Abstract: A device and methods for monitoring status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment of the present invention, the device includes a first substrate having a first surface and an opposite second surface, a second substrate supported by the first substrate, the second substrate having a first surface, an opposite second surface, a body portion between the first surface and the second surface, a first side surface and an opposite second side surface, wherein the body portion defines a first passage between the first side surface and the second side surface and an opening on the first surface of the second substrate and in fluid communication with the first passage, and sidewalls positioned above the first surface of the second substrate.

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: Systems, methods and apparatus provide flexible and efficient high throughput electroporation systems. An electrical pulse may be transmitted to any number of channels of a multi-channel sample plate. Drivers can provide the selection of which channels to transmit the electrical pulse. To provide efficient transitions between electrical pulses, discharge circuits provide efficient means achieve a desired voltage.

Abstract: The present invention relates to the field of cell and tissue culture. In particular, the invention provides a novel device for organotypic culture. The device comprises a medium conduit having one open end and one end closed by a porous membrane fused across it; and a frame holding the medium conduit in a substantially vertical orientation; wherein the medium conduit is adapted to permit retention by capillarity of a sufficient volume of liquid culture medium in the medium conduit to contact the surface of the porous membrane and thus supply nutrients to cells that may be grown on the porous membrane. The device may further comprise a cell suspension conduit having one end open and one end closed by the surface of said porous membrane contralateral to the surface of said porous membrane sealed to said medium conduit; wherein said frame holds the medium conduit and the cell suspension conduit in a substantially vertical orientation.

Abstract: The invention relates to an optical method for targeted transfer of molecules, preferably of DNA, RNA, peptides, amino acids and proteins, into vital cells by means of laser radiation and to an arrangement for implementing the method. The object of the invention, to find a novel possibility for targeted molecule transfer into the interior of vital cells, particularly the transfer of DNA, RNA, peptides, amino acids and proteins, which achieves a high transfer efficiency while extensively excluding destructive side effects such as a lethal effect on a treated cell, is met according to the invention in that cellular membranes are opened transiently for the molecule transfer by multiple laser pulses in the microjoule range or less and a pulsed, near-infrared laser beam with a pulse width in the femtosecond range is directed in each instance to a submicrometer spot of a membrane of the vital cell for an irradiation period of less than one second.

Abstract: The present invention includes methods for effecting phenotype conversion in a cell by transfecting the cell with phenotype-converting nucleic acid. Expression of the nucleic acids results in a phenotype conversion in the transfected cell. Preferably the phenotype-converting nucleic acid is a transcriptome, and more preferably an mRNA transcriptome.

Abstract: The present invention is directed to methods of delivering macromolecules to a target cell or tissue by microwave irradiation. A target cell or tissue is exposed to one or more macromolecules to be delivered into the desired cell or tissue and irradiated with microwave radiation. The strength or power of the microwave radiation is such that the macromolecules are delivered into the target cell or tissue. Preferably, the strength of the microwave radiation does not significantly impact cell viability in a negative manner (e.g., apoptosis).

Abstract: A method of delivering a reagent into a cell comprising positioning at least one cell, and at least one magnetically susceptible particle attached to the reagent, in the magnetic field of a Halbach array such that the magnetically susceptible particle is attracted to and contacts the cell is described together with an apparatus for delivery of the reagent.

Abstract: Systems and methods for magnetic targeting of therapeutic particles are provided. Therapeutic particles comprise one or more magnetic or magnetizable materials and at least one therapeutic agent. Therapeutic particles are specifically targeted using uniform magnetic fields capable of magnetizing magnetizable materials, and can be targeted to particular locations in the body, or can be targeted for capture, containment, and removal. Therapeutic particles can comprise antioxidant enzymes, and can be targeted to cells to protect the cells from oxidative damage.

Abstract: The present invention provides a peptide comprising amino acid sequences R I, F I and R I G C and containing 25 or fewer amino acid residues, and capable of transporting a functional molecule into a cell, and also into a nucleus, more efficiently than a previous PTD.

Abstract: The present invention provides a sonoporation-based method that can be universally applied for delivery of compounds into Gram positive bacteria. Gram positive bacteria which can be transformed by sonoporation include, for example, Bacillus, Streptococcus, Acetobacterium, and Clostridium. Compounds which can be delivered into Gram positive bacteria via sonoporation include nucleic acids (DNA or RNA), proteins, lipids, carbohydrates, viruses, small organic and inorganic molecules, and nano-particles.

