WO2019076353A1 - 一种流式电穿孔装置 - Google Patents

一种流式电穿孔装置 Download PDF

Info

Publication number
WO2019076353A1
WO2019076353A1 PCT/CN2018/110915 CN2018110915W WO2019076353A1 WO 2019076353 A1 WO2019076353 A1 WO 2019076353A1 CN 2018110915 W CN2018110915 W CN 2018110915W WO 2019076353 A1 WO2019076353 A1 WO 2019076353A1
Authority
WO
WIPO (PCT)
Prior art keywords
flow electroporation
electroporation device
electrode
flow
rhodium
Prior art date
Application number
PCT/CN2018/110915
Other languages
English (en)
French (fr)
Inventor
戴晓兵
Original Assignee
苏州壹达生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州壹达生物科技有限公司 filed Critical 苏州壹达生物科技有限公司
Priority to US16/756,269 priority Critical patent/US20200318055A1/en
Priority to EP18868610.9A priority patent/EP3699262A1/en
Publication of WO2019076353A1 publication Critical patent/WO2019076353A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/327—Applying electric currents by contact electrodes alternating or intermittent currents for enhancing the absorption properties of tissue, e.g. by electroporation

Definitions

  • This invention relates to the field of electroporation, and more particularly to a flow electroporation device, a method and apparatus for delivering a flowing biologically active substance into a flowing living cell by an electric field.
  • Electroporation was discovered in the 1970s. The electric field was used to treat cells to form micropores, but did not cause permanent damage to the cells. The micropores were self-resetting. This discovery made it possible for biologically active substances to enter cells. Previous studies have found that four phenomena may play a role in electroporation. The first phenomenon is insulation breakdown. Insulation breakdown refers to the ability of a high electric field to create small pores or holes in the cell membrane. Once micropores are produced, the cells can carry bioactive substances. The second phenomenon is the insulating bunching effect, which refers to the mutual self-attraction generated by the stacking of vesicles in a uniform electric field. The third phenomenon is vesicle fusion.
  • Vesicle fusion refers to the tendency of a film of a biological vesicle that produces micropores by dielectric breakdown to bond together at its insulation breakdown site at close range.
  • the fourth phenomenon is the tendency of cells to line up along one of their axes in the presence of a high frequency electric field.
  • electroporation involves the application of a predetermined position in the vesicle rotation, vesicle perforation, and the dielectric constant of the vesicle.
  • Flow electroporation refers to a process comprising: transferring a suspension of cells and a suspension of exogenous biologically active material into a device consisting of a fluid chamber or fluid flow channel; the fluid chamber or fluid
  • the flow path is formed by an electrode disposed along a side of the fluid chamber or fluid flow path and configured to cause a mixed liquid in the fluid chamber or fluid flow path to be subjected to an electric field suitable for electroporation; and to be electroporated A device in which the cell suspension is removed from the device.
  • This method is particularly effective for large volume cells.
  • Patent CN1195997A discloses a flow electroporation chamber using an elastically deformable material as a passage for fluid flow.
  • the deformation of the elastic material can be used as a buffer for the increase of the instantaneous pressure in the fluid flow passage, but it has not studied the suitable flow pattern. Electroporation of electrode materials, so the patent disclosed that nearly 20 years later, there is still no product on the market.
  • the device for flow electroporation comprises two planar electrodes, and the cell suspension to be electroporated continuously and steadily flows between the two electrodes until the entire cell suspension is electroporated.
  • the flow electroporation device comprises an electroporation chamber with an electrode and an interface through which the cell suspension can be injected.
  • the electrodes are connected to a circuit that provides a high voltage pulse to the electrodes.
  • the high voltage pulse is controlled by a program control computer.
  • Flow electroporation systems have been developed which are capable of electroporating larger volumes of cells to obtain viable cells that are introduced into the molecule of interest.
  • the current successful commercial flow electroporation device is extremely rare, on the one hand, directly related to the market demand, and on the other hand, the efficiency of electroporation is related to the stability of the product.
  • the conditions of flow electroporation vary depending on the cell type, and also differ depending on the type of molecule desired to be introduced into the cell.
  • an optimal processing condition that includes optimal voltage, pulse width, optimal time interval, and number of pulses for pulse.
  • the cell suspension can be selected through the electroporation chamber, the flow rate between the electrodes, and the frequency of the pulses when using the flow electroporation device described in the art.
  • the optimal number of pulses is applied to a volume of cell suspension.
  • the optimal number of pulses applied to each cell is known to be 2, and the volume through the electroporation chamber is 1 ml, then the flow rate is set to 1 ml per unit time, each unit The number of pulses applied during the time is 2. If electroporation is performed in this way, each cell will be pulsed twice in this example. However, the hydrodynamic flow of the cell suspension through the electroporation chamber causes the cells to pass through the chamber at different rates and between the electrodes. Since the flow rate is higher than the wall near the chamber wall, the time required for the cells flowing between the electrodes to flow through the center of the electrode to pass through the area between the electrodes is lower than the unit time, and the number of pulses received.
  • the cells flowing near the wall may take longer than the unit time to pass through the area between the electrodes, and the number of pulses may be higher than 2 times. Since the optimal number of pulses per cell is 2 in this example, it is clear that not every cell is pulsed optimally, so the overall electroporation effect of the cell suspension is not optimal. of.
  • the optimal conditions for electroporation vary depending on the particular cell type of electroporation and the type of molecule that is desired to be introduced into the cell by electroporation. It is possible to solve this problem by a test method, using a static electroporation chamber to systematically change the conditions of electroporation, and then applying the optimum conditions determined for these tests to the flow electroporation system.
  • This method has two drawbacks. First, since it takes a lot of time to perform electrostatic perforation of a static chamber for a large number of samples, cells for each static electroporation also change during the test. Second, this method is used only because it is believed that the optimal conditions for electroporation of static chambers are the same as for flow electroporation, but it is not known whether this is true.
  • the electric rotor is mainly in the form of a cuvette inserted into a metal electrode for electroporation or electrofusion.
  • Most of the containers used for this purpose are small containers with a closed bottom, an open top, and an internal space formed by two pairs of side walls arranged in parallel opposite each other.
  • the function of the internal space is to receive a cell suspension in which the cells to be treated are suspended, i.e., usually a buffered aqueous solution or a cell culture medium.
  • Most of such cuvettes contain a pair of electrodes that are placed adjacent to the bottom of a pair of oppositely disposed sidewalls. During discharge, current flows through the cell suspension between the two electrodes, allowing nucleic acids or other molecules to enter the cell or cause cell fusion depending on the selected conditions.
  • the electrodes are made of metal, of which aluminum is most commonly used.
  • these known, commercially available cuvettes have the disadvantage that metal ions are emitted into the buffer solution during discharge, causing undesirable stimulation of the cells and poisoning the cells.
  • the use of an aluminum cuvette can illustrate the side effects due to the release of Al 3+ ions.
  • undesired precipitation occurs, which is also due to the release of metal ions from the electrodes. This precipitation can be a combination of a metal hydroxide or metal ion with a biomacromolecule in a buffer solution.
  • another disadvantage of the aluminum cuvette is that the resistance of the cuvette decreases during discharge, presumably because the aluminum oxide layer having a higher electrical resistance is detached from the electrode by current.
  • the patent CN03805811.1 discloses an electrode made of a conductive synthetic material of a plastic doped with at least one conductive substance, and a dopant in the plastic.
  • the total concentration is 20-80% w/w
  • the dopant is composed of carbon fiber, graphite, carbon black and/or carbon nanotubes, and can effectively treat cells and cell derivatives with current.
  • Patent CN201610044870.8 discloses a cell electrical transfection device having a nanowire structure on a surface thereof, the nanowire being selected from the group consisting of copper oxide nanowires, titanium dioxide nanowires, gold nanowires, silver nanowires, copper nanowires, titanium nanowires, Any of iron nanowires, platinum nanowires, titanium-platinum alloy nanowires, iron oxide nanowires, tin dioxide nanowires, and metal-coated silicon nanowires, using a transfection voltage of 10 V or less, using an electrode surface
  • the microstructure increases the local field strength by 3-4 orders of magnitude, but the electrotransfection device is only a static electroporation for adherent cells.
  • the main technical problems existing in the current flow transfection process are the pH change of the solution around the cathode caused by electrolyzed water, the oxidation of the surface of the anode electrode to produce harmful substances, the Joule effect of current causing the temperature of the solution to rise and the electric rotation to occur.
  • Foreign matter such as bubbles causes changes in electric field strength and its distribution.
  • electrode development is the core content, in which the electrode material is more critical, the electrode material is directly in contact with the cell solution, and an electrochemical reaction occurs during electroporation, resulting in an electric field change, affecting the result.
  • the unstable anode material will precipitate metal ions, causing cell death or contaminating the cell solution, causing greater harm. Therefore, the biocompatible stable electrode material developed by the present invention is the primary problem to solve the commercial application of flow electroporation.
  • the electrode In the electroporation process of the present invention, the electrode is in contact with the cell solution during the electroporation process, and the electrolyzed water reacts easily on the surface of the electrode to generate bubbles. Ideally, the bubble will quickly discharge from the inside of the electrode as the liquid flows. However, due to the influence of the electrode structure and the surface roughness of the electrode material, if the bubble stays inside the electroporation chamber, the electric field intensity distribution in the electrode changes due to the non-conduction of the bubble; while the bubble occupies the chamber, the solution flow rate is changed, causing the cell to flow. The number of times and the intensity of the current pulse signal received by the electrode region changes, causing a change in the electroporation result. Therefore, the present invention needs to solve the technical problem of bubbles generated during electroporation and how to discharge the bubbles.
  • a high pressure pulse is applied to the cells as the cell suspension flows steadily between the two electrodes.
  • Repeated application of high-voltage pulses to the electrodes in accordance with Joule's law results in the generation of heat, so heat must be removed by the cooling device to prevent excessive temperature of the electrodes and cell suspension.
  • the cooling device Since the flow is stable and continuous in the flow electroporation method, the heat generated is also continuous. Therefore, the method of removing heat from the electroporation chamber must be able to balance the generated heat to avoid the temperature rise of the electroporation chamber. To an unacceptable level. Therefore, maintaining the temperature in the flow electroporation chamber and the temperature of the planar electrode within a controllable range is also a technical problem to be solved.
  • Another object of the present invention is to provide a device for flow electroporation that enables fluid to flow through a flow electroporation chamber at a relatively constant velocity.
  • Another object of the present invention is to provide a device for flow electroporation, which optimizes the generation and discharge of bubbles by the optimization of the electrode structure and the control of the electric field, thereby preventing the bubble from remaining inside the electrode from affecting the result of the electrotransfer.
  • Another object of the present invention is to provide a device for flow electroporation which can effectively control the temperature of a planar electrode during electrical transfection to fluctuate within a certain range.
  • Another object of the present invention is to provide a method for preparing a device for flow electroporation, which is simple in steps, convenient in operation, and easy to standardize in processing.
  • Another object of the present invention is to provide a method of using a device for flow electroporation for treating cells in large volumes.
  • Another object of the present invention is to provide a system for flow electroporation that controls the environment surrounding the flow electroporation device.
  • a flow electroporation device comprising a stent and an electrode device, the electrode device being a planar electrode, the electrode device being a planar electrode, the bracket and the planar electrode collectively defining a channel for fluid flow; preferably the plane The surface of the electrode is smooth and flat, and the contact with the bracket is smooth and without gap and sealed;
  • the material of the electrode device comprises one or more of titanium-plated platinum, platinum, rhodium-plated molybdenum, rhodium-plated rhodium, rhodium-plated rhodium, and platinum rhodium.
  • the stent of the flow electroporation device of the present invention is used to fix a planar electrode, and the stent material is selected from medical grade engineering plastics.
  • the flow electroporation device further includes an inflow port communicating with the passage and an outflow port communicating with the passage.
  • the flow electroporation device further includes a power source, and the electrode device is electrically connected to the power source.
  • the surface of the planar electrode material may be coated or plated, and the coating or plating material is different from the material of the substrate;
  • the coating or plating material of the planar electrode is one or more of gold, silver, titanium, cerium oxide, lanthanum, platinum, lanthanum, cerium, molybdenum, niobium, tungsten, and conductive ceramic materials.
  • the material of the planar electrode is platinum gold plating, platinum silver plating, platinum rhodium plating, platinum rhodium plating, platinum rhodium plating, platinum platinum plating, platinum rhodium plating, platinum plating conductive ceramic.
  • the material of the planar electrode is titanium gold plating, titanium silver plating, titanium rhodium plating, titanium platinum plating, titanium rhodium plating, titanium rhodium plating, titanium molybdenum plating, titanium rhodium plating, titanium plating conductive ceramics.
  • the material of the planar electrode is ruthenium gold plating, ruthenium silver plating, rhodium platin plating, rhodium rhodium plating, rhodium rhodium plating, rhodium chrome plating, rhodium rhodium plating, rhodium plating electroconductive ceramic.
  • the material of the planar electrode is ruthenium gold plating, iridium silver plating, ruthenium rhodium plating, rhodium platypium plating, rhodium rhodium plating, rhodium rhodium plating, rhodium chrome plating, rhodium plating electroconductive ceramic.
  • the plating layer of the planar electrode has a thickness of from 0.1 ⁇ m to 10 ⁇ m.
  • the flow electroporation device of the present invention mainly comprises a support and a planar electrode defining a fluid flow channel, and two planar electrodes are embedded in parallel to form an electroporation chamber, wherein the planar electrode comprises a power source for placing and a high voltage pulse power supply.
  • the two planar electrodes are respectively connected to the positive and negative poles of the high-voltage pulse power supply output electrical signal, and after the electrical signal is turned on, a uniform electric field is formed in the electroporation chamber between the two planar electrodes, thereby The fluid flow channel moves a fluid flowing through the electroporation chamber to experience a pulsed electric field.
  • the flow electroporation electrode of the invention adopts a metal material with good electrical conductivity and needs stable electrochemical performance, and cannot chemically react with the cell solution during electroporation to avoid generating harmful substances.
  • the electrode materials to be selected are aluminum, stainless steel, gold, silver, titanium, platinum, rhodium, ruthenium, iridium, osmium, molybdenum, tungsten, conductive ceramics, and the like.
  • the electrode material is gold, titanium, platinum, rhodium, ruthenium, osmium, molybdenum, tungsten, rhenium.
  • the shape of the planar electrode is one or more of a parallelogram, a circle, and an ellipse.
  • the shape of the planar electrode is a right-angled parallelogram. Further preferably, the planar electrode has a rectangular shape.
  • the length of the planar electrode is not less than 8 mm, and the length of the planar electrode is not more than 25 mm.
  • the planar electrode has a length of 8 mm.
  • the planar electrode has a length of 12.5 mm.
  • the planar electrode has a length of 15 mm.
  • the planar electrode has a length of 25 mm.
  • the planar electrode has a width of not less than 2 mm, and the planar electrode has a width of not more than 10 mm.
  • the planar electrode has a width of 2 mm.
  • the planar electrode has a width of 4 mm.
  • the planar electrode has a width of 10 mm.
