device

Abstract

The present invention provides a neuronal device that enables observation of early neural activity (spikes, bursts, etc.) by culturing neurons on a cell scaffold, and measures electrical intensity with enhanced accuracy. The use of the neuronal device of the present invention also enables intracellular signal imaging.

Description

[Technical field]

[0001] The present invention relates to a novel neuronal device for measuring neural activity. [Background technology]

[0002] In safety evaluation in drug development, neurotoxicity, along with cardiotoxicity and hepatotoxicity, is one of the main reasons drug development is discontinued. Therefore, accurate neurotoxicity evaluation is necessary from the early stage of drug development. However, evaluation of neurotoxicity in non-clinical trials is mainly performed using in vivo evaluation systems such as symptom observation in animal experiments and pathological brain evaluation, and a simpler in vitro system for evaluating neurotoxicity has not been established. Neuroactivity is based on electrical activity expressed by the interaction of multiple nerve cells. In recent years, as an in vitro test system, a method has been developed in which isolated nerve cells or nerve cells induced from pluripotent stem cells are dispersed and cultured on a multi-electrode array (hereinafter abbreviated as MEA) to reconstruct a neural network and observe neural activity by measuring the extracellular potential (Non-Patent Document 1), and studies on evaluating neurotoxicity using this method have been reported (Non-Patent Documents 2 and 3). As a method for observing nerve cell activity, a method of measuring intracellular signals such as the membrane potential and intracellular calcium of nerve cells and visualizing (imaging) them is also used. It has been reported that changes in membrane potential and fluctuations in intracellular calcium concentration can be measured using voltage-sensitive dyes or calcium-sensitive dyes incorporated into neurons; it has also been reported that they can be measured using voltage-sensitive fluorescent proteins or calcium-sensitive fluorescent proteins expressed in neurons (Non-Patent Documents 4 to 6).

[0003] Although it is generally considered difficult to culture nerve cells, various means have been reported to provide a scaffold on which nerve cells can adhere and promote the proliferation of nerve cells. For example, it has been reported that the proliferation of nerve cells is promoted by seeding nerve cells on a scaffold in which microfibers made of polycaprolactone or polycaprolactone mixed with gelatin are aligned and embedded in a porous three-dimensional hydrogel (Non-Patent Document 7). In addition, there are several reports on scaffold materials used as cell scaffolds for the purpose of application to medical materials, etc. For example, cell culture or tissue regeneration is effectively performed using nanofibers composed of polyolefin, polyamide, polyurethane, polyester, fluoropolymer, polylactic acid, polyvinyl alcohol, etc., or a cell scaffold material composed of the nanofibers adsorbed with protein components (Patent Document 1); 3D cell culture using a cell scaffold material having a hollow fiber membrane mesh and a nanofiber layer is performed to supply nutrients and oxygen to cultured cells and remove metabolic waste products from cultured cells with high efficiency (Patent Document 2); A cell scaffold material composed of nanofibers containing gelatin, collagen, or cellulose, or crosslinked nanofibers is used to mass-supply pluripotent stem cells and suppress cell death (Patent Document 3); A cell scaffold material coated with nanofibers composed of polyglycolic acid or gelatin, etc., on a support body made of polyglycolic acid, is used to improve the proliferation rate of human pluripotent stem cells (Patent Document 4), etc. However, there have been no reports on means for detecting electrical signals early after the start of neuronal culture in extracellular potential measurement using MEA, and means for obtaining enhanced electrical signals. Furthermore, there have been no reports of a means for detecting changes in intracellular signals in individual cells at an early stage after the initiation of neuronal culture. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2006-254722 [Patent Document 2] Japanese Patent Application Laid-Open No. 2011-239756 [Patent Document 3] Japanese Patent Application Laid-Open No. 2013-247943 [Patent Document 4] WO2016 / 068266 [Non-Patent Document]

[0005] [Non-Patent Document 1] Odawara, A., et al. Scientific Reports 2016 May 18;6:26181. [Non-Patent Document 2] Kraushaar, U., et al. Stem Cell-Derived Models in Toxicology, Methods in Pharmacology and Toxicology, 293-308, Mike Clements and Liz Roquemore (eds.), Springer Science New York 2017. [Non-Patent Document 3] Schmidt, B. Z., et al. Arch Toxicol (2017) 91:1-33. [Non-Patent Document 4] Grienberger, C. and Konnerth, A., Neuron 73, 862-885, 2012. [Non-Patent Document 5] Antic, S. D., et al. J Neurophysiol. 116: 135-152, 2016. [Non-Patent Document 6] Miller, E. W., Curr Opin Chem Biol. 33: 74-80, 2016. [Non-Patent Document 7] Lee, S-. J., et al. Tissue Eng Part A. 2017 Jun;23(11-12):491-502. [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] There are various problems when evaluating neural activity using MEAs. For example, in order to directly seed neurons on a measurement probe, long-term culture of 5 to 6 weeks or more is required before they can be used to measure neural activity; in the case of direct seeding on a conventional measurement probe, cells tend to detach or aggregate, leading to a loss of uniformity in terms of cell maintenance, which can lead to unstable situations in evaluating neural activity; with long-term cell culture, there is an increased possibility of undesirable events (cell detachment, aggregation, contamination, etc.) occurring during culture, and if such events occur, the costs and time spent on cell culture up to that point will be wasted; long-term cell culture requires high maintenance costs; and with the current cell culture format, it is impossible to supply cells in a state that can be used immediately for observing neural activity. Problems associated with long-term culture of neurons also occur when evaluating neural activity using intracellular signal imaging methods. An object of the present invention is to overcome the above-mentioned problems of the prior art. [Means for solving the problem]

[0007] The inventors conducted intensive research aimed at overcoming the above-mentioned problems of the conventional technology, and as a result, discovered that it is possible to provide a neuronal device for culturing neurons on a cell scaffold, thereby completing the present invention.

