WO2019082621A1 - Cell stimulation device, cell culture device, and cell stimulation method - Google Patents

Cell stimulation device, cell culture device, and cell stimulation method

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Publication number
WO2019082621A1
WO2019082621A1 PCT/JP2018/037268 JP2018037268W WO2019082621A1 WO 2019082621 A1 WO2019082621 A1 WO 2019082621A1 JP 2018037268 W JP2018037268 W JP 2018037268W WO 2019082621 A1 WO2019082621 A1 WO 2019082621A1
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WO
WIPO (PCT)
Prior art keywords
electrode
culture medium
charge
cell
culture
Prior art date
Application number
PCT/JP2018/037268
Other languages
French (fr)
Japanese (ja)
Inventor
平川　弘幸
治男 山本
岩松　正
坂本　泰宏
山地　博之
新川　幸治
まい 高崎
翔平 鴻丸
文亮 杉森
Original Assignee
シャープ株式会社
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
Priority claimed from JP2018143848A external-priority patent/JP7032263B2/en
Application filed by シャープ株式会社 filed Critical シャープ株式会社
Priority to CN201880067680.5A priority Critical patent/CN111225972B/en
Priority to US16/756,080 priority patent/US11485950B2/en
Publication of WO2019082621A1 publication Critical patent/WO2019082621A1/en

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    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

Definitions

  • the present invention relates to a cell stimulation device, a cell culture device and a cell stimulation method.
  • Patent Document 1 There is known a cell culture method of providing electrical stimulation to cultured cells that are responsive to electrical stimulation (see, for example, Patent Document 1).
  • electrical stimulation promotes cell metabolism and can accelerate cell proliferation.
  • an electron-emitting device is known in which electrons are made to flow in the intermediate layer by applying a voltage between the lower electrode and the surface electrode to emit the electrons (see, for example, Patent Document 2).
  • an anion generating apparatus which generates anions of oxygen by electrons emitted by the electron emitting element and places the anions on a flow of gas and jets them toward a living body (for example, Patent Document 3) reference).
  • a conventional cell culture method of providing electrical stimulation to cultured cells at least two electrodes are placed in a medium, and a voltage is applied between the two electrodes. For this reason, an electrochemical reaction may occur at the interface between the electrode and the culture solution, resulting in the electrolysis of water or the decomposition / precipitation of the components of the culture solution.
  • the conventional anion generator it is difficult to uniformly supply the charge to the cells to which the electrical stimulation is to be applied, because the anions are ejected together with the gas.
  • the present invention has been made in view of such circumstances, and it is possible to uniformly apply electrical stimulation to cells to which electrical stimulation is to be given, and to suppress cell decomposition and deposition of culture medium components. I will provide a.
  • the present invention is a cell stimulation device for stimulating cells cultured in a culture medium, wherein the cell stimulation device comprises an electron emitting element for supplying a charge to the culture medium via a gas phase, and a charge from the culture medium
  • the electron emission device includes a lower electrode, a surface electrode, and an intermediate layer disposed between the lower electrode and the surface electrode, and the charge recovery electrode includes the charge recovery electrode.
  • a cell stimulation device characterized in that it is provided so as to be able to contact a culture medium.
  • the electron emitting element included in the cell stimulation device of the present invention includes the lower electrode, the surface electrode, and the intermediate layer disposed between the lower electrode and the surface electrode, the electron emitting device is interposed between the lower electrode and the surface electrode By applying a voltage, electrons flowing through the intermediate layer can be emitted from the surface electrode side. The emitted electrons can generate charges such as anions of oxygen in the gas phase between the electron emitting element and the culture medium.
  • the charge collection electrode included in the cell stimulation device of the present invention is provided so as to be able to contact the medium for culturing the cells, so that electrons can flow from the medium to the charge collection electrode. Therefore, the potential difference between the charge collection electrode and the culture medium can be reduced.
  • an electric field can be generated in the gas phase between the surface electrode of the electron-emitting device and the surface of the culture medium.
  • the generated anions can be transported to the surface of the culture medium along the electric field lines (gradient of the electric field strength) in this electric field, and the culture medium can be supplied with the anions.
  • the anion can provide electrical stimulation to cells cultured in the medium.
  • this anion flows to the charge collection electrode, it is possible to suppress the culture medium from being charged.
  • a gas flow mechanism of nitrogen gas or argon gas is not required in order to supply anions to the culture medium using the electric field generated in the gas phase. For this reason, there is no concern that drying of the culture medium or cell suspension will occur when the cells are electrically stimulated, and the gas composition of the culture atmosphere will not be affected.
  • the electron-emitting device included in the cell stimulation device of the present invention can emit electrons from the surface electrode, so that uniform anions can be generated in the gas phase between the surface of the culture medium and the surface electrode. it can. Therefore, anions can be uniformly supplied to the culture medium located below the surface electrode, and electrical stimulation can be uniformly applied to cells located below the surface electrode.
  • the charge generated by the electron emitting element is supplied to the culture medium through the gas phase, and therefore, the electrode contacting the culture medium is only the charge collection electrode. For this reason, it can suppress that the water contained in a culture medium electrolyzes.
  • the charge collection electrode has a positive polarity, precipitation of mineral components (K + , Ca 2+ , Na + , Mg 2+ etc.) in the medium can be suppressed on the charge collection electrode. It is possible to suppress the change in the component composition of
  • FIG. 1 is a schematic exploded view of a cell stimulation device according to an embodiment of the present invention. It is a schematic top view of the electron-emitting device contained in the cell stimulation apparatus of one Embodiment of this invention. It is a schematic sectional drawing of the cell culture apparatus of one Embodiment of this invention. It is a schematic sectional drawing of the cell culture apparatus of one Embodiment of this invention.
  • the cell stimulation apparatus of the present invention is a cell stimulation apparatus for stimulating cells cultured in a culture medium, wherein the cell stimulation apparatus comprises an electron emitting element for supplying a charge to the culture medium via a gas phase, and A charge recovery electrode for recovering charge from the culture medium, the electron emitting device comprising a lower electrode, a surface electrode, and an intermediate layer disposed between the lower electrode and the surface electrode, the charge recovery electrode An electrode is characterized in that it is provided so as to be able to contact the culture medium.
  • the lower electrode and the surface electrode included in the electron emitting element preferably include a plurality of elongated electrodes, and it is preferable that the lower electrode and the surface electrode be arranged in a grid in which one of the lower electrode and the surface electrode is a row and the other is a column. With such a configuration, it is possible to emit electrons from any intersection of the lower electrode and the surface electrode, and it is possible to apply electrical stimulation only to cells present at desired locations in the culture medium. In addition, it becomes possible to electrically stimulate cells in the culture medium with a desired distribution of the amount of electrical stimulation.
  • the cell stimulator of the present invention comprises a power supply.
  • the power supply device is preferably provided such that a voltage can be applied between the lower electrode and the surface electrode. By this, an electric field can be generated in the intermediate layer, electrons can flow in the intermediate layer, and electrons can be emitted from the surface electrode. In addition, negative ions can be generated in the gas phase by the electrons.
  • the power supply device is preferably provided so that a potential difference can be generated between the electron emitting element and the charge collection electrode. By this, an electric field can be generated between the surface electrode and the surface of the culture medium, and the anion can be transported to the culture medium by this electric field.
  • the power supply device emits electrons so that the current generated in the gas phase by supplying charges from the electron emitting element to the medium and the current generated in the medium by collecting charges from the medium by the charge recovery electrode become loop currents. It is preferable to electrically connect the element and the charge collection electrode. As a result, charges can be efficiently supplied to the medium, and cells can be appropriately stimulated.
  • the cell stimulation device of the present invention preferably comprises an insulating member and a needle-like terminal fixed to the insulating member. Further, it is preferable that the electron emitting element be fixed to the insulating member. Furthermore, the needle-like terminal preferably contacts the lower electrode at its tip. With such a configuration, a voltage can be applied to the lower electrode through the needle-like terminal. Moreover, it can suppress that the contact defect of a needle-like terminal and a lower electrode arises.
  • the charge collection electrode included in the cell stimulation device of the present invention is preferably fixed to the insulating member. With such a configuration, when the insulating member is placed on the culture medium, the charge collection electrode can be brought into contact with the culture medium.
  • the present invention also provides a cell culture apparatus comprising the cell stimulator of the present invention and a culture vessel for containing a culture medium.
  • the cell stimulator comprises an insulating member.
  • the electron emitting element is fixed to the insulating member.
  • the insulating member is disposed on the culture vessel such that the surface electrode is opposed to the surface of the culture medium via the gas phase.
  • the insulating member is preferably provided such that the distance between the surface electrode and the surface of the culture medium is 0.5 mm or more and 3 mm or less. This allows anions generated in the gas phase to be easily supplied to the culture medium.
  • the charge recovery electrode is preferably fixed to a culture vessel. This can suppress the flow of a leak current between the charge collection electrode and the electron emitting element.
  • a voltage is applied between the lower electrode and the surface electrode of an electron-emitting device including the lower electrode, the surface electrode, and the intermediate layer disposed between the lower electrode and the surface electrode.
  • a method of stimulating a cell comprising the step of providing an electrical charge to a medium for culturing cells via the gas phase.
  • the step of supplying the charge is a step of supplying a charge using an electric field between the electron emitter and the culture medium.
  • FIG. 1 is a schematic cross-sectional view of a cell culture device 35 including the cell stimulation device 30 of the present embodiment
  • FIG. 2 is a schematic circuit diagram of the cell culture device 35.
  • the cell stimulation device 30 of the present embodiment is a cell stimulation device 30 for providing stimulation to the cells 3 cultured in the culture medium 2, and the cell stimulation device 30 supplies charge to the culture medium 2 via the gas phase 21.
  • An electron emitting element 4 and a charge collection electrode 9 for recovering charge from the culture medium 2 are provided.
  • the electron emitting element 4 is disposed between the lower electrode 6, the surface electrode 8, the lower electrode 6 and the surface electrode 8
  • the charge recovery electrode 9 is provided so as to be able to contact the culture medium 2.
  • the cell culture device 35 of the present embodiment includes the cell stimulation device 30 of the present embodiment and a culture container 20 for containing the culture medium 2.
  • the lower electrode 6 and the surface of the electron emitting element 4 provided with the lower electrode 6, the surface electrode 8, and the intermediate layer 7 disposed between the lower electrode 6 and the surface electrode 8
  • the step of supplying a charge to the culture medium 2 for culturing the cell 3 through the gas phase 21 by applying a voltage between the electrode 8 and the charge storage step includes the step of supplying the electron emission element 4 and the culture medium 2 Supplying an electric charge using an electric field between them.
  • the charge includes electrons, ions in the gas phase, and ions in the liquid.
  • the cell stimulation device 30, the cell culture device 35, and the cell stimulation method of the present embodiment will be described.
  • the cell stimulation device 30 is a device for applying an electric stimulation to the cells 3 cultured in the medium 2 for the purpose of cell proliferation, differentiation induction and the like.
  • the cells 3 cultured in the medium 2 are, for example, cells / tissues of multicellular organisms, cells into which a gene has been introduced, cells into which a gene has been recombined, or a microorganism.
  • Cell 3 can be a cell responsive to electrical stimulation. Examples of cells responsive to electrical stimulation include muscle cells, nerve cells, osteoblasts, osteoclasts, chondrocytes, osteocytes, fibroblasts and the like.
  • cells precultured in advance can be used for cells 3 cultured in advance can be used.
  • the precultured cells can be seeded on the medium 2 such that the medium 2 contains 10 3 to 10 6 cells / cm 2 of cells 3.
  • Medium 2 provides cells 3 with a growth environment.
  • the medium 2 can be used without particular limitation as long as it is a commonly used cell culture medium.
  • the medium 2 may be a liquid medium, or may be a solid medium in which the liquid medium is solidified with a gelling agent such as agar.
  • the medium 2 may be a combination of a liquid medium and a solid medium.
  • Medium 2 may be, for example, MEM medium (Eagle minimal essential medium), D-MEM medium (Dulbecco modified Eagle medium), ⁇ -MEM medium (Eagle minimal essential medium ⁇ modified), GMEM (Glasgow minimal essential medium), Ham's F- 12 (Nutrient Mixture F-12 Ham), IMDM (Iscove's Modified Dulbecco's Medium), RPMI-1640 Medium, D-PBS (Dulbecco's Phosphate Buffered Saline), HBSS (Hanks Balanced Salt Solution), and the like.
  • MEM medium Eagle minimal essential medium
  • D-MEM medium Dulbecco modified Eagle medium
  • ⁇ -MEM medium Eagle minimal essential medium ⁇ modified
  • GMEM Gasgow minimal essential medium
  • Ham's F- 12 Nutrient Mixture F-12 Ham
  • IMDM Iscove's Modified Dulbecco's Medium
  • RPMI-1640 Medium D-PBS (Dulbec
  • the medium 2 is accommodated in the culture vessel 20.
  • the culture vessel 20 is, for example, a multiwell plate provided with a plurality of wells 12, a culture dish, or the like. Furthermore, by combining the culture container 20 and the cell stimulation device 30, the cell culture device 35 can be formed.
  • a material of the culture vessel 20 glass, plastic or the like which is an existing material can be used.
  • the culture vessel 20 is preferably one provided with a surface treatment suitable for the fixation of the cells 3 on the bottom of the vessel.
  • the surface treatment can be generally known, such as matrix protein, collagen, fibronectin, laminin, etc., applied to the bottom of the container.
  • Cell culture apparatus 35 can be placed in incubator 18. By this, the temperature of culture medium 2, atmosphere gas, etc. can be controlled, and cell 3 can be cultured stably.
  • the culture atmosphere is, for example, air containing temperature: 37 ° C., relative humidity: 95%, atmosphere gas: 5% carbon dioxide gas.
  • the cell stimulator 30 comprises an electron emitting element 4 for supplying a charge to the culture medium 2 via the gas phase 21.
  • the electron emitting device 4 is a device having a planar shape and capable of emitting electrons.
  • the electron emitter 4 includes a lower electrode 6, a surface electrode 8, and an intermediate layer 7 disposed between the lower electrode 6 and the surface electrode 8. Further, a voltage can be applied between the surface electrode 8 and the lower electrode 6 by the power supply 11 b to generate an electric field in the intermediate layer 7. Electrons can flow in the intermediate layer 7 by this electric field, and electrons can be emitted from the surface electrode 8 of the electron emitting element 4. The electrons generate charges such as oxygen anions in the gas phase 21. In addition, this anion is irradiated to the culture medium 2.