  • the planar electrode has a length of 15 mm and the planar electrode has a width of 10 mm.
  • the thickness of the planar electrode is not less than 0.1 mm, and the thickness of the planar electrode is not more than 5 mm.
  • the planar electrode has a thickness of not less than 0.5 mm.
  • the thickness of the planar electrode is not less than 1 mm, and the thickness of the planar electrode is not more than 3 mm.
  • the planar electrode has a thickness of 2 mm.
  • the surface area of the planar electrode is not less than 10 mm 2 , and the surface area of the planar electrode is not more than 2500 mm 2 .
  • the surface area of the planar electrode is not less than 50 mm 2 , and the surface area of the planar electrode is not more than 1000 mm 2 .
  • the two planar electrodes are each made of the same or different materials.
  • the distance between the two planar electrodes is not less than 0.5 mm, and the distance between the two planar electrodes is not more than 8 mm.
  • the distance between the two planar electrodes is not less than 1 mm, and the distance between the two planar electrodes is not more than 5 mm.
  • the flow electroporation device further comprises a conduit fixedly connected to the bracket, the conduit comprising a liquid inlet tube connected to the flow inlet, the conduit further comprising the outflow conduit Connected outlet tubes.
  • the fluid flows into the chamber through the liquid inlet, completes electroporation under the action of the electric field, and finally flows out through the liquid outlet.
  • the diameter of the liquid outlet tube is not less than the diameter of the liquid inlet tube.
  • the diameter of the conduit is no less than 2 mm and the diameter of the conduit is no greater than 10 mm.
  • the catheter is selected from medical engineering plastics or medical silica gel.
  • the manner of connecting the catheter to the bracket includes, but is not limited to, a threaded connection, a snap connection, a cladding connection, and ultrasonic welding.
  • the inlet pipe is in a straight line shape
  • the outlet pipe is in a straight line shape
  • the angle between the axial direction of the inflow port and the longitudinal direction of the channel is between 0 and 90 degrees.
  • the axial direction of the outflow port and the longitudinal direction of the channel are between 0 and 90 degrees.
  • the flow electroporation device of the present invention specifically sets a liquid inlet mode in which the fluid flows in a direction parallel to the direction of the electroporation chamber.
  • the axial direction of the inflow port is parallel to the longitudinal direction of the channel.
  • the axial direction of the outflow port is parallel to the longitudinal direction of the passage.
  • the bracket is provided with at least one fluid buffer region, the fluid buffer region being located between the inflow port and the electrode; and/or the fluid buffer region is located at the outflow port and the electrode between.
  • the cross section of the fluid buffer region is in the form of a triangle, a positive trapezoid, an inverted trapezoid, a square, a semicircle, a semi-ellipse, a circular arc, and an S curved arc.
  • the flow electroporation device further comprises a fluid drive device that drives the fluid in a positive pressure and/or a negative pressure manner.
  • the fluid drive device is a peristaltic pump or a magnetic pump.
  • the fluid drive device drives the fluid in a negative pressure manner.
  • the flow electroporation device further comprises a cooling temperature control device comprising a temperature-controlled cooling module for cooling the electrode device. Further preferably, the temperature-controlled cooling module is disposed outside the planar electrode.
  • the cooling temperature control device includes one of a coolant, a cooling gas, a cooling pipe, a cooling fin, and a cooling fan.
  • the flow electroporation device further comprises an electrical pulse generating device and a control system.
  • the flow electroporation device further comprises at least one fluid storage device.
  • the flow electroporation device further comprises temperature adjustment means for adjusting the ambient temperature of the flow electroporation device.
  • the temperature regulating device controls the ambient temperature of the flow electroporation device to be between 0 and 40 °C.
  • the flow electroporation device further comprises a carbon dioxide generating device for adjusting the concentration of carbon dioxide in the surrounding environment of the flow electroporation device.
  • the carbon dioxide generating device controls the concentration of carbon dioxide around the flow electroporation device to be between 3 and 8%.
  • the invention also provides a flow electroporation device for transfecting a substance to be transfected into a cell, the substance to be converted comprises a biologically active substance and a non-biologically active substance, and the biologically active substance and the non-biologically active substance respectively comprise biological activity Molecular and non-biologically active molecules, the flow electroporation device of any of the flow electroporation devices described herein.
  • the biologically active substance includes, but is not limited to, a pharmaceutical agent, genetic material DNA and RNA, cytokines, antibodies or other proteins.
  • the non-biologically active substance comprises cytokines, toner nanoparticles.
  • the species of the cell includes, but is not limited to, prokaryotic cells, eukaryotic cells, plant protoplasts.
  • the prokaryotic cells include bacteria, fungi, yeasts.
  • the eukaryotic cells include animal cell line cells, primary cells, and egg cells.
  • the inflow port is located on a lower side of the flow electroporation device, and the outflow port is located on an upper side of the flow electroporation device.
  • the invention also provides a preparation method of a flow electroporation device, comprising the following steps:
  • a stent having an inflow port and a flow outlet by injection molding or computer control technology (CNC) or 3D printing technology, the bracket being provided with two recessed windows;
  • CNC computer control technology
  • 3D printing technology the bracket being provided with two recessed windows;
  • the material of the flat electrode is selected from the group consisting of titanium platinized platinum, platinum, rhodium-plated molybdenum, rhodium-plated rhodium, rhodium-plated rhodium, and platinum rhodium-plated Species or several
  • Two of the planar electrodes are respectively mounted in the recessed window, and then the planar electrode is packaged.
  • the coating or coating of the planar electrode is prepared by painting or electroplating, and the preparation process includes the steps of pretreatment, pretreatment, painting or electroplating, and drying.
  • the pretreatment includes selecting a suitable substrate for shape processing, surface blasting, selecting different blasting meshes according to the thickness of the plating layer, and cleaning the surface oxide layer with acid.
  • the pretreatment includes ultrasonic cleaning with a cleaning agent and deionized water washing to remove the cleaning agent residue.
  • the application consists of applying a uniform coating on the surface of the substrate by dipping or brushing or spraying/smoothing.
  • the electroplating involves electroplating a uniform coating on the surface of the substrate, smearing or plating and then rinsing with deionized water. Remove the application solution or plating solution and dry for use.
  • the invention also provides a method for using a flow electroporation device, wherein a cell and a substance to be transferred are placed in a solution, and the solution is transferred into a flow electroporation device, and the substance to be transferred is transferred to the cell,
  • the flow electroporation device comprises:
  • the bracket including a passage for fluid flow, an inflow port communicating with the passage, and an outflow port communicating with the passage;
  • the electrode device and the The power supply is electrically connected, and the electrode device is located in the channel, and the material of the electrode device comprises one or more of titanium platinized platinum, platinum, rhodium-plated molybdenum, rhodium-plated rhodium, rhodium-plated rhodium, platinum rhodium-plated;
  • a voltage is applied to the planar electrode, which produces a current having a density of up to 120 A/cm 2 , and the biologically active substance is transferred to the cell by the principle of electroporation.
  • the invention also provides a method for using a flow electroporation device, wherein a cell and a substance to be transferred are placed in a solution, and the solution is transferred into a flow electroporation device, and the substance to be transferred is transferred to the cell,
  • the flow electroporation device comprises:
  • the bracket including a passage for fluid flow, an inflow port communicating with the passage, and an outflow port communicating with the passage;
  • the electrode device and the The power supply is electrically connected, and the electrode device is located in the channel, and the material of the electrode device comprises one or more of titanium platinized platinum, platinum, rhodium-plated molybdenum, rhodium-plated rhodium, rhodium-plated rhodium, platinum rhodium-plated;
  • a voltage is applied to the electrode, the electrode generates a current, and a current is applied to the electrode, the current having a density of 2-14 A/cm 2 .
  • the current has a pulse width of 0.01 to 100 ms.
  • the invention also provides a method for using a flow electroporation device, wherein a cell and a substance to be transferred are placed in a solution, and the solution is transferred into a flow electroporation device, and the substance to be transferred is transferred to the cell,
  • the flow electroporation device comprises:
  • the bracket including a passage for fluid flow, an inflow port communicating with the passage, and an outflow port communicating with the passage;
  • the electrode device and the The power supply is electrically connected, and the electrode device is located in the channel, and the material of the electrode device comprises one or more of titanium platinized platinum, platinum, rhodium-plated molybdenum, rhodium-plated rhodium, rhodium-plated rhodium, platinum rhodium-plated;
  • a voltage is applied to the electrodes, and an electric field is generated in the channels, the field strength of the voltage being 0.2-10 kV/cm.
  • the voltage has a pulse width of 0.01-100 ms.
  • the flow electroporation device of the present invention achieves significant progress and unexpected technical effects:
  • planar electrode made of the preferred planar electrode material of the invention can provide a continuous and stable current, can provide a stable uniform electric field in the presence of a buffer solution, can significantly improve the electroporation efficiency, and at the same time ensure high cell survival rate;
  • the preferred planar electrode of the present invention even after a high pulse voltage after contact with the buffer, surprisingly finds that the electrolytic reaction generated is very small, and at the same time, very few bubbles are generated, which is advantageous for continuously applying pulses to the planar electrode. conditions of;
  • the planar electrode of the invention has less OH - (cathode effect) in the working state, does not change the pH value of the cell liquid, has less damage to cells, and has high cell survival rate;
  • the flow electroporation device of the invention has simple production process, low preparation cost, standardized production of product preparation, larger volume of batch processing cell liquid, higher transfection efficiency and cell survival rate, better stability, convenient operation method. .
  • the electroporation cells treated by the flow electroporation device of the present invention have a cell electrotransfection rate of at least 60% and a cell survival rate of at least 60%.
  • the flow electroporation device of the invention is the most advanced flow electroporation product at present, and can be used for actual transfection of the laboratory and production links, and has a huge commercial space.
  • Figure 2 shows the current stability of a conductive ceramic coated titanium electrode, an aluminum electrode and a stainless steel gold plated electrode
  • Figure 3 shows the color change of the solution after the electroplating of the titanium planar electrode with the conductive ceramic coating
  • Figure 4 shows the color change of the solution after the aluminum electrode is electrically rotated
  • Figure 5 shows the color change of the solution after the electroplating of the stainless steel gold-plated electrode
  • Figure 6 is the color of the solution after the electro-rotation of the pure titanium electrode
  • Figure 8 shows the surface change of the electrode after the electroplating of the titanium planar electrode with conductive ceramic coating (anode on the left and cathode on the right);
  • Figure 9 shows the surface change of the electrode after the aluminum electrode is rotated (the anode on the left and the cathode on the right);
  • Figure 10 shows the surface changes of the electrode after the stainless steel gold-plated electrode is electrically rotated (the anode on the left and the cathode on the right);
  • Figure 11 shows the surface change of the electrode after the electric rotation of the pure titanium electrode (the anode on the left and the cathode on the right);
  • Figure 12 shows the surface change of the electrode after the electroplating of the titanium-plated platinum electrode (the anode on the left and the cathode on the right);
  • Figure 13 is a schematic diagram of the fluid forward and forward exit mode
  • Figure 14 is a schematic diagram of the fluid side-in and side-out mode
  • Figure 15 is a schematic view showing the structure of a fluid buffer region
  • Figure 16 is a schematic diagram of an electrode cooling temperature control device
  • Figure 17 is a schematic exploded view of the overall structure of the device.
  • 1-catheter outlet tube
  • 2-bracket outlet 3-first buffer zone
  • 4-electroporation chamber 5-first plane electrode
  • 6-first plane electrode cooling device 7-catheter
  • 8-conduit inlet pipe
  • 9-second buffer zone 10-bracket
  • 11-second plane electrode cooling device 12-second plane electrode
  • 13-coolant input port - Coolant outlet
  • 15 - Coolant inlet 16 - Coolant outlet.
  • the flow electroporation device of the present invention adopts a planar electrode, and the size of the planar electrode determines the speed of processing the cells per unit time, and the length, width and spacing of the electroporation chamber have different effects.
  • the length of the planar electrode affects the time during which the solution flows inside the electrode. Increasing the length of the electrode increases the contact time with the electrode surface when the bubble flows through the electrode region, increasing the probability of bubble retention.
  • the width of the planar electrode affects the velocity of the solution flowing inside the electrode. The flow velocity in the middle region of the fluid is fast, the flow velocity in the edge region is slow, and the electrode width is larger, and the deviation is more obvious.
  • a flow electroporation device was fabricated using planar electrodes with different length and width ratios.
  • the current data was tested with an empty buffer, and the bubble generation and discharge were observed, and the relationship between the length and width of the planar electrode was optimized.
  • the shape of the planar electrode is selected to be rectangular or square.
  • the plane electrode test material was selected from aluminum, and the test sample length, width and thickness were respectively set to be 15 ⁇ 10 ⁇ 2 mm, 25 ⁇ 4 ⁇ 2 mm, 25 ⁇ 2 ⁇ 2 mm, and 12.5 ⁇ 4 ⁇ 2 mm.
  • a stent with two recessed windows is formed by CNC or 3D printing, the size of the recessed window being the same as the size of the planar electrode described above.
  • the planar electrode is embedded in the recessed window and the stent is then packaged to form an electroporation chamber with a fluid flow inlet and a flow outlet.
  • the two electrodes of the above device are respectively connected to the positive and negative electrodes of the output end of the pulse power source.
  • the electric rotation condition is 150V/11ms/1 time, and the corresponding volume of EL buffer solution (sold by Suzhou Yuda Company and its agents, item number: H10305) is added to record the current data changes during the electric shock (see Figure 1). Observe the bubble generation and discharge throughout the process.
  • the electrode with a length of 25 mm is easy to accumulate in the electrode during the electric shock, and adheres to the surface of the electrode, and gradually grows into a large bubble and then discharges, causing the current to fluctuate periodically, resulting in a decrease in electric field stability.
  • the electrode with a length of 12.5mm, the small bubbles generated during the electric shock are discharged with the solution, rarely accumulate in the electrode, and the current stability is good, but the total volume of the electroporation chamber with the plane electrode of 2 ⁇ 4mm is only half of 2 ⁇ 8mm.
  • the 2 ⁇ 8mm specification is selected under the same conditions to increase the throughput per unit time.
  • the electrode width is changed separately, and the larger the width, the larger the processing amount per unit time, and the smaller the width, the smaller the processing amount per unit time.
  • the difference in solution flow velocity between the two sides of the electrode and the intermediate portion is also increased, and 15 ⁇ 10 ⁇ 2 mm is considered as the preferred electrode size.
  • the flow type electroporation device of the present invention the development of the planar electrode material as the core content, the surface of the planar electrode should be smooth and flat, the contact with the bracket is smooth and no difference is formed, and the sealed chamber is formed to minimize the possibility of bubble retention.
  • planar electrode materials to be selected in this embodiment are aluminum, 304 stainless steel, pure gold, stainless steel gold plating, pure titanium, conductive ceramic coating titanium, pure platinum, titanium platinized platinum, pure tantalum;
  • Aluminum widely used as an electroporation material in the field of electrical transfection
  • Stainless steel gold plating gold has stable chemical and electrical properties and good biocompatibility
  • Pure titanium a metal material with good biocompatibility, widely used in biomedical fields;
