TOP > 外国特許検索 > Formation and use of neuronal network, and neuron seeding device

Formation and use of neuronal network, and neuron seeding device NEW

外国特許コード F170009208
整理番号 AF19-04WO
掲載日 2017年9月12日
出願国 欧州特許庁(EPO)
出願番号 13838905
公報番号 2899262
公報番号 2899262
出願日 平成25年3月21日(2013.3.21)
公報発行日 平成27年7月29日(2015.7.29)
公報発行日 平成29年8月9日(2017.8.9)
国際出願番号 JP2013057976
国際公開番号 WO2014045618
国際出願日 平成25年3月21日(2013.3.21)
国際公開日 平成26年3月27日(2014.3.27)
優先権データ
  • 特願2012-205561 (2012.9.19) JP
  • 2013WO-JP57976 (2013.3.21) WO
発明の名称 (英語) Formation and use of neuronal network, and neuron seeding device NEW
発明の概要(英語) (EP2899262)
Provided are a culture device for neuronal network formation that makes it possible to construct a neuronal network while restricting movement of neurons grown in the culture medium, and a means for the use thereof.
A culture device for neuronal network formation in which is formed a cell fixation part surrounded by a plurality of projections on a flat substrate that can be filled with cell culture medium, and in which (1) there are established spaces having widths such that the neuron cells cannot pass through between the plurality of projections, (2) the inner diameter is such that the cell fixation part can house the cells of from one to a number of neurons, (3) the substrate surface, which is the bottom of the cell fixation part, is coated by an extracellular matrix-forming substance and/or provided with minute through-holes for suctioning culture medium by a suction device on the lower side of the substrate surface.
特許請求の範囲(英語) [claim1]
1. A culturing device for formation of a neuron network, wherein cell plating sections surrounded by a plurality of protrusions are formed on a flat plate that can be filled with a cell culture medium, characterized in that the cell plating section satisfying the following conditions (1) to (3). (1) gaps are set between the plurality of protrusions which defines the cell plating sections, wherein the gaps are wide but do not allow a neuronal cell body to pass through; (2) the inner diameter of the cell plating sections defined by the plurality of protrusions is of sizes capable of accommodating one to several neuronal cell bodies; (3) the plate surface forming the bottom of each cell plating section comprises at least one constituent of the following (a) and (b): (a) the plate surface is coated with an extracellular matrix-forming substance. (b) fine through-holes for suction of medium by an aspirator provided below the plate surface are provided, wherein the hole diameters is such that the neurons cannot pass through.
[claim2]
2. The culturing device for formation of a neuron network according to claim 1, characterized in that the culturing device corresponds to any one of the following (1) to (3): (1) cell plating sections are formed as selected regions on the plate, and one to several neurons are placed in the cell plating sections as the selected cells, while other neurons are simply seeded on the plate; (2) multiple cell plating sections are formed on the plate with appropriate gaps between them, and one to several neurons are placed in the cell plating sections, while one cell plating section is used as a selected region in which the neuron is placed; (3) a culturing device for high-throughput analysis of a neuron network, wherein the cell plating sections are formed dispersedly at appropriate locations so that several or multiple units of neuron networks according to (1) or (2) can be formed on the plate.
[claim3]
3. The culturing device for formation of a neuron network according to claim 1 or claim 2, wherein the culturing device for formation of a neuron network is a planar patch-clamp device designed for a neuron network, characterized in that: (1) the plate is an electrical insulating plate, wherein fine through-holes are formed so as to pass through both sides of the plate surface, which composes the bottom of the cell plating section of the electrical insulating plate; (2) liquid pool sections and electrode sections are provided on the neuron network-formed side, as the first surface side of the fine through-holes, and on the second surface side which is the opposite side, respectively, wherein the liquid pool sections hold the conducting liquid as the cell culture medium, and the electrode sections are disposed to be electrically conductive to the conducting liquid of the liquid pool section; and (3) the liquid pool sections on the first surface side are liquid pool sections for neurons plated in the cell plating sections.
[claim4]
4. The culturing device for formation of a neuron network according to claim 3, characterized in that the electrode sections on the first surface side and second surface side disposed in the planar patch-clamp device comprise the following constituents (a) to (c): (a) an electrode receptacle, wherein at least a portion of the receptacle wall that is in contact with the conducting liquid, when the conducting liquid is introduced into the liquid pool sections, is composed of an inorganic porous material. (b) an electrode having a precious metal chloride (NmCl) layer formed on a surface layer section of the precious metal (Nm), and housed in the electrode receptacle. (c) an electrode solution filled into the electrode receptacle, wherein the precious metal chloride (NmCl) and an alkali metal chloride are dissolved at saturated concentration.
[claim5]