  • a rectangular wave AC voltage with a frequency of 500 to 10000 Hz can be applied between the surface electrode 8 and the lower electrode 6 by the power supply 11 b with a peak value of 14V 0-p to 24V 0-p .
  • the on / off duty ratio can be variable from 1 to 100%.
  • the ion irradiation dose from the electron emitter 4 can be 0.5 to 5.0 ⁇ A / cm 2 . If it is less than 0.5 ⁇ A / cm 2 , the culture is slowed, and if it is more than 5.0 ⁇ A / cm 2 , the possibility of death is large and the culture is slow.
  • the ion irradiation dose during driving is set to a substantially constant value (the fluctuation range is within ⁇ 10%, preferably within ⁇ 5%), and the drive voltage peak value or the rectangular wave duty ratio is used as a parameter to satisfy the irradiation dose. Control can be performed.
  • the culture can be stably advanced by setting the ion irradiation amount to a substantially constant value.
  • the surface electrode 8 is an electrode located on the surface of the electron emitter 4.
  • the surface electrode 8 can have a thickness of 5 nm or more and 100 nm or less, preferably 40 nm or more and 100 nm or less.
  • the material of the surface electrode 8 is, for example, gold or platinum. This can suppress oxidation of the surface electrode 8 by autoclave sterilization or the like.
  • the surface electrode 8 may be comprised from several metal layers.
  • the surface electrode 8 may have a plurality of openings, gaps, and portions thinned to a thickness of 10 nm or less, even in the case of the preferable 40 nm or more.
  • Electrons having flowed through the intermediate layer 7 can pass through or pass through the opening, the gap, the thinned portion, and can emit electrons from the surface electrode 8.
  • Such openings, gaps, and thinned portions can be formed by applying a voltage between the lower electrode 2 and the surface electrode 4 (forming process, initial voltage application).
  • the lower electrode 6 is an electrode facing the surface electrode 8 via the intermediate layer 7.
  • the lower electrode 6 may be a metal plate, or may be a plate made of an insulator having a metal layer or a conductor layer. When the lower electrode 6 is made of a metal plate, the metal plate may be a substrate of the electron emitting element 4.
  • the material of the lower electrode 6 is, for example, aluminum, stainless steel, nickel or the like.
  • the thickness of the lower electrode is, for example, 200 ⁇ m or more and 1 mm or less.
  • the intermediate layer 7 is a layer through which electrons flow due to an electric field formed by applying a voltage to the surface electrode 8 and the lower electrode 6.
  • the intermediate layer 7 can have semiconductivity.
  • the intermediate layer 7 contains at least one of an insulating resin, a conductive resin, and an insulating fine particle.
  • middle layer 7 contains electroconductive fine particles.
  • the thickness of the intermediate layer 7 can be, for example, 1 ⁇ m or more and 1.8 ⁇ m or less. Since the electrons flowing through the intermediate layer 7 are emitted from the surface electrode 8, the electron emitting element 4 can emit electrons from the surface electrode 8. Therefore, electrons can be uniformly emitted to the gas phase 21 on the surface electrode 8, and charges such as anions of oxygen can be generated by the electrons.
  • the electron emitter 4 may have an insulating layer 5 between the surface electrode 8 and the lower electrode 6.
  • the insulating layer 5 can have an opening.
  • the opening of the insulating layer 5 is provided to correspond to the region of the surface electrode where it is desired to emit electrons. Since electrons can not flow in the insulating layer 5, electrons flow in the intermediate layer 7 corresponding to the opening of the insulating layer 5, and electrons are emitted from the surface electrode 8. Therefore, the electron emission region can be formed on the surface electrode 8 by providing the insulating layer 5.
  • the electron emitting element 4 can be removably fixed to the insulating member 13.
  • the surface electrode 8 of the electron emitting element 4 is opposed to the surface of the culture medium 2 in the culture vessel 20 via the gas phase 21. 4 can be arranged.
  • the electron-emitting device 4 By arranging the electron-emitting device 4 in this manner, the electrons emitted from the surface electrode 8 side of the electron-emitting device 4 cause oxygen anions and the like in the gas phase 21 between the electron-emitting device 4 and the culture medium 2. It can generate charge.
  • the electron emitting element 4 can be arranged such that the surface electrode 8 and the surface of the culture medium 2 are substantially parallel.
  • the insulating member 13 may be integrated with the lid member 10.
  • the insulating member 13 can be provided so that the insulating member 13 protrudes from the lid member 10 to the culture medium 2 side (in the well 12).
  • the electron emitting element 4 can be fixed to the tip end surface of the insulating member 13 protruding from the lid member 2 so as to be parallel to the surface of the culture medium 2.
  • the material of the insulating member 13 and the lid member 10 is preferably a PEEK material resistant to heat and a fluorine-based material. The parts facing the cells are previously sterilized using an autoclave. Because of the treatment with high temperature steam above 150 ° C., it is necessary to withstand repeated thermal deformation of the material.
  • the lid member 10 is provided so that the cell stimulator 30 can be placed on the culture vessel 20. Moreover, the cover member 10 can have insulation.
  • the insulating member 13 or the lid member 10 can be provided such that the distance between the surface electrode 8 and the surface of the culture medium 2 is 0.5 mm or more and 3 mm or less. As a result, anions generated in the gas phase 21 can be easily supplied to the culture medium 2.
  • the distance between the surface electrode 8 and the surface of the culture medium 2 can be preferably 1 mm or more and 2 mm or less. For example, the distance between the surface electrode 8 and the surface of the culture medium 2 by adjusting the depth of the well of the culture vessel 20, the depth of the culture medium 2, the height of the insulating member 13 projecting from the lid member 10, etc. Can be adjusted.
  • the electron emitting element 4 by causing the electron emitting element 4 to face the surface of the culture medium 2 via the gas phase 21, it is possible to suppress inhibition of the supply of oxygen gas from the gas phase 21 to the culture medium 2. For this reason, it becomes possible to maintain the amount of dissolved oxygen of the culture medium 2. Further, the electron emitting device 4 can be easily replaced by detachably fixing the electron emitting device 4 to the insulating member 3.
  • the cell stimulator 30 can have a first terminal 15 and a second terminal 16 fixed to the insulating member 13.
  • the first terminal 15 and the second terminal 16 each have a contact point in contact with the electron emitter 4.
  • the electron-emitting device 4 is attached to the insulating member 3
  • the first terminal 15 is electrically connected to the lower electrode 6
  • the second terminal 16 is electrically connected to the surface electrode 8.
  • a second terminal and an electron emitter 4 can be provided.
  • a voltage can be applied between the surface electrode 8 and the lower electrode 6 through the first and second terminals.
  • the charge recovery electrode 9 is provided so as to be able to contact the medium 2 for culturing the cells 3. Therefore, electrons can flow from the culture medium 2 to the charge collection electrode 9, and the potentials of the charge collection electrode 9 and the culture medium 2 can be made equal or almost equal. For example, if the charge recovery electrode 9 is connected to ground, the culture medium 2 can be prevented from being charged.
  • the charge generated by the electron emitting element 4 is supplied to the culture medium 2 via the gas phase 21, so the electrode contacting the culture medium 2 is only the charge collection electrode 9. For this reason, it can suppress that the water contained in the culture medium 2 electrolyzes.
  • the charge collection electrode 9 has a positive polarity, the mineral components (K + , Ca 2+ , Na + , Mg 2+, etc.) in the culture medium 2 are not precipitated on the charge collection electrode 9. It is possible to suppress the change in component composition.
  • the charge collection electrode 9 can be fixed to, for example, the insulating member 13 or the lid member 10.
  • the charge collection electrode 9 can be provided so that the charge collection electrode 9 contacts the culture medium 2 when the insulating member 13 and the lid member 10 are installed on the culture vessel 20.
  • the shape of the charge collection electrode 9 is, for example, a bar, a plate, a mesh, or a punching metal.
  • the charge collection electrode 9 may have a structure in which a flat plate, a mesh or a punching metal is attached to the end of a rod.
  • the flat plate, mesh or punching metal attached to the tip of the rod included in the charge recovery electrode 9 may be disposed at the bottom of the well 12 so that the cell 3 is located between the charge recovery electrode 9 and the electron emitting element 4 it can.
  • the anions generated on the surface of the culture medium 2 by the electron emission of the electron emitter 4 flow toward the bottom of the well 12. For this reason, it is possible to efficiently provide electrical stimulation to the cells 3 cultured in the medium 2.
  • an electric field can be generated in the gas phase 21 between the surface electrode 8 of the electron-emitting device 4 and the surface of the culture medium 2.
  • the anions generated by the electron emitting element 4 emitting electrons can be transported to the surface of the culture medium 2 along electric lines of force (gradient of the electric field strength) in the electric field of the gas phase 21 ( current can flow to the gas phase 21), the medium 2 in the anion (OH -, Cl -, O 2 - , etc.) can be generated. Electrical stimulation can be given to the cells 3 cultured in the medium 2 by this anion. Moreover, since this anion moves the culture medium 2 and flows to the charge collection electrode 9 (a current flows to the culture medium 2), it is possible to suppress the culture medium 2 from being charged.
  • the charge collection electrode 9 can be connected to ground, and a DC voltage of -1000 V to -50 V can be applied between the electron-emitting device 4 and the ground by the power supply 11a.
  • the distance between the surface electrode 8 of the electron emitting element 4 and the surface of the culture medium 2 can be 0.5 mm to 3 mm (preferably 1 mm or more and 2 mm or less). Since the charge collection electrode 9 is connected to ground, the potential of the culture medium 2 becomes almost 0 V, and the potential of the surface electrode 8 becomes ⁇ 1000 V to ⁇ 50 V. Further, since the distance between the culture medium 2 and the surface electrode 8 is 0.5 mm to 3 mm, an electric field of strong electric field strength can be generated in the gas phase 21 between the culture medium 2 and the surface electrode 8. The anions generated by the electron emitter 4 emitting electrons using this electric field can be supplied to the culture medium 2.
  • the power supply devices 11a and 11b supply a charge from the electron-emitting device 4 to the culture medium 2, and a current generated in the gas phase 21 by the charge recovery electrode 9 recovering the charge from the culture medium 2 It can be electrically connected to the electron emitting element 4 and the charge collection electrode 9 so as to be a loop current. For example, as shown in FIGS.
  • the ion B gas phase 21 by an electric field caused by applying a voltage V e between the surface electrode 8 with the power supply 11a and the charge collection electrode 9 - (ion B - by electrons emitted from the surface electrode 8 occurs in the gas phase 21) current is generated in the gas phase 21 by move, ion B reaching the medium 2 - penetration into the medium 2, the cells It stimulates signal transduction system involving the various elements forming, finally ion C - (ion C - ion B - itself and / or ion B - another ionic species derived from) moving to the charge collection electrode 9 as Generates a current in the medium 2. Therefore, a loop current can be supplied to the circuit including the power supply device 11 a, the surface electrode 8, the gas phase 21 and the culture medium 2.
  • anions can be uniformly generated in the gas phase 21 between the surface of the culture medium 2 and the surface electrode 8. Since this anion can be supplied to the culture medium 2 by the electric field, the anion can be uniformly generated in the culture medium 2 located below the surface electrode 8 and the cells 3 located below the surface electrode 8 Can be given electrical stimulation uniformly. For this reason, electrical stimulation can be uniformly applied to cells to which electrical stimulation is desired.
  • the electrical stimulation (ion irradiation) by the cell stimulator 30 can be started, for example, from the timing when the seeded cells start cell division.
  • the ion irradiation to the medium 2 in which the cells 3 are fixed can be performed by setting the irradiation amount, the irradiation interval, and the irradiation timing suitable for the cell type.
  • the time for which electrons are emitted from the electron emission element 4 can be, for example, 5 seconds or more and 1 minute or less.
  • FIG. 3 is a schematic perspective view of a cell stimulation device 30 of the present embodiment
  • FIG. 4 is a schematic exploded view of the cell stimulation device 30 of the present embodiment.
  • the electron-emitting device 4 included in the cell stimulation device 30 as shown in FIG. 3 is provided so as to be removable from the insulating member 13 as shown in the exploded view of FIG.
  • the intermediate layer 7 and the surface electrode 8 are stacked on the metal plate which is the lower electrode 6.
  • the lower electrode 6 is a metal plate of 12 mm ⁇ 24 mm square and 0.5 mm thick.
  • the material of the lower electrode 6 is, for example, aluminum, stainless steel, nickel or the like.
  • the thickness of the intermediate layer 7 is, for example, 1.0 ⁇ m to 1.8 ⁇ m.
  • an insulating layer 5 having an opening in a portion where the electron emission region 25 is formed is laminated between the lower electrode 6 and the surface electrode 8. Since no current flows in the insulating layer 5, electrons are emitted only from the electron emitting region 25 of the surface electrode 8 corresponding to the opening of the insulating layer 5.
  • the opening of the insulating layer 5 can be, for example, 5 mm square.
  • inorganic materials such as metal oxides and metal nitrides, and organic materials such as silicone resins and phenol resins can be used.
  • the surface electrode 8 can have a thickness of 40 nm or more and 100 nm or less.
  • the size of the surface electrode 8 can be 18 mm ⁇ 8.5 mm square. Also, the size of the electron emission region can be 5 mm square.
  • the material of the surface electrode 8 is, for example, gold or platinum.
  • the first terminals 15 a and 15 b are disposed in the openings provided in the insulating member 13 and have a needle shape, and their tip end portions are in contact with the back surface of the lower electrode 6 of the electron emitting element 4. Therefore, a voltage can be applied to the lower electrode 6 through the first terminal 15. Further, by setting the tip end portion of the first terminal 15 as a contact, it is possible to suppress the occurrence of a contact failure between the first terminal 15 and the lower electrode 6. Further, the first terminal 15 may be a spring type needle-like terminal. This can suppress the occurrence of contact failure between the first terminal 15 and the lower electrode 6.
  • the cell stimulator 30 is sterilized using an autoclave before culturing the cells 3 in the cell culture device 35.
  • a thin oxide film may be formed on the surface of a metal member (for example, a metal plate which is the lower electrode 6) included in the cell stimulation device 30 by this treatment.
  • This oxide film causes the contact failure.
  • the second terminal 16 is provided to fix the electron emitting element 4 to the insulating member 13, and contacts the surface electrode 8 of the electron emitting element 4. Therefore, a voltage can be applied to the surface electrode 8 through the second terminal 16.