  • Titanium with conductive ceramic coating Professor Su Daxin provides that the coating material is conductive ceramic with good oxidation resistance;
  • Platinum Platinum Stable anode material, widely used in the electrolysis industry, and has good biocompatibility
  • Pure gold It has extremely high corrosion resistance, good electrical conductivity and thermal conductivity, is easy to form and is easily plated on other metals and ceramic and glass surfaces.
  • Pure platinum chemically stable, insoluble in strong acid and alkali solution, catalytically active, can be used as an anticancer drug.
  • Pure bismuth chemically stable, insoluble in aqua regia, widely used in high-tech fields, such as aerospace technology, pharmaceutical and automotive industries.
  • the above material was made into a planar electrode having a length ⁇ width ⁇ thickness of 15 ⁇ 10 ⁇ 2 mm.
  • a bracket having two recessed windows is formed by CNC or 3D printing, and the size of the recessed window is the same as the size of the above-mentioned planar electrode.
  • a planar electrode made of the above material is embedded in the recessed window, and the stent is then packaged to form an electroporation chamber with a fluid inflow port and an outflow port.
  • the two electrodes embedded in the bracket are respectively connected to the positive and negative electrodes of the output end of the pulse power source to form an electroporation device.
  • the current of the stainless steel gold-plated electrode is gradually increased from 4A to 5A; the aluminum electrode current is rapidly increased from 4.5A to 5A, and the current is always fluctuating around 5A; the titanium planar electrode current with conductive ceramic coating starts from It was very stable at the end and stayed at 4.8A.
  • the current of the titanium electrode, the aluminum electrode and the titanium platinized electrode with conductive ceramic coating is relatively stable, the current of the stainless steel gold-plated electrode gradually rises, the initial current of the pure titanium electrode decreases, then slowly rises, the latter gradually becomes stable, and the current waveform changes. .
  • the change of the solution after treatment of various materials can be seen from Fig. 3-7.
  • the titanium planar electrode with conductive ceramic coating reacts with the chloride ion in the solution, and the solution turns yellow after the electrophoresis (Fig. 3), and the precipitated ion component will be The cell survival rate after electroporation was affected, and there was no obvious precipitation after centrifugation.
  • the electrode After electrospinning, the electrode was disassembled. It can be seen from Fig. 8-12 that the surface of the titanium planar electrode with conductive ceramic coating after electrorotation is not obviously oxidized (Fig. 8), while aluminum (Fig. 9) and stainless steel plated planar electrode anode have Oxidation, especially the gold plating of stainless steel plated planar electrodes, has fallen off ( Figure 10).
  • the pure titanium electrode was anodized to form an oxide layer (Fig. 11), and no oxidation was observed on the surface of the titanium plated electrode (Fig. 12).
  • the pMAX plasmid purchased from Lonza Human T cell Nucleofector Kit, item number: VAPA-1002
  • CHO-S cells Professor Zhou from Peking University Medical College, Professor Zhou purchased in Life, item number R80007
  • EL Buffer EL Buffer
  • the buffer fluid is gradually injected into the electroporation chamber from the bottom of the electroporation device, and different electrode materials are selected, respectively, at a voltage of 150 V, a pulse width of 11 ms, a number of electric shocks of one time, an interval of 2352 ms, and a voltage of 150 V, 5 ms.
  • the width was 3 times, the electric field was treated under the condition of 784 ms, and then flowed out from the top opening to culture the cells, and the electrotransfection rate and cell survival rate were measured.
  • TE electrotransfer efficiency
  • VE cell viability
  • MIX overall efficiency
  • MFI mean fluorescence intensity
  • the titanium-plated electrode and the conductive ceramic-coated titanium electrode have little difference in transfection efficiency, but the survival rate of the titanium-plated electrode is 15% higher than that of the coated electrode, and the fluorescence intensity of the two is basically the same.
  • the overall efficiency of the titanium-plated platinum electrode is higher than that of the titanium electrode with the conductive ceramic coating.
  • the electric rotation result of pure titanium electrode is better than that of aluminum and stainless steel gold-plated electrode, the anode of pure titanium electrode will be oxidized, the electric field stability will change, and the black matter will be observed in the solution after electro-rotation, so pure titanium is not suitable as anode material.
  • the titanium electrode electro-transfer solution with conductive ceramic coating will yellow.
  • the electroplating efficiency of the titanium-plated platinum electrode and the conductive ceramic-coated titanium electrode is close, and the cell survival rate of the titanium-plated platinum electrode is higher. Therefore, the overall efficiency of the titanium-plated platinum electrode is higher than that of the conductive ceramic-coated titanium electrode.
  • the angle between the horizontal angle of the fluid entering the electroporation chamber and the horizontal angle when flowing through the electroporation chamber can be between 0° and 90°.
  • the fluid When the angle is at 0°, the fluid is positive and positive.
  • the angle When the angle is at 90°, the fluid is side-in and out.
  • the main way for the fluid to enter and exit is that the inlet and outlet of the stent extend linearly, so that the silicone tube at the proximal end of the electrode is connected in parallel with the electrode.
  • the main way of the fluid side-in and side-out is that the liquid inlet and the liquid outlet of the stent are at a 90° angle with the plane electrode, so that the silicone tube at the proximal end of the electrode is vertically connected with the electrode.
  • the main difference between the side-in and out-outs in the inverted “U”-type collection piping system is only the difference in the corners of the electrode outlet end.
  • the corners of the side entry side are 90°, and the foam group has a long period of stagnation and a large accumulation at the corners.
  • the angle of the forward and backward is relieved to more than 90° due to the curvature of the silicone tube. At this time, the stagnation and aggregation of the foam group are alleviated, and the stability of the electrical conversion efficiency of each section is improved compared with the lateral side.
  • the retention zone is prone to occur, and the solution flow rate in the retention zone is zero.
  • This zone is an invalid electrical transition zone, and Accumulate bubbles. Therefore, it is necessary to increase the buffer zone in the inlet port and the plane electrode region, to smoothly transition, to avoid the occurrence of the retention zone, and to ensure the solution flow rate in the planar electrode region is stable. In theory, the larger the buffer volume, the more stable the electrode area flow rate. Similarly, a buffer zone should be added between the planar electrode area and the stent outlet to avoid rapid closing, and the pressure change affects the flow rate.
  • the pressure in the buffer solution gradually becomes larger, which is convenient for accumulating bubbles on the tube wall when the bubbles generated by the electrolysis flow. If the bubbles cannot be discharged in time, the volume gradually becomes larger, the effect of the buffer zone is offset, and the effect of stabilizing the flow rate cannot be achieved. .
  • the experimental results show that the electric perforation device in the conical buffer area has less accumulated bubbles at the liquid outlet, and the remaining bubbles accumulate at the right angle of the inner side of the electrode; the square buffer area electroporation device accumulates a large number of bubbles at the liquid outlet and accumulates When discharged at a certain amount, the liquid flow is uneven; the electro-perforation device in the arc buffer region accumulates the least bubbles, the current is the most stable, and the transfection efficiency is the highest; the bubble accumulation and transfection efficiency of the trapezoidal buffer region electroporation device are middle.
  • the flow electroporation device of the present invention drives fluid flow in a positive pressure and/or negative pressure manner, preferably in a negative pressure manner.
  • the fluid enters the electroporation chamber to receive electric shock, and the cells after the electric shock treatment and other substances generated by the electric rotation are taken out of the electric field to ensure the precise and orderly electrical rotation.
  • the entire hydrodynamic form can be divided into positive and negative pressure modes.
  • the positive pressure mode is to place the peristaltic pump on the front side of the electroporation chamber, and the thrust generated by the rotary peristaltic pump pushes the liquid into the electroporation chamber in one direction in the piping system.
  • the negative pressure mode is to place the peristaltic pump on the back side of the electroporation chamber, and the sample in the sample tube is drawn by rotating the suction generated by the peristaltic pump and flowing through the electroporation chamber in one direction in the piping system.
  • Experimental scheme design using the positive-in and forward-out arc-shaped buffer electroporation device to monitor the current stability during the driving process of the positive and negative pressure modes, and observe the generation and discharge of the foam group during the electric shock.
  • the stability of the electric field strength and the flow stability of the fluid in the pipeline are compared between the two power modes.
  • the CHO-S cells were electrically converted to pMAX in two working modes, and the electroporation efficiency was obtained by selecting a titanium plated platinum electrode at a voltage of 150 V, a pulse width of 5 ms, a number of electric shocks of 3 times, and an interval of 784 ms. Survival rate, comprehensive efficiency and fluorescence expression were compared to compare the electroporation effects and stability of cells in the two dynamic modes.
  • the electric field strength is consistent under the two dynamic modes of positive pressure and negative pressure.
  • the measured current in positive pressure dynamic mode is 5.20 ⁇ 0.39.
  • A the measured current in the negative pressure dynamic mode is 4.95 ⁇ 0.55A, the difference between the two is small, and the stability is good.
  • the generation and discharge of the foam on the surface of the electrode are basically the same, both of which are firstly produced by a small volume of foam, and most of them are continuously discharged without any retention at the electrode.
  • the sheet and the joint seam are decelerated to form a larger foam mass, and then decelerate again at the outlet end of the electrode to collect into a larger foam mass, and finally enter the collection pipe system.
  • the discharge of foam in the collection pipeline is quite different: under positive pressure conditions, the foam group is easy to accumulate at the corner of the pipeline. When sampling, the foam is not collected in the front section, and a large amount of foam is collected in the latter stage.
  • the flow rate is not uniform during the electrotransfer process, and the volume of the cell fluid collected in different sections is not uniform.
  • the accumulation of foam in the corner of the pipeline is lighter; when sampling in each section, the distribution of foam in the collection tube and the volume of cell liquid are consistent.
  • the negative fluid power mode the fluid suspension is more stable in the pipeline, and the discharge of the foam group is more uniform and smooth.
  • a cooling temperature control device is disposed outside the planar electrode (see FIG. 16), and the temperature control cooling module is adhered to the outside of the planar electrode by using a thermal conductive glue, and the cooling temperature control module is internally connected with the circulation pump and the semiconductor refrigeration chip;
  • the circulation pump drives the coolant in the cooling tube to take away heat transferred from the planar electrode to the cooling module to maintain the temperature of the planar electrode.
  • the semiconductor refrigeration chip is used to cool the coolant flowing back from the cooling module.
  • the experimental results show that the monitoring results of the planar electrode temperature in the working process show that the temperature of the outer surface of the planar electrode with cooling device is small during the transfection process, and the average temperature is 38.6 °C ⁇ 2.3 ° C, compared with no cooling.
  • the temperature of the planar electrode of the device was 9.5 ° C lower on average, the transfection efficiency was 9.3%, and the cell survival rate was 11.2%.
  • Single-tube fluid refers to the peristaltic pump pushing the cell fluid into and filling the electroporation chamber, and then suspending the work. After the electroporation, the cell fluid is electroporated, the peristaltic pump continues to work, and the new cell fluid is pushed into the electroporation chamber until the new cell
  • the liquid fills the electroporation chamber, that is, the action of repeating the liquid-suspension-shock;
  • the continuous liquid inlet means that the peristaltic pump continuously works to push the cell liquid continuously through the electroporation chamber, and the pulse power source electroporates the cell liquid by applying an electric field at a designed frequency.
  • the electroporation volume is 10ml.
  • Single tube feed and continuous feed were compared under multiple electroporation conditions to compare current stability and bubble generation and removal.
  • the electroporation efficiency, survival rate, comprehensive efficiency and fluorescence expression of CHO-S cells electroporated DNA in two working modes were tested to compare the electroporation effects and stability of the cells in the two infusion modes.
  • Table 7 Electrochemical conversion effect and current change of single-tube liquid and continuous liquid-filled cells with conductive ceramic coating
  • the average current of single tube inlet and continuous feed is basically the same, but due to the single tube inlet, the bubbles generated in the 3 shocks are accumulated first and then eliminated again. , resulting in a decrease in current stability and an increase in the error value.
  • the results of cell electrophoresis showed that continuous feed was much better than single-tube feed, and the effect of continuous feed was better than single-tube feed in the electrode tube structure of positive-in and out-side.
  • Experimental design select aluminum, conductive ceramic coating titanium, stainless steel gold plating, titanium, gold, tantalum, niobium, tantalum, niobium, molybdenum, tungsten, titanium platinized platinum, platinum as a planar electrode, which is coated with conductive ceramic coating Titanium is supplied by the Materials Physics Laboratory of Suzhou University. Titanium, gold and titanium platinum and platinum are purchased from Zhongnuo New Materials Beijing Co., Ltd., and aluminum and stainless steel gold plating are purchased by Beijing Gao Dewei Metal Technology Co., Ltd., ⁇ , ⁇ , ⁇ , mo Tungsten is supplied by Xiamen Tungsten Group.
  • the electrode sheet spacing was 1.5 mm, and the solution contact area size was 4 mm x 8 mm.
  • the structure of the inlet and outlet of the bracket is forward and forward, the fluid buffer area is set in the bracket, and the direct plane transition to the 2 mm round hole at the liquid outlet, and the creep mode of the continuous pressure in the negative pressure.
  • the electroporation volume is 40ml.
  • the flow electroporation device after comprehensive optimization conditions of the present invention has a significantly improved overall efficiency than the initially simple flow electroporation device.
  • the aluminum electrode under the same electroporation conditions, the transfection efficiency was 50.15%, and the white salt precipitated out in the solution, which was toxic to the cells, and the survival rate was 44.69%;
  • the titanium electrode with conductive ceramics had a transfection efficiency of 59.97% under the same electroporation conditions. Although the solution turned yellowish, it was slightly toxic to cells, but the cell survival rate was 59.06%.
  • the stainless steel gold-plated electrode has a transfection efficiency of 44.49% and a cell survival rate of 52.48%. During the electrotransfer process, the gold falls off, the solution turns yellow, and the electrode oxidation is significant;
  • the electrotransfection efficiency and cell viability were higher than those of the conductive ceramic titanium electrode, reaching 66.03% and 74.93%, respectively, but after centrifugation, the solution was centrifuged and found to have material falling off to form a precipitate;
  • the gold electrode Under the same electrospinning conditions, the gold electrode has a transfection efficiency of 77.59% and a survival rate of 64.05%.
  • the solution has no color change and no precipitation, and the electrode sheet with gold as the anode is easily oxidized during the electrotransfer process;
  • the electrodes of ruthenium, osmium, iridium, osmium, molybdenum and tungsten were relatively stable. There was no significant change in the surface of the solution and the electrode after electroporation. The transfection efficiency and cell survival rate were between 58-71% and 57-76%, respectively. The efficiency is better than that of aluminum electrodes and stainless steel gold-plated electrodes.
  • the titanium-plated platinum electrode has a transfection efficiency of 92.25%, and the survival rate is 90.65%, which is much higher than other material electrodes.
  • the solution has no color change and precipitation, and the stability is very good.
  • Titanium gold plating, platinum rhodium plating, rhodium rhodium plating, rhodium rhodium plating, rhodium rhodium plating, rhodium plating molybdenum are stable and anti-oxidation, achieving good results, and the electrical transfection rate and cell survival rate are between 77-86%. Between 69-80%, the overall efficiency is higher, which can meet the actual needs of flow electroporation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Sustainable Development (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Electromagnetism (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Thermal Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Water Treatment By Electricity Or Magnetism (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