Abstract: A cell isolating device and method is provided to concentrate or isolate cells with specific characteristics from a mixture of different cell types. One embodiment may comprise two subtypes of antibodies that are directly conjugated to biotin (Abb) and conjugated to a fluorescent molecule (Abf). The conjugated antibodies (Abb+Abf) bind to the target cells in a mixed cell suspension. The cell suspension is then passed over an immobilized avidin or streptavidin substrate on a glass microscope slide. The biotinylated target cells adhere to the avidin/streptavidin substrate, while the unbound cells are washed off and collected in a wicking member. Captured cells on the avidin/streptavidin substrate may then be visualized directly using a fluorescent microscope or detected and enumerated via an on-board fluorescent detection device.

Abstract: In method of injecting a substance into a living cell having a cell membrane, the substance, the cell and a liquid are placed into a tapering passage. Energy is applied to the cell, thereby inducing poration. To sort cells, a cellular suspension is placed in a tapering passage, including a narrow end that defines an opening that has a dimension corresponding to a cell size. An acoustic wave is applied, thereby forcing cells having a cell size smaller than the selected cell size through the opening, with a portion of the cells having a cell size not smaller than the selected cell size not forced through the opening. To extract material from a cell, an electric field and an acoustic wave are applied, thereby causing the cell membrane to allow the material to pass out of the cell.

Abstract: Optoinjection method for transiently permeabilizing a target cell by (a) illuminating a population of cells contained in a frame; (b) detecting at least one property of light directed from the frame; (c) locating a target cell by the property of light; and (d) irradiating the target cell with a pulse of radiation.

Abstract: A method of optoperforation of the membrane of a cell by application of laser pulses characterized by focusing the pulsed laser beam onto the cell membrane to be perforated, applying a series of laser pulses of predetermined pulse energy, measuring the oscillation time of the bubbles formed in the laser focus from the change in laser intensity of a test laser beam transmitted through the laser focus and caused by the bubbles in the laser focus, and increasing the pulse energy to a level at which the oscillation time of the bubbles attains a predetermined value.

Abstract: The invention relates to a reagent and a process for the identification and counting of biological cells in a sample. This reagent comprises a cell lysing agent selected from at least one detergent in a concentration capable of specifically lysing a given type of cells in the sample, and a stain capable of marking the intracellular nucleic acids of the remaining unlysed cells. Application in particular for the identification and counting of cells using an automated analysis system based on flow cytometry.

Abstract: Electro-poration device for the permeabilization of cells contained in a substrate comprising a signal generator for generating a stimulating signal applied by means of electrodes to the substrate wherein an electric field permeabilizing the cells membranes is induced. The device calculates and monitors the instantaneous value or the ratio GT between the current flowing through the substrate and the voltage of the stimulating signal applied to the substrate; in particular, the stimulating signal is applied in a modified manner according to the shape of an initial portion of the waveform of a curve CGT representing the value of ratio GT in successive instants after the application of the stimulating signal. The system will achieve permeabilization of cells in substrate with no or minimum damage, allowing to introduce or to extract molecules in or from the electropermeabilized living cells.

Abstract: An apparatus and method for manipulating, effecting interaction of, photochemically transforming and/or sorting small dielectric particles or other materials. The apparatus and method involves use of one or more diffractive optical elements which each receive a laser beam and form a plurality of laser beams. These laser beams are operated on by a telescope lens system and then an objective lens element to create an array of optical traps for manipulating, effecting interaction of, photochemically transforming and/or sorting small dielectric particles or other materials.

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: The present invention includes devices, systems, and methods for assaying cells using cell-substrate impedance monitoring. In one aspect, the invention provides cell-substrate impedance monitoring devices that comprise electrode arrays on a nonconducting substrate, in which each of the arrays has an approximately uniform electrode resistance across the entire array. In another aspect, the invention provides cell-substrate monitoring systems comprising one or more cell-substrate monitoring devices comprising multiple wells each having an electrode array, an impedance analyzer, a device station that connects arrays of individual wells to the impedance analyzer, and software for controlling the device station and impedance analyzer. In another aspect, the invention provides cellular assays that use impedance monitoring to detect changes in cell behavior or state. The methods can be used to test the effects of compounds on cells, such as in cytotoxicity assays.