  • the second terminal 16 and the first terminal 15 described above may be a metal plate having a gold plating layer on the surface. By this, it can suppress that an oxide film is formed in the surface of the 2nd terminal 16 by autoclave sterilization, and can suppress that a contact failure arises.
  • the insulating member 13 has a slit structure on its surface. Therefore, leakage current (edge surface leakage) is generated between the metal member electrically connected to the charge collection electrode 9 and the electrode 9 and the metal member electrically connected to the electron emitting element 4 and the element 4. It can be suppressed.
  • the other configuration is the same as that of the first embodiment.
  • the description of the first embodiment described above applies to the second embodiment as long as there is no contradiction.
  • the slit structure on the surface of the insulating member 13 can be applied to other structures such as the first embodiment.
  • the electron-emitting device 4 has a rectangular shape (the electron-emitting regions 25a and 25b are square). However, depending on the situation, a circular shape or a star-like shape is also possible.
  • to use the medium 2 as efficiently as possible in the form of the medium 2 (however, that part is missing because contact with the charge recovery electrode 9 is impossible) Is also possible.
  • FIG. 5 is a schematic top view of the electron-emitting device 4 included in the cell stimulation device 30 of the present embodiment.
  • the lower electrode 6 included in the electron emitting element 4 is composed of a plurality of elongated electrodes, and in FIG. 5, for example, is composed of electrodes 6a to 6j.
  • the electrodes 6a to 6j can be arranged to be parallel. These electrodes 6a to 6j may be, for example, metal layers or conductor layers formed on an insulating substrate. This allows the electrodes 6a to 6j to be electrically separated. Further, different terminals can be connected to the electrodes 6a to 6j.
  • the surface electrode 8 included in the electron emitting element 4 is composed of a plurality of elongated electrodes, and in FIG.
  • the electrodes 8a to 8j are composed of electrodes 8a to 8j.
  • the electrodes 8a to 8j can be arranged in parallel.
  • the electrodes 8a to 8j may be, for example, a metal layer or a conductor layer formed on the intermediate layer 7. Further, different terminals can be connected to the electrodes 8a to 8j.
  • the lower electrode 6 and the surface electrode 8 are arranged in a grid so that one of the electrodes 6a to 6j and the electrodes 8a to 8j is a row and the other is a column.
  • An insulating layer 5 having an opening at the intersection of the electrodes 6a to 6j and the electrodes 8a to 8j is stacked between the lower electrode 6 and the intermediate layer 7.
  • the electron emission region 25 can be formed on the surface electrode 8 at a portion where the electrodes 6a to 6j and the electrodes 8a to 8j intersect (a portion corresponding to the opening of the insulating layer 5).
  • These electron emission regions 25 are arranged in a matrix.
  • Electrons can be emitted in the electron emission region 25 of the For example, when a voltage is applied only between the electrodes 6a to 6e and the electrodes 8a to 8e, electrons are emitted in the electron emission region 25 at the intersection of these electrodes, but no electrons are emitted in the other electron emission regions 25. . Therefore, electrical stimulation can be applied only to cells in the culture medium located below the electron emission region 25 that emits electrons. That is, it becomes possible to apply electrical stimulation only to the cells 3 present in the selected area of the medium 2.
  • the amount of electron emission can be changed in the plurality of electron emission regions 25 arranged in a matrix.
  • an AC voltage of 20 V is applied between the electrodes 6a-6e and the electrodes 8a-8e
  • an AC voltage of 10 V is applied between the electrodes 6f-6j and the electrodes 8f-8j.
  • a potential difference of 15 V can be applied between ⁇ 6e and electrodes 8f ⁇ 8j and between electrodes 6f 6 6j and electrodes 8a 8 8e.
  • the amount of electron emission is relatively large in the electron emission region 25 at the intersection of the electrodes 6a to 6e and the electrodes 8a to 8e, and electron emission in the electron emission region 25 at the intersection of the electrodes 6f to 6j and the electrodes 8f to 8j.
  • the amount is relatively small.
  • the amount of electron emission in the electron emission region 25 at the intersection of the electrodes 6a to 6e and the electrodes 8f to 8j and in the electron emission region 25 at the intersection of the electrodes 6f to 6j and the electrodes 8a to 8e is about middle.
  • the intensity of the electrical stimulation applied to the cells 3 in the medium 2 also changes corresponding to the amount of emitted electrons.
  • FIG. 6 is a schematic cross-sectional view of a cell culture apparatus 35 of the present embodiment.
  • the charge collection electrode 9 is fixed to the culture vessel 20.
  • the electron emitter 4 is fixed to the insulating member 13. With such a configuration, it is possible to suppress a leak current from flowing between the charge collection electrode 9 and the electron emitting element 4.
  • the charge recovery electrode 9 is disposed at the bottom of the well 12, and the cell 3 is located between the charge recovery electrode 9 and the electron-emitting device 4. Therefore, anions generated on the surface of the medium 2 by the ion irradiation of the cell stimulation device 30 flow toward the bottom of the well 12. For this reason, it is possible to efficiently provide electrical stimulation to the cells 3 cultured in the medium 2.
  • the other configuration is the same as in the first to third embodiments.
  • the descriptions of the first to third embodiments also apply to the fourth embodiment unless there is a contradiction.
  • FIG. 7 is a schematic cross-sectional view of a cell culture apparatus 35 of the present embodiment.
  • the culture vessel 20 is provided with a plurality of wells 12a to 12c.
  • the cell stimulation device 30 is provided with insulating members 13a to 13c corresponding to the respective wells 12a to 12c, electron emitting elements 4a to 4c, and charge collecting electrodes 9a to 9c.
  • electrical stimulation can be given to the cells cultured in each well 12a to 12c of the culture container 20.
  • the switching circuit it is possible to perform the electrical stimulation of the cells only at the necessary places among the plurality of wells 12 at the necessary time and at the necessary timing.
  • the number of wells 12 provided in the culture vessel 20 is not particularly limited.
  • the culture vessel 20 is a 6 well plate.
  • the cell stimulator 30 may include the insulating member 13 corresponding to all the wells 12 of the culture vessel 20, the electron emitting element 4 and the charge collection electrode 9, and corresponds to a part of the wells 12 of the culture vessel 20. Insulating member 13, electron emitting element 4 and charge collection electrode 9 may be provided.
  • the cell stimulation device 30 can have a structure in which a plurality of insulating members 13 are fixed to one lid member 10. The other configuration is the same as in the first to fourth embodiments.
  • the descriptions of the first to fourth embodiments described above also apply to the fifth embodiment unless there is a contradiction.

Abstract

The cell stimulation device according to the present invention is for stimulating a cell cultured in a culture medium, and is characterized by being provided with an electron emission element which feeds electric charge to the culture medium via a gas phase, and an electric charge recovery electrode which recovers electric charge from the culture medium, wherein the electron emission element is provided with a lower electrode, a surface electrode, and an intermediate layer disposed between the lower electrode and the surface electrode, and the electric charge recovery electrode is disposed so as to be capable of making contact with the culture medium.

Description

細胞刺激装置、細胞培養装置及び細胞刺激方法Cell stimulation device, cell culture device and cell stimulation method
　本発明は、細胞刺激装置、細胞培養装置及び細胞刺激方法に関する。 The present invention relates to a cell stimulation device, a cell culture device and a cell stimulation method.
　電気刺激に対して反応のある培養細胞に電気刺激を与える細胞培養方法が知られている（例えば、特許文献１参照）。この細胞培養方法では、電気刺激により細胞の代謝が促進され、細胞の増殖速度を速めることができる。
　一方、下部電極と表面電極との間に電圧を印加することにより中間層に電子を流し、電子を放出させる電子放出素子が知られている（例えば、特許文献２参照）。また、電子放出素子により放出させた電子により酸素の陰イオンを発生させ、この陰イオンを気体の流れにのせて生体に向けて噴出する陰イオン生成装置が知られている（例えば、特許文献３参照）。 There is known a cell culture method of providing electrical stimulation to cultured cells that are responsive to electrical stimulation (see, for example, Patent Document 1). In this cell culture method, electrical stimulation promotes cell metabolism and can accelerate cell proliferation.
On the other hand, an electron-emitting device is known in which electrons are made to flow in the intermediate layer by applying a voltage between the lower electrode and the surface electrode to emit the electrons (see, for example, Patent Document 2). Further, there is known an anion generating apparatus which generates anions of oxygen by electrons emitted by the electron emitting element and places the anions on a flow of gas and jets them toward a living body (for example, Patent Document 3) reference).
特開昭６０－１１０２８７号公報Japanese Patent Application Laid-Open No. 60-110287 特開２０１６－１３６４８５号公報JP, 2016-136485, A 特開２００６－３２５４９３号公報Japanese Patent Application Laid-Open No. 2006-325493
　培養細胞に電気刺激を与える従来の細胞培養方法では、培地内に少なくとも２枚の電極を設置し、この２枚の電極の間に電圧を印加する。このため、電極と培養液との界面において電気化学反応が生じ、水の電気分解や培養液成分の分解・析出などを生じる場合がある。また、電極間距離の短い部分に局所的に電流が流れやすく、電気刺激を与えたい培養細胞に一様に電気刺激を与えることが難しい。
　また、従来の陰イオン生成装置では、陰イオンを気体と共に噴出させるため、電気刺激を与えたい細胞に一様に電荷を供給することが難しい。また、陰イオン生成装置から噴出させた気体により培地の水分が蒸発しやすく、培地が乾燥するという問題がある。また、細胞を培養する雰囲気ガスのガス種条件を維持することが難しい。
　本発明は、このような事情に鑑みてなされたものであり、電気刺激を与えたい細胞に一様に電気刺激を与えることができ、培地成分の分解・析出を抑制することができる細胞刺激装置を提供する。 In a conventional cell culture method of providing electrical stimulation to cultured cells, at least two electrodes are placed in a medium, and a voltage is applied between the two electrodes. For this reason, an electrochemical reaction may occur at the interface between the electrode and the culture solution, resulting in the electrolysis of water or the decomposition / precipitation of the components of the culture solution. In addition, it is easy for current to flow locally in a portion where the distance between the electrodes is short, and it is difficult to uniformly apply electrical stimulation to cultured cells to which electrical stimulation is desired.
Moreover, in the conventional anion generator, it is difficult to uniformly supply the charge to the cells to which the electrical stimulation is to be applied, because the anions are ejected together with the gas. In addition, there is a problem that the water of the culture medium is easily evaporated by the gas ejected from the anion generator, and the culture medium is dried. In addition, it is difficult to maintain the gas species conditions of the atmosphere gas for culturing the cells.
The present invention has been made in view of such circumstances, and it is possible to uniformly apply electrical stimulation to cells to which electrical stimulation is to be given, and to suppress cell decomposition and deposition of culture medium components. I will provide a.
　本発明は、培地で培養する細胞に刺激を与えるための細胞刺激装置であって、前記細胞刺激装置は、前記培地に気相を介して電荷を供給する電子放出素子と、前記培地から電荷を回収する電荷回収電極とを備え、前記電子放出素子は、下部電極と、表面電極と、前記下部電極と前記表面電極との間に配置された中間層とを備え、前記電荷回収電極は、前記培地に接触することができるように設けられたことを特徴とする細胞刺激装置を提供する。 The present invention is a cell stimulation device for stimulating cells cultured in a culture medium, wherein the cell stimulation device comprises an electron emitting element for supplying a charge to the culture medium via a gas phase, and a charge from the culture medium The electron emission device includes a lower electrode, a surface electrode, and an intermediate layer disposed between the lower electrode and the surface electrode, and the charge recovery electrode includes the charge recovery electrode. Provided is a cell stimulation device characterized in that it is provided so as to be able to contact a culture medium.
　本発明の細胞刺激装置に含まれる電子放出素子は、下部電極と、表面電極と、下部電極と表面電極との間に配置された中間層とを備えるため、下部電極と表面電極との間に電圧を印加することにより中間層を流れた電子を表面電極側から放出させることができる。この放出させた電子により、電子放出素子と培地との間の気相に酸素の陰イオンなどの電荷を発生させることができる。
　本発明の細胞刺激装置に含まれる電荷回収電極は、細胞を培養する培地に接触することができるように設けられるため、培地から電荷回収電極へ電子が流れることができる。このため、電荷回収電極と培地との電位差を小さくすることができる。また、電子放出素子又は電荷回収電極に電圧を印加することにより、電子放出素子の表面電極と培地の表面との間の気相に電界を生じさせることができる。この電界の中の電気力線（電界強度の勾配）に沿って、発生させた陰イオンを培地の表面に搬送することができ、培地に陰イオンを供給することができる。この陰イオンにより培地で培養する細胞に電気刺激を与えることができる。また、この陰イオンは、電荷回収電極へと流れるため、培地が帯電することを抑制することができる。また、本発明の細胞刺激装置では、気相に生じさせた電界を利用して陰イオンを培地に供給するため、窒素ガスやアルゴンガスのガスフロー機構を必要としない。このため、細胞の電気刺激時に、培地の乾燥や細胞浮遊を引き起こす心配がない上、培養雰囲気のガス組成に影響を与える事がない。 Since the electron emitting element included in the cell stimulation device of the present invention includes the lower electrode, the surface electrode, and the intermediate layer disposed between the lower electrode and the surface electrode, the electron emitting device is interposed between the lower electrode and the surface electrode By applying a voltage, electrons flowing through the intermediate layer can be emitted from the surface electrode side. The emitted electrons can generate charges such as anions of oxygen in the gas phase between the electron emitting element and the culture medium.
The charge collection electrode included in the cell stimulation device of the present invention is provided so as to be able to contact the medium for culturing the cells, so that electrons can flow from the medium to the charge collection electrode. Therefore, the potential difference between the charge collection electrode and the culture medium can be reduced. In addition, by applying a voltage to the electron-emitting device or the charge collection electrode, an electric field can be generated in the gas phase between the surface electrode of the electron-emitting device and the surface of the culture medium. The generated anions can be transported to the surface of the culture medium along the electric field lines (gradient of the electric field strength) in this electric field, and the culture medium can be supplied with the anions. The anion can provide electrical stimulation to cells cultured in the medium. In addition, since this anion flows to the charge collection electrode, it is possible to suppress the culture medium from being charged. In addition, in the cell stimulating apparatus of the present invention, in order to supply anions to the culture medium using the electric field generated in the gas phase, a gas flow mechanism of nitrogen gas or argon gas is not required. For this reason, there is no concern that drying of the culture medium or cell suspension will occur when the cells are electrically stimulated, and the gas composition of the culture atmosphere will not be affected.