公开了一种流式电穿孔装置，包括支架及电极装置，所述电极装置为平面电极，所述支架和所述平面电极共同限定供流体流动的通道；所述电极装置的材料包含钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种；所述流式电穿孔装置生产工艺简单，产品制作能够标准化生产，批次处理细胞液体积更大，转染效率和细胞存活率更高，稳定性更好，使用方法操作方便。

Description

一种流式电穿孔装置 技术领域
本发明涉及电穿孔领域，特别是涉及流式电穿孔装置，通过电场将流动的生物活性物质递送进流动的活细胞的方法和装置。
背景技术
电穿孔现象是20世纪70年代发现的，利用电场处理细胞，形成微孔，但是不对细胞造成永久性的损伤，微孔可自行复位。这项发现使生物活性物质进入细胞成为可能。已有的研究发现，有四个现象可能在电穿孔的过程中起作用。第一个现象是绝缘击穿。绝缘击穿指的是高电场在细胞膜上产生小的微孔或洞的能力。一旦产生微孔，细胞就可以荷载生物活性物质。第二个现象是绝缘聚束效应，它指的是在均匀的电场中通过泡囊的堆放产生相互自吸引。第三个现象是泡囊融合。泡囊融合指的是通过绝缘击穿产生微孔的生物泡囊的膜在近距离内与其在绝缘击穿位点处结合在一起的趋势。第四个现象是在高频电场存在下细胞沿着它们的一个轴排列成一条直线的趋势。因此，为了荷载和卸下细胞泡囊，电穿孔涉及在泡囊旋转预定位，泡囊穿孔和泡囊的介电常数中的应用。
在现有技术中可得到的电穿孔室大多被设计为仅在静态使用，这些静态的电穿孔室受体积的限制，不适于处理大量的样品，也不能使用高压或重复的充电。为了进行大体积处理细胞，发展出了流式电穿孔室。
流式电穿孔指的是这样的过程，其包括：将细胞的悬浮液和外源的生物活性物质的混悬液转移到由流体室或流体流道构成的设备中；所述流体室或流体流道由沿着流体室或流体流道的侧面设置、并且被配置为、使所述流体室或流体流道内的混合液受到适合于电穿孔的电场作用的电极构成；以及将经过电穿孔的细胞悬浮液从设备中转移出的装置。该方法对于大规模体积的细胞特别有效。专利CN1195997A公开了一种流式电穿孔室，采用可弹性形变的材料作为流体流动的通道，弹性材料的形变可以作为流体流动通道内瞬时压力的增加的部分缓冲，但是其并未研究适宜流式电穿孔的电极材料，所以该专利公开了近20年后，仍没有产品上市。
一般来说，用于流式电穿孔的装置包含两个平面电极，要进行电穿孔的细胞悬液持续而稳定地流过两个电极之间，直到整个细胞悬液都进行了电穿孔。流式电穿孔装置包含带电极和接口的电穿孔室，细胞悬液可通过接口注入。电极与能给电极提供高压脉冲的电路相连。高压脉冲由程序控制计算机控制。现在已开发出了流式电穿孔系统，该系统能够对较大体积的细胞进行电穿孔，获得导入目的分子的活细胞。但是，目前成功商业化的流式电穿孔仪却极为少见，一方面与市场的需求有直接的关联，另一方面是由于电穿孔的效率和产品的稳定性有关。
流式电穿孔的条件因细胞类型的不同而不同，还因期望导入到细胞内的分子的类型不同而不同。对于任何一个特定类型的细胞来说，都存在一个最佳的处理条件，其中包括最佳电压、脉冲宽度，最佳时间间隔、脉冲次数进行脉冲。为了使施加到细胞上的电脉冲次数达到最佳，在使用本领域所描述的流式电穿孔装置时，细胞悬液通过电穿孔室、在电极之间的流速以及脉冲的频率都是可以选择的，从而对一定体积的细胞悬液进行最佳次数的脉冲。例如，对于一个特定细胞来说，如果已知每个细胞上施加的最佳脉冲数是2，通过电穿孔室的体积是1ml，那么，流速就设定为每一单位时间1ml，每一单位时间内施加的脉冲次数为2。如果以这种方式进行电穿孔，在这个例子中每个细胞就会受到2次脉冲。但是，细胞悬液通过电穿孔室的流体动力学流动会导致细胞以不同的速率通过室以及在电极之间流动。由于离室壁远的地方比靠近室壁的地方流速高，因此，在电极之间向液流中心流动的细胞通过电极之间的区域所需要的时间会低于单位时间，其受到的脉冲次数可能会少于2次，而靠近室壁流动的细胞通过电极之间的区域所需要的时间会高于单位时间，其受到的脉冲次数可能高于2次。由于在这个例子中每个细胞受到的最佳脉冲次数为2次，但是，很明显并不是每个细胞都受到了最佳次数的脉冲，因此，细胞悬液的整体电穿孔效果就不是最佳的。
电穿孔的最佳条件根据电穿孔的特定细胞类型以及想要通过电穿孔导入到细胞内的分子类型的不同而不同。利用试验的方法解决这个问题是可能的，利用静态的电穿孔室有组织地改变电穿孔 的条件，然后将确定于这些试验的最佳条件应用到流式电穿孔系统中。这个方法有两个缺点。第一，由于进行大量样品的静态室电穿孔需要耗费大量的时间，因此，用于每次静态电穿孔的细胞在试验中也会发生改变。第二，使用这种方法只是因为人们认为静态室电穿孔的最佳条件与流式电穿孔是一样的，但是并不知道这是否真实。
我们期望的是利用一个细胞样品就能够确定使用同一个装置进行所有电穿孔试验的最佳条件，这个条件可用于治疗或其他目的所需的细胞大规模电穿孔。
电转杯主要是以插入金属电极的比色杯形式，用于电穿孔或电融合。用于这种用途的容器多半是小容器，其底部封闭，顶部开口，且内部空间通过两对平行相对布置的侧壁构成。内部空间的作用是接收其中悬浮了待处理细胞的细胞悬浮液，即通常是缓冲水溶液或细胞培养介质。这种比色杯多半包含布置在一对相对排布的侧壁底部附近的施加电压的一对电极。放电期间，电流流过两个电极之间的细胞悬浮液，能使核酸或其他分子进入细胞或根据所选择的条件而导致细胞融合。电极多半由金属制成，其中铝最常用。但这些公知的、可商购的比色杯有一个缺点，就是金属离子在放电期间会发射到缓冲溶液中，对细胞产生不希望有的刺激，使细胞中毒。例如，采用铝制比色杯，可说明由于Al 3+离子的释放产生的副作用。此外，采用具有金属制成的电极的比色杯，会发生不希望有的沉淀，这也是由于从电极释放金属离子产生的。这种沉淀可以是金属氢氧化物或金属离子与缓冲溶液中的生物大分子的结合物。最后，铝制比色杯的另一个缺点是，比色杯的电阻在放电期间降低，大概是由于具有较高电阻的氧化铝层通过电流而从电极脱落。
现有技术中对电极材料做了尝试性的改进，例如，专利CN03805811.1公开了一种掺杂至少一种导电物质的塑料的导电合成材料制成的电极，并且塑料中的掺杂物的总浓度为20-80％w/w，掺杂物由碳纤维、石墨、碳黑和/或碳纳米管组成，能够用电流有效处理细胞、细胞衍生物。专利CN201610044870.8公开了一种细胞电转染装置，电极表面具有纳米线结构，纳米线选自氧化铜纳米线、二氧化钛纳米线、金纳米线、银纳米线、铜纳米线、钛纳米线、铁纳米线、铂纳米线、钛铂合金纳米线、氧化铁纳米线、二氧化锡纳米线、金属包覆的硅纳米线中的任一种，使用的转染电压小于等于10V，利用电极表面微观结构使局部场强增加3-4个数量级，但是该电转染装置仅仅是用于贴壁细胞的静态电穿孔。
根据研究人员的研究，目前流式电转染过程中存在的主要技术问题是电解水造成的阴极周围溶液pH变化、阳极电极表面氧化产生有害物质、电流的焦耳效应导致溶液温度升高以及电转产生的气泡等异物引起电场强度及其分布的变化。
本发明的流式电穿孔室的研发过程中，电极研发作为核心内容，其中电极材料更是关键，电极材料直接与细胞溶液接触，在电穿孔过程中发生电化学反应，导致电场变化，影响结果，同时不稳定的阳极材料会有金属离子析出，引起细胞死亡或污染细胞溶液，造成更大的危害。因此，本发明研发的具有生物兼容性的稳定电极材料是解决流式电穿孔商业化应用的首要问题。
本发明的流式电穿孔室在电穿孔过程，电极与细胞溶液接触，在电极表面易发生电解水反应，产生气泡，理想情况下，气泡会随着液体流动，快速从电极内部排出。但是由于电极结构和电极材料表面粗糙度的影响，如果气泡在电穿孔室内部滞留，由于气泡不导电，电极内电场强度分布发生变化；同时气泡占据腔室，会改变溶液流速，导致细胞流过电极区域接受到的电流脉冲信号次数和强度的改变，造成电穿孔结果发生变化。因此，本发明需要解决电穿孔时产生的气泡以及如何将气泡排出的技术问题。
在细胞悬液稳定地流过两个电极之间时，高压脉冲被施加到细胞上。在电极上重复施加高压脉冲按照焦耳定律会导致热量的产生，因此必须通过冷却装置将热量带走，防止电极和细胞悬液的温度过高。由于在流式电穿孔法中液流是稳定而持续的，其产生的热量也是持续的，因此，从电穿孔室中带走热量的方法必须能够平衡产生的热量从而避免电穿孔室的温度上升到不可接受的程度。因此，保持流式电穿孔室内的温度和平面电极的温度在可控的范围内波动也是亟需解决的技术问题。
流体进入电穿孔室时，由于导管与电穿孔室的横截面积的差异，会产生瞬间的压强改变，流体在电穿孔室流动的过程中，如何保持液体横截面流动相对速度的稳定，使流体的流速与脉冲的次数 稳定，也是本发明需要解决的技术问题。
发明内容
本发明的目的在于提供一种用于流式电穿孔的装置的改进电极，其能够替代铝电极的使用，并比铝电极具有更高的电转染效率和细胞存活率。
本发明的另一目的在于提供一种流式电穿孔的装置，其能够使流体以相对恒定的速度流过流式电穿孔室。
本发明的另一目的在于提供一种流式电穿孔的装置，通过电极结构的优化及电场的控制来优化气泡的产生及排出，避免气泡在电极内部滞留影响电转结果。
本发明的另一目的在于提供一种流式电穿孔的装置，其能够有效控制平面电极在电转染时的温度在一定的范围内波动。
本发明的另一目的在于提供一种流式电穿孔的装置的制备方法，该方法步骤简单，操作方便，产品易于标准化处理。
本发明的另一目的在于提供一种流式电穿孔的装置的使用方法，用于大体积处理细胞。
本发明的另一目的在于提供一种流式电穿孔的系统，控制流式电穿孔装置周围的环境。
一种流式电穿孔装置，包括支架及电极装置，所述电极装置为平面电极，所述电极装置为平面电极，所述支架和所述平面电极共同限定供流体流动的通道；优选所述平面电极的表面光滑平整，与支架接触平缓无断差且密封；
所述电极装置的材料包含钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种。
优选地，本发明流式电穿孔装置的支架用于固定平面电极，所述支架材料选用医疗级的工程塑料。所述流式电穿孔装置还包括与所述通道连通的流入口及与所述通道连通的流出口。
优选地，所述流式电穿孔装置还包括电源，所述电极装置与所述电源电连接。
优选所述平面电极材料表面可以添加涂层或镀层，涂层或镀层材料与基材的材料不同；
优选所述平面电极的涂层或镀层材料为金、银、钛、氧化铱、铱、铂、铌、钌、钼、铑、钨、导电陶瓷材料中的一种或几种。优选所述平面电极的材料为铂镀金、铂镀银、铂镀铱、铂镀铌、铂镀钌、铂镀钼、铂镀铑、铂镀导电陶瓷。优选所述平面电极的材料为钛镀金、钛镀银、钛镀铱、钛镀铂、钛镀铌、钛镀钌、钛镀钼、钛镀铑、钛镀导电陶瓷。优选所述平面电极的材料为铱镀金、铱镀银、铱镀铂、铱镀铌、铱镀钌、铱镀钼、铱镀铑、铱镀导电陶瓷。优选所述平面电极的材料为铑镀金、铑镀银、铑镀铱、铑镀铂、铑镀铌、铑镀钌、铑镀钼、铑镀导电陶瓷。
优选所述平面电极的镀层的厚度为0.1μm-10μm。
本发明的流式电穿孔装置，主要包括支架和限定流体流动通道的平面电极，两片平面电极平行嵌入支架，组成一个电穿孔腔室，所述平面电极包括用于放置与高压脉冲电源有电联系的装置，两片平面电极分别接入高压脉冲电源输出电信号的正、负极，接通电信号后，在两片平面电极之间的所述电穿孔腔室形成均匀的电场，从而使沿所述流体流动通道移动流经所述电穿孔腔室的流体经历脉冲电场。
本发明的流式电穿孔电极选用导电性能良好的金属材料，同时需要稳定的电化学性能，在电穿孔过程中不能与细胞溶液发生化学反应，避免产生有害物质。
可供选择的电极材料有铝、不锈钢、金、银、钛、铂、铱、铌、钌、铑、钼、钨、导电陶瓷等。优选所述的电极材料为金、钛、铂、铱、铌、钌、钼、钨、铑。
进一步优选地，所述平面电极的形状为平行四边形、圆形、椭圆形中的一种或几种。
进一步优选地，所述平面电极的形状为直角平行四边形。进一步优选地，所述平面电极的形状为长方形。
进一步优选地，所述平面电极的长度不小于8mm，所述平面电极的长度不大于25mm。
进一步优选地，所述平面电极的长度为8mm。
进一步优选地，所述平面电极的长度为12.5mm。
进一步优选地，所述平面电极的长度为15mm。
进一步优选地，所述平面电极的长度为25mm。
进一步优选地，所述平面电极的宽度不小于2mm，所述平面电极的宽度不大于10mm。
进一步优选地，所述平面电极的宽度为2mm。
进一步优选地，所述平面电极的宽度为4mm。
进一步优选地，所述平面电极的宽度为10mm。
进一步优选地，所述平面电极的长度为15mm，所述平面电极的宽度为10mm。
进一步优选地，所述平面电极的厚度不小于0.1mm，所述平面电极的厚度不大于5mm。
进一步优选地，所述平面电极的厚度不小于0.5mm。
进一步优选地，所述平面电极的厚度不小于1mm，所述平面电极的厚度不大于3mm。
进一步优选地，所述平面电极的厚度为2mm。
进一步优选地，所述平面电极的表面积不小于10mm 2，所述平面电极的表面积不大于2500mm 2。
进一步优选地，所述平面电极的表面积不小于50mm 2，所述平面电极的表面积不大于1000mm 2。
进一步优选地，两片所述平面电极分别由相同或者不同材料制成。
进一步优选地，两片所述平面电极之间的距离不小于0.5mm，两片所述平面电极之间的距离不大于8mm。
进一步优选地，两片所述平面电极之间的距离不小于1mm，两片所述平面电极之间的距离不大于5mm。
优选地，所述流式电穿孔装置还包括导管，所述导管与所述支架固定连接，所述导管包括与所述流入口相连接的进液管，所述导管还包括与所述流出口相连接的出液管。流体通过进液口流入腔室，在电场的作用下，完成电穿孔，最后通过出液口流出。
进一步优选地，所述出液管的直径不小于所述进液管的直径。
优选所述导管的直径在不小于2mm，所述导管的直径在不大于10mm。优选所述导管选用医疗工程塑料或医用硅胶。进一步优选地，所述导管与所述支架的连接方式包括但不限于螺纹连接，卡扣连接，包覆连接，超声焊接。
进一步优选地，所述进液管呈直线型，所述出液管呈直线型。
进一步优选地，所述流入口的轴线方向与所述通道的长度方向的夹角介于0-90°。
进一步优选地，所述流出口的轴线方向与所述通道的长度方向的夹角介于0-90°。