Abstract: A surface of a cell is irradiated with laser beam to make an opening in the cell membrane, and a drug fluid is inserted in the cell through the opening.

Abstract: A method for introducing a molecule into the cytosol of a cell in which the cell is contacted with a photosensitizing agent, the cell is irradiated with light of a wavelength effective to activate the photosensitizing agent and, substantially at the same time or after the irradiation, the cell is contacted with the molecule to be introduced, particularly for use in cancer treatment, gene therapy and vaccination.

Abstract: The electroporation array is comprised of three technologies: microwire glass electrodes, microelectronic multiplexer stimulator chips and microfluidic flow chamber. Various substances, such as genes, gene silencing RNAi, gene inhibition agents or drugs, can be perfused into the microfluidic flow chamber. The entry of the various substances into the cells will be facilitated by electroporation. An applied electric potential causes nanoscale pores to open in the cell membrane allowing substances in the solution to freely diffuse into the cell. The specific cells selected for electroporation are defined using the computer controlled microelectronic stimulator array. An “image” of which electrodes within the array to apply the electric potential to, and thus electroporate, is de-multiplexed onto the array. All the selected electrodes deliver a current pulse varied by the intensity of the de-multiplexed “image”.

Abstract: Biological cells and other membranous structures are transfected in a flow-through system by first rendering the structures magnetically active such that they respond to a magnetic field, suspending the structures in a solution of an exogenous species with which the structures are to be transfected, then placing the suspension in a channel and using a moving magnetization pattern along the channel wall to cause the structures to travel through the channel. Along their path of travel, the structures pass a transmitter that emits transfection energy sufficient to cause the exogenous species in the suspension to permeate the structure membranes and enter the interiors of the structures.

Abstract: A device for measuring an extracellular potential of a test cell includes a substrate having a well formed in a first surface thereof and a first trap hole formed therein. The well has a bottom. The first trap hole includes a first opening formed in the bottom of the well and extending toward a second face of the substrate, a first hollow section communicating with the first opening via a first connecting portion, and a second opening extending reaching the second surface and communicating with the first hollow section via a second connecting portion. The first connecting portion has a diameter smaller than a maximum diameter of the first hollow section, greater than a diameter of the second connecting portion, and smaller than a diameter of the test cell. The device can retain the test cell securely and accept chemicals and the test cell to be put into the device easily.

Abstract: A device for detecting cells and/or molecules on an electrode surface is disclosed. The device detects cells and/or molecules through measurement of impedence changes resulting from the cells and/or molecules. A disclosed embodiment of the device includes a substrate having two opposing ends along a longitudinal axis. A plurality of electrode arrays are positioned on the substrate. Each electrode array includes at least two electrodes, and each electrode is separated from at least one adjacent electrode in the electrode array by an expanse of non-conductive material. The electrode has a width at its widest point of more than about 1.5 and less than about 10 times the width of the expanse of non-conductive material. The device also includes electrically conductive traces extending substantially longitudinally to one of the two opposing ends of the substrate without intersecting another trace. Each trace is in electrical communication with at least one of the electrode arrays.

Abstract: A system and method for processing a lipid membrane bound structure, the method comprising the steps of providing the structure to be processed in a liquid medium; and heating the liquid medium containing the structure at a rate and through a range sufficient to cause a discrete phase transition in at least one of the membranes, such that the membranes fuse. The method may be used to fuse the structure with another structure, or to reduce the integrity of the structure. The apparatus atomizes a medium containing the structure into small droplets and subjects them to an environment containing steam vapor while moving at high velocity, to rapidly increase the droplet temperature to the steam temperature by release of the latent heat of vaporization.

Abstract: A device and methods for monitoring status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment of the present invention, the device includes a first substrate having a first surface and an opposite second surface, a second substrate supported by the first substrate, the second substrate having a first surface, an opposite second surface, a body portion between the first surface and the second surface, a first side surface and an opposite second side surface, wherein the body portion defines a first passage between the first side surface and the second side surface and an opening on the first surface of the second substrate and in fluid communication with the first passage, and sidewalls positioned above the first surface of the second substrate.

Abstract: A method and system for electrically wounding and/or monitoring cell activity in vitro. The invention comprises methods and systems for wounding and/or monitoring cells that place a cell culture on a well that has an exposed electrode. The cell culture can then be wounded and/or monitored using the electrode.