　本発明の細胞刺激装置に含まれる電子放出素子は、表面電極から電子を面放出させることができるため、培地の表面と表面電極との間の気相に一様に陰イオンを発生させることができる。このため、表面電極の下部に位置する培地にも一様に陰イオンを供給することができ、表面電極の下部に位置する細胞に一様に電気刺激を与えることができる。
　本発明の細胞刺激装置では、電子放出素子により発生させた電荷を気相を介して培地に供給するため、培地に接触する電極は電荷回収電極だけである。このため、培地に含まれる水が電気分解することを抑制することができる。また、電荷回収電極は正極性となるため、培地中のミネラル成分（K+, Ca2+, Na+, Mg2+等）が電荷回収電極上に析出することを抑制することができ、培地の成分組成が変化することを抑制することができる。 The electron-emitting device included in the cell stimulation device of the present invention can emit electrons from the surface electrode, so that uniform anions can be generated in the gas phase between the surface of the culture medium and the surface electrode. it can. Therefore, anions can be uniformly supplied to the culture medium located below the surface electrode, and electrical stimulation can be uniformly applied to cells located below the surface electrode.
In the cell stimulation apparatus of the present invention, the charge generated by the electron emitting element is supplied to the culture medium through the gas phase, and therefore, the electrode contacting the culture medium is only the charge collection electrode. For this reason, it can suppress that the water contained in a culture medium electrolyzes. In addition, since the charge collection electrode has a positive polarity, precipitation of mineral components (K + , Ca 2+ , Na + , Mg 2+ etc.) in the medium can be suppressed on the charge collection electrode. It is possible to suppress the change in the component composition of
本発明の一実施形態の細胞培養装置の概略断面図である。It is a schematic sectional drawing of the cell culture apparatus of one Embodiment of this invention. 本発明の一実施形態の細胞培養装置の概略回路図である。It is a schematic circuit diagram of the cell culture device of one embodiment of the present invention. 本発明の一実施形態の細胞刺激装置の概略斜視図である。It is a schematic perspective view of the cell stimulator of one embodiment of the present invention. 本発明の一実施形態の細胞刺激装置の概略分解図である。FIG. 1 is a schematic exploded view of a cell stimulation device according to an embodiment of the present invention. 本発明の一実施形態の細胞刺激装置に含まれる電子放出素子の概略上面図である。It is a schematic top view of the electron-emitting device contained in the cell stimulation apparatus of one Embodiment of this invention. 本発明の一実施形態の細胞培養装置の概略断面図である。It is a schematic sectional drawing of the cell culture apparatus of one Embodiment of this invention. 本発明の一実施形態の細胞培養装置の概略断面図である。It is a schematic sectional drawing of the cell culture apparatus of one Embodiment of this invention.
　本発明の細胞刺激装置は、培地で培養する細胞に刺激を与えるための細胞刺激装置であって、前記細胞刺激装置は、前記培地に気相を介して電荷を供給する電子放出素子と、前記培地から電荷を回収する電荷回収電極とを備え、前記電子放出素子は、下部電極と、表面電極と、前記下部電極と前記表面電極との間に配置された中間層とを備え、前記電荷回収電極は、前記培地に接触することができるように設けられたことを特徴とする。 The cell stimulation apparatus of the present invention is a cell stimulation apparatus for stimulating cells cultured in a culture medium, wherein the cell stimulation apparatus comprises an electron emitting element for supplying a charge to the culture medium via a gas phase, and A charge recovery electrode for recovering charge from the culture medium, the electron emitting device comprising a lower electrode, a surface electrode, and an intermediate layer disposed between the lower electrode and the surface electrode, the charge recovery electrode An electrode is characterized in that it is provided so as to be able to contact the culture medium.
　電子放出素子に含まれる下部電極及び表面電極は、それぞれ複数の細長い電極を含み、下部電極及び表面電極のうち一方が行となり他方が列となる格子状に配置されることが好ましい。このような構成とすると、下部電極と表面電極の任意の交点から電子を放出することが可能になり、培地の所望の箇所に存在する細胞にだけ電気刺激を与えることが可能である。また、電気刺激量に所望の分布を持たせて培地中の細胞を電気刺激することが可能になる。 The lower electrode and the surface electrode included in the electron emitting element preferably include a plurality of elongated electrodes, and it is preferable that the lower electrode and the surface electrode be arranged in a grid in which one of the lower electrode and the surface electrode is a row and the other is a column. With such a configuration, it is possible to emit electrons from any intersection of the lower electrode and the surface electrode, and it is possible to apply electrical stimulation only to cells present at desired locations in the culture medium. In addition, it becomes possible to electrically stimulate cells in the culture medium with a desired distribution of the amount of electrical stimulation.
　本発明の細胞刺激装置は電源装置を備えることが好ましい。電源装置は、下部電極と表面電極との間に電圧を印加することができるように設けられることが好ましい。このことにより、中間層に電界を生じさせることができ、この電界により、中間層に電子を流すことができ、表面電極から電子を放出することができる。また、この電子により気相に陰イオンを発生させることができる。また、電源装置は、電子放出素子と電荷回収電極との間に電位差を生じさせることができるように設けられることが好ましい。このことにより、表面電極と培地の表面との間に電界を生じさせることができ、この電界により陰イオンを培地に搬送することができる。
　前記電源装置は、電子放出素子から培地へ電荷を供給することにより気相に生じる電流と、電荷回収電極が培地から電荷を回収することにより培地に生じる電流とがループ電流となるように電子放出素子及び電荷回収電極と電気的に接続することが好ましい。このことにより、電荷を効率よく培地に供給することができ、適切に細胞を刺激することができる。 Preferably, the cell stimulator of the present invention comprises a power supply. The power supply device is preferably provided such that a voltage can be applied between the lower electrode and the surface electrode. By this, an electric field can be generated in the intermediate layer, electrons can flow in the intermediate layer, and electrons can be emitted from the surface electrode. In addition, negative ions can be generated in the gas phase by the electrons. In addition, the power supply device is preferably provided so that a potential difference can be generated between the electron emitting element and the charge collection electrode. By this, an electric field can be generated between the surface electrode and the surface of the culture medium, and the anion can be transported to the culture medium by this electric field.
The power supply device emits electrons so that the current generated in the gas phase by supplying charges from the electron emitting element to the medium and the current generated in the medium by collecting charges from the medium by the charge recovery electrode become loop currents. It is preferable to electrically connect the element and the charge collection electrode. As a result, charges can be efficiently supplied to the medium, and cells can be appropriately stimulated.
　本発明の細胞刺激装置は、絶縁性部材と絶縁性部材に固定された針状端子とを備えることが好ましい。また、電子放出素子は絶縁性部材に固定されることが好ましい。さらに、針状端子は、その先端部で下部電極と接触することが好ましい。このような構成により、針状端子を介して下部電極に電圧を印加することができる。また、針状端子と下部電極との接点不良が生じることを抑制することができる。
　本発明の細胞刺激装置に含まれる電荷回収電極は、絶縁性部材に固定されることが好ましい。このような構成により、絶縁性部材を培地上に配置した際に電荷回収電極と培地とを接触させることができる。 The cell stimulation device of the present invention preferably comprises an insulating member and a needle-like terminal fixed to the insulating member. Further, it is preferable that the electron emitting element be fixed to the insulating member. Furthermore, the needle-like terminal preferably contacts the lower electrode at its tip. With such a configuration, a voltage can be applied to the lower electrode through the needle-like terminal. Moreover, it can suppress that the contact defect of a needle-like terminal and a lower electrode arises.
The charge collection electrode included in the cell stimulation device of the present invention is preferably fixed to the insulating member. With such a configuration, when the insulating member is placed on the culture medium, the charge collection electrode can be brought into contact with the culture medium.
　本発明は、本発明の細胞刺激装置と、培地を収容するための培養容器とを備えた細胞培養装置も提供する。細胞刺激装置は絶縁性部材を備える。前記電子放出素子は前記絶縁性部材に固定される。絶縁性部材は表面電極が培地の表面と気相を挟んで対向するように培養容器上に配置される。
　前記絶縁性部材は、表面電極と培地の表面との間の距離が０．５ｍｍ以上３ｍｍ以下となるように設けられることが好ましい。このことにより、気相に発生させた陰イオンを培地に容易に供給することができる。
　前記電荷回収電極は培養容器に固定されることが好ましい。このことにより、電荷回収電極と電子放出素子との間にリーク電流が流れることを抑制することができる。
　本発明は、下部電極と、表面電極と、下部電極と表面電極との間に配置された中間層とを備えた電子放出素子の下部電極と表面電極との間に電圧を印加することにより、気相を介して細胞を培養する培地に電荷を供給するステップを含む細胞刺激方法も提供する。前記電荷を供給するステップは、電子放出素子と培地との間の電界を利用して電荷を供給するステップである。 The present invention also provides a cell culture apparatus comprising the cell stimulator of the present invention and a culture vessel for containing a culture medium. The cell stimulator comprises an insulating member. The electron emitting element is fixed to the insulating member. The insulating member is disposed on the culture vessel such that the surface electrode is opposed to the surface of the culture medium via the gas phase.
The insulating member is preferably provided such that the distance between the surface electrode and the surface of the culture medium is 0.5 mm or more and 3 mm or less. This allows anions generated in the gas phase to be easily supplied to the culture medium.
The charge recovery electrode is preferably fixed to a culture vessel. This can suppress the flow of a leak current between the charge collection electrode and the electron emitting element.
According to the present invention, a voltage is applied between the lower electrode and the surface electrode of an electron-emitting device including the lower electrode, the surface electrode, and the intermediate layer disposed between the lower electrode and the surface electrode. Also provided is a method of stimulating a cell comprising the step of providing an electrical charge to a medium for culturing cells via the gas phase. The step of supplying the charge is a step of supplying a charge using an electric field between the electron emitter and the culture medium.
　以下、複数の実施形態を参照して本発明をより詳細に説明する。図面や以下の記述中で示す構成は、例示であって、本発明の範囲は、図面や以下の記述中で示すものに限定されない。 The invention will now be described in more detail with reference to a plurality of embodiments. The configurations shown in the drawings and the following description are exemplifications, and the scope of the present invention is not limited to those shown in the drawings and the following description.
第１実施形態
　図１は本実施形態の細胞刺激装置３０を含む細胞培養装置３５の概略断面図であり、図２は、細胞培養装置３５の概略回路図である。
　本実施形態の細胞刺激装置３０は、培地２で培養する細胞３に刺激を与えるための細胞刺激装置３０であって、細胞刺激装置３０は、培地２に気相２１を介して電荷を供給する電子放出素子４と、培地２から電荷を回収する電荷回収電極９とを備え、電子放出素子４は、下部電極６と、表面電極８と、下部電極６と表面電極８との間に配置された中間層７とを備え、電荷回収電極９は、培地２に接触することができるように設けられたことを特徴とする。
　本実施形態の細胞培養装置３５は、本実施形態の細胞刺激装置３０と、培地２を収容するための培養容器２０とを備える。
　本実施形態の細胞刺激方法は、下部電極６と、表面電極８と、下部電極６と表面電極８との間に配置された中間層７とを備えた電子放出素子４の下部電極６と表面電極８との間に電圧を印加することにより、気相２１を介して細胞３を培養する培地２に電荷を供給するステップを含み、電荷を供給するステップは、電子放出素子４と培地２との間の電界を利用して電荷を供給するステップである。
　本明細書において、電荷には、電子、気相中のイオン、液中のイオンが含まれる。
　以下、本実施形態の細胞刺激装置３０、細胞培養装置３５及び細胞刺激方法について説明する。 First Embodiment FIG. 1 is a schematic cross-sectional view of a cell culture device 35 including the cell stimulation device 30 of the present embodiment, and FIG. 2 is a schematic circuit diagram of the cell culture device 35.
The cell stimulation device 30 of the present embodiment is a cell stimulation device 30 for providing stimulation to the cells 3 cultured in the culture medium 2, and the cell stimulation device 30 supplies charge to the culture medium 2 via the gas phase 21. An electron emitting element 4 and a charge collection electrode 9 for recovering charge from the culture medium 2 are provided. The electron emitting element 4 is disposed between the lower electrode 6, the surface electrode 8, the lower electrode 6 and the surface electrode 8 The charge recovery electrode 9 is provided so as to be able to contact the culture medium 2.
The cell culture device 35 of the present embodiment includes the cell stimulation device 30 of the present embodiment and a culture container 20 for containing the culture medium 2.
In the cell stimulation method of the present embodiment, the lower electrode 6 and the surface of the electron emitting element 4 provided with the lower electrode 6, the surface electrode 8, and the intermediate layer 7 disposed between the lower electrode 6 and the surface electrode 8 The step of supplying a charge to the culture medium 2 for culturing the cell 3 through the gas phase 21 by applying a voltage between the electrode 8 and the charge storage step includes the step of supplying the electron emission element 4 and the culture medium 2 Supplying an electric charge using an electric field between them.
As used herein, the charge includes electrons, ions in the gas phase, and ions in the liquid.
Hereinafter, the cell stimulation device 30, the cell culture device 35, and the cell stimulation method of the present embodiment will be described.
　細胞刺激装置３０は、細胞の増殖や分化誘導等を目的として培地２で培養する細胞３に電気刺激を与えるための装置である。
　培地２で培養する細胞３は、例えば、多細胞生物の細胞・組織、遺伝子を導入した細胞、遺伝子を組み換えた細胞、微生物などである。細胞３は、電気刺激に反応のある細胞とすることができる。電気刺激に反応のある細胞としては、筋細胞、神経細胞、骨芽細胞、破骨細胞、軟骨細胞、骨細胞、繊維芽細胞等が挙げられる。
　また、培地２で培養する細胞３には、事前に予備培養した細胞を用いることができる。また、予備培養した細胞は、培地２に103～106cells/cm2で細胞３が含まれるように培地２に播種することができる。
　培地２は、細胞３に生育環境を提供するものである。培地２は、通常用いられている細胞培養用培地であれば特に制限なく用いる事ができる。培地２は、液体培地であってもよく、寒天などのゲル化剤で液体培地を固めた固体培地であってもよい。また、培地２は、液体培地と固体培地とを組み合わせたものであってもよい。培地２は、例えばMEM培地（イーグル最小必須培地）、D-MEM培地（ダルベッコ改変イーグル培地）、α-MEM培地（イーグル最小必須培地 α改変型）、GMEM（グラスゴー最小必須培地）、Ham‘s F-12（栄養混合物 F-12ハム）、IMDM（イスコフ改変ダルベッコ培地）、RPMI-1640培地、D-PBS（ダルベッコ リン酸緩衝生理食塩水）、HBSS（ハンクス平衡塩類溶液）等である。 The cell stimulation device 30 is a device for applying an electric stimulation to the cells 3 cultured in the medium 2 for the purpose of cell proliferation, differentiation induction and the like.