本发明的流式电穿孔装置为了控制流体进入电穿孔腔体和流出电穿孔腔体的流向，具体设置了与流体流经电穿孔室方向平行流向和垂直流向的进液方式。
进一步优选地，所述流入口的轴线方向与所述通道的长度方向平行。
进一步优选地，所述流出口的轴线方向与所述通道的长度方向平行。
优选地，所述支架上设置有至少一个流体缓冲区域，所述流体缓冲区域位于所述流入口与所述电极之间；和/或所述流体缓冲区域位于所述流出口与所述电极之间。
进一步优选地，所述流体缓冲区域的横截面所呈图形包括但不限于三角形、正梯形、倒梯形、方形、半圆形、半椭圆形、圆弧形、S弯弧形。
优选地，所述流式电穿孔装置还包括流体驱动装置，所述流体驱动装置采用正压和/或负压的方式驱动所述流体。
优选地，所述流体驱动装置为蠕动泵或磁力泵。
进一步优选地，所述流体驱动装置采用负压的方式驱动所述流体。
进一步优选地，所述流式电穿孔装置还包括用于冷却所述电极装置的含有控温冷却模块的冷却控温装置。进一步优选地，所述控温冷却模块设置在所述平面电极的外侧。
进一步优选地，所述冷却控温装置包括冷却液、冷却气体、冷却管、冷却片及冷却扇中的一种。
优选地，所述流式电穿孔装置还包括电脉冲发生装置及控制系统。
优选地，所述流式电穿孔装置还包括至少一个流体装储装置。
优选地，所述流式电穿孔装置还包括用于调节所述流式电穿孔装置的周围环境温度的温度调节 装置。
进一步优选地，所述温度调节装置控制所述流式电穿孔装置的周围环境温度在0-40℃之间。
优选地，所述流式电穿孔装置还包括用于调节所述流式电穿孔装置的周围环境二氧化碳的浓度的二氧化碳发生装置。进一步优选地，所述二氧化碳发生装置控制所述流式电穿孔装置的周围二氧化碳的浓度在3-8％之间。
本发明还提供一种流式电穿孔装置，用于将待转物质转染进细胞，所述待转物质包括生物活性物质和非生物活性物质，生物活性物质和非生物活性物质分别包括生物活性分子和非生物活性分子，所述流式电穿孔装置为本申请中提供的任一所述的流式电穿孔装置。
进一步优选地，所述生物活性物质包括但不限于药剂、基因物质DNA和RNA、细胞因子、抗体或其他的蛋白质。
进一步优选地，所述非生物活性物质包括造细胞因子、影剂纳米颗粒。
进一步优选地，所述细胞的种类包括但不限于原核细胞、真核细胞、植物原生质体。
进一步优选地，所述原核细胞包括细菌、真菌、酵母菌。进一步优选地，所述真核细胞包括动物细胞系细胞、原代细胞、卵细胞。
优选地，所述流入口位于所述流式电穿孔装置的下侧，所述流出口位于所述流式电穿孔装置的上侧。
本发明还提供一种流式电穿孔装置的制备方法，包括如下步骤：
通过注模法或计算机控制技术(CNC)或3D打印技术制作具有流入口和流出口的支架，所述支架设有两个凹陷窗；
用电镀或涂抹的方式在平面电极的金属基体表层涂覆金属层，所述平板电极的材料选自钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种；
将两块所述平面电极分别安装到所述凹陷窗中，然后再将所述平面电极封装。
优选所述平面电极的涂层或镀层的制备方法为涂抹或电镀，制备工艺：包括前处理、预处理、涂抹或电镀、烘干的步骤。
前处理包括选取合适基材进行外形加工、表面喷砂处理，根据镀层厚度选取不同的喷砂目数、使用酸清洗表面氧化层。
预处理包括使用清洗剂进行超声清洗，去离子水冲洗去除清洗剂残留。
涂抹包括使用浸泡或毛刷或喷枪喷/涂抹后烘干将基材表面涂上均匀的涂层，电镀包括使用电解法在基材表面镀上均匀的镀层，涂抹或电镀后用去离子水冲洗，去除涂抹液或电镀液，烘干备用。
本发明还提供一种流式电穿孔装置的使用方法，将细胞和待转物质置于溶液中，并将溶液转移到流式电穿孔装置内，所述待转物质向细胞中转移，所述流式电穿孔装置包括：
电源、支架、电极装置、电脉冲发生装置及控制系统，所述支架含有供流体流动的通道、与所述通道连通的流入口及与所述通道连通的流出口；所述电极装置与所述电源电连接，所述电极装置位于所述通道内，所述电极装置的材料包含钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种；
向所述平面电极施加电压，所述电极产生电流，所述电流的密度最高达到120A/cm 2，通过电穿孔原理将生物活性物质转入细胞。
本发明还提供一种流式电穿孔装置的使用方法，将细胞和待转物质置于溶液中，并将溶液转移到流式电穿孔装置内，所述待转物质向细胞中转移，所述流式电穿孔装置包括：
电源、支架、电极装置、电脉冲发生装置及控制系统，所述支架含有供流体流动的通道、与所述通道连通的流入口及与所述通道连通的流出口；所述电极装置与所述电源电连接，所述电极装置位于所述通道内，所述电极装置的材料包含钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种；
向所述电极施加电压，所述电极产生电流，向所述电极施加电流，所述电流的密度为2-14A/cm 2。
优选地，所述电流的脉冲宽度为0.01-100ms。
本发明还提供一种流式电穿孔装置的使用方法，将细胞和待转物质置于溶液中，并将溶液转移到流式电穿孔装置内，所述待转物质向细胞中转移，所述流式电穿孔装置包括：
电源、支架、电极装置、电脉冲发生装置及控制系统，所述支架含有供流体流动的通道、与所述通道连通的流入口及与所述通道连通的流出口；所述电极装置与所述电源电连接，所述电极装置位于所述通道内，所述电极装置的材料包含钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种；
向所述电极施加电压，所述通道内产生电场，所述电压的场强为0.2-10kV/cm。
其中，所述电压的脉冲宽度0.01-100ms。
与现有技术相比，本发明的流式电穿孔装置取得了显著的进步以及预料不到的技术效果：
本发明优选的平面电极材料制成的平面电极，能够提供持续稳定的电流，在缓冲溶液存在下，能提供稳定的均匀电场，能够显著提高电穿孔效率，同时保证细胞高存活率；
本发明优选的平面电极在与缓冲液接触后，即使较高的脉冲电压，令人意外的发现产生的电解反应非常小，同时产生的气泡非常少，对能够持续对平面电极施加脉冲提供了有利的条件；
本发明的平面电极在工作状态产生的OH -(阴极效应)少，不改变细胞液的pH值,对细胞伤害小，细胞存活率高；
本发明的流式电穿孔装置生产工艺简单，制备成本低，产品制作能够标准化生产，批次处理细胞液体积更大，转染效率和细胞存活率更高，稳定性更好，使用方法操作方便。利用本发明所述流式电穿孔装置经过处理的电穿孔细胞，细胞电转染率至少60％以上，细胞存活率至少60％以上。通过优化支架结构中的出入口形状，使细胞-生物活性物质混悬液能够均匀地进入电穿孔室，同时电击处理后的细胞及电转产生的其他物质也能够顺利地从电穿孔室排出。本发明所述的流式电穿孔装置能够用于超大体积细胞液处理，显著提高电转染效率的同时显著提高细胞的存活率。
本发明的流式电穿孔装置是目前最先进的流式电穿孔产品，能够用于实际的实验室及生产环节进行电转染，具有巨大的商业化空间。
附图说明
图1不同尺寸铝电极的电流稳定性；
图2加导电陶瓷涂层的钛电极、铝电极和不锈钢镀金电极的电流稳定性；
图3加导电陶瓷涂层的钛平面电极电转后溶液颜色变化；
图4铝电极电转后溶液颜色变化；
图5不锈钢镀金电极电转后溶液颜色变化；
图6纯钛电极的电转后溶液颜色；
图7钛镀铂电极的电转后溶液颜色；
图8加导电陶瓷涂层的钛平面电极电转后电极表面变化(左边为阳极、右边为阴极)；
图9铝电极电转后电极表面变化(左边为阳极、右边为阴极)；
图10不锈钢镀金电极电转后电极表面变化(左边为阳极、右边为阴极)；
图11纯钛电极电转后电极表面变化(左边为阳极、右边为阴极)；
图12钛镀铂电极电转后电极表面变化(左边为阳极、右边为阴极)；
图13流体正进正出方式示意图；
图14流体侧进侧出方式示意图；
图15流体缓冲区域结构示意图；
图16电极冷却控温装置示意图；
图17装置整体结构分解示意图；
注释：1-导管(出液管)；2-支架出液口；3-第一缓冲区域；4-电穿孔室；5-第一平面电极；6-第一平面电极冷却装置；7-导管(进液管)；8-导管(进液管)；9-第二缓冲区域；10-支架；11-第二平面电极冷却装置；12-第二平面电极；13-冷却液输入口；14-冷却液输出口；15-冷却液输入口；16-冷却液输出口。
具体实施例
下面将结合具体实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照本领域常规条件或制造商建议的条件进行。
实施例一
电极的形状和尺寸的选择
本发明的流式电穿孔装置采用平面电极，平面电极的尺寸大小决定了单位时间内处理细胞的速度，电穿孔腔室的长度、宽度和间距分别有不同的影响。在流速恒定的情况下，平面电极的长度影响电极内部溶液流动的时间，增加电极长度会增加气泡流过电极区域时与电极表面接触时间，增加气泡滞留的概率。平面电极的宽度影响电极内部溶液流动的速度，流体中间区域流速快，边缘区域流速慢，电极宽度越大，偏差越明显。平面电极的间距影响电场的强度，根据公式E＝U/d，相同电压下，间距越大，电场越小。增加电极间距，如果要达到最佳的电转强度，就需要提高电压，增加仪器的输出。
实验方案设计：使用不同长度与宽度的比例关系的平面电极制作流式电穿孔装置，用空缓冲液测试电流数据变化，同时观测气泡产生和排出情况，优化平面电极的长度与宽度比例关系。平面电极的形状选择长方形或正方形。平面电极测试材料选择铝，测试样本长宽厚尺寸分别设定为：15×10×2mm，25×4×2mm，25×2×2mm，12.5×4×2mm。用CNC或3D打印制作带有两个凹陷窗口的支架，所述凹陷窗口的尺寸与上述平面电极的尺寸一致。平面电极嵌入凹陷窗口中，再将支架封装，形成一个带有流体流入口和流出口的电穿孔室。将上述装置的两片电极分别连接脉冲电源输出端的正负极。电转条件150V/11ms/1次，加入相应体积的EL缓冲溶液(苏州壹达公司及其代理商有售产品，货号：H10305)，记录电击过程中的电流数据变化情况(参见附图1)，全程观察气泡产生与排出情况。
电击过程检测结果：长度25mm的电极，电击过程中产生的小气泡容易在电极内累积，附着在电极表面，逐渐成长为大气泡再排出，引起电流周期性波动，造成电场稳定性下降。长度12.5mm的电极，电击过程中产生的小气泡随溶液流动排出，很少在电极内累积，电流稳定性好，但平面电极为2×4mm的电穿孔室总体积只有2×8mm的一半，相同条件下选取2×8mm规格，以提高单位时间内的处理量。长度和电极间距不变的情况下，单独改变电极宽度，宽度越大，单位时间处理量越大，反之宽度越小，单位时间处理量越小。但是加大宽度后，电极两侧区域与中间区域内溶液流速差异也会变大，综合考虑15×10×2mm为优选的电极尺寸。
实施例二
平面电极材料的选择
本发明的流式电穿孔装置，平面电极材料研发作为核心内容，平面电极表面应光滑平整，与支架接触平缓无段差且形成密封腔室，尽量减少气泡滞留可能性。通过电极材料的选择、电极结构的优化及电场强度的控制，来消减电击过程中电化学反应产物对电转染效果的不利影响。
实验方案设计：本实施例可供选择的平面电极材料的种类为铝、304不锈钢、纯金、不锈钢镀金、纯钛、加导电陶瓷涂层的钛，纯铂、钛镀铂、纯铱；
铝：作为电穿孔材料广泛用于电转染领域；
304不锈钢：生物亲和性好，成本低，最常用的医用金属材料；
不锈钢镀金：金具有稳定的化学和导电性能，具有良好的生物兼容性；
纯钛：具有良好生物兼容性的金属材料，广泛应用于生物医疗领域；
加导电陶瓷涂层的钛：苏大辛教授提供，涂层材料为导电陶瓷，具有良好的抗氧化性能；
钛镀铂：稳定的阳极材料，广泛用于电解工业，并具有良好的生物兼容性；
纯金：具有极高的抗腐蚀的稳定性，良好的导电性和导热性，极易加工成形，并很容易镀到其它金属和陶器及玻璃的表面上
纯铂：化学性质极稳定，不溶于强酸强碱溶液，具有催化活性，可做抗癌药物。
纯铱：化学性质非常稳定，不溶于王水，铱在高技术领域中得到广泛的使用，如航天技术，制药和汽车行业。
将上述材料制成长×宽×厚为15×10×2mm的平面电极。同实施例一，用CNC或3D打印制作带有两个凹陷窗口的支架，所述凹陷窗口的尺寸与上述平面电极的尺寸一致。将上述材料制成的平面电极嵌入凹陷窗口中，再将支架封装，形成一个带有流体流入口和流出口的电穿孔室。将上述已嵌入支架中的两片电极分别连接脉冲电源输出端的正负极，制成一个电穿孔装置。
试验一，在空缓冲液条件下(是指缓冲液中没有细胞和生物活性物质)，选择加导电陶瓷涂层的钛平面电极、铝电极、不锈钢镀金电极、纯钛电极、钛镀铂电极，使用150V电压，11ms脉宽，电击次数为1次，间隔为2352ms电转条件，30ml的电转体积电击测试组装电极稳定性，记录电击过程中的电流数据，实验结果参见图2，同时观察电转后溶液变化，参见图3-7，和平面电极表面变化，参见图8-12。
由图2可见，不锈钢镀金电极的电流从4A逐渐升高到5A；铝电极电流从4.5A迅速升高到5A，后电流一直在5A上下波动；加导电陶瓷涂层的钛平面电极电流从开始到结束都非常稳定，保持在4.8A。加导电陶瓷涂层的钛电极、铝电极和钛镀铂电极电流相对稳定，不锈钢镀金电极电流逐渐上升，纯钛电极初期电流会下降，然后缓慢上升，后面逐步趋于平稳，并且电流波形发生变化。
各种材料处理后溶液的变化由图3-7可见，加导电陶瓷涂层的钛平面电极会与溶液中的氯离子发生反应，电转后溶液变黄(图3)，析出的离子成分会对电转后细胞存活率造成影响，离心后无明显沉淀。
电击过程中，铝和不锈钢镀金材料，阳极发生剧烈氧化反应。铝电极的Al 3+会与溶液中OH -等反应产生白色沉淀(图4)，因此，产生氧化物会脱落到细胞溶液中，不可用作阳极材料。而不锈钢镀金平面电极表面的金脱落(图10)，溶液变成微黄(图5)。
上述三种材料均有产生白色泡沫团，其中铝电极产生最多，并且有白色沉淀物析出(图4)。
纯钛平面电极电击后，溶液未变色，但离心后有明显颗粒沉淀，且全部过程都存在沉淀(图6)。
钛镀铂平面电极电击后，溶液未变色，未见明显沉淀，离心后也无沉淀物(图7)。
电转后，将电极拆开，由图8-12可见，电转后加导电陶瓷涂层的钛平面电极表面未见明显氧化(图8)，而铝(图9)和不锈钢镀金平面电极阳极均有氧化，尤其不锈钢镀金平面电极的镀金层已脱落(图10)。纯钛电极阳极氧化形成氧化层(图11)，钛镀铂电极表面未见氧化(图12)。
