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Formation and use of neuronal network, and neuron seeding device

Foreign code F170009194
File No. AF19-04WO
Posted date Sep 12, 2017
Country Republic of Korea
Application number 20157006609
Gazette No. 20150056551
Gazette No. 102027808
Date of filing Mar 16, 2015
Gazette Date May 26, 2015
Gazette Date Oct 2, 2019
International application number JP2013057976
International publication number WO2014045618
Date of international filing Mar 21, 2013
Date of international publication Mar 27, 2014
Priority data
  • P2012-205561 (Sep 19, 2012) JP
  • 2013JP57976 (Mar 21, 2013) WO
Title Formation and use of neuronal network, and neuron seeding device
Abstract 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.
(From EP2899262 A1)
Outline of related art and contending technology BACKGROUND ART
Conventionally, for practical purposes or research purposes (in particular a liquid medium) in the media from the cells and maintenance of nerves, nerve cells in a living state in vitro neuronal network consisting of proposals have been made to the embodiment of the present invention.
For example the following non Patent document 1 shown in Fig. 2 as a substrate are provided in a plurality of protrusions Si is surrounded by a doped region is formed, wherein the peripheral nerve cells of the card (stagnalis) aggregate (ganglion) is arranged on the large ganglia, nerve cells and detecting a change in potential of the present invention. In the following non patent document 2 shown in Fig. 3 on the substrate will be referred to as the 'cage' substantially disk shaped enclosures (about 9 micro m height) and form a plurality of, in the space of the central portion of each cage as well as neuronal n=best, the cage is provided through some of the tunnel or the like of the axon of the nerve cells in the adjacent cage extend toward neurons to neurotrophins are provided with a chip.
Scope of claims [claim1]
1. An electrically insulating substrate having through holes, wherein the electrically insulating substrate 1 is formed on the surface side of the first and second liquid storing part 1, formed on a surface side opposite to the first 2 and second liquid storing part 2, the first and second liquid storing portion 1 is introduced to the liquid storing portion 2 the conductive liquid disposed in each of the electrode can be energized to a culture medium for neuronal network including a planar patch clamp device type, the first surface side of the electrically insulating substrate 1 to the peripheral edge of the through hole of the plurality of protrusions is surrounded by a cell and a fixing unit, wherein, a plurality of protrusions of the neural cells that do not pass through the cell at a spacing, 그리고전극부가 (a) less than or equal to (c) said ∼ liquid storing portion (a) when the conductive liquid is introduced into the conductive liquid contacts the inorganic porous material is at least a portion of the container wall of the container with the electrode; (b) the electrode is housed in the surface layer of noble metal of the noble metal chloride Nm NmCl layer formed on an electrode; (c) wherein the electrode is placed in the container and the chloride of the noble metal halide and alkali metal salts of saturated NmCl dissolved to a concentration of the solution to the electrode provided with a neural network for the cultured cells to a planar patch clamp device type.

[claim2]
2. Method according to claim 1, wherein the plurality of protrusions defined by the inner diameter of the fixing unit 1 ∼ cell or several cells of the neural cell can accommodate the neuronal network is of a size in the planar patch clamp device in the form of a cell culture.

[claim3]
3. Method according to claim 1, wherein the cells constituting the fixing unit to a substrate surface coated with an extracellular matrix forming material is a nerve cell culture type planar patch clamp device to the network.

[claim4]
4. Method according to claim 1, wherein the electrically insulating layer on a substrate having a plurality of through holes and the fixing portion and a cell, a neural cell network to enable the formation of the fixing part is arranged at an interval of the cell culture in the planar patch clamp type for neuronal network device.

[claim5]
5. Any one of claims 1 to 4 in the first method according to claim, wherein the neuronal network is cultured for a planar patch clamp device in the form of a lower one (A) ∼ (C) is used for the purpose of nerve cells in a culture medium for a planar patch clamp device in the form of a network. (A) the neuronal network of neurons in the ion channel current measurement, and to analysis for the purpose of analyzing the at least one imaging Ca (B), synaptophysin or display a marker of synaptic site by imaging analysis synaptic marker, a marker of a spine imaging analysis of the marking MAP2, and endosome or exosomes labeling by FM1 field 43 or imaging analysis FM4 field 64 imaging analysis comprises using a neural cell network for the purpose of high throughput (C) for use in a screening system for the purpose of

[claim6]
6. Method according to claim 5, wherein said neural cell culture type planar patch clamp device is a network of the first imaging described in claim 5 (B) used in the analysis in the case of the 1 ∼ (D) (F) with at least two neuronal network is cultured for a planar patch clamp device type. (D) Provided in an upper portion of the substrate, the light receiving device of the light emitted from the nerve cells of the substratE provided in an upper portion, a nerve cell or a light source which irradiates the surface of the substrate and the irradiation of the devicE (F) device, only a single predetermined in order to investigate neuronal focusing system for focusing the light irradiation apparatus

[claim7]
7. The method of any one of claims 1-4 neuronal network is cultured for a planar patch clamp device type using a neural network to form a cell culture method comprising the following, (1) neural cell culture and the cells are seeded onto the substrate under the filling step and the fixing unit, (2) cells of the extracellular matrix forming material and/or fixing the cells to the bottom of the liquid medium from the fine holes of the fixing by suction of the neuronal cell disposed at each of several 1 ∼, and a fixing step of fixing unit fixing a nerve cell, (3) the cell movement of a plurality of protrusions and a plurality of protrusions while constrained by the spacing relative to one another by a portion of a neuronal axon or dendrites to recognize the presence of the synaptic junctions between nerve cell comprising the steps of the method of the neuronal network.

[claim8]
8. Method according to claim 7, wherein the neural cells of neural network formation method (1) and the cells are seeded in the further step of glial cells seeded in the portions other than the fixing portion of said method of forming a neural cell network.

[claim9]
9. AMEND STATUS: Delete

[claim10]
10. AMEND STATUS: deletion

[claim11]
11. AMEND STATUS: deletion

[claim12]
12. AMEND STATUS: deletion

[claim13]
13. AMEND STATUS: deletion
  • Applicant
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • URISU TSUNEO
  • WANG ZHI HONG
  • UNO HIDETAKA
  • SAITOH MIHO
  • NAGAOKA YASUTAKA
IPC(International Patent Classification)
Reference ( R and D project ) CREST Creation of Nanosystems with Novel Functions through Process Integration AREA
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