresponding to a pre-selected voltage dependent state of a target ion channel.

Abstract: A process and system for electrical extraction of intracellular matter from biological matter, and intracellular matter products formed thereby, based on preparing a mixture of biological matter featuring cells, and an electro-conductive liquid, and electrifying the mixture by transmitting controlled cycles of pulses and pauses of electrical current into the mixture by using electrodes, whereby the pulses of electrical current pierce holes into or perforate the cell membranes of the cells, enabling the release of intracellular matter for collecting and separating into target intracellular matter extract and solid waste. Pauses included in each cycle of transmitting pulses of electrical current enable firm control of electrical extraction processing conditions, including extent of extraction, temperature effects, and pressure effects, during the electrical extraction process.

Abstract: The present invention relates to a linear DNA vector and to a method for identifying chromosomal regions having a physical proximity within a living cell, and/or for locating in chromosomes of a living cell a DNA double strand break having a physical proximity with a known non-repetitive DNA sequence. The linear DNA vector has a first end which comprises a nucleotide sequence capable of homologous recombination to a first region of a cell chromosome which comprises a known non-repetitive DNA sequence. The linear DNA vector also has a second end which comprises a nucleotide sequence non-homologous to the chromosomes of the cell and being capable of illegitimate integration with a second region of a chromosome in physical proximity to the first chromosomal region.

Abstract: Method of making an electrotransport device containing an analog of a parent polypeptide having one or more amino acid residues substituted relative to the parent polypeptide with an amino acid residue selected from the group consisting of proline, glycine and asparagine.

Abstract: Electroporation is performed in a controlled manner in either individual or multiple biological cells or biological tissue by monitoring the electrical impedance, defined herein as the ratio of current to voltage in the electroporation cell. The impedance detects the onset of electroporation in the biological cell(s), and this information is used to control the intensity and duration of the voltage to assure that electroporation has occurred without destroying the cell(s). This is applicable to electroporation in general. In addition, a particular method and apparatus are disclosed in which electroporation and/or mass transfer across a cell membrane are accomplished by securing a cell across an opening in a barrier between two chambers such that the cell closes the opening. The barrier is either electrically insulating, impermeable to the solute, or both, depending on whether pore formation, diffusive transport of the solute across the membrane, or both are sought.

Abstract: An electroporation apparatus for introducing exogenous material into cells is described herein. The apparatus comprises first a base member configured for holding a cell support, the cell support having a top surface portion, with the top surface portion configured for carrying adherent cells. The apparatus further comprises an electrode carrier operably associated with the base member, the electrode carrier having a bottom surface portion, a first electrode connected to the electrode carrier, and a second electrode also connected to the electrode carrier. The electrode carrier has a channel formed therein, with the channel positioned between the first electrode and the second electrode, so that exogenous material may be introduced through the channel and into contact with the cells. Methods for introducing exogenous compounds into a cell and for visually detecting the location of binding events within a cell are also disclosed.