试验二，将含有pMAX质粒(购于Lonza Human T cell Nucleofector Kit，货号：VAPA-1002)和CHO-S细胞(北京大学医学院周教授惠赠，周教授购于Life，货号R80007)及EL Buffer电转缓冲液的流体从上述电穿孔装置底部逐渐注入电穿孔室中，选择不同的电极材料，分别经过在150V电压，11ms脉宽，电击次数为1次，间隔为2352ms，以及在150V电压，5ms脉宽，电击次数为3次，间隔为784ms条件下电场处理，然后从顶部开口处流出，对细胞进行培养，测定电转染率，细胞存活率等指标。
表1加导电陶瓷涂层的钛电极、铝电极、不锈钢镀金电极和纯钛电极在150V 11ms 1次条件下处理细胞实验结果
Figure PCTCN2018110915-appb-000001
注释：TE：电转效率；VE：细胞存活率；MIX：综合效率；MFI：平均荧光强度；
上述四种电极材料电转结果表明，30ml电转体积，转染效率基本维持不变，有5％左右的波动，电转效率：纯钛>加导电陶瓷涂层的钛>铝>不锈钢镀金；细胞存活率：纯钛>加导电陶瓷涂层的钛>不锈钢镀金>铝；综合效率：纯钛>加导电陶瓷涂层的钛>铝＝镀金不锈钢；荧光强度：纯钛>铝>加导电陶瓷涂层的钛>不锈钢镀金。
表2加导电陶瓷涂层的钛电极和钛镀铂电极在150V 5ms 3次条件下电转细胞实验结果
Figure PCTCN2018110915-appb-000002
钛镀铂电极和加导电陶瓷涂层的钛电极在转染效率上个体间差异较小,但钛镀铂电极的存活率要比涂层电极高15％，二者的荧光强度基本一致，因此钛镀铂电极综合效率要高于加导电陶瓷涂层的钛电极。
虽然纯钛电极电转结果优于铝、不锈钢镀金电极，但纯钛电极的阳极会发生氧化，电场稳定性发生变化，而且在电转后溶液中观察到黑色物脱落，因此纯钛不适合作为阳极材料；而加导电陶瓷涂层的钛电极电转溶液会发黄。钛镀铂电极和加导电陶瓷涂层的钛电极电转效率接近，钛镀铂电极的细胞存活率更高，因此钛镀铂电极综合效率要高于加导电陶瓷涂层的钛电极。
实施例三
流体不同进出电穿孔室方式的选择
实验方案设计：流体进入电穿孔室时的水平角度与在电穿孔室流动时的水平角度的夹角可以在0°至90°之间，当夹角在0°时，流体为正进正出，当角度在90°时，流体为侧进侧出。流体正进正出的主要方式为支架的进液口和出液口呈直线延伸，使得电极近端的硅胶管与电极呈平行连接状态。而流体侧进侧出的主要方式为支架的进液口和出液口与平面电极呈90°转角，使得电极近端的硅胶管与电极呈垂直连接状态。
选择钛镀铂的平面电极，测试流体正进正出(参见附图13)和侧进侧出(参见附图14)两种方式，进行上述CHO-S细胞电转染上述pMAX质粒实验，在150V电压，11ms脉宽，电击次数为1次，间隔为2352ms下，以及在150V电压，5ms脉宽，电击次数为3次，间隔为784ms下通过监测电击过程中电流的稳定性、泡沫团(气泡及死细胞的混合物)的产生与排出情况，对比两种类型平面电极的电转染过程稳定性差异；同时通过检测不同时间段内细胞的电转效率(TE)、细胞存活率(VE)及平均荧光强度(MFI)等，对比两种类型电极的电转染效果的差异。
表3.正进正出和侧进侧出电极在150V 11ms 1次条件下电转效果
Figure PCTCN2018110915-appb-000003
表4正进正出和侧进侧出电极在150V 5ms 3次电转条件下的电转效果
Figure PCTCN2018110915-appb-000004
从平均实测电流大小及标准差可以推测：正进正出和侧进侧出两种电极电流稳定性均较好。从细胞电转效果的各项结果值可以得出：正进正出和侧进侧出两种电极在不同进样模式下的电转效 率、细胞存活率及综合效率相差不大，但正进正出电极所得阳性细胞的MFI值高于侧进侧出。
侧进侧出和正进正出在倒“U”型收集管道系统中的主要差别仅是电极出口端转角的差异。侧进侧出的转角为90°，泡沫团在转角处存在较长时间的停滞和大量的累积。而正进正出的转角因硅胶管的弧度而缓解为大于90°，此时泡沫团的停滞和聚集现象有所缓解，且各段电转效率的稳定性较侧进侧出有所改善。
正进正出与侧进侧出在电场强度与稳定性上及电转效果上无显著差异，但正进正出更有利于气泡排出，正进正出电极所得细胞电转效果稳定性较好。
实施例四
设置流体缓冲区域
如果支架进液口截面为圆形，而平面电极腔室区域截面为方形，直接从圆形变为方形，则容易产生滞留区，滞留区溶液流速为零，此区域为无效电转区，且易累积气泡。因此需要在进液口和平面电极区域增加缓冲区，平缓过渡，避免产生滞留区，确保平面电极区域内溶液流速稳定。理论上，缓冲区体积越大，则电极区流速越稳定。同理，在平面电极区和支架出液口之间也要增加缓冲区，避免快速收口，压强变化影响流速。缓冲区溶液内压强逐渐变大，方便电解产生的气泡流过时，容易在管壁上累积气泡，如果气泡无法及时排出，体积逐渐变大，缓冲区的作用被抵消，无法起到稳定流速的效果。这时需要考虑缓冲区的形状，首先收口的方式可以增加溶液内压强，有利于气泡排出。其次不能有拐角产生滞留区，最后外形过渡应平滑，溶液内压强不能有剧烈变化。
实验方案设计：选择钛镀铂的平面电极，在正进正出的电穿孔室的顶部和底部分别设置流体缓冲区域(参见图13)，用于克服流体从支架进液口进入电穿孔室时的瞬间压强的改变，缓冲区域的形状设置成圆锥形、方形、弧形和梯形(参见图15)。
表5不同缓冲区形状电极在150V 5ms 3次电转条件下的电转效果
Figure PCTCN2018110915-appb-000005
实验结果表明：圆锥形缓冲区域电穿孔装置在出液口处累积气泡变少，剩余气泡多累积在电极内部侧边直角处；方形缓冲区域电穿孔装置在出液口积累大量气泡并在积累到一定量时一次排出，造成液流不均匀；弧形缓冲区域电穿孔装置积累的气泡最少，电流最稳定，转染效率最高；梯形缓冲区域电穿孔装置的气泡积累情况和转染效率则居中。
实施例五
正压或负压驱动流体
本发明的流式电穿孔装置以正压和/或负压的方式驱动流体流动，优选以负压的方式驱动流体流动。主要以蠕动泵的转动作为流体流动的动力，促使流体进入电穿孔室接受电击，并将电击处理后的细胞及电转产生的其他物质带出电场，确保电转精确有序的进行。根据蠕动泵放置的位置，可将整个液体动力形式分为正压和负压两种模式。正压动力模式是将蠕动泵放置在电穿孔室的前侧，通过转动蠕动泵产生的推力推动液体在管道系统内单向流动进入电穿孔室。负压动力模式是将蠕动泵放置在电穿孔室的后侧，通过转动蠕动泵产生的吸力抽取样品管内的样品并使其在管道系统内单向流动通过电穿孔室。
实验方案设计：采用正进正出弧形缓冲区电穿孔装置，监测正压、负压两种动力模式下，驱动流体进行电转过程中的电流稳定性及观察电击过程中泡沫团的产生与排出情况，比较两种动力模式下电场强度的稳定性和流体在管道内的流动稳定性。检测两种工作模式下CHO-S细胞电转pMAX，在150V电压，5ms脉宽，电击次数为3次，间隔为784ms条件下进行电转，选择钛镀铂平面电极，所得的细胞电转染效率、存活率、综合效率和荧光表达量，比较两种动力模式下细胞的电转效果及稳定性。
表6正压和负压动力模式下细胞电转效果
Figure PCTCN2018110915-appb-000006
结果分析：从平均实测电流大小及标准差可以推测：正压和负压两种动力模式下电场强度大小较一致，CHO-S细胞电转DNA测试中，正压动力模式下实测电流为5.20±0.39A，负压动力模式下实测电流为4.95±0.55A，二者差异较小，且稳定性均较好。
从流体和泡沫团在管道系统内的运动状况可以看出，电极表面泡沫团的产生与排出情况基本相同，均表现为先是较小体积泡沫团产生，大部分为无滞留连续快速排出，在电极片与支架接缝处减速汇集成较大泡沫团，随后在电极出口端收口处再次出现减速汇集成更大的泡沫团，最后进入收集管道系统。但泡沫团在收集管道内的排出情况差异较大：正压条件下，泡沫团易在管道转角处停滞累积，分段取样时，前段收集不到泡沫团，后段收集到大量泡沫团,导致电转过程中流速不均匀，不同段收集到的细胞液体积量不均一。负压条件下，泡沫团在管道转角处停滞累积程度较轻；各段取样时，收集管内泡沫团分布和细胞液体积量均较一致。相对于正压，负压动力模式下，流体混悬液在管道内的运输更稳定，且泡沫团的排出更均匀顺畅。
从细胞电转效果的各项结果值可以得出：负压动力模式下，细胞电转染效率、存活率、综合效率及MFI均比正压动力模式下所得结果高。且电转效率与存活率，负压动力模式更稳定。负压动力模式可以有效的促进泡沫团在管道系统内的流动和排出，此时样品管可以正立放置，避免了因样品管密封不良等原因造成的漏液等现象，同时便于后期进样管道系统的改造。
实施例六
平面电极冷却控温装置
在平面电极外侧设置冷却控温装置(参见附图16)，将控温冷却模块用导热胶粘贴在平面电极外侧，所述控温冷却模块内部有冷却管与循环泵及半导体制冷芯片连接；所述循环泵驱动冷却管内的冷却液，带走由平面电极传导给冷却模块的热量，维持平面电极的温度稳定；所述半导体制冷芯片用于将从冷却模块回流的冷却液降温。
实验方案设计：采用与实施例二相同的细胞和实验条件，同时用带有冷却控温装置的平面电极和不带冷却控温装置的加导电陶瓷涂层的钛平面电极进行细胞转染对比实验。
实验结果表明：对工作中的平面电极温度的监测结果显示，带冷却装置的平面电极在转染过程中，其外表面的温度变化较小，平均温度为38.6℃±2.3℃，比不带冷却装置的平面电极的温度平均低9.5℃，其转染效率高9.3％，细胞存活率高11.2％。
实施例七
单管进液和连续进液比较
单管进液是指蠕动泵推动细胞液进入并充满电穿孔室，然后暂停工作，待电穿孔室内该部分细胞液完成电穿孔，蠕动泵继续工作，将新细胞液推进电穿孔室内直至新细胞液充满电穿孔室，即重复进液-暂停-电击的动作；连续进液是指蠕动泵连续工作推动细胞液连续通过电穿孔室，脉冲电源按设计的频率对细胞液施加电场进行电穿孔。
实验方案设计：根据前述实施例二至实施例六的实验结果，选择优化的实验条件，选择加导 电陶瓷涂层的钛电极，电极片间距为1.5mm，溶液接触区尺寸为4mm×8mm。支架进液口和出液口的结构为正进正出和侧进侧出，正进正出支架设置流体缓冲区域，出液口处直接平面过渡到2mm圆孔，负压连续进液的蠕动模式。通过稳定的电转体系：EL buffer，电压150V，脉宽5ms，次数3次，间隔784ms，5E+06细胞/ml，pMAX质粒20μg/ml进行验证测试，电转体积为10ml。单管进液与连续进液在多次电转条件下对比，比较电流稳定性以及气泡产生和排除情况。检测两种工作模式下CHO-S细胞电转DNA所得的细胞电转染效率、存活率、综合效率和荧光表达量，以比较两种进液模式下细胞的电转效果及稳定性。
表7加导电陶瓷涂层的钛电极单管进液与连续进液细胞电转效果及电流变化
Figure PCTCN2018110915-appb-000007
电流监测结果，在150V 5ms 3次电转条件下，单管进液与连续进液的平均电流基本一致，但是由于单管进液，在3次电击过程中，产生的气泡先累积后再一次排除，导致电流稳定性下降，误差值增加。细胞电转结果表明，连续进液远好于单管进液，正进正出和侧进侧出的电极管结构中，均是连续进液的效果优于单管进液的效果。
实施例八
不同电极材料在优化条件下进行电穿孔后实验
实验方案设计：选择铝、加导电陶瓷涂层的钛、不锈钢镀金、钛、金、铱、铑、铌、钌、钼、钨、钛镀铂、铂作为平面电极，其中加导电陶瓷涂层的钛由苏州大学材料物理实验室提供，钛、金、钛镀铂、铂从中诺新材北京有限公司采购,铝、不锈钢镀金由北京高德威金属科技有限公司采购，铱、铑、铌、钌、钼、钨由厦门钨业集团提供。电极片间距为1.5mm，溶液接触区尺寸为4mm×8mm。支架进液口和出液口的结构为正进正出，支架设置流体缓冲区域，出液口处直接平面过渡到2mm圆孔，负压连续进液的蠕动模式。通过稳定的电转体系：EL buffer，电压150V，脉宽5ms，次数3次，间隔784ms，1.00E+07细胞/ml，pMAX质粒20μg/ml进行验证测试，电转体积为40ml。
表8不同流式电穿孔仪电转效果
Figure PCTCN2018110915-appb-000008
Figure PCTCN2018110915-appb-000009
根据表8与表1和表2中的实验数据比较，本发明综合优化条件后的流式电穿孔装置，比最初简易的流式电穿孔装置的综合效率有显著提升。
铝电极，在相同电转条件下，转染效率为50.15％，并且溶液中有白色盐沉淀析出，对细胞产生毒性，存活率为44.69％；
加导电陶瓷的钛电极，在相同的电转条件下，转染效率为59.97％，虽然溶液变成微黄色，对细胞产生轻微毒性，但是细胞存活率为59.06％；
不锈钢镀金电极，转染效率为44.49％，细胞存活率为52.48％，在电转过程中，金脱落，溶液变黄，电极氧化显著；
钛电极，在电转过程中，电转染效率和细胞存活率都较加导电陶瓷的钛电极要高，分别达到66.03％和74.93％，但是电转完对溶液进行离心，发现有物质脱落形成沉淀；
金电极，在相同电转条件下，转染效率达77.59％，存活率达64.05％，溶液未见颜色变化，也没有沉淀析出，金作为阳极的电极片在电转过程中极易被氧化；
铱、铑、铌、钌、钼、钨电极相对稳定，电转后溶液和电极表面都没有显著变化，转染效率和 细胞存活率分别在58-71％之间和57-76％之间，综合效率均优于铝电极和不锈钢镀金电极。
铂电极，在相同电转条件下，转染效率达86.93％，存活率达63.55％，电转过程中溶液颜色未变化，无脱落，电转后进行电极阴阳极未发生变化，稳定性好。
钛镀铂电极在转染效率上达到92.25％，存活率达90.65％远高于其他材料电极，溶液未见颜色变化和沉淀析出，稳定性非常好。
钛镀金、铂镀铱、铱镀铑、铱镀铌、铑镀钌、铑镀钼性能稳定并抗氧化，达到很好的效果，电转染率和细胞存活率分别在77-86％之间和69-80％之间，综合效率较高，能够满足流式电穿孔的实际需要。
尽管已经用具体实施例来说明和描述了本发明，然而应意识到，在不背离本发明的精神和范围的情况下，可以作出许多其它的更改和修改。因此，这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。