Abstract: A method of perforating a membrane 10 is presented. The method comprises: bringing a membrane-denaturing substance into contact with or close proximity to at least a site of said membrane 10, said substance inducing a membrane-denaturing reaction by a stimulus; providing said stimulus to said substance so as to denature said membrane 10; perforating said membrane with a membrane-destroying member 1. The stimulus is carried through said membrane-destroying member 1. The present invention eliminates the influence of membrane-denaturing agent to the substance to be injected into the cell. The present invention also enables applying the stimulus locally, with a simple construction.

Abstract: An electroporation device having a signal generating circuit (3) connectable at the output to electrodes (5) fittable to a substrate (35) having cells. The electrodes (5) produce an electric field which induces permeabilization of the cell membranes to facilitate introduction of substances into the cells. The amplitude of the signal (12) applied to the electrodes is closed-loop controlled on the basis of the impedance (Z(?)) of the substrate, so as to regulate the amplitude of the signal following a reduction in impedance caused by permeabilization of the cell membranes.

Abstract: Optoinjection method for transiently permeabilizing a target cell by (a) illuminating a population of cells contained in a frame; (b) detecting at least one property of light directed from the frame; (c) locating a target cell by the property of light; and (d) irradiating the target cell with a pulse of radiation.

Abstract: Methods for delivering nucleic acid molecules into cells and methods for measuring nucleic acid delivery into cells and the expression of the nucleic acids are provided. The methods are designed for introduction of large nucleic acid molecules, including artificial chromosomes, into cells, and are practiced in vitro and in vivo.

Abstract: The present invention provides a method for introducing a molecule into the cytosol of a cell in which the cell is contacted with a photosentistising agent, the cell is irradiated with light of a wavelength effective to activate the photosentisitising agent and, substantially at the same time or after the irradiation, the cell is contacted with the molecule to be introduced, particularly for use in cancer treatment, gene therapy and vaccination.

Abstract: A method is described for treating biological cells and/or their cell components with electrical fields in a reaction medium, in which an inhibitor is added to the reaction medium to counteract the action of enzymes that break down protein.

Abstract: This invention relates generally to the field of cell separation or isolation. In particular, the invention provides a method for separating cells, which method comprises: a) selectively staining cells to be separated with a dye so that there is a sufficient difference in a separable property of differentially stained cells; and b) separating said differentially stained cells via said separable property. Preferably, the separable property is dielectrophoretic property of the differentially stained cells and the differentially stained cells are separated or isolated via dielectrophoresis. Methods for separating various types of cells in blood samples are also provided. Centrifuge tubes useful in density gradient centrifugation and dielectrophoresis isolation devices useful for separating or isolating various types of cells are further provided.

Abstract: Methods and compositions are provided for detecting changes in membrane potential in membranes biological systems. In one aspect, the method comprises; a) providing a living cell with a first reagent comprising a charged hydrophobic molecule which is typically a fluorescence resonance energy transfer (FRET) acceptor or donor, or is a quencher and is capable of redistributing within the membrane of a biological membrane in response to changes in the potential across the membrane; b) providing the cell with a second reagent that can label the first face or the second face of a biological membrane within the cell; c) detecting light emission from the first reagent or the second reagent. One aspect of this method involves monitoring membrane potential changes in subcellular organelle membranes in a living cells. Another aspect of the invention is the use of certain embodiments of the method for the screening of test chemicals for activity to modulate the activity of a target ion channel.

Abstract: Methods and compositions are provided for determining the potential of a membrane. In one aspect, the method comprises: (a) introducing a first reagent comprising a hydrophobic fluorescent ion capable of redistributing from a first face of the membrane to a second face of the membrane in response to changes in the potential of the membrane, as described by the Nernst equation, (b) introducing a second reagent which labels the first face or the second face of the membrane, which second reagent comprises a chromophore capable of undergoing energy transfer by either (i) donating excited state energy to the fluorescent ion, or (ii) accepting excited state energy from the fluorescent ion, (c) exposing the membrane to radiation; (d) measuring energy transfer between the fluorescent ion and the second reagent, and (e) relating the energy transfer to the membrane potential. Energy transfer is typically measured by fluorescence resonance energy transfer.

Abstract: Methods and apparatuses for treating fluids to inactivate microorganisms which may be present therein, said fluid containing one or more components selected from the group consisting of protein, blood and blood constituents are provided. The methods comprise adjusting the percentage of plasma in said fluid to a desired value; mixing an inactivation-effective, substantially non-toxic amount of an endogenous photosensitizer or endogenously-based derivative photosensitizer to said fluid; exposing said fluid to photoradiation of sufficient wavelength and energy to activate the photosensitizer, whereby said microorganisms are inactivated.