[0008] That is, the object of the present invention is achieved by the following invention. (1) A neuronal device containing a cell scaffold and neuronal cells. (2) The neural device described in (1), in which the neural cells are oriented. (3) The neuronal device described in (1), wherein the cell scaffold is a fiber sheet formed of a polymeric material. (4) The neuronal device described in (3), wherein the fiber sheet has an oriented structure, a non-oriented structure, or a mixed structure of oriented and non-oriented structures. (5) The neuronal device described in (3), in which the fiber sheet is coated with an extracellular matrix protein selected from polylysine, polyornithine, laminin, fibronectin, Matrigel (registered trademark), and Geltrex (registered trademark). (6) A neuronal device described in any one of (1) to (5), in which neuronal cells form a three-dimensional structure on and / or within the cell scaffold. (7) The neuronal device according to any one of (1) to (6), wherein the neuronal cells are primary cultured cells or neuronal cells derived from pluripotent stem cells. (8) The neuronal device described in (7), wherein the neuronal cells derived from the primary cultured cells or pluripotent stem cells are mammalian-derived neuronal cells. (9) The neuronal device according to any one of (1) to (8), wherein the neuronal cells include glutamatergic, dopaminergic, γ-aminobutyric acidergic, monoaminergic, histaminergic or cholinergic neuronal cells. (10) Neurons were added to the cell scaffold at 1 × 10 4 cells / cm 2 ~4×10 6 cells / cm 2 The neuronal device according to any one of (1) to (9), wherein the cells are seeded at a density of 1000×1000 μg / ml. (11) The neuronal device according to any one of (1) to (10), further comprising a frame for supporting the periphery of the neuronal device. (12) The neuronal device according to (11), wherein the length and width of the frame are 2 mm x 2 mm to 15 mm x 15 mm, respectively, and the frame is circular or polygonal. (13) A method for evaluating neural activity using the neural cell device described in any one of (1) to (12). (14) A method for evaluating neural activity, comprising contacting a neural cell device described in any one of (1) to (12) with a multielectrode array and measuring the extracellular potential of a neuron contained in the neural cell device. (15) A method for evaluating neural activity using an intracellular signal imaging substance, the method using a neural cell device described in any one of (1) to (12). (16) The method according to (15), wherein the intracellular signal imaging substance is a fluorescent calcium indicator or a fluorescent voltage indicator. (17) A neuronal device-mounted dish having the neuronal device according to any one of (1) to (12). (18) A neuronal device-mounted plate, comprising a multiwell plate having a plurality of wells, at least one of the wells having the neuronal device described in any one of (1) to (12). Effect of the Invention

[0009] According to the present invention, by culturing nerve cells using a cell scaffold, cell adhesion is improved, and stable culture including reduced cell detachment during culture is possible. As a result, by using the nerve cell device of the present invention, the seeded nerve cells do not detach from the electrodes of the multi-electrode array, and the time until it becomes possible to measure nerve activities such as spikes and bursts is shortened. Furthermore, since aggregation of nerve cells is suppressed, high-frequency spikes can be observed. The nerve cell device of the present invention also enables intracellular signal imaging. The nerve cell device of the present invention is useful for neurotoxicity evaluation, drug screening for nerve diseases, and the like. [Brief description of the drawings]