Claims (64)

  1. 一种流式电穿孔装置，包括：
    支架，
    电极装置，所述电极装置为平面电极，所述支架和所述平面电极共同限定供流体流动的通道；其特征在于，
    所述电极装置的材料包含钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种。
  2. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述流式电穿孔装置还包括与所述通道连通的流入口及与所述通道连通的流出口。
  3. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述电极装置的镀层的厚度为0.1μm-10μm。
  4. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述流式电穿孔装置还包括电源，所述电极装置与所述电源电连接。
  5. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述平面电极的形状为平行四边形、圆形、椭圆形中的一种或几种。
  6. 根据权利要求5所述的流式电穿孔装置，其特征在于，所述平面电极的形状为直角平行四边形。
  7. 根据权利要求6所述的流式电穿孔装置，其特征在于，所述平面电极的形状为长方形。
  8. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述平面电极的长度不小于8mm，所述平面电极的长度不大于25mm。
  9. 根据权利要求8所述的流式电穿孔装置，其特征在于，所述平面电极的长度为8mm。
  10. 根据权利要求8所述的流式电穿孔装置，其特征在于，所述平面电极的长度为12.5mm。
  11. 根据权利要求8所述的流式电穿孔装置，其特征在于，所述平面电极的长度为15mm。
  12. 根据权利要求8所述的流式电穿孔装置，其特征在于，所述平面电极的长度为25mm。
  13. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述平面电极的宽度不小于2mm，所述平面电极的宽度不大于10mm。
  14. 根据权利要求13所述的流式电穿孔装置，其特征在于，所述平面电极的宽度为2mm。
  15. 根据权利要求13所述的流式电穿孔装置，其特征在于，所述平面电极的宽度为4mm。
  16. 根据权利要求13所述的流式电穿孔装置，其特征在于，所述平面电极的宽度为10mm。
  17. 根据权利要求16所述的流式电穿孔装置，其特征在于，所述平面电极的长度为15mm。
  18. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述平面电极的厚度不小于0.1mm，所述平面电极的厚度不大于5mm。
  19. 根据权利要求18所述的流式电穿孔装置，其特征在于，所述平面电极的厚度不小于0.5mm。
  20. 根据权利要求19所述的流式电穿孔装置，其特征在于，所述平面电极的厚度不小于1mm，所述平面电极的厚度不大于3mm。
  21. 根据权利要求20所述的流式电穿孔装置，其特征在于，所述平面电极的厚度为2mm。
  22. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述平面电极的表面积不小于10mm 2，所述平面电极的表面积不大于2500mm 2。
  23. 根据权利要求22所述的流式电穿孔装置，其特征在于，所述平面电极的表面积不小于50mm 2，所述平面电极的表面积不大于1000mm 2。
  24. 根据权利要求1所述的流式电穿孔装置，其特征在于，两片所述平面电极分别由相同材料制成。
  25. 根据权利要求1所述的流式电穿孔装置，其特征在于，两片所述平面电极分别由不同材料制成。
  26. 根据权利要求1所述的流式电穿孔装置，其特征在于，两片所述平面电极之间的距离不小于0.5mm，两片所述平面电极之间的距离不大于8mm。
  27. 根据权利要求26所述的流式电穿孔装置，其特征在于，两片所述平面电极之间的距离不小于1mm，两片所述平面电极之间的距离不大于5mm。
  28. 根据权利要求2所述的流式电穿孔装置，其特征在于，所述流式电穿孔装置还包括导管，所述导管与所述支架固定连接，所述导管包括与所述流入口相连接的进液管，所述导管还包括与所述流出口相连接的出液管。
  29. 根据权利要求28所述的流式电穿孔装置，其特征在于，所述导管的直径在不小于2mm，所述导管的直径在不大于10mm。
  30. 根据权利要求28所述的流式电穿孔装置，其特征在于，所述导管选用医疗工程塑料或医用硅胶。
  31. 根据权利要求28所述的流式电穿孔装置，其特征在于，所述出液管的直径不小于所述进液管的直径。
  32. 根据权利要求28所述的流式电穿孔装置，其特征在于，所述导管与所述支架的连接方式包括但不限于螺纹连接，卡扣连接，包覆连接，超声焊接。
  33. 根据权利要求28所述的流式电穿孔装置，其特征在于，所述进液管呈直线型，所述出液管呈直线型。
  34. 根据权利要求2所述的流式电穿孔装置，其特征在于，所述流入口的轴线方向与所述通道的长度方向的夹角介于0-90°。
  35. 根据权利要求34所述的流式电穿孔装置，其特征在于，所述流入口的轴线方向与所述通道的长度方向平行。
  36. 根据权利要求2所述的流式电穿孔装置，其特征在于，所述流出口的轴线方向与所述通道的长度方向的夹角介于0-90°。
  37. 根据权利要求36所述的流式电穿孔装置，其特征在于，所述流出口的轴线方向与所述通道的长度方向平行。
  38. 根据权利要求2所述的流式电穿孔装置，其特征在于，所述支架上设置有至少一个流体缓冲区域，所述流体缓冲区域位于所述流入口与所述电极之间；和/或
    所述流体缓冲区域位于所述流出口与所述电极之间。
  39. 根据权利要求38所述的流式电穿孔装置，其特征在于，所述流体缓冲区域的横截面所呈图形包括但不限于三角形、正梯形、倒梯形、方形、半圆形、半椭圆形、圆弧形、S弯弧形。
  40. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述流式电穿孔装置还包括流体驱动装置，所述流体驱动装置采用正压和/或负压的方式驱动所述流体。
  41. 根据权利要求40所述的流式电穿孔装置，其特征在于，所述流体驱动装置采用负压的方式驱动所述流体。
  42. 根据权利要求40所述的流式电穿孔装置，其特征在于，所述流体驱动装置为蠕动泵或磁力泵。
  43. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述流式电穿孔装置还包括用于冷却所述电极装置的含有控温冷却模块的冷却控温装置。
  44. 根据权利要求43所述的流式电穿孔装置，其特征在于，所述控温冷却模块设置在所述平面电极的外侧。
  45. 根据权利要求43所述的流式电穿孔装置，其特征在于，所述冷却控温装置包括冷却液、冷却气体、冷却管、冷却片及冷却扇中的一种。
  46. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述流式电穿孔装置还包括电脉冲发生装置及控制系统。
  47. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述流式电穿孔装置还包括至少一个流体装储装置。
  48. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述流式电穿孔装置还包括用于调节所述流式电穿孔装置的周围环境温度的温度调节装置。
  49. 根据权利要求48所述的流式电穿孔装置，其特征在于，所述温度调节装置控制所述流式电穿孔装置的周围环境温度在0-40℃之间。
  50. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述流式电穿孔装置还包括用于调节所述流式电穿孔装置的周围环境二氧化碳的浓度的二氧化碳发生装置。
  51. 根据权利要求50所述的流式电穿孔装置，其特征在于，所述二氧化碳发生装置控制所述流式电穿孔装置的周围二氧化碳的浓度在3-8％之间。
  52. 一种流式电穿孔装置，用于将待转物质转染进细胞，所述待转物质包括生物活性物质和非生物活性物质，其特征在于，所述流式电穿孔装置为权利要求1-51任一项所述的流式电穿孔装置。
  53. 根据权利要求52所述的流式电穿孔装置，其特征在于，所述生物活性物质包括但不限于药剂、基因物质DNA和RNA、细胞因子、抗体或其他的蛋白质。
  54. 根据权利要求52所述的流式电穿孔装置，其特征在于，所述非生物活性物质包括造影剂纳米颗粒。
  55. 根据权利要求52所述的流式电穿孔装置，其特征在于，所述细胞的种类包括但不限于原核细胞、真核细胞、植物原生质体。
  56. 根据权利要求55所述的流式电穿孔装置，其特征在于，所述原核细胞包括细菌、真菌、酵母菌。
  57. 根据权利要求55所述的流式电穿孔装置，其特征在于，所述真核细胞包括动物细胞系细胞、原代细胞、卵细胞。
  58. 根据权利要求1所述的流式电穿孔装置，其特征在于，所述支架材料选用医疗级的工程塑料。
  59. 根据权利要求2所述的流式电穿孔装置，其特征在于，所述流入口位于所述流式电穿孔装置的下侧，所述流出口位于所述流式电穿孔装置的上侧。
  60. 一种流式电穿孔装置的制备方法，其特征在于，包括如下步骤：
    通过注模法或计算机控制技术或3D打印技术制作具有流入口和流出口的支架，所述支架设有两个凹陷窗；
    制作电极装置，所述电极装置为平面电极，所述电极装置的材料选自钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种；
    将两块所述平面电极分别安装到所述凹陷窗中，然后再将所述平面电极封装。
  61. 一种流式电穿孔装置的使用方法，其特征在于，将细胞和待转物质置于溶液中，并将溶液转移到流式电穿孔装置内，所述待转物质向细胞中转移，所述流式电穿孔装置包括：
    电源、支架、电极装置、电脉冲发生装置及控制系统，所述支架含有供流体流动的通道、与所述通道连通的流入口及与所述通道连通的流出口；所述电极装置与所述电源电连接，所述电极装置位于所述通道内，所述电极装置的材料包含钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种；
    向所述电极施加电压，所述电极产生电流，所述电流最高达到120A/cm 2。
  62. 一种流式电穿孔装置的使用方法，其特征在于，将细胞和待转物质置于溶液中，并将溶液转移到流式电穿孔装置内，所述待转物质向细胞中转移，所述流式电穿孔装置包括：
    电源、支架、电极装置、电脉冲发生装置及控制系统，所述支架含有供流体流动的通道、与所述通道连通的流入口及与所述通道连通的流出口；所述电极装置与所述电源电连接，所述电极装置位于所述通道内，所述电极装置的材料包含钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种；
    向所述电极施加电压，所述电极产生电流，所述电流的密度为2-14A/cm 2。
  63. 一种流式电穿孔装置的使用方法，其特征在于，将细胞和待转物质置于溶液中，并将溶液转移到流式电穿孔装置内，所述待转物质向细胞中转移，所述流式电穿孔装置包括：
    电源、支架、电极装置、电脉冲发生装置及控制系统，所述支架含有供流体流动的通道、与所述通道连通的流入口及与所述通道连通的流出口；所述电极装置与所述电源电连接，所述电极装置位于所述通道内，所述电极装置的材料包含钛镀铂、铂、铑镀钼、铑镀钌、铱镀铌、铂镀铱中的一种或几种；
    向所述电极施加电压，所述通道内产生电场，所述电场的场强为0.2-10kV/cm。
  64. 根据权利要求62或63任一所述的流式电穿孔装置的使用方法，其特征在于，所述电压的脉冲宽度0.01-100ms。
PCT/CN2018/110915 2017-10-19 2018-10-19 一种流式电穿孔装置 WO2019076353A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US16/756,269 US20200318055A1 (en) 2017-10-19 2018-10-19 A flow electroporation device
EP18868610.9A EP3699262A1 (en) 2017-10-19 2018-10-19 Flow electroporation device