The cells 3 cultured in the medium 2 are, for example, cells / tissues of multicellular organisms, cells into which a gene has been introduced, cells into which a gene has been recombined, or a microorganism. Cell 3 can be a cell responsive to electrical stimulation. Examples of cells responsive to electrical stimulation include muscle cells, nerve cells, osteoblasts, osteoclasts, chondrocytes, osteocytes, fibroblasts and the like.
In addition, for cells 3 cultured in medium 2, cells precultured in advance can be used. Alternatively, the precultured cells can be seeded on the medium 2 such that the medium 2 contains 10 3 to 10 6 cells / cm 2 of cells 3.
Medium 2 provides cells 3 with a growth environment. The medium 2 can be used without particular limitation as long as it is a commonly used cell culture medium. The medium 2 may be a liquid medium, or may be a solid medium in which the liquid medium is solidified with a gelling agent such as agar. In addition, the medium 2 may be a combination of a liquid medium and a solid medium. Medium 2 may be, for example, MEM medium (Eagle minimal essential medium), D-MEM medium (Dulbecco modified Eagle medium), α-MEM medium (Eagle minimal essential medium α modified), GMEM (Glasgow minimal essential medium), Ham's F- 12 (Nutrient Mixture F-12 Ham), IMDM (Iscove's Modified Dulbecco's Medium), RPMI-1640 Medium, D-PBS (Dulbecco's Phosphate Buffered Saline), HBSS (Hanks Balanced Salt Solution), and the like.
　培地２は、培養容器２０に収容される。培養容器２０は、例えば、複数のウェル１２を備えたマルチウェルプレート、培養ディッシュなどである。また、培養容器２０と細胞刺激装置３０とを組み合わせることにより、細胞培養装置３５を形成することができる。培養容器２０の材質は、既存の材料であるガラス、プラスチック等が利用できる。培養容器２０は、容器底面に細胞３の定着に適した表面処理を付与したものが好ましい。表面処理は一般的に知られている、マトリックスタンパク質、コラーゲン、フィブロネクチン、ラミニン等を容器底面に施す処理とすることができる。
　細胞培養装置３５は、インキュベーター１８中に配置することができる。このことにより、培地２の温度、雰囲気ガスなどを制御することができ、細胞３を安定して培養することができる。培養雰囲気は、例えば、温度：３７℃、相対湿度：９５％、雰囲気ガス：５％炭酸ガスを含む空気である。 The medium 2 is accommodated in the culture vessel 20. The culture vessel 20 is, for example, a multiwell plate provided with a plurality of wells 12, a culture dish, or the like. Furthermore, by combining the culture container 20 and the cell stimulation device 30, the cell culture device 35 can be formed. As a material of the culture vessel 20, glass, plastic or the like which is an existing material can be used. The culture vessel 20 is preferably one provided with a surface treatment suitable for the fixation of the cells 3 on the bottom of the vessel. The surface treatment can be generally known, such as matrix protein, collagen, fibronectin, laminin, etc., applied to the bottom of the container.
Cell culture apparatus 35 can be placed in incubator 18. By this, the temperature of culture medium 2, atmosphere gas, etc. can be controlled, and cell 3 can be cultured stably. The culture atmosphere is, for example, air containing temperature: 37 ° C., relative humidity: 95%, atmosphere gas: 5% carbon dioxide gas.
　細胞刺激装置３０は、培地２に気相２１を介して電荷を供給する電子放出素子４を備える。電子放出素子４は、平面形状を有し、電子を放出することが可能な素子である。電子放出素子４は、下部電極６と、表面電極８と、下部電極６と表面電極８との間に配置された中間層７とを備える。また、電源装置１１ｂにより表面電極８と下部電極６との間に電圧を印加し中間層７に電界を生じさせることができる。この電界により、中間層７に電子を流すことができ、電子放出素子４の表面電極８から電子を放出することができる。この電子は、酸素の陰イオンなどの電荷を気相２１中に生じさせる。また、この陰イオンは培地２に照射される。 The cell stimulator 30 comprises an electron emitting element 4 for supplying a charge to the culture medium 2 via the gas phase 21. The electron emitting device 4 is a device having a planar shape and capable of emitting electrons. The electron emitter 4 includes a lower electrode 6, a surface electrode 8, and an intermediate layer 7 disposed between the lower electrode 6 and the surface electrode 8. Further, a voltage can be applied between the surface electrode 8 and the lower electrode 6 by the power supply 11 b to generate an electric field in the intermediate layer 7. Electrons can flow in the intermediate layer 7 by this electric field, and electrons can be emitted from the surface electrode 8 of the electron emitting element 4. The electrons generate charges such as oxygen anions in the gas phase 21. In addition, this anion is irradiated to the culture medium 2.
　例えば、電源装置１１ｂにより、波高値１４Ｖ0-p～２４Ｖ0-pで周波数５００～１００００Ｈｚの矩形波交流電圧を表面電極８と下部電極６との間に印加することができる。また、on, offデューティー比は 1～100%までの可変とすることができる。また、電子放出素子４からのイオン照射量は、0.5～5.0μA/cm2とすることができる。0.5μA/cm2より小さいと培養が遅くなり、5.0μA/cm2より大きいと死滅する可能性が大きく培養が遅い。
また、駆動中のイオン照射量はほぼ一定値（振れ幅は±10％以内、好ましくは±５%以内）とし、照射量を満足するために、駆動電圧波高値あるいは矩形波デューティー比をパラメータとして制御を行うことができる。イオン照射量はほぼ一定値にすることにより、安定的に培養を進めることができる。 For example, a rectangular wave AC voltage with a frequency of 500 to 10000 Hz can be applied between the surface electrode 8 and the lower electrode 6 by the power supply 11 b with a peak value of 14V 0-p to 24V 0-p . Also, the on / off duty ratio can be variable from 1 to 100%. The ion irradiation dose from the electron emitter 4 can be 0.5 to 5.0 μA / cm 2 . If it is less than 0.5 μA / cm 2 , the culture is slowed, and if it is more than 5.0 μA / cm 2 , the possibility of death is large and the culture is slow.
Further, the ion irradiation dose during driving is set to a substantially constant value (the fluctuation range is within ± 10%, preferably within ± 5%), and the drive voltage peak value or the rectangular wave duty ratio is used as a parameter to satisfy the irradiation dose. Control can be performed. The culture can be stably advanced by setting the ion irradiation amount to a substantially constant value.
　表面電極８は、電子放出素子４の表面に位置する電極である。表面電極８は、５ｎｍ以上１００ｎｍ以下、好ましくは４０ｎｍ以上１００ｎｍ以下の厚さを有することができる。また、表面電極８の材質は、例えば、金、白金である。このことにより、オートクレーブ滅菌などにより表面電極８が酸化されることを抑制することができる。また、表面電極８は、複数の金属層から構成されてもよい。
　表面電極８は、好ましい４０ｎｍ以上の場合であっても、複数の開口、すき間、１０ｎｍ以下の厚さに薄くなった部分を有してもよい。中間層７を流れた電子がこの開口、すき間、薄くなった部分を通過又は透過することができ、表面電極８から電子を放出することができる。このような開口、すき間、薄くなった部分は、下部電極２と表面電極４との間に電圧を印加すること（フォーミング処理、初期電圧印加）により形成することができる。 The surface electrode 8 is an electrode located on the surface of the electron emitter 4. The surface electrode 8 can have a thickness of 5 nm or more and 100 nm or less, preferably 40 nm or more and 100 nm or less. The material of the surface electrode 8 is, for example, gold or platinum. This can suppress oxidation of the surface electrode 8 by autoclave sterilization or the like. Moreover, the surface electrode 8 may be comprised from several metal layers.
The surface electrode 8 may have a plurality of openings, gaps, and portions thinned to a thickness of 10 nm or less, even in the case of the preferable 40 nm or more. Electrons having flowed through the intermediate layer 7 can pass through or pass through the opening, the gap, the thinned portion, and can emit electrons from the surface electrode 8. Such openings, gaps, and thinned portions can be formed by applying a voltage between the lower electrode 2 and the surface electrode 4 (forming process, initial voltage application).
　下部電極６は、中間層７を介して表面電極８と対向する電極である。下部電極６は、金属板であってもよく、金属層又は導電体層を有した絶縁体から成る板であってもよい。また、下部電極６が金属板からなる場合、この金属板は電子放出素子４の基板であってもよい。下部電極６の材質は、例えば、アルミニウム、ステンレス鋼、ニッケルなどである。下部電極の厚さは、例えば２００μｍ以上１ｍｍ以下である。 The lower electrode 6 is an electrode facing the surface electrode 8 via the intermediate layer 7. The lower electrode 6 may be a metal plate, or may be a plate made of an insulator having a metal layer or a conductor layer. When the lower electrode 6 is made of a metal plate, the metal plate may be a substrate of the electron emitting element 4. The material of the lower electrode 6 is, for example, aluminum, stainless steel, nickel or the like. The thickness of the lower electrode is, for example, 200 μm or more and 1 mm or less.
　中間層７は、表面電極８と下部電極６とに電圧を印加することにより形成される電界により電子が流れる層である。中間層７は、半導電性を有することができる。中間層７は、絶縁性樹脂、導電性樹脂、絶縁性微粒子のうち少なくとも１つを含む。また、中間層７は導電性微粒子を含むことが好ましい。中間層７の厚さは、例えば、１μｍ以上１．８μｍ以下とすることができる。中間層７を流れた電子を表面電極８から放出するため、電子放出素子４は、表面電極８から電子を面放出することができる。このため、表面電極８上の気相２１に電子を一様に放出することができ、この電子により酸素の陰イオンなどの電荷を発生させることができる。 The intermediate layer 7 is a layer through which electrons flow due to an electric field formed by applying a voltage to the surface electrode 8 and the lower electrode 6. The intermediate layer 7 can have semiconductivity. The intermediate layer 7 contains at least one of an insulating resin, a conductive resin, and an insulating fine particle. Moreover, it is preferable that the intermediate | middle layer 7 contains electroconductive fine particles. The thickness of the intermediate layer 7 can be, for example, 1 μm or more and 1.8 μm or less. Since the electrons flowing through the intermediate layer 7 are emitted from the surface electrode 8, the electron emitting element 4 can emit electrons from the surface electrode 8. Therefore, electrons can be uniformly emitted to the gas phase 21 on the surface electrode 8, and charges such as anions of oxygen can be generated by the electrons.
　電子放出素子４は、表面電極８と下部電極６との間に絶縁層５を有してもよい。この絶縁層５は、開口を有することができる。絶縁層５の開口は、表面電極の電子を放出させたい領域に対応するように設けられる。絶縁層５には電子が流れることができないため、絶縁層５の開口に対応する中間層７に電子が流れ表面電極８から電子が放出される。従って、絶縁層５を設けることにより、表面電極８に電子放出領域を形成することができる。 The electron emitter 4 may have an insulating layer 5 between the surface electrode 8 and the lower electrode 6. The insulating layer 5 can have an opening. The opening of the insulating layer 5 is provided to correspond to the region of the surface electrode where it is desired to emit electrons. Since electrons can not flow in the insulating layer 5, electrons flow in the intermediate layer 7 corresponding to the opening of the insulating layer 5, and electrons are emitted from the surface electrode 8. Therefore, the electron emission region can be formed on the surface electrode 8 by providing the insulating layer 5.
　電子放出素子４は、絶縁性部材１３に取り外し可能に固定することができる。この絶縁性部材１３を培養容器２０の上に配置することにより、電子放出素子４の表面電極８が、培養容器２０内の培地２の表面に気相２１を介して対向するように電子放出素子４を配置することができる。このように電子放出素子４を配置することにより、電子放出素子４の表面電極８側から放出させた電子から、電子放出素子４と培地２との間の気相２１に酸素の陰イオンなどの電荷を発生させることができる。また、電子放出素子４は、表面電極８と培地２の表面とが実質的に平行となるように配置することができる。
　絶縁性部材１３は、蓋部材１０と一体化されていてもよい。この場合、絶縁性部材１３が蓋部材１０から培地２側（ウェル１２中）に突出するように絶縁性部材１３を設けることができる。また、電子放出素子４を、蓋部材２から突出した絶縁性部材１３の先端面に培地２の表面と平行となるように固定することができる。
　絶縁性部材１３及び蓋部材１０の材質は熱に耐性のあるＰＥＥＫ材、フッ素系の材料が好ましい。細胞と相対する部材は、事前にオートクレーブを用いて滅菌処理される。150℃を超える高温水蒸気による処理であるため、材料の繰り返し熱変変形に耐える必要がある。 The electron emitting element 4 can be removably fixed to the insulating member 13. By arranging the insulating member 13 on the culture vessel 20, the surface electrode 8 of the electron emitting element 4 is opposed to the surface of the culture medium 2 in the culture vessel 20 via the gas phase 21. 4 can be arranged. By arranging the electron-emitting device 4 in this manner, the electrons emitted from the surface electrode 8 side of the electron-emitting device 4 cause oxygen anions and the like in the gas phase 21 between the electron-emitting device 4 and the culture medium 2. It can generate charge. In addition, the electron emitting element 4 can be arranged such that the surface electrode 8 and the surface of the culture medium 2 are substantially parallel.
The insulating member 13 may be integrated with the lid member 10. In this case, the insulating member 13 can be provided so that the insulating member 13 protrudes from the lid member 10 to the culture medium 2 side (in the well 12). In addition, the electron emitting element 4 can be fixed to the tip end surface of the insulating member 13 protruding from the lid member 2 so as to be parallel to the surface of the culture medium 2.
The material of the insulating member 13 and the lid member 10 is preferably a PEEK material resistant to heat and a fluorine-based material. The parts facing the cells are previously sterilized using an autoclave. Because of the treatment with high temperature steam above 150 ° C., it is necessary to withstand repeated thermal deformation of the material.