[0010] [Figure 1] Optical microscope photographs (magnification ×4) showing the state of the cell sheet obtained after seeding iPS cell-derived neurons on an oriented fiber sheet (a) or a random fiber sheet (b) and culturing for 6 days. (c) Immunostained image (magnification ×4) of neurons seeded on the oriented fiber sheet using an anti-MAP2 antibody. (d) Number of spikes and bursts of neurons seeded on the oriented fiber sheet or random fiber sheet. [Diagram 2]These are optical microscope photographs (magnification ×4) showing the state of a cell sheet obtained by seeding iPS cell-derived neurons onto a culture dish (a) or onto an electrode (b) and then culturing them for 1-2 days. [Diagram 3] 253G1 iPS cell-derived neurons and XCL-1 NEURONS were seeded on a cell scaffold, and the 253G1 iPS cell-derived neurons were cultured in culture medium A or B, and XCL-1 NEURONS was cultured in culture medium C for 1 to 3 weeks. These are optical microscopy photographs (magnification x4) showing the state of the cell sheet obtained. Figure 3a, b, and c: Oriented PLGA fibers were used as the cell scaffold. Figure 3d, e, and f: A probe (electrode) coated with 0.003% poly-L-lysine and 20 μg / ml laminin after immersion treatment in culture medium containing 10% bovine serum was used as the cell scaffold. [Figure 4] Figure 4 shows the effect of cell scaffold on the extracellular potential of iPS cell-derived neurons XCL-1 NEURONS measured using MEA probe. NFD: Cells were seeded on an oriented PLGA fiber sheet. probe: Cells were seeded directly on the MEA probe. Figure 4a: Spike counts were measured 2 weeks after culture under high density seeding conditions. Figure 4b: Potential was measured 2 weeks after culture under high density seeding conditions. Figure 4c: Spike counts were measured 4 weeks after culture under low or high density seeding conditions. Figure 4d: Spike counts were measured 6 weeks after culture under low or high density seeding conditions. [Diagram 5] Primary cultured rat cerebral cortical neurons were seeded on an oriented PLGA fiber sheet at 9.0×105 cells / cm2 and cultured for 4 weeks to obtain a cell sheet, and the neural activity of the cell sheet was measured using an MEA probe. The numbers in the figure indicate the MEA channel numbers. [Figure 6]Figure 6 shows the photographs taken by an optical microscope and the neural activity measured by an MEA probe for the cell sheet obtained by culturing iPS cell-derived neurons iCell GlutaNeurons on an oriented polystyrene fiber sheet. Figure 6a: Optical microscope photograph (magnification x20) of the cell sheet obtained by seeding at 12 x 105 cells / cm2 and culturing for 4 weeks. Figure 6b: The neural activity (spikes and synchronous bursts) was measured by an MEA probe for the cell sheet obtained by seeding at 12 x 105 cells / cm2 and culturing for 2 weeks. [Figure 7] This figure shows the effect of cell scaffolds on the drug (4-aminopyridine) responsiveness of neurons. NFD: Cells were seeded on an oriented PLGA fiber sheet. Probe: Cells were seeded directly on an MEA probe. Figure 7a: The spike count was measured after 4 weeks of culture of 253G1 hiPS cell-derived neurons. Figure 7b: The burst count was measured after 6 weeks of culture of iPS cell-derived neurons XCL-1 NEURONS. [Figure 8] This figure shows the drug (4-aminopyridine) responsiveness of iPS cell-derived nerve cells XCL-1 NEURONS seeded at high density (24 x 105 cells / cm2) on an oriented PLGA fiber sheet. Figure 8a shows the results of analyzing the spike count for a cell sheet obtained after 4 weeks of culture. Figure 8b shows the results of analyzing the synchronous burst count for a cell sheet obtained after 4 weeks of culture. Figure 8c shows the results of analyzing the spike count and synchronous burst count by raster plot for a cell sheet obtained after 6 weeks of culture. [Figure 9] This is a fluorescence micrograph (magnification x10) of rat cerebral cortical neurons cultured on an oriented polystyrene fiber sheet stained with a calcium indicator reagent (Fluo-8). The arrows in the figure indicate examples of positions where changes in fluorescence intensity due to changes in calcium ion concentration are observed as blinking. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The cell scaffold used in the neuronal device of the present invention is composed of fibers produced from a polymeric material. The cell scaffold is preferably a fiber sheet having the shape of a sheet in which fibers are accumulated. The fiber sheet can have an oriented structure, a non-oriented structure, or a mixed structure of oriented and non-oriented structures. The oriented structure is a structure in which the fibers constituting the fiber sheet are arranged in one direction, and when the angle of the one direction is 0°, 80% or more of the fibers are present within a range of ±30°. In the oriented structure, the distance between the fibers is not particularly limited, but is preferably 5 to 50 μm. The non-oriented structure is a structure in which the direction of the fibers is randomly arranged. The polymeric material constituting the fibers is preferably a biodegradable or non-biodegradable polymeric material, and examples thereof include, but are not limited to, PLGA (polylactic acid polyglycolic acid), polystyrene (PS), polysulfone (PSU), and polytetrafluoroethylene (PTFE). The cross-sectional diameter of the fibers constituting the fiber sheet is not particularly limited, but is, for example, 0.1 to 8 μm, preferably 0.5 to 7 μm, and more preferably 1 to 6 μm. The thickness of the fiber sheet is, for example, 1 to 40 μm, preferably 5 to 35 μm, and more preferably 10 to 30 μm. The porosity of the fibers constituting the fiber sheet may vary depending on the polymer material used. The porosity is not particularly limited, but is, for example, 10 to 50%, preferably 15 to 45%, and more preferably 20 to 40%. Here, the porosity refers to the ratio of the area where no fibers exist to a certain area of ​​the plane of the fiber sheet.

[0012] The fiber sheet can be produced, for example, by electrospinning a solution containing a polymeric material. When producing a fiber sheet having an oriented structure, the method is not particularly limited, but for example, a rotating drum is used, and while rotating the drum, a solution containing a polymeric material is sprayed from a nozzle onto the rotating surface of the drum, and the fiber formed on the rotating drum is wound up to produce the fiber sheet. When producing a fiber sheet having a non-oriented structure, the fiber sheet can be produced by spraying a solution containing a polymeric material onto a flat plate. When producing a fiber sheet having a mixed structure of an oriented structure and a non-oriented structure, for example, the above-mentioned production methods for producing fiber sheets having an oriented structure and a non-oriented structure can be combined. As the polytetrafluoroethylene (PTFE) sheet, for example, commercially available Poreflon (registered trademark) from Sumitomo Electric Industries, Ltd. can be used. The polymer material solution may be an organic solvent that dissolves the polymer material to be used at 10 to 30% by weight at room temperature, such as 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or N,N-dimethylformamide (DMF).

[0013] The fiber sheet can be fixed or held by a frame around its periphery. When the frame is fixed or held to the fiber sheet, there is no particular limitation as long as it does not affect the cell culture, but for example, a commercially available biocompatible adhesive, such as silicone one-component condensation type RVT rubber (Shin-Etsu Chemical, catalog number KE-45), can be used to bond the frame and the fiber sheet. The material of the frame is not particularly limited as long as it does not affect cell culture. Examples include polydimethylsiloxane (PDMS), PS, polycarbonate, stainless steel, etc. The thickness of the frame is not particularly limited, but is 0.1 to 4 mm, preferably 0.25 to 3 mm, and more preferably 0.5 to 2 mm. The shape of the frame can be changed depending on the purpose of use, and preferably has a length x width of 2 mm x 2 mm to 15 mm x 15 mm, and is circular or polygonal.