Abstract: Methods and compositions are provided for detecting changes in membrane potential in membranes biological systems. In one aspect, the method comprises; a) providing a living cell with a first reagent comprising a charged hydrophobic molecule which is typically a fluorescence resonance energy transfer (FRET) acceptor or donor, or is a quencher and is capable of redistributing within the membrane of a biological membrane in response to changes in the potential across the membrane; b) providing the cell with a second reagent that can label the first face or the second face of a biological membrane within the cell; c) detecting light emission from the first reagent or the second reagent. One aspect of this method involves monitoring membrane potential changes in subcellular organelle membranes in a living cells. Another aspect of the invention is the use of certain embodiments of the method for the screening of test chemicals for activity to modulate the activity of a target ion channel.

Abstract: A ready-to-use electroporation cuvette is provided that includes a cuvette, first and second electrodes positioned within the cuvette and electroporation competent cells frozen in a suspension solution within the cuvette, wherein the electroporation cuvette is configured to permit electroporation of the cells when the cells are thawed. The electroporation cuvette may be sealed with a cap that may be color coded to aid the user.

Abstract: The present invention provides biological fertilizer compositions that comprise yeast cells and garbage. The yeast cells of the invention have an enhanced ability to fix atmospheric nitrogen, decompose phosphorus minerals and compounds, decompose potassium minerals and compounds, decompose complex carbon compounds, overproduce growth factors, overproduce ATP, decompose undesirable chemicals, suppress growth of pathogenic microorganisms, or reduce undesirable odor. The biological fertilizer composition of the invention can replace mineral fertilizers in supplying nitrogen, phosphorus, and potassium to crop plants. Methods of manufacturing biological fertilizer compositions, and methods of uses are also encompassed.

Abstract: The present invention provides biological fertilizer compositions that comprise yeast cells that have an enhanced ability to fix atmospheric nitrogen, decompose phosphorus minerals and compounds, decompose potassium minerals and compounds, decompose complex carbon compounds, over produce growth factors, and over produce ATP. The biological fertilizer composition of the invention can replace mineral fertilizers in supplying nitrogen, phosphorus, and potassium to crop plants. Methods of manufacturing the biological fertilizer compositions and methods of uses are also encompassed.

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: A technique for controlling membrane denaturation reactions other than physical shear force was developed. For example, the present invention provides, a method for causing membrane disruption at a specific site by reacting a stimulus such as light with a compound that is activated by the stimulus, where the reaction occurs on a membrane such as a biomembrane. It also provides a membrane structure such as cells in which a specific site has been disrupted, which are obtained by the present method. Introduction of substances such as genes also became possible by using this membrane structure. Further provided is a membrane-destroying member for disrupting a membrane at a specific site. Thus, the present invention enabled, for example, easy membrane penetration using components constituting microelectrodes, micromanipulators, and microinjectors, which were conventionally hardly usable in penetrating cell membranes.

Abstract: A method for counting leukocytes which comprises liberating elastase from granulocytes contained in a specimen, adding an anti-granulocyte elastase antibody to the thus liberated elastase, measuring the antibody bonded to the elastase to thereby determine the concentration of the elastase, and then calculating therefrom the number of leukocytes contained in the specimen with the use of the ratio of the leukocyte count to the elastase concentration. This method makes it possible to conveniently and less expensively count leukocytes at a high accuracy comparable to the one established by using a conventional automatic blood cell counter, as the figure shows.

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 system and method for processing a lipid membrane bound structure, the method comprising the steps of providing the structure to be processed in a liquid medium; and heating the liquid medium containing the structure at a rate and through a range sufficient to cause a discrete phase transition in at least one of the membranes, such that the membranes fuse. The method may be used to fuse the structure with another structure, or to reduce the integrity of the structure. The apparatus atomizes a medium containing the structure into small droplets and subjects them to an environment containing steam vapor while moving at high velocity, to rapidly increase the droplet temperature to the steam temperature by release of the latent heat of vaporization.

Abstract: Biological agents in solution within an underwater treatment zone are transferred by infusion to living cells under an acoustic energy field of limited duration and certain accompanying water flow and temperature conditions to maximize processing efficiency.