　蓋部材１０は、細胞刺激装置３０を培養容器２０上に載せることができるように設けられる。また、蓋部材１０は、絶縁性を有することができる。
　絶縁性部材１３又は蓋部材１０は、表面電極８と培地２の表面との間の距離が０．５ｍｍ以上３ｍｍ以下となるように設けることができる。このことにより、気相２１に発生させた陰イオンを培地２に容易に供給することができる。表面電極８と培地２の表面との間の距離は、好ましくは１ｍｍ以上２ｍｍ以下とすることができる。
　例えば、培養容器２０のウェルの深さと、培地２の深さ、蓋部材１０から突出した絶縁性部材１３の高さなどを調整することにより、表面電極８と培地２の表面との間の距離を調整することができる。
　このように電子放出素子４を気相２１を介して培地２の表面と対向させることにより、気相２１から培地２への酸素ガスの供給が阻害されることを抑制することができる。このため、培地２の溶存酸素量を保持することが可能になる。
　また、電子放出素子４を絶縁性部材３に取り外し可能に固定することにより、電子放出素子４を容易に交換することができる。 The lid member 10 is provided so that the cell stimulator 30 can be placed on the culture vessel 20. Moreover, the cover member 10 can have insulation.
The insulating member 13 or the lid member 10 can be provided such that the distance between the surface electrode 8 and the surface of the culture medium 2 is 0.5 mm or more and 3 mm or less. As a result, anions generated in the gas phase 21 can be easily supplied to the culture medium 2. The distance between the surface electrode 8 and the surface of the culture medium 2 can be preferably 1 mm or more and 2 mm or less.
For example, the distance between the surface electrode 8 and the surface of the culture medium 2 by adjusting the depth of the well of the culture vessel 20, the depth of the culture medium 2, the height of the insulating member 13 projecting from the lid member 10, etc. Can be adjusted.
As described above, by causing the electron emitting element 4 to face the surface of the culture medium 2 via the gas phase 21, it is possible to suppress inhibition of the supply of oxygen gas from the gas phase 21 to the culture medium 2. For this reason, it becomes possible to maintain the amount of dissolved oxygen of the culture medium 2.
Further, the electron emitting device 4 can be easily replaced by detachably fixing the electron emitting device 4 to the insulating member 3.
　細胞刺激装置３０は、絶縁性部材１３に固定された第１端子１５及び第２端子１６を有することができる。第１端子１５及び第２端子１６は、それぞれ電子放出素子４と接触する接点を有する。電子放出素子４を絶縁性部材３に取り付けている場合に、第１端子１５が下部電極６と電気的に接続し、第２端子１６が表面電極８と電気的に接続するように第１及び第２端子並びに電子放出素子４を設けることができる。この第１及び第２端子を介して表面電極８と下部電極６との間に電圧を印加することができる。 The cell stimulator 30 can have a first terminal 15 and a second terminal 16 fixed to the insulating member 13. The first terminal 15 and the second terminal 16 each have a contact point in contact with the electron emitter 4. When the electron-emitting device 4 is attached to the insulating member 3, the first terminal 15 is electrically connected to the lower electrode 6, and the second terminal 16 is electrically connected to the surface electrode 8. A second terminal and an electron emitter 4 can be provided. A voltage can be applied between the surface electrode 8 and the lower electrode 6 through the first and second terminals.
　電荷回収電極９は、細胞３を培養する培地２に接触することができるように設けられる。このため、培地２から電荷回収電極９へ電子が流れることができ、電荷回収電極９と培地２との電位を等しくあるいはほぼ等しくすることができる。例えば、電荷回収電極９を接地接続すれば、培地２が帯電することを抑制することができる。
　本実施形態の細胞刺激装置３０では、電子放出素子４により発生させた電荷を気相２１を介して培地２に供給するため、培地２に接触する電極は電荷回収電極９だけである。このため、培地２に含まれる水が電気分解することを抑制することができる。また、電荷回収電極９は正極性となるため、培地２中のミネラル成分（K+, Ca2+, Na+, Mg2+等）が電荷回収電極９上に析出することなく、培地２の成分組成が変化することを抑制することができる。
　電荷回収電極９は、例えば、絶縁性部材１３又は蓋部材１０に固定することができる。電荷回収電極９は、絶縁性部材１３及び蓋部材１０を培養容器２０上に設置した際に電荷回収電極９が培地２と接触するように設けることができる。
　電荷回収電極９の形状は、例えば、棒状、平板状、メッシュ、パンチングメタルである。また、電荷回収電極９は、棒の先に平板、メッシュ又はパンチングメタルが取り付けられた構造を有してもよい。電荷回収電極９に含まれる棒の先に取り付けた平板、メッシュ又はパンチングメタルは、細胞３が電荷回収電極９と電子放出素子４との間に位置するようにウェル１２の底に配置することができる。このような構成とすると、電子放出素子４の電子放出により培地２の表面で生じた陰イオンは、ウェル１２の底に向かって流れる。このため、培地２で培養する細胞３に効率よく電気刺激を与えることが可能になる。 The charge recovery electrode 9 is provided so as to be able to contact the medium 2 for culturing the cells 3. Therefore, electrons can flow from the culture medium 2 to the charge collection electrode 9, and the potentials of the charge collection electrode 9 and the culture medium 2 can be made equal or almost equal. For example, if the charge recovery electrode 9 is connected to ground, the culture medium 2 can be prevented from being charged.
In the cell stimulation device 30 of the present embodiment, the charge generated by the electron emitting element 4 is supplied to the culture medium 2 via the gas phase 21, so the electrode contacting the culture medium 2 is only the charge collection electrode 9. For this reason, it can suppress that the water contained in the culture medium 2 electrolyzes. In addition, since the charge collection electrode 9 has a positive polarity, the mineral components (K + , Ca 2+ , Na + , Mg 2+, etc.) in the culture medium 2 are not precipitated on the charge collection electrode 9. It is possible to suppress the change in component composition.
The charge collection electrode 9 can be fixed to, for example, the insulating member 13 or the lid member 10. The charge collection electrode 9 can be provided so that the charge collection electrode 9 contacts the culture medium 2 when the insulating member 13 and the lid member 10 are installed on the culture vessel 20.
The shape of the charge collection electrode 9 is, for example, a bar, a plate, a mesh, or a punching metal. Further, the charge collection electrode 9 may have a structure in which a flat plate, a mesh or a punching metal is attached to the end of a rod. The flat plate, mesh or punching metal attached to the tip of the rod included in the charge recovery electrode 9 may be disposed at the bottom of the well 12 so that the cell 3 is located between the charge recovery electrode 9 and the electron emitting element 4 it can. In such a configuration, the anions generated on the surface of the culture medium 2 by the electron emission of the electron emitter 4 flow toward the bottom of the well 12. For this reason, it is possible to efficiently provide electrical stimulation to the cells 3 cultured in the medium 2.
　電源装置１１ａで電子放出素子４又は電荷回収電極９に電圧を印加することにより、電子放出素子４の表面電極８と培地２の表面との間の気相２１に電界を生じさせることができる。この気相２１の電界の中の電気力線（電界強度の勾配）に沿って、電子放出素子４が電子を放出することにより発生させた陰イオンを培地２の表面に搬送することができ（気相２１に電流を流すことができる）、培地２に陰イオン（ＯＨ-、Ｃｌ-、Ｏ2 -など）を発生させることができる。この陰イオンにより培地２で培養する細胞３に電気刺激を与えることができる。また、この陰イオンは、培地２を移動し電荷回収電極９へと流れる（培地２に電流が流れる）ため、培地２が帯電することを抑制することができる。 By applying a voltage to the electron-emitting device 4 or the charge collection electrode 9 by the power supply device 11a, an electric field can be generated in the gas phase 21 between the surface electrode 8 of the electron-emitting device 4 and the surface of the culture medium 2. The anions generated by the electron emitting element 4 emitting electrons can be transported to the surface of the culture medium 2 along electric lines of force (gradient of the electric field strength) in the electric field of the gas phase 21 ( current can flow to the gas phase 21), the medium 2 in the anion (OH -, Cl -, O 2 - , etc.) can be generated. Electrical stimulation can be given to the cells 3 cultured in the medium 2 by this anion. Moreover, since this anion moves the culture medium 2 and flows to the charge collection electrode 9 (a current flows to the culture medium 2), it is possible to suppress the culture medium 2 from being charged.
　例えば、電荷回収電極９を接地接続し、電源装置１１ａにより電子放出素子４と接地との間に－１０００Ｖ～－５０Ｖの直流電圧を印加することができる。また、電子放出素子４の表面電極８と培地２の表面との間隔は０．５ｍｍ～３ｍｍ（好ましくは１ｍｍ以上２ｍｍ以下）とすることができる。電荷回収電極９が接地接続しているため培地２の電位はほぼ０Ｖになり、表面電極８の電位は－１０００Ｖ～－５０Ｖとなる。また、培地２と表面電極８との間隔は０．５ｍｍ～３ｍｍであるため、培地２と表面電極８との間の気相２１に強い電界強度の電界を生じさせることができる。この電界を利用して電子放出素子４が電子を放出することにより発生させた陰イオンを培地２に供給することができる。 For example, the charge collection electrode 9 can be connected to ground, and a DC voltage of -1000 V to -50 V can be applied between the electron-emitting device 4 and the ground by the power supply 11a. Further, the distance between the surface electrode 8 of the electron emitting element 4 and the surface of the culture medium 2 can be 0.5 mm to 3 mm (preferably 1 mm or more and 2 mm or less). Since the charge collection electrode 9 is connected to ground, the potential of the culture medium 2 becomes almost 0 V, and the potential of the surface electrode 8 becomes −1000 V to −50 V. Further, since the distance between the culture medium 2 and the surface electrode 8 is 0.5 mm to 3 mm, an electric field of strong electric field strength can be generated in the gas phase 21 between the culture medium 2 and the surface electrode 8. The anions generated by the electron emitter 4 emitting electrons using this electric field can be supplied to the culture medium 2.
　電源装置１１ａ、１１ｂは、電子放出素子４から培地２へ電荷を供給することにより気相２１に生じる電流と、電荷回収電極９が培地２から電荷を回収することにより培地２に生じる電流とがループ電流となるように電子放出素子４及び電荷回収電極９と電気的に接続することができる。
　例えば、図１、２に示したように、電源装置１１ａを用いて表面電極８と電荷回収電極９との間に電圧Ｖeを印加することにより生じさせた電界により気相２１をイオンＢ-（イオンＢ-は表面電極８から放出された電子により気相２１中に生じる）が移動することにより気相２１に電流が生じ、培地２に達したイオンＢ-は培地２中に溶け込み、細胞の各種要素形成に関わるシグナル伝達系を刺激し、最終的にイオンＣ-（イオンＣ-はイオンＢ-そのものand／orイオンＢ-由来の別イオン種）として電荷回収電極９へと移動することにより培地２に電流が生じる。従って、電源装置１１ａ、表面電極８、気相２１及び培地２を含む回路にループ電流を流すことができる。 The power supply devices 11a and 11b supply a charge from the electron-emitting device 4 to the culture medium 2, and a current generated in the gas phase 21 by the charge recovery electrode 9 recovering the charge from the culture medium 2 It can be electrically connected to the electron emitting element 4 and the charge collection electrode 9 so as to be a loop current.
For example, as shown in FIGS. 1 and 2, the ion B gas phase 21 by an electric field caused by applying a voltage V e between the surface electrode 8 with the power supply 11a and the charge collection electrode 9 - (ion B - by electrons emitted from the surface electrode 8 occurs in the gas phase 21) current is generated in the gas phase 21 by move, ion B reaching the medium 2 - penetration into the medium 2, the cells It stimulates signal transduction system involving the various elements forming, finally ion C - (ion C - ion B - itself and / or ion B - another ionic species derived from) moving to the charge collection electrode 9 as Generates a current in the medium 2. Therefore, a loop current can be supplied to the circuit including the power supply device 11 a, the surface electrode 8, the gas phase 21 and the culture medium 2.
　電子放出素子４は、表面電極８から電子を面放出させることができるため、培地２の表面と表面電極８との間の気相２１に一様に陰イオンを発生させることができる。この陰イオンを電界により培地２に供給することができるため、表面電極８の下部に位置する培地２にも一様に陰イオンを発生させることができ、表面電極８の下部に位置する細胞３に一様に電気刺激を与えることができる。このため、電気刺激を与えたい細胞に一様に電気刺激を与えることができる。
　細胞刺激装置３０による電気刺激（イオン照射）は、例えば、播種された細胞が細胞分裂を始めるタイミングから開始することができる。
　また、細胞３が定着した培地２に対するイオン照射は、細胞種に適した照射量、照射間隔、照射タイミングを定めて行うことができる。
　電子放出素子４から電子を放出させる時間（電源装置１１ａ、１１ｂにより電圧を印加する時間）は、例えば、５秒間以上１分間以内とすることができる。 Since the electron emitting element 4 can cause surface emission of electrons from the surface electrode 8, anions can be uniformly generated in the gas phase 21 between the surface of the culture medium 2 and the surface electrode 8. Since this anion can be supplied to the culture medium 2 by the electric field, the anion can be uniformly generated in the culture medium 2 located below the surface electrode 8 and the cells 3 located below the surface electrode 8 Can be given electrical stimulation uniformly. For this reason, electrical stimulation can be uniformly applied to cells to which electrical stimulation is desired.
The electrical stimulation (ion irradiation) by the cell stimulator 30 can be started, for example, from the timing when the seeded cells start cell division.
In addition, the ion irradiation to the medium 2 in which the cells 3 are fixed can be performed by setting the irradiation amount, the irradiation interval, and the irradiation timing suitable for the cell type.
The time for which electrons are emitted from the electron emission element 4 (the time for which a voltage is applied by the power supply devices 11a and 11b) can be, for example, 5 seconds or more and 1 minute or less.