[0014] A neuronal device using a fiber sheet, or the fiber sheet fixed or held around its periphery by a frame, as a cell scaffold can be placed directly in at least one of the wells contained in a cell culture dish or a multiwell plate having multiple wells.

[0015] In this specification, a nerve cell means a nerve unit composed of a cell body, a dendrite and an axon, and is also called a neuron.Nerve cells can be classified according to the difference in the neurotransmitters they produce, and examples of the neurotransmitters include monoamines such as dopamine, noradrenaline, adrenaline and serotonin, non-peptide neurotransmitters such as acetylcholine, γ-aminobutyric acid and glutamic acid, and peptide neurotransmitters such as adrenocorticotropic hormone (ACTH), α-endorphin, β-endorphin, γ-endorphin and vasopressin.For example, nerve cells that use dopamine, acetylcholine and glutamic acid as transmitters are called dopaminergic neurons, cholinergic neurons and glutamatergic neurons, respectively.

[0016] As the nerve cells, primary cultured cells can be used. Primary cultured cells retain many of the cellular functions inherent to the living body, and are therefore important as a system for evaluating the effects of drugs, etc., in the living body. As primary cultured cells, mammalian, e.g., rodent mouse or rat, or monkey or human primate central and peripheral nervous system nerve cells can be used. When preparing and culturing these nerve cells, the animal dissection method, tissue collection method, nerve separation and isolation method, nerve cell culture medium, culture conditions, etc. can be selected from known methods depending on the type of cells to be cultured and the purpose of the cells. As commercially available primary cultured nerve cell products, for example, rat brain nerve cells from Lonza and human brain nerve cells from ScienCell Research Laboratories can be used.

[0017] As the nerve cells, nerve cells derived from pluripotent stem cells can be used. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and iPS cells. By inducing the differentiation of pluripotent stem cells using known nerve differentiation induction methods, various types of nerve cells can be obtained. For example, nerve cells can be obtained by a differentiation induction method using a low molecular weight compound described in the literature (Honda et al. Biochemical and biophysical Research Communications 469 (2016) 587-592). Alternatively, commercially available pluripotent stem cell-derived neural cell products, such as iCell Neurons from Cellular Dynamics International and XCL-1 Neurons from XCell Science, can be used. These commercially available neural cells are cultured using the accompanying culture medium. Neurons can be cultured together with astrocytes derived from mammalian brains, or the culture medium after culturing astrocytes (astrocyte culture supernatant) can be added to the culture medium for neuronal cells at a final concentration of 5-30%.

[0018] Neurons suspended in culture medium were placed on the fiber sheet or the fiber sheet fixed or held by a frame at 1 × 10 4 cells / cm 2 ~4×10 6 cells / cm 2 , preferably 5 x 104 cells / cm 2 ~3×10 6 cells / cm 2 , more preferably 1×10 5 cells / cm 2 ~2×10 6 cells / cm 2 A neuronal device in which the neurons uniformly form a three-dimensional structure can be obtained by seeding them at a density of 100x100x100 and culturing them for 7 to 14 days while changing the culture medium every 1 to 7 days. Forming a three-dimensional structure means that the neurons adhere along the fibers that make up the fiber sheet and grow on one or both sides of the fiber sheet and inside the fiber sheet. When nerve cells are seeded on a fiber sheet and cultured, the cells are maintained in a uniformly spread state with almost no detachment or aggregation of cells during culture, which is observed when cells are seeded directly on a culture dish without using a fiber sheet.

[0019] The neuronal device of the present invention is brought into contact with a multi-electrode array, and the extracellular potential of the neuronal cell contained in the neuronal device is measured by measuring the extracellular potential of the neuronal cell in a 5% CO 2 The measurements can be performed in an environment of 37°C. Multi-electrode arrays have many planar microelectrodes arranged on a substrate, allowing electrical signals from multiple cells to be observed simultaneously.

[0020] In addition to the multi-electrode array, other means for measuring neural activity include measuring changes in various intracellular ions and potentials. For this purpose, fluorescent calcium indicators such as calcium-sensitive dyes and calcium-sensitive fluorescent proteins, and fluorescent voltage indicators such as voltage-sensitive dyes and voltage-sensitive fluorescent proteins are used (Grienberger, C. and Konnerth, A., Neuron 73, 862-885, 2012; Antic, SD, et al. J Neurophysiol. 116: 135-152, 2016; Miller, EW, Curr Opin Chem Biol. 33: 74-80, 2016). Using these indicators, calcium and potential fluctuations of the neural cells contained in the neural cell device of the present invention can also be measured by a cell imaging device. Examples of fluorescent calcium indicators include calcium-sensitive dyes such as Quin-2, Fura-2, Fluo-3, -4 and -8, Indo-1, Rhod-2 and -3, X-Rhod-1, Cal-520, Calbryte®, and CaTM. These are usually used as acetoxymethyl (AM) esters to impart permeability to nerve cells. The acetoxymethyl group is hydrolyzed and eliminated by intracellular esterases. Genetically encoded calcium probes such as Camgaroo-1 and -2, GCaMP-2, -3, -5 and -6, CaMPARI, and Case12 are known as calcium-sensitive fluorescent proteins. Examples of fluorescent potential indicators include potential-sensitive dyes such as Merocyanine 540, Rh1692, di-4-ANEPPS, JPW-1114, ANNINE-6, Indocyanine Green, Dipicrylamine, and FluoVolt (registered trademark). In addition, genetically encoded membrane potential probes such as VSFP-1 and -2, FlaSh, and SPARC based on Green Fluorescent Protein (GFP) are known as voltage-sensitive fluorescent proteins (Siegel, MS and Isacoff, EY, Neuron 19, 735-741, 1997; Sakai, R., Repunte-Canonigo, V., et al. Eur. J Neurosci. 13, 2314-2318, 2001; Ataka, K. and Pieribone, VA, Biophys J. 82, 509-516, 2002; Akemann, W. Mutoh, H., et al. Nature Methods, 7, 643-649, 2010). EXAMPLES

