Formation and use of neuronal network, and neuron seeding device
|発明の名称 （英語）||Formation and use of neuronal network, and neuron seeding device|
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.
1. A neural cell network-forming culture device, characterized in, that is filled with cell culture medium on a flat substrate in a device formed by wrapping the cell adhesion portion is surrounded by a plurality of protrusions, the unit that the cells-(3) have the following(1) conditions,
The portion of the plurality of protrusions forming the(1) predetermined cell is set between the wider spacing, a width in the neural cells does not allow the bodies within limits,
The portion is defined by the(2) plurality of protrusions inner diameter capable of accommodating a 1 cell-cell size of the plurality of nerve cells,
The bottom surface of the substrate surface is to constitute the cell(3) unit has the following (i) or (ii) at least one element,
(I) is coated with extracellular matrix-forming material,
(Ii) is provided with fine holes, the fine through hole is provided with a substrate surface with the lower suction aspirated fine through holes, having nerve cells cannot pass through the aperture.
2. Neural cell network forming culture apparatus according to claim 1, characterized in, the(1) corresponding in-(3) any one of the culture device,
Formed on the substrate(1) as the unit cells of selected timing, the predetermined cells is disposed in the 1-the number of neural cells as selected, and the, other neural cells only seeding on the substrate,
On a substrate(2) appropriate form a plurality of mutually spaced fixed cell adhesion portion, the 1 portion is disposed in the predetermined cell-number of nerve cells, and the, wherein the 1 cells are used as the unit equipped with the predetermined selected areas of the nerve cell,
Can be formed on the substrate or the complex 3) one of the unit is equivalent to or(1)(2) neural cell network, which is formed in place of the discrete cell of high throughput analysis for neural network unit cell culture device.
3. According to claim 1 or a cell culture device for forming a network according to claim 2, characterized in, neural cells cultured neuronal cell network to network formation plane of the object is of a patch clamp device,
The substrate is(1) an electrically insulating material, provided with fine holes, the fine through hole such that the electrically insulating substrate the substrate is a bottom portion of the cells constituting the communicating lateral surface of the side surface of the fine holes, (2) 1st, and its opposite side that neural cell network formed side, i.e. side surface is provided for respectively 2nd as conductive liquid cell culture medium liquid reservoirs for holding, and the liquid reservoir according to the energization of the conductive liquid to the electrode part is disposed a liquid reservoir, (3) the side surface 1st the unit cell constant in the predetermined liquid reservoirs for neural cells.
4. Neural cell network-forming culture apparatus according to claim 3, characterized in, a planar patch clamp device, and a surface side surface side of the electrode unit 1st 2nd has the following (a)-(c) elements,
(A) electrode container, the electrode reservoir, the reservoir portion in the conductive liquid in contact with conductive liquid into a wall of the container is at least one is composed of inorganic porous material,
(B) electrodes housed within the container, the surface layer portion formed with the noble metal Nm chloride NmCl noble metal electrode layer,
(C) filling the electrode within the container, to saturation concentration of the dissolved precious metal and the alkali metal chloride solution electrode NmCl chloride.
5. 1 according to claim-claim 4 for forming a network according to any one neural cell culture device, characterized in, a neural network for forming a cell culture device in the following (A)-(C) according to any of the purposes,
(A) nerve cell networks in nerve cells in the measurement of ion channel current · used for analysis,
(B) Ca imaging analysis comprising at least, based on the presynaptic site as a marker or a synapsin synaptophysm labeled imaging analysis, based on the marker as dendrites based on imaging resolution and MAP2 labeled endosomes, exosomes or imaging resolution imaging labeled FM 1-43 FM 4-64 used in analysis,
(C) in high throughput screening system for use in a neural cell network.
6. Neural cell network forming culture apparatus according to claim 5, characterized in, the neural cells for culture device for forming a network (B) imaging analysis is, also has a (D)-(F) more elements of 1,
(D) at an upper portion receiving the light emitted by the nerve cell is provided with a light receiving device,
(E) is provided at an upper portion for irradiating light irradiation device substrate surface or neural cell,
(F) (E) illuminating means is equipped with only a single nerve cell of the predetermined light-collecting system.
7. A neuron network forming method, characterized in, claim 1 using-claim 6 in the neural cell network-forming culture device of any one of, based on the research purposes forming medium at any neuronal cell network, comprising:
Seeding filled with(1) cell culture medium of the step of nerve cells on a flat substrate,
Positioning the part cell with(2) extracellular matrix-forming material, the part is from a cell and the bottom/or through the fine through holes through the liquid medium, the predetermined cells is disposed for each of the 1 · given-number of nerve cells step, and
A plurality of protrusions that settle in the interaction with(3) the neural cell migration and cell portion, and with the plurality of protrusions spaced-apart portions to each other to identify the presence of nerve cell partner, to form a nerve axons or dendrites based intercellular synaptic connections step.
8. Neural cell network formation method according to claim 7, characterized in, in neuronal cells in a method for forming a network, in the step of seeding in(1) neural cells, glial cells seeded in the cell the portion other than the predetermined.
9. A nerve cell seeding device, a neural cell seeding device is provided in neural cells cultured by the culture apparatus or network forming the plane of the device in a patch clamp apparatus capable of cell culture medium of the device substrate by flat filling, a nerve cell seeding to wrapping the plurality of protrusions in the device substrate from the portion is surrounded by a plurality of cells,
Plate-like device on the substrate can be positioned in the device body in the installed state of the given portion covering a plurality of cells with the extent, and wherein the predetermined cells with the planar bottom portion of the plurality of plurality of protrusions contact tip,
A device body with a density from an external supply for :(1) suspending a suspension of the cell suspension a supply port nerve neural cells inside of the body from the suspension supply port in the device ;(2) a dendrimer suspension flow path extending to a plurality of fine; suspension flow path and at(3) each end, open to the device bottom surface of the body, cell suspension into a nerve cell suspension inlet end of the respective determined.
10. A cell seeding device according to claim 9, characterized in, a cell seeding device, a plurality of suspension flow is set to substantially the same length, and is designed to: the nerve cells are seeded device is provided in neural cells cultured network formation on the substrate is planar patch clamp device or a container, each suspension inlet position of the unit is precisely corresponding to each cell.
11. According to claim 9 or a cell seeding device according to claim 10, characterized in, that the device body is a plate provided with a suspension inlet suspension supply port upper plate and arranged in a tightly adhering state that joins the lower plate, and the upper plate and lower plate constituting at least one of the suspension flow path is formed engagement surface.
12. 9 according to claim-claim 11 seeded device according to any one of nerve cells, characterized in, a cell culture device for forming a network of neural cell culture device for forming a network according to claim 1, and a planar patch clamp/or planar patch clamp device of claim 3 means.
13. 9 according to claim-claim 12 neural cell seeding device claims, characterized in, the device body suspension the flow channel system has 2nd, 2nd suspension the flow path system for neural cell network formed of planar patch clamp device substrate for culture device or the predetermined cells beyond the device in the neural cell suspension injected into the region.
|参考情報 （研究プロジェクト等）||CREST Creation of Nanosystems with Novel Functions through Process Integration AREA|
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