第２実施形態
　図３は本実施形態の細胞刺激装置３０の概略斜視図であり、図４は本実施形態の細胞刺激装置３０の概略分解図である。
　本実施形態では、図３に示したような細胞刺激装置３０に含まれる電子放出素子４が、図４の分解図のように絶縁性部材１３から取り外し可能に設けられている。
　電子放出素子４は、下部電極６である金属板上に中間層７及び表面電極８が積層されている。例えば、下部電極６は、１２ｍｍ×２４ｍｍ角、厚さ０．５ｍｍの金属板である。また、下部電極６の材質は、例えば、アルミニウム、ステンレス鋼、ニッケルなどである。また、中間層７の厚さは、例えば１．０μｍ～１．８μｍである。
　また、電子放出領域２５が形成される部分に開口を有する絶縁層５が下部電極６と表面電極８との間に積層されている。絶縁層５には電流が流れないため、絶縁層５の開口に対応した表面電極８の電子放出領域２５だけから電子が放出される。絶縁層５の開口は例えば、５ｍｍ角とすることができる。絶縁層５には、金属酸化物・金属窒化物などの無機材料、シリコーン系樹脂、フェノール系樹脂などの有機材料を使用することができる。
　表面電極８は、４０ｎｍ以上１００ｎｍ以下の厚さを有することができる。このことにより、絶縁層５の開口に対応する表面電極８の電子放出領域２５から中間層７を流れた電子を放出させることができる。例えば、表面電極８の大きさは１８ｍｍ×８．５ｍｍ角とすることができる。また、電子放出領域の大きさは５ｍｍ角とすることができる。表面電極８の材質は例えば金又は白金である。 Second Embodiment FIG. 3 is a schematic perspective view of a cell stimulation device 30 of the present embodiment, and FIG. 4 is a schematic exploded view of the cell stimulation device 30 of the present embodiment.
In the present embodiment, the electron-emitting device 4 included in the cell stimulation device 30 as shown in FIG. 3 is provided so as to be removable from the insulating member 13 as shown in the exploded view of FIG.
In the electron emitting element 4, the intermediate layer 7 and the surface electrode 8 are stacked on the metal plate which is the lower electrode 6. For example, the lower electrode 6 is a metal plate of 12 mm × 24 mm square and 0.5 mm thick. The material of the lower electrode 6 is, for example, aluminum, stainless steel, nickel or the like. The thickness of the intermediate layer 7 is, for example, 1.0 μm to 1.8 μm.
In addition, an insulating layer 5 having an opening in a portion where the electron emission region 25 is formed is laminated between the lower electrode 6 and the surface electrode 8. Since no current flows in the insulating layer 5, electrons are emitted only from the electron emitting region 25 of the surface electrode 8 corresponding to the opening of the insulating layer 5. The opening of the insulating layer 5 can be, for example, 5 mm square. For the insulating layer 5, inorganic materials such as metal oxides and metal nitrides, and organic materials such as silicone resins and phenol resins can be used.
The surface electrode 8 can have a thickness of 40 nm or more and 100 nm or less. As a result, it is possible to emit the electrons having flowed through the intermediate layer 7 from the electron emission region 25 of the surface electrode 8 corresponding to the opening of the insulating layer 5. For example, the size of the surface electrode 8 can be 18 mm × 8.5 mm square. Also, the size of the electron emission region can be 5 mm square. The material of the surface electrode 8 is, for example, gold or platinum.
　第１端子１５ａ、１５ｂは、絶縁性部材１３に設けられた開口中に配置されており、針形状を有し、その先端部が電子放出素子４の下部電極６の裏面との接点となる。このため、第１端子１５を介して下部電極６に電圧を印加することができる。また、第１端子１５の先端部を接点とすることにより、第１端子１５と下部電極６との接点不良が生じることを抑制することができる。また、第１端子１５はバネ式針状端子であってもよい。このことにより、第１端子１５と下部電極６との接点不良が生じることを抑制することができる。
　通常、細胞刺激装置３０は、細胞培養装置３５で細胞３を培養する前にオートクレーブを用いて滅菌処理される。オートクレーブ滅菌は高温水蒸気による処理であるため、この処理により細胞刺激装置３０に含まれる金属部材（例えば、下部電極６である金属板）の表面に薄い酸化膜が形成される場合がある。この酸化膜は接点不良を引き起こす原因となる。針状の第１端子１５の先端部を接点とすることにより、この先端部が酸化膜を貫通することができ、第１端子１５と下部電極６との電気的導通を確実に確保することができる。 The first terminals 15 a and 15 b are disposed in the openings provided in the insulating member 13 and have a needle shape, and their tip end portions are in contact with the back surface of the lower electrode 6 of the electron emitting element 4. Therefore, a voltage can be applied to the lower electrode 6 through the first terminal 15. Further, by setting the tip end portion of the first terminal 15 as a contact, it is possible to suppress the occurrence of a contact failure between the first terminal 15 and the lower electrode 6. Further, the first terminal 15 may be a spring type needle-like terminal. This can suppress the occurrence of contact failure between the first terminal 15 and the lower electrode 6.
Generally, the cell stimulator 30 is sterilized using an autoclave before culturing the cells 3 in the cell culture device 35. Since autoclave sterilization is a treatment with high temperature steam, a thin oxide film may be formed on the surface of a metal member (for example, a metal plate which is the lower electrode 6) included in the cell stimulation device 30 by this treatment. This oxide film causes the contact failure. By using the tip of needle-like first terminal 15 as a contact, the tip can penetrate the oxide film, and electrical continuity between first terminal 15 and lower electrode 6 can be reliably ensured. it can.
　第２端子１６は、絶縁性部材１３に電子放出素子４を固定するように設けられており、電子放出素子４の表面電極８に接触する。このため、第２端子１６を介して表面電極８に電圧を印加することができる。また、第２端子１６および前述の第１端子１５は表面に金めっき層を有する金属板であってもよい。このことにより、オートクレーブ滅菌により第２端子１６の表面に酸化膜が形成されることを抑制することができ、接触不良が生じることを抑制することができる。 The second terminal 16 is provided to fix the electron emitting element 4 to the insulating member 13, and contacts the surface electrode 8 of the electron emitting element 4. Therefore, a voltage can be applied to the surface electrode 8 through the second terminal 16. Further, the second terminal 16 and the first terminal 15 described above may be a metal plate having a gold plating layer on the surface. By this, it can suppress that an oxide film is formed in the surface of the 2nd terminal 16 by autoclave sterilization, and can suppress that a contact failure arises.
　絶縁性部材１３はその表面にスリット構造を有する。このため、電荷回収電極９及び電極９と電気的に接続した金属部材と、電子放出素子４及び素子４と電気的に接続した金属部材との間に漏れ電流（縁面漏れ）が生じることを抑制することができる。
　他の構成は第１実施形態と同様である。また、上記の第１実施形態についての記載は、矛盾がない限り第２実施形態についても当てはまる。また、上記絶縁性部材１３の表面のスリット構造については第１実施形態等他の構造にも適用が可能である。
　本実施形態では電子放出素子４は長方形（電子放出領域２５ａ、２５ｂは正方形）の形状をしているが、状況により、円形や星型等の形状も可能である。１例として培地２をできるだけ効率よく使用するために培地２の形状に合わせた形状（ただし、電荷回収電極９との接触は不可であるために、その部分は欠けた形状になる）にすることも可能である。 The insulating member 13 has a slit structure on its surface. Therefore, leakage current (edge surface leakage) is generated between the metal member electrically connected to the charge collection electrode 9 and the electrode 9 and the metal member electrically connected to the electron emitting element 4 and the element 4. It can be suppressed.
The other configuration is the same as that of the first embodiment. In addition, the description of the first embodiment described above applies to the second embodiment as long as there is no contradiction. The slit structure on the surface of the insulating member 13 can be applied to other structures such as the first embodiment.
In the present embodiment, the electron-emitting device 4 has a rectangular shape (the electron-emitting regions 25a and 25b are square). However, depending on the situation, a circular shape or a star-like shape is also possible. As an example, to use the medium 2 as efficiently as possible in the form of the medium 2 (however, that part is missing because contact with the charge recovery electrode 9 is impossible) Is also possible.
第３実施形態
　図５は、本実施形態の細胞刺激装置３０に含まれる電子放出素子４の概略上面図である。
　電子放出素子４に含まれる下部電極６は、複数の細長い電極からなり、例えば図５では電極６ａ～６ｊから構成される。電極６ａ～６ｊは平行となるように配置することができる。これらの電極６ａ～６ｊは、例えば、絶縁性基板上に形成した金属層又は導電体層であってもよい。このことにより、電極６ａ～６ｊを電気的に分離することができる。また、電極６ａ～６ｊにそれぞれ異なる端子を接続することができる。
　電子放出素子４に含まれる表面電極８は、複数の細長い電極からなり、例えば図５では電極８ａ～８ｊから構成される。電極８ａ～８ｊは平行となるように配置することができる。これらの電極８ａ～８ｊは、例えば、中間層７上に形成した金属層又は導電体層であってもよい。また、電極８ａ～８ｊにそれぞれ異なる端子を接続することができる。 Third Embodiment FIG. 5 is a schematic top view of the electron-emitting device 4 included in the cell stimulation device 30 of the present embodiment.
The lower electrode 6 included in the electron emitting element 4 is composed of a plurality of elongated electrodes, and in FIG. 5, for example, is composed of electrodes 6a to 6j. The electrodes 6a to 6j can be arranged to be parallel. These electrodes 6a to 6j may be, for example, metal layers or conductor layers formed on an insulating substrate. This allows the electrodes 6a to 6j to be electrically separated. Further, different terminals can be connected to the electrodes 6a to 6j.
The surface electrode 8 included in the electron emitting element 4 is composed of a plurality of elongated electrodes, and in FIG. 5, for example, is composed of electrodes 8a to 8j. The electrodes 8a to 8j can be arranged in parallel. The electrodes 8a to 8j may be, for example, a metal layer or a conductor layer formed on the intermediate layer 7. Further, different terminals can be connected to the electrodes 8a to 8j.
　下部電極６及び表面電極８は、電極６ａ～６ｊと電極８ａ～８ｊの一方が行となり他方が列となるように格子状に配置される。また、電極６ａ～６ｊと電極８ａ～８ｊとが交わる箇所に開口を有する絶縁層５が下部電極６と中間層７との間に積層されている。このことにより、電極６ａ～６ｊと電極８ａ～８ｊとが交わる箇所（絶縁層５の開口に対応する箇所）の表面電極８に電子放出領域２５を形成することができる。これらの電子放出領域２５はマトリクス状に並んでいる。
　電圧を印加する電極６ａ～６ｊと電極８ａ～８ｊとの組み合わせを変えることにより、マトリクス状に並んだ複数の電子放出領域２５のうち、一部の電子放出領域２５で電子を放出させず、他の電子放出領域２５で電子を放出することができる。例えば、電極６ａ～６ｅと電極８ａ～８ｅとの間にだけ電圧を印加すると、これらの電極の交点の電子放出領域２５では電子が放出されるが、他の電子放出領域２５では電子が放出されない。従って、電子を放出させる電子放出領域２５の下部に位置する培地中の細胞にだけ電気刺激を与えることが可能になる。つまり、培地２のうち選択した領域に存在する細胞３にのみ電気刺激を与えることが可能になる。 The lower electrode 6 and the surface electrode 8 are arranged in a grid so that one of the electrodes 6a to 6j and the electrodes 8a to 8j is a row and the other is a column. An insulating layer 5 having an opening at the intersection of the electrodes 6a to 6j and the electrodes 8a to 8j is stacked between the lower electrode 6 and the intermediate layer 7. As a result, the electron emission region 25 can be formed on the surface electrode 8 at a portion where the electrodes 6a to 6j and the electrodes 8a to 8j intersect (a portion corresponding to the opening of the insulating layer 5). These electron emission regions 25 are arranged in a matrix.
By changing the combination of the electrodes 6a to 6j and the electrodes 8a to 8j to which a voltage is applied, electrons are not emitted from a part of the electron emission regions 25 among the plurality of electron emission regions 25 arranged in a matrix, Electrons can be emitted in the electron emission region 25 of the For example, when a voltage is applied only between the electrodes 6a to 6e and the electrodes 8a to 8e, electrons are emitted in the electron emission region 25 at the intersection of these electrodes, but no electrons are emitted in the other electron emission regions 25. . Therefore, electrical stimulation can be applied only to cells in the culture medium located below the electron emission region 25 that emits electrons. That is, it becomes possible to apply electrical stimulation only to the cells 3 present in the selected area of the medium 2.
　電極６ａ～６ｊと電極８ａ～８ｊとの組み合わせにより印加する電圧を変化させることにより、マトリクス状に並んだ複数の電子放出領域２５で電子放出量を変化させることができる。例えば、電極６ａ～６ｅと電極８ａ～８ｅとの間に波高値２０Ｖの交流電圧を印加し、電極６ｆ～６ｊと電極８ｆ～８ｊとの間に波高値１０Ｖの交流電圧を印加し、電極６ａ～６ｅと電極８ｆ～８ｊとの間および電極６ｆ～６ｊと電極８ａ～８ｅとの間に１５Ｖの電位差を与えることができる。この場合、電極６ａ～６ｅと電極８ａ～８ｅとの交点の電子放出領域２５では電子放出量が比較的大きくなり、電極６ｆ～６ｊと電極８ｆ～８ｊとの交点の電子放出領域２５では電子放出量が比較的小さくなる。また、電極６ａ～６ｅと電極８ｆ～８ｊとの交点の電子放出領域２５および電極６ｆ～６ｊと電極８ａ～８ｅとの交点の電子放出領域２５での電子放出量は中間程度になる。これらの電子放出量に対応して培地２中の細胞３に与える電気刺激の強度も変化する。従って、電気刺激量に所望の分布を持たせて培地２中の細胞３を電気刺激することが可能になる。また、１つのウェル１２中で電気刺激量を変化させることによりスクリーニング試験をすることが可能になる。
　他の構成は第１又は第２実施形態と同様である。また、上記の第１又は第２実施形態についての記載は、矛盾がない限り第３実施形態についても当てはまる。 By changing the voltage applied by the combination of the electrodes 6a to 6j and the electrodes 8a to 8j, the amount of electron emission can be changed in the plurality of electron emission regions 25 arranged in a matrix. For example, an AC voltage of 20 V is applied between the electrodes 6a-6e and the electrodes 8a-8e, and an AC voltage of 10 V is applied between the electrodes 6f-6j and the electrodes 8f-8j. A potential difference of 15 V can be applied between ～ 6e and electrodes 8f ～ 8j and between electrodes 6f 6 6j and electrodes 8a 8 8e. In this case, the amount of electron emission is relatively large in the electron emission region 25 at the intersection of the electrodes 6a to 6e and the electrodes 8a to 8e, and electron emission in the electron emission region 25 at the intersection of the electrodes 6f to 6j and the electrodes 8f to 8j. The amount is relatively small. The amount of electron emission in the electron emission region 25 at the intersection of the electrodes 6a to 6e and the electrodes 8f to 8j and in the electron emission region 25 at the intersection of the electrodes 6f to 6j and the electrodes 8a to 8e is about middle. The intensity of the electrical stimulation applied to the cells 3 in the medium 2 also changes corresponding to the amount of emitted electrons. Therefore, it becomes possible to electrically stimulate the cells 3 in the medium 2 with the desired distribution of the amount of electrical stimulation. In addition, it is possible to conduct a screening test by changing the amount of electrical stimulation in one well 12.