[0021] The present invention will now be described in detail with reference to examples, but the present invention is not limited thereto. EXAMPLES

[0022] [Fabrication of fiber device] (1) Preparation of random fiber sheet PLGA (SIGMA P1941) or PSU (SIGMA 182443) was dissolved at room temperature in HFIP (wako 089-04233) to a concentration of 20 wt%, and the solution was loaded into a syringe (Norm-Ject Syringes 5 ml capacity, Osaka Chemical), which was then placed in a nanofiber electrospinning device NANON-03 (Mec Co., Ltd.) and a fiber sheet was produced on a plate collector under the following conditions: needle diameter 22 G, voltage: 20 kV, feed rate: 1 ml / h for PLGA, and needle diameter 27 G, voltage: 15 kV, feed rate: 1 ml / h for PSU. (2) Preparation of oriented fiber sheet PLGA (SIGMA P1941) was dissolved at room temperature in HFIP (wako 089-04233) to a concentration of 20 wt%, and loaded into a syringe (Norm-Ject Syringes 5 ml capacity, Osaka Chemical). The syringe was then placed in a nanofiber electrospinning device NANON-03 (Mec Co., Ltd.) and a PLGA fiber sheet was produced on a drum collector under the following conditions: needle diameter 22 G, voltage: 20 kV, feed rate: 1 ml / h, and rotation speed: 750 rpm. To prepare a PS fiber sheet, PS (Fluka) was dissolved in DMF (N,N-dimethylformamide, Wako Pure Chemical Industries) at room temperature to a concentration of 30% by weight, and the solution was loaded into a syringe (Norm-Ject Syringes, 5 ml capacity, Osaka Chemical Co., Ltd.). The syringe was then placed in a nanofiber electrospinning device NANON-03 (Mec Co., Ltd.) and a fiber sheet was prepared on a drum collector under the following conditions: needle diameter: 25 G, voltage: 10 kV, feed rate: 1.5 ml / h, and rotation speed: 2000 rpm. (3) Frame attachment to fiber sheet A polycarbonate frame (15 mm × 15 mm) or a stainless steel circular frame (outer diameter 6 mm, inner diameter 3 mm) was attached to the prepared fiber sheet using a silicone one-component condensation type RVT rubber (Shin-Etsu Chemical, catalog number KE-45) to prepare a fiber device. EXAMPLES

[0023] [Preparation of nerve cells] (1) Induction of neural differentiation from human iPS cells (253G1 cell line) We used neural cells produced by inducing neural differentiation from human iPS cells 253G1 as previously reported (Honda et al. Biochemical and biophysical Research Communications 469 (2016) 587-592). Undifferentiated hiPS cells were seeded on culture dishes coated with poly-L-lysine (PLL, Sigma-Aldrich) and laminin 111 (LM, Sigma-Aldrich) and cultured for 7 to 10 days in N2B27 neural medium (a 1:1 mixture of DMEM / F-12 medium containing N2 supplement (GIBCO, 17502048) and Neurobasal medium containing B27 minus vitamin A supplement (GIBCO, 12587001)) containing 100 nM LDN193189 (Cellagen Technology) and 1 μM SB431542 (Sigma-Aldrich). The cell colonies were then dissociated into small cell clusters with collagenase (GIBCO) and cultured in N2B27 neural medium containing only LDN1939189 for another 7-10 days. The resulting cell colonies were again dissociated into cell clusters with collagenase and cultured in N2B27 medium. After 5-7 days, neurons were obtained and can be used for the next experiment. The obtained neurons can be cryopreserved, and the cryopreserved neurons can also be used. To cryopreserve the neurons, the neuronal clusters were dissociated into single cells with Accutase (Innovative Cell Technologies), suspended in Bambanker (Nihon Genetics), frozen at -80°C, and then stored in a deep-freezer at -150°C for long-term storage. (2) Commercially available hiPS cell-derived neurons Commercially available hiPS cell-derived neurons, XCL-1 NEURONS (XCell Science, Inc., catalog number XN-001-1V), were thawed from frozen cells according to XCell Science's protocol to obtain a neuronal cell suspension. EXAMPLES

[0024] [Culture of human iPS cell-derived nerve cells] Using the fiber sheet prepared in Example 1 as a cell scaffold, 1.3 × 10 iPS cell-derived nerve cells suspended in N2B27 culture medium were cultured. 6 cells / cm 2 The cells were seeded on oriented PLGA, random PLGA, and random PSU fiber sheets and on a PTFE sheet (Sumitomo Electric Industries, Ltd.) at a density of 100 μg / ml and incubated at 5% CO 2The cells were cultured in an incubator at 37°C for 6 days. The results of observing the obtained cell sheet (neuronal device) with an optical microscope are shown in Figure 1a (using an oriented PLGA fiber sheet) and Figure 1b (using a random PSU fiber sheet). As a control, iPS cell-derived nerve cells were seeded in a culture dish (Nunc (registered trademark) Cell-Culture Treated Multidishes) or on a commonly used electrode (Alpha Med Scientific / MED Probe 16 / MED-RG515A) in the same manner and cultured for 1 to 2 days. The results of observing the obtained cells with an optical microscope are shown in Figure 2a (on a culture dish) and Figure 2b (on an electrode). As a result, it was shown that the cell sheet was not maintained in the control group even after 1 day of culture. On the other hand, it was shown that the cells were growing uniformly on the fiber sheet even after 6 days of culture (Figures 1 and 2). iPS cell-derived neurons were seeded, and after 5 weeks, the cells were fixed by treating with 4% paraformaldehyde (Nacalai Tesque) at room temperature for 15-30 minutes. After washing with PBS, the cells were treated with 0.2% TritonX-100 (Sigma) at room temperature. After 5 minutes, the cells were washed with PBS and blocked with 1% bovine serum albumin at room temperature for 1 hour. The cells were washed with PBS and treated with anti-MAP2 antibody (Abcom). After 1 hour, the cells were washed with PBS and treated with Alexa Fluor 546-anti-rabbit IgG antibody (Thermofisher) for 1 hour. After washing with PBS, the cells were observed under a fluorescent microscope (Keyence) (Figure 1c). As shown in Figure 1c, the neurons were oriented by staining with MAP2 antibody, which can stain the axons of neurons. Figure 1d shows the results of seeding on random PLGA fiber sheets and oriented PLGA fiber sheets, and measuring the number of spikes and bursts of neurons using MEA after 4 weeks. Figure 1d shows that the oriented fiber sheet is effective in increasing the number of spikes and bursts. EXAMPLES