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710997572 2017-10-19
CN201710997572.5 2017-10-19

Publications (1)

Publication Number Publication Date
WO2019076353A1 true WO2019076353A1 (zh) 2019-04-25

Family

ID=66174317

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/110915 WO2019076353A1 (zh) 2017-10-19 2018-10-19 一种流式电穿孔装置

Country Status (4)

Country Link
US (1) US20200318055A1 (zh)
EP (1) EP3699262A1 (zh)
CN (2) CN116987589A (zh)
WO (1) WO2019076353A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USD965170S1 (en) 2020-10-23 2022-09-27 Life Technologies Corporation Electroporation device

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112779154A (zh) * 2019-11-05 2021-05-11 广州中国科学院先进技术研究所 一种电穿孔装置及系统
CN114686371A (zh) * 2020-12-29 2022-07-01 苏州壹达生物科技有限公司 一种流式电转染装置
US20230109873A1 (en) * 2021-10-13 2023-04-13 CyteQuest, Inc. Devices, methods, and systems for electroporation using controlled parameters
CN116288197A (zh) * 2023-01-06 2023-06-23 广东海洋大学 电极、高压脉冲电场处理室及高压脉冲电场处理设备
CN118147127A (zh) * 2024-05-10 2024-06-07 深圳市赛特罗生物医疗技术有限公司 一种大规模连续电转染方法及系统

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998024490A1 (en) * 1996-12-05 1998-06-11 Entremed, Inc. Improved electrodes and method of use
WO2006037527A1 (en) * 2004-09-30 2006-04-13 Pamgene Bv Masked solid porous supports allowing fast and easy reagent exchange to accelerate electrode-based microarrays
CN101928666A (zh) * 2010-07-30 2010-12-29 北京大学 一种流式电穿孔装置及系统
CN102174387A (zh) * 2011-01-25 2011-09-07 重庆大学 一种低压直流控制的连续流细胞电融合芯片
JP4910716B2 (ja) * 2006-04-03 2012-04-04 東ソー株式会社 細胞融合装置及びそれを用いた細胞融合方法
CN103555574A (zh) * 2013-11-11 2014-02-05 苏州文曲生物微系统有限公司 一种流式电穿孔装置
CN103865794A (zh) * 2014-03-18 2014-06-18 苏州文曲生物微系统有限公司 一种手持电穿孔装置
CN103937669A (zh) * 2014-03-18 2014-07-23 苏州文曲生物微系统有限公司 一种手持电穿孔装置
WO2017040995A1 (en) * 2015-09-04 2017-03-09 Rutgers, The State University Of New Jersey High throughput, feedback-controlled electroporation microdevice for efficient molecular delivery into single cells

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4910140A (en) * 1988-04-18 1990-03-20 Bio-Rad Laboratories, Inc. Electroporation of prokaryotic cells
WO2001070928A1 (en) * 2000-03-21 2001-09-27 Skwarczuk, Vanda Method and apparatus for electroporation of cells using electrical pulses of long duration
DE60143064D1 (de) * 2000-07-10 2010-10-21 Vertex Pharma San Diego Llc Ionenkanal-Assayverfahren
DE10208188B4 (de) * 2002-02-20 2006-05-24 Amaxa Gmbh Behälter mit zumindest einer Elektrode
CA2565316C (en) * 2004-05-12 2017-05-30 Maxcyte, Inc. Methods and devices related to a regulated flow electroporation chamber
AU2006342101A1 (en) * 2005-12-07 2007-10-25 Genetronics, Inc Variable volume electroporation chamber and methods therefore
US8222014B2 (en) * 2007-03-01 2012-07-17 Queen's University At Kingston Planar electroporation apparatus and method
WO2011050009A1 (en) * 2009-10-19 2011-04-28 Rational Biotechnology Inc. Method, device and apparatus for inducing self-adjusting cell electroporation
EP2720731B1 (en) * 2011-06-15 2020-11-18 The Charles Stark Draper Laboratory, Inc. Systems, methods, and devices relating to a biomimetic cellularized nephron unit
CN102533547B (zh) * 2011-12-15 2013-11-13 北京航空航天大学 一种剪切力-均匀电场联合刺激的细胞培养装置
CN104073428A (zh) * 2014-07-09 2014-10-01 北京大学 一种细胞分离微结构系统
EP3277368B1 (en) * 2015-03-31 2020-05-20 OncoSec Medical Incorporated Systems for improved tissue-sensing based electroporation

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998024490A1 (en) * 1996-12-05 1998-06-11 Entremed, Inc. Improved electrodes and method of use
WO2006037527A1 (en) * 2004-09-30 2006-04-13 Pamgene Bv Masked solid porous supports allowing fast and easy reagent exchange to accelerate electrode-based microarrays
JP4910716B2 (ja) * 2006-04-03 2012-04-04 東ソー株式会社 細胞融合装置及びそれを用いた細胞融合方法
CN101928666A (zh) * 2010-07-30 2010-12-29 北京大学 一种流式电穿孔装置及系统
CN102174387A (zh) * 2011-01-25 2011-09-07 重庆大学 一种低压直流控制的连续流细胞电融合芯片
CN103555574A (zh) * 2013-11-11 2014-02-05 苏州文曲生物微系统有限公司 一种流式电穿孔装置
CN103865794A (zh) * 2014-03-18 2014-06-18 苏州文曲生物微系统有限公司 一种手持电穿孔装置
CN103937669A (zh) * 2014-03-18 2014-07-23 苏州文曲生物微系统有限公司 一种手持电穿孔装置
WO2017040995A1 (en) * 2015-09-04 2017-03-09 Rutgers, The State University Of New Jersey High throughput, feedback-controlled electroporation microdevice for efficient molecular delivery into single cells

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USD965170S1 (en) 2020-10-23 2022-09-27 Life Technologies Corporation Electroporation device

Also Published As

Publication number Publication date
CN109679844B (zh) 2023-08-22
EP3699262A1 (en) 2020-08-26
US20200318055A1 (en) 2020-10-08
CN116987589A (zh) 2023-11-03
CN109679844A (zh) 2019-04-26

Similar Documents

Publication Publication Date Title
WO2019076353A1 (zh) 一种流式电穿孔装置
US10982182B2 (en) Flow electroporation device
US20040115784A1 (en) Apparatus and method for streaming electroporation
AU2020311908B2 (en) Electroporation devices and methods of cell transfection
US20070128708A1 (en) Variable volume electroporation chamber and methods therefore
JP2016526923A (ja) マイクロ流体ボルテックス支援電気穿孔システム及び方法
CN208250332U (zh) 一种流式电穿孔装置
CA2801028A1 (en) Electroporation electrode configuration and methods
CN111315892B (zh) 细胞内递送及其方法
CN208250333U (zh) 一种流式电穿孔装置
US8450102B2 (en) Device and method for stabilising the flow through a chamber
US11390840B2 (en) Systems and methods including porous membrane for low-voltage continuous cell electroporation
CN110872559A (zh) 一种流式电转染装置
WO2008051169A1 (en) Tip electrode chamber for small volume electroporation
US20190338235A1 (en) Method and Device for Exosomes Electroporation
CN208250334U (zh) 一种流式电穿孔装置
WO2021000844A1 (zh) 外源物质递送至真核细胞内的装置和方法及其应用
JP2004147517A (ja) 遺伝子導入装置
CN208577721U (zh) 一种结合高压和低压模式的流式电转染装置
CN217173754U (zh) 一种利用六孔板分割式转染装置
US20230295553A1 (en) Substance introduction unit and substance introduction device
KR20210015249A (ko) 전기천공장치
CN118147127A (zh) 一种大规模连续电转染方法及系统
AU2022379018A1 (en) Electroporation devices and methods of cell transfection
CN111575181A (zh) 一种可加压的流式电穿孔装置

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18868610

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2018868610

Country of ref document: EP

Effective date: 20200519