5. The culturing device for formation of a neuron network according to any one of claims 1 to 4, characterized in that the culturing device for formation of a neuron network is used for a purpose according to any one of the following (A) to (C): (A) for measurement and analysis of neuronal ion channel current in a neuron network; (B) for imaging analysis, including at least Ca imaging analysis, imaging analysis with synaptophysin or synapsin labeling as synaptic site markers, imaging analysis with MAP2 as a dendrite marker, and imaging analysis with FM1-43 or FM4-64 which labels endosomes or exosomes; (C) for high throughput screening systems for neuron networks.
[claim6]
6. The culturing device for formation of a neuron network according to claim 5 characterized in that when the culturing device for formation of a neuron network is used for imaging analysis according to (B), the culturing device comprises one or more of the following constituents (D) to (F). (D) a photodetector for detecting light emitted by neurons is set above the plate; (E) an irradiating device that irradiates light onto the neurons or plate surface is set above the plate; (F) the irradiating device of (E) is equipped with an optical focusing system for irradiation of light only on a prescribed single neuron.
[claim7]
7. A method of forming a neuron network under culturing for any research purpose by means of a culturing device for formation of a neuron network according to claims 1 to 6, the method comprising: (1) seeding neurons on said flat plate that is filled with cell culture medium, (2) placing and/or plating one or several neurons in each cell plating section by an extracellular matrix-forming substance in the cell plating section, and/or by suctioning liquid medium from the fine through-holes at the bottom of the cell plating section, and (3) forming synaptic junctions between neurons by axons or dendrites, wherein while movement of the neurons plated in the cell plating section is restricted by the plurality of protrusions, the presence of adjacent neurons is mutually recognized through the gap sections of the plurality of protrusions.
[claim8]
8. The method of forming a neuron network according to claim 7, characterized in that during seeding of the neurons in step (1), glial cells are also seeded at sections other than the cell plating sections.
[claim9]
9. A neuron seeding device for seeding neurons in multiple cell plating sections surrounded by a plurality of protrusions on a flat apparatus plate, wherein the neuron seeding device is set on the flat apparatus plate that can be filled with cell culture medium in a culturing device for formation of a neuron network or in a planar patch-clamp device utilizing such a culturing device, characterized in that: a board-shaped device body that can be set on the apparatus plate has a size covering the multiple cell plating sections and the flat bottom of the device body is in contact with the apexes of the plurality of protrusions at the multiple cell plating sections in the set situation, the device body is provided with (1) a suspension supply port for externally supplying a neuron suspension having neurons suspended at a fixed density, (2) a plurality of fine suspension flow channels extending in a branched manner from the suspension supply port, inside the device body, and (3) a suspension injection port for injecting the neuron suspension into each cell plating section, opened at the bottom of the device body at the end of each suspension flow channel.
[claim10]
10. The neuron seeding device according to claim 9, characterized in that the plurality of suspension flow channels are set to have substantially the same lengths, and the design is such that when the neuron seeding device is placed on the apparatus plate of the culturing device for formation of a neuron network or the planar patch-clamp device, the individual suspension injection ports are located to precisely correspond with the individual cell plating sections.
[claim11]
11. A neuron seeding device according to claim 9 or claim 10, characterized in that the board-shaped device body comprises an upper board provided with the suspension supply ports and a lower board provided with the suspension injection ports, joined in a closely bonded state, and grooves composing the suspension flow channels are formed on at least one of the bonding surfaces of the upper board and lower board.
[claim12]
12. A neuron seeding device according to any one of claims 9 to 11, wherein the culturing device for formation of a neuron network is a culturing device for formation of a neuron network according to claim 1, and/or the planar patch-clamp device is a planar patch-clamp device according to claim 3.
[claim13]
13. A neuron seeding device according to any one of claims 9 to 12, wherein the device body further comprises a second suspension flow channel system for injection of the neuron suspension into regions other than the cell plating sections of the apparatus plate of the culturing device for formation of a neuron network or the planar patch-clamp device.
  • 出願人(英語)
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • URISU TSUNEO
  • WANG ZHI-HONG
  • UNO HIDETAKA
  • SAITOH MIHO
  • NAGAOKA YASUTAKA
国際特許分類(IPC)
指定国 (EP2899262)
Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
参考情報 (研究プロジェクト等) CREST Creation of Nanosystems with Novel Functions through Process Integration AREA
ライセンスをご希望の方、特許の内容に興味を持たれた方は、問合せボタンを押してください。

PAGE TOP

close
close
close
close
close
close