[0025] [Effect of the type of culture medium used on neural cell adhesion to the cell scaffold] Three different culture media were used: N2B27 medium containing 20% ​​Astrocyte Conditioned Medium (ACM) (Astrocyte Conditioned Medium-Serum Free (ACM-sf) / 11811-sf, ScienCell Research Laboratories) (Culture Media A), Neurobasal medium containing CultureOne Supplement (Gibco) and B27 minus vitamin A supplement (Culture Media B), and BrainPhys Neuronal Medium (05792, STEMCELL TECHNOLOGIES) containing 20% ​​ACM (Culture Media C). 253G1 iPSC-derived neurons and XCL-1 NEURONS (XCell Science, Catalog No. XN-001-1V) were cultured at 1.2 × 10 cells / well on the cell scaffolds. 6 cells / cm 2 The growth of cells seeded at a density of 100 μg / ml was observed. For the 253G1 iPS cell-derived neurons, oriented PLGA fibers coated with 0.002% poly-L-lysine and 20 μg / ml laminin were used as cell scaffolds, and for the XCL-1 NEURONS, oriented PLGA fibers coated with 0.002% poly-D-lysine and 10 μg / ml laminin were used. The seeded cells were incubated at 4°C for 1 h in a 5% CO 2The cells were cultured for 1 to 3 weeks in a 37°C incubator. The results of observing the obtained cell sheet (neuronal device) with an optical microscope are shown in Figure 3a, b, and c. As a control, the probe (electrode) was immersed in a culture medium containing 10% bovine serum based on the conventional method, and then neurons were seeded on the probe (electrode) coated with 0.003% poly-L-lysine (Sigma-Aldrich) and 20 μg / ml laminin (Sigma-Aldrich) for 253G1 iPS cell-derived neurons, or on the probe (electrode) coated with 0.002% poly-D-lysine and 10 μg / ml laminin for XCL-1 NEURONS, and cultured for 1 to 3 weeks. The results of observing the obtained cell sheet with an optical microscope are shown in Figure 3d, e, and f. The cell seeding density was 1.3 × 10 6 cells / cm 2 (Figure 3d) or 3 × 10 5 cells / cm 2 (Figures 3e and f). As a result, in the control group, when culture medium A was used, the neurons detached from the electrode, and the detached neurons were observed to form cell masses. In the control group, when culture medium B was used, the cells formed small colonies, and when culture medium C was used, cell detachment was observed at the edge of the area where the cells were seeded. On the other hand, when cells were cultured on oriented PLGA fibers, it was shown that a uniform cell sheet was maintained regardless of the type of culture medium (Figure 3). EXAMPLES

[0026] [Extracellular potential measurement of neurons grown on oriented PLGA fiber sheets as cell scaffolds using MEA] iPS cell-derived neuronal cells, XCL-1 NEURONS (XCell Science, Inc., Catalog No. XN-001-1V), were cultured at 3 × 10 5 cells / cm 2 (low density seeding condition) or 12 × 10 5 cells / cm 2(high density seeding condition) and incubated for 2, 4, and 6 weeks in BrainPhys® Neuronal Medium (05792, STEMCELL TECHNOLOGIES) supplemented with or without 20% ACM at 5% CO 2 The fibers were placed on a multi-electrode array (MEA) probe (MED64 system, Alpha Med Scientific, Inc.) to bring the cells into contact with the electrodes of the MEA probe, and neural activity (spikes and bursts) was measured. In parallel, 3 × 10 neurons were directly placed on the MEA probe. 5 cells / cm 2 and 5% CO for 2, 4, and 6 weeks. 2 The cells were cultured in a 37°C incubator and neural activity was measured in the same manner. The results are shown in Figure 4. As a result, it was shown that, compared to the conventional method (cells seeded on the MEA probe), the cells seeded at high density on the oriented PLGA fiber sheet showed high frequency spikes as early as two weeks after culture (Figure 4a). This phenomenon was particularly noticeable in the neurons on the fiber sheet cultured in a culture medium containing ACM, and an increase in the average potential was also observed (Figure 4b). Furthermore, at four weeks after cell seeding, even the neurons on the fiber sheet seeded at low density showed high frequency spikes and bursts compared to the conventional method (Figure 4c). Even more frequent spikes and bursts were observed in the neurons at six weeks (Figure 4d).