The other configuration is the same as that of the first or second embodiment. The description of the first or second embodiment also applies to the third embodiment unless there is a contradiction.
第４実施形態
　図６は、本実施形態の細胞培養装置３５の概略断面図である。
　本実施形態では、電荷回収電極９が培養容器２０に固定されている。一方、電子放出素子４は、絶縁性部材１３に固定されている。このような構成とすると、電荷回収電極９と電子放出素子４との間にリーク電流が流れることを抑制することができる。
　また、電荷回収電極９は、ウェル１２の底に配置されており、細胞３は電荷回収電極９と電子放出素子４との間に位置する。このため、細胞刺激装置３０のイオン照射により培地２の表面で生じた陰イオンは、ウェル１２の底に向かって流れる。このため、培地２で培養する細胞３に効率よく電気刺激を与えることが可能になる。
　他の構成は第１～３実施形態と同様である。また、上記の第１～３実施形態についての記載は、矛盾がない限り第４実施形態についても当てはまる。 Fourth Embodiment FIG. 6 is a schematic cross-sectional view of a cell culture apparatus 35 of the present embodiment.
In the present embodiment, the charge collection electrode 9 is fixed to the culture vessel 20. On the other hand, the electron emitter 4 is fixed to the insulating member 13. With such a configuration, it is possible to suppress a leak current from flowing between the charge collection electrode 9 and the electron emitting element 4.
In addition, the charge recovery electrode 9 is disposed at the bottom of the well 12, and the cell 3 is located between the charge recovery electrode 9 and the electron-emitting device 4. Therefore, anions generated on the surface of the medium 2 by the ion irradiation of the cell stimulation device 30 flow toward the bottom of the well 12. For this reason, it is possible to efficiently provide electrical stimulation to the cells 3 cultured in the medium 2.
The other configuration is the same as in the first to third embodiments. The descriptions of the first to third embodiments also apply to the fourth embodiment unless there is a contradiction.
第５実施形態
　図７は、本実施形態の細胞培養装置３５の概略断面図である。
　本実施形態では、培養容器２０が複数のウェル１２ａ～１２ｃを備えている。また、細胞刺激装置３０が各ウェル１２ａ～１２ｃに対応する絶縁性部材１３ａ～１３ｃ、電子放出素子４ａ～４ｃ及び電荷回収電極９ａ～９ｃを備えている。このような構成にすると、培養容器２０の各ウェル１２ａ～１２ｃで培養する細胞にそれぞれ電気刺激を与えることができる。
　また、スイッチング回路を用いて電源装置１１による電圧印加を制御することにより、複数のウェル１２のうち必要な箇所にのみ、必要な時間、必要なタイミングで細胞の電気刺激を行う事ができる。 Fifth Embodiment FIG. 7 is a schematic cross-sectional view of a cell culture apparatus 35 of the present embodiment.
In the present embodiment, the culture vessel 20 is provided with a plurality of wells 12a to 12c. Further, the cell stimulation device 30 is provided with insulating members 13a to 13c corresponding to the respective wells 12a to 12c, electron emitting elements 4a to 4c, and charge collecting electrodes 9a to 9c. With such a configuration, electrical stimulation can be given to the cells cultured in each well 12a to 12c of the culture container 20.
Further, by controlling the voltage application by the power supply device 11 using the switching circuit, it is possible to perform the electrical stimulation of the cells only at the necessary places among the plurality of wells 12 at the necessary time and at the necessary timing.
　培養容器２０が備えるウェル１２の数は特に限定されない。例えば、培養容器２０は６ウェルプレートである。
　細胞刺激装置３０は、培養容器２０が有するすべてのウェル１２に対応する絶縁性部材１３、電子放出素子４及び電荷回収電極９を備えてもよく、培養容器２０が有する一部のウェル１２に対応する絶縁性部材１３、電子放出素子４及び電荷回収電極９を備えてもよい。
　また、細胞刺激装置３０は、１つの蓋部材１０に複数の絶縁性部材１３が固定された構造を有することができる。
　他の構成は第１～４実施形態と同様である。また、上記の第１～４実施形態についての記載は、矛盾がない限り第５実施形態についても当てはまる。 The number of wells 12 provided in the culture vessel 20 is not particularly limited. For example, the culture vessel 20 is a 6 well plate.
The cell stimulator 30 may include the insulating member 13 corresponding to all the wells 12 of the culture vessel 20, the electron emitting element 4 and the charge collection electrode 9, and corresponds to a part of the wells 12 of the culture vessel 20. Insulating member 13, electron emitting element 4 and charge collection electrode 9 may be provided.
In addition, the cell stimulation device 30 can have a structure in which a plurality of insulating members 13 are fixed to one lid member 10.
The other configuration is the same as in the first to fourth embodiments. In addition, the descriptions of the first to fourth embodiments described above also apply to the fifth embodiment unless there is a contradiction.
　２、２ａ～２ｃ：培地　　３、３ａ～３ｃ：細胞　　４、４ａ～４ｃ：電子放出素子　　５：絶縁層　　６、６ａ～６ｊ：下部電極　　７：中間層　　８、８ａ～８ｊ：表面電極　　９、９ａ～９ｃ：電荷回収電極　　１０：蓋部材　　１１、１１ａ、１１ｂ：電源装置　　１２、１２ａ～１２ｃ：ウェル　　１３、１３ａ～１３ｃ：絶縁性部材　　１５、１５ａ、１５ｂ：第１端子　　１６：第２端子　　１８：インキュベーター　　２０：培養容器　　２１：気相　　２５、２５ａ、２５ｂ：電子放出領域　　３０： 細胞刺激装置　　３５：細胞培養装置 2, 2a-2c: culture medium 3, 3a-3c: cell 4, 4a-4c: electron emitting element 5: insulating layer 6, 6a-6j: lower electrode 7: intermediate layer 8, 8a-8j: surface electrode 9, 9a 9c: Charge collection electrode 10: Lid member 11, 11a, 11b: Power supply device 12, 12a to 12c: Well 13, 13a to 13c: Insulating member 15, 15a, 15b: First terminal 16: Second terminal 18: Incubator 20: culture vessel 21: gas phase 25, 25a, 25b: electron emission region 30: cell stimulator 35: cell culture device

Claims (13)

  1. 　培地で培養する細胞に刺激を与えるための細胞刺激装置であって、
    前記細胞刺激装置は、前記培地に気相を介して電荷を供給する電子放出素子と、前記培地から電荷を回収する電荷回収電極とを備え、
    前記電子放出素子は、下部電極と、表面電極と、前記下部電極と前記表面電極との間に配置された中間層とを備え、
    前記電荷回収電極は、前記培地に接触することができるように設けられたことを特徴とする細胞刺激装置。     A cell stimulator for stimulating cells cultured in a culture medium, comprising:
    The cell stimulator includes an electron emitting element that supplies charge to the culture medium via a gas phase, and a charge collection electrode that collects charge from the culture medium,
    The electron emission element includes a lower electrode, a surface electrode, and an intermediate layer disposed between the lower electrode and the surface electrode.
    The cell stimulation device, wherein the charge recovery electrode is provided to be able to contact the culture medium.
  2. 　前記下部電極及び前記表面電極は、それぞれ複数の細長い電極を含み、前記下部電極及び前記表面電極のうち一方が行となり他方が列となる格子状に配置された請求項１に記載の細胞刺激装置。 The cell stimulating device according to claim 1, wherein the lower electrode and the surface electrode each include a plurality of elongated electrodes, and one of the lower electrode and the surface electrode is arranged in a grid in which one is a row and the other is a column. .
  3. 　電源装置をさらに備え、
    前記電源装置は、前記下部電極と前記表面電極との間に電圧を印加することができるように設けられ、かつ、前記電子放出素子と前記電荷回収電極との間に電位差を生じさせることができるように設けられた請求項１又は２に記載の細胞刺激装置。     Further equipped with a power supply,
    The power supply device is provided to be able to apply a voltage between the lower electrode and the surface electrode, and can generate a potential difference between the electron emitting element and the charge collection electrode. The cell stimulating device according to claim 1 or 2, provided as follows.
  4. 　前記電源装置は、前記電子放出素子から前記培地へ電荷を供給することにより前記気相に生じる電流と、前記電荷回収電極が前記培地から電荷を回収することにより前記培地に生じる電流とがループ電流となるように前記電子放出素子及び前記電荷回収電極と電気的に接続する請求項３に記載の細胞刺激装置。 The power supply device generates a loop current by generating a charge in the gas phase by supplying a charge from the electron emitting element to the culture medium, and a current generated in the culture medium by collecting the charge from the culture medium by the charge recovery electrode. The cell stimulation device according to claim 3 electrically connected to said electron emission element and said charge recovery electrode so that it becomes.
  5. 　前記電荷回収電極を接地接続する請求項３又は４に記載の細胞刺激装置。 The cell stimulation device according to claim 3 or 4, wherein the charge recovery electrode is connected to ground.
  6. 　絶縁性部材と、前記絶縁性部材に固定された針状端子とをさらに備え、
    前記電子放出素子は、前記絶縁性部材に固定され、
    前記針状端子は、その先端部で前記下部電極と接触する請求項１～５のいずれか１つに記載の細胞刺激装置。     It further comprises an insulating member, and a needle-like terminal fixed to the insulating member,
    The electron emitting device is fixed to the insulating member,
    The cell stimulation device according to any one of claims 1 to 5, wherein the needle-like terminal contacts the lower electrode at its tip.
  7. 　前記絶縁性部材は、その表面にスリット構造を有する請求項６に記載の細胞刺激装置。 The cell stimulating device according to claim 6, wherein the insulating member has a slit structure on its surface.
  8. 　前記電荷回収電極は、前記絶縁性部材に固定された請求項６又は７に記載の細胞刺激装置。 The cell stimulation device according to claim 6, wherein the charge collection electrode is fixed to the insulating member.
  9. 　請求項１～７のいずれか１つに記載の細胞刺激装置と、前記培地を収容するための培養容器とを備え、
    前記細胞刺激装置は、絶縁性部材を備え、
    前記電子放出素子は、前記絶縁性部材に固定され、
    前記絶縁性部材は、前記表面電極が前記培地の表面と気相を挟んで対向するように前記培養容器上に配置された細胞培養装置。     A cell stimulator according to any one of claims 1 to 7 and a culture vessel for containing said culture medium,
    The cell stimulator comprises an insulating member,
    The electron emitting device is fixed to the insulating member,
    The cell culture device, wherein the insulating member is disposed on the culture vessel such that the surface electrode faces the surface of the culture medium via a gas phase.
  10. 　前記絶縁性部材は、前記表面電極と前記培地の表面との間の距離が０．５ｍｍ以上３ｍｍ以下となるように設けられた請求項９に記載の細胞培養装置。 The cell culture device according to claim 9, wherein the insulating member is provided such that a distance between the surface electrode and the surface of the culture medium is 0.5 mm or more and 3 mm or less.
  11. 　前記電荷回収電極は、前記培養容器に固定された請求項９又は１０に記載の細胞培養装置。 The cell culture device according to claim 9 or 10, wherein the charge recovery electrode is fixed to the culture vessel.
  12. 　下部電極と、表面電極と、前記下部電極と前記表面電極との間に配置された中間層とを備えた電子放出素子の前記下部電極と前記表面電極との間に電圧を印加することにより、気相を介して細胞を培養する培地に電荷を供給するステップを含み、
    前記電荷を供給するステップは、前記電子放出素子と前記培地との間の電界を利用して電荷を供給するステップである細胞刺激方法。     By applying a voltage between the lower electrode and the surface electrode of the electron-emitting device provided with the lower electrode, the surface electrode, and the intermediate layer disposed between the lower electrode and the surface electrode, Supplying a charge to the culture medium for culturing the cells via the gas phase,
    The step of supplying the charge is a step of supplying a charge using an electric field between the electron-emitting device and the culture medium.
  13. 　前記電荷を供給するステップは、前記電子放出素子から前記培地へ電荷を供給することにより前記気相に生じる電流と、電荷回収電極が前記培地から電荷を回収することにより前記培地に生じる電流とがループ電流として流れるステップである請求項１２に記載の細胞刺激方法。 In the step of supplying the charge, a current generated in the gas phase by supplying the charge from the electron emitting element to the culture medium, and a current generated in the culture medium by the charge collection electrode recovering the charge from the culture medium are The cell stimulation method according to claim 12, which is a step of flowing as a loop current.
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JPH01179629A (en) * 1988-01-11 1989-07-17 Nissin Electric Co Ltd Method for treating fungi-culturing medium for growth acceleration
JP2003000225A (en) * 2001-06-25 2003-01-07 Hakuju Inst For Health Science Co Ltd Cell culture apparatus and cell culture method
JP2006325493A (en) * 2005-05-26 2006-12-07 Matsushita Electric Works Ltd Organism-activating method
JP2016136485A (en) * 2015-01-23 2016-07-28 シャープ株式会社 Electron emission element and electron emission apparatus
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* Cited by examiner, † Cited by third party
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JPS5736980A (en) * 1980-08-11 1982-02-27 Iwatani & Co Acceleration of modifying properties of microorganism
JPS60110287A (en) * 1983-11-21 1985-06-15 Yamasa Shoyu Co Ltd Method and apparatus for cultivation of cell with electrical stimulation
JPH01179629A (en) * 1988-01-11 1989-07-17 Nissin Electric Co Ltd Method for treating fungi-culturing medium for growth acceleration
JP2003000225A (en) * 2001-06-25 2003-01-07 Hakuju Inst For Health Science Co Ltd Cell culture apparatus and cell culture method
JP2006325493A (en) * 2005-05-26 2006-12-07 Matsushita Electric Works Ltd Organism-activating method
JP2016136485A (en) * 2015-01-23 2016-07-28 シャープ株式会社 Electron emission element and electron emission apparatus
WO2017033898A1 (en) * 2015-08-25 2017-03-02 旭硝子株式会社 Cell culture device and biological sample production method

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