[0027] Primary cultured rat cerebral cortical neurons (ThermoFisher, product number A10840) were used as the nerve cells, and 1.0 × 10 5 cells / cm 2 , 3.0×10 5 cells / cm 2 or 9.0×10 5 cells / cm 2The cells were cultured at a cell density of 1000 x 1000 in Neurobasal® Medium (GIBCO, Cat. No. 21103-049) containing 1x B-27® Supplement / serum-free (GIBCO, Cat. No. 17504-044) at 5% CO 2 The cells were cultured in a 37°C incubator for 1, 2 and 4 weeks. The resulting cell sheet (neuronal device) was placed on an MEA probe (MED64 system, Alpha Med Scientific) and the cells were brought into contact with the electrodes of the MEA probe to measure neural activity (spikes and bursts). As a result, spikes and bursts were observed in the neuron device obtained after 2 and 4 weeks of culture. Figure 5 shows the results of a 9.0×10 5 cells / cm 2 The extracellular potential of the cell sheets (neuronal device) obtained by seeding cells in the cells at 100 nm and culturing them for four weeks was measured using an MEA. Spikes and synchronous bursts were observed at each electrode. EXAMPLES

[0028] [Measurement of extracellular potential of neurons grown on oriented polystyrene fiber sheets as a cell scaffold using MEA] iPS cell-derived neurons, iCell® GlutaNeurons (glutamate-sensitive neurons, Cellular Dynamics International, product number GNC-301-030-000.5), were cultured at 8 × 10 cells / well on an oriented polystyrene fiber sheet held in a circular frame with an outer diameter of 6 mm and an inner diameter of 3 mm as a cell scaffold. 5 cells / cm 2 , 12×10 5 cells / cm 2 or 16×10 5 cells / cm 2 The cells were cultured at a cell density of 100 x 100 cm in iCell Glutamine Neuronal Medium (Cellular Dynamics International, Inc.) supplemented with or without 20% ACM at 5% CO 2 The cells were cultured in an incubator at 37°C for 1, 2, and 4 weeks.5 cells / cm 2 Figure 6a is a photograph of a cell sheet (neuronal device) obtained by seeding neurons with 10% ACM and culturing them for 4 weeks in iCell Glutamine Neuronal Medium without ACM, observed under an optical microscope. Figure 6a shows that neurons adhere well to the polystyrene fiber sheet, and even after 4 weeks of continued culturing, a uniform cell sheet without cell detachment is maintained. This was also the case when a medium containing 20% ​​ACM was used. Also, 12×10 5 cells / cm 2 The cells were seeded at 1000 nm and cultured for 2 weeks in iCell Glutamine Neuronal Medium supplemented with 20% ACM to obtain a cell sheet (neuronal device). The cell was placed on an MEA probe (MED64 System, Alpha Med Scientific) and the cells were brought into contact with the electrodes of the MEA probe to measure neural activity (spikes and synchronous bursts). Spikes and synchronous bursts were detected by raster plotting using a MED64 Burstscope (Alpha Med Scientific). The results are shown in Figure 6b. Synchronized bursts can be confirmed even after 2 weeks of culture. EXAMPLES

[0029] [Confirmation of drug responsiveness of nerve cells grown on oriented PLGA fiber sheets as cell scaffolds] iPS cell-derived neurons (253G1 hiPS cell-derived neurons or XCL-1 NEURONS (XCell Science, Inc., Cat. No. XN-001-1V)) were cultured at 13 × 10 5 cells / cm 2 253G1 hiPS cell-derived neurons were seeded in N2B27 culture medium or N2B27 culture medium containing 20% ​​ACM, and XCL-1 neurons were seeded in BrainPhys® Neuronal Medium (05792, STEMCELL TECHNOLOGIES) culture medium or BrainPhys® Neuronal Medium containing 20% ​​ACM at 37 °C under 5% CO2 The fibers were then cultured in a 37°C incubator for 4 or 6 weeks. The fiber sheet was placed on an MEA probe (MED64 system, Alpha Med Scientific), and the cells were brought into contact with the electrodes of the multi-electrode array probe to measure neural activity (spikes). As a control, neurons were seeded directly on a probe (electrode) based on the conventional method in the same manner, and cultured in a 37°C incubator for 4 or 6 weeks in 5% CO 2 The cells were cultured in a 37°C incubator. The results were compared with those of the conventional method (direct seeding on the probe). After 4 weeks of culture, more spikes were confirmed in the 253G1 hiPS cell-derived neurons on the fiber sheet (Figure 7a). Compared with the neurons on the probe, the spike number increase rate in the neurons cultured on the fiber sheet of the present invention was 6 times, and further in the fiber sheet group cultured in a culture medium containing 20% ​​ACM, it was 9 times, and the number of spikes was significantly increased by using the fiber sheet. In addition, 4-aminopyridine (4-AP), which is known to have an effect of inhibiting various potassium channels in neurons and to sustain neuronal action potentials, was added to this measurement system at a final concentration of 100 μM to the neurons cultured for 4 weeks, and spikes were measured. As a result, an increase in spikes was observed in both the control group and the fiber sheet group by the addition of 4-aminopyridine, and appropriate responsiveness to the drug was also confirmed (Figure 7a). The spike increase rate was 156% in the control group cultured on the probe, whereas it was 167% in the fiber sheet group of the present invention, and furthermore, the fiber sheet group cultured in a culture medium containing 20% ​​ACM showed an increase rate of 194%. 5 cells / cm 2 When XCL-1 NEURONS seeded at a density of 100 μM and cultured in ACM-containing medium for 6 weeks were treated with 4-AP at a final concentration of 100 μM, the number of bursts was increased 2.6-fold (Figure 7b). EXAMPLES

[0030] [Drug responsiveness of neurons seeded at high density on oriented PLGA fiber sheets as cell scaffolds] iPS cell-derived nerve cells XCL-1 NEURONS (XCell Science, Inc., Catalog No. XN-001-1V) were cultured on an oriented PLGA fiber sheet as a cell scaffold at a density of 24 × 10 5 cells / cm 2 The cells were cultured in BrainPhys® Neuronal Medium (05792, STEMCELL TECHNOLOGIES) containing 20% ​​ACM at 37 °C under 5% CO 2 The cells were cultured in a 37°C incubator for 2, 4, and 6 weeks. The obtained cell sheet (neuronal device) was placed on an MEA probe (MED64 system, Alpha Med Scientific), and neural activity (spikes and bursts) was measured by contacting the cells with the electrodes of the MEA probe. 4-aminopyridine (4-AP), which is known to sustain neural action potentials, was added to the measurement system at a final concentration of 100 μM to examine the responsiveness of the neural cells to drugs. A MED64 Burstscope (Alpha Med Scientific) was used to analyze neural activity. The results of analyzing the number of spikes and the number of synchronized bursts for the cell sheet obtained after 4 weeks of culture are shown in FIG. 8a and FIG. 8b, respectively. The number of spikes increased by 499% and the number of synchronized bursts increased by 375% by 4-AP treatment. Furthermore, the results of analyzing the neural activity of the cell sheet obtained after 6 weeks of culture by raster plot are shown in FIG. 8c. The raster plot also showed a significant increase in the number of spikes and the number of synchronized bursts by 4-AP treatment. These results demonstrated that the neuronal device of the present invention enhances the responsiveness of neurons to the drug (4-AP). EXAMPLES

[0031] [Observation of neural activity using calcium-sensitive dyes in neurons grown on oriented polystyrene fiber sheets as a cell scaffold] Primary cultured rat cerebral cortical neurons (ThermoFisher, product number A10840) were cultured at 1.0 × 10 5 cells / cm 2 , 3.0×10 5 cells / cm 2 or 9.0×10 5 cells / cm 2 The cells were cultured at a cell density of 100 x 100 cm at 20 °C in Neurobasal® Medium (GIBCO, Cat. No. 21103-049) containing B-27® Supplement (x50) / serum-free (GIBCO, Cat. No. 17504-044) at 5% CO 2 The cells were cultured for 4 weeks in an incubator at 37°C. The obtained cell sheet (neuronal device) was transferred to a culture medium containing 5 μM of Fluo-8-AM (AAT Bioquest, catalog number 21081), a calcium indicator reagent, and incubated in a 5% CO 2 The cells were incubated for 1 hour in a 37°C incubator. The cell sheet incorporating Fluo-8 was placed on a warming plate attached to a fluorescence microscope (Olympus IX73) and the fluorescence of Fluo-8 was observed. Changes in fluorescence intensity due to changes in calcium ion concentration were observed as synchronous blinking (blinking period: approximately 1.83 seconds) in individual neurons. Figure 9 shows an image observed with a fluorescence microscope (magnification x10). It is shown that the neuronal device of the present invention makes it possible to visualize (image) fluctuations in intracellular calcium concentration of neurons.

Claims

1. A neuronal device comprising a cell scaffold and neurons, the cell scaffold being a fiber sheet formed of a polymeric material, the fibers constituting the fiber sheet being arranged in one direction, and having an oriented structure in which 80% or more of the fibers are within a range of ±30° when the angle of the one direction is set to 0°, the distance between the fibers being 5-50 μm, the cross-sectional diameter of the fibers constituting the fiber sheet being 0.1-8 μm, the thickness of the fiber sheet being 1-40 μm, the porosity of the fibers constituting the fiber sheet being 10-50%, the neurons forming a three-dimensional structure on and / or within the cell scaffold, the neurons being seeded on the cell scaffold at a density of 1 x 10 4 cells / cm 2 to 4 x 10 6 cells / cm 2, and the neuronal device further comprising a frame that holds the periphery of the neuronal device.

2. 2. The neuronal device according to claim 1, wherein the fiber sheet is coated with an extracellular matrix protein selected from polylysine, polyornithine, laminin, fibronectin, Matrigel (registered trademark), and Geltrex (registered trademark).

3. The neuronal device according to claim 1 or 2, wherein the neuronal cells are primary cultured cells or neuronal cells derived from pluripotent stem cells.

4. The neuronal device according to claim 3 , wherein the neuronal cells derived from primary cultured cells or pluripotent stem cells are mammalian-derived neuronal cells.

5. The neuronal device according to any one of claims 1 to 4, wherein the neuronal cells include glutamatergic, dopaminergic, γ-aminobutyric acidergic, monoaminergic, histaminergic or cholinergic neuronal cells.

6. The neuronal device according to claim 1 , wherein the length and width of the frame are 2 mm×2 mm to 15 mm×15 mm, respectively, and the frame is circular or polygonal.

7. A method for evaluating neural activity, comprising using the neural cell device according to any one of claims 1 to 6.

8. A method for evaluating neural activity, comprising contacting the neural cell device described in any one of claims 1 to 6 with a multi-electrode array and measuring the extracellular potential of a neuron contained in the neural cell device.

9. A method for evaluating neural activity using an intracellular signal imaging substance, comprising using the neural cell device according to any one of claims 1 to 6.

10. The method of claim 9 , wherein the intracellular signal imaging substance is a fluorescent calcium indicator or a fluorescent voltage indicator.

11. A neuronal device-mounted dish comprising the neuronal device according to any one of claims 1 to 6.

12. A neuronal device-mounted plate, comprising a multiwell plate having a plurality of wells, at least one of the wells comprising the neuronal device according to any one of claims 1 to 6.

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