Method for forming a neuronal network structure and method for evaluating neurons in vitro

Abstract

To provide a method for forming neural cell network structure, capable of preventing neural cell detachment from a base material and neural cell aggregation, associated with long-term culture in a maturation process of immature neural cells, and maintaining structure of neural cell network on the base material so as to stably provide a neural cell model.SOLUTION: A neural cell network structure forming method is a method for forming a neural cell network structure, which comprises: a maturation step of maturing immature neural cells as a neural cell agglomerate; an arrangement step of arranging a plurality of the neural cell agglomerates obtained by the maturation step on a surface of a substrate in a manner where the neural cell agglomerates are separated each other in a plan view; and a network forming step of connecting via neurites at least two of the nerve cell agglomerates among the plurality of the neural cell agglomerates that have been arranged in the arrangement step.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] The present invention relates to a method for forming a neuronal network structure, a neuronal network structure, and a method for evaluating neurons in vitro. [Background technology]

[0002] By culturing animal or human cells and using them in vitro, the effects of drugs and toxicants can be evaluated in vitro. Traditionally, drugs have been added to individual organisms or tissue slices or cells extracted from them, and their responses observed. However, issues arise with animal cells due to species differences, and with human cells, issues arise regarding the ethics of obtaining cells and individual reproducibility, so alternative methods are needed. Currently, advances in stem cell technology, such as iPS cells, have made it possible to generate cells corresponding to various organs, and have made it possible to utilize cells such as nerve cells, which were previously difficult to extract from living organisms and could not be propagated. By culturing and maturing nerve cells outside of a living body, cell tissue models have been developed that can exhibit responses similar to those of humans, and it is already known that some of these models are being used for toxicity assessment and drug efficacy evaluation by electrical measurement (for example, Patent Document 1).

[0003] Patent Document 2 and Non-Patent Document 1 disclose that, for the purpose of improving the handling of tissue fragments for nerve transplantation, nerve bundles for transplantation, which are made by bundling cells by encapsulating them in microgel fibers, have sufficient flexibility and strength, are a structure that can supply sufficient nutrients to the cells inside, and can maintain a high cell density, and disclose that neural stem cells inside the microfibers can be differentiated and then combined. Also known is a method of inducing axonal outgrowth from cell clusters and using this for electrical evaluation (e.g., Patent Documents 3 and 4). Summary of the Invention [Problem to be solved by the invention]

[0004] In recent years, attempts have been made to perform high-throughput drug screening using neural network models constructed using stem cell-derived immature neurons. However, conventional neural cell models using stem cell-derived immature neurons require a long culture period of more than one month for maturation, making it difficult to maintain long-term cultures on a substrate. Specifically, the creation of neural cell models using stem cells requires the processes of maturation of immature neurons and the construction of functional neural networks. These two processes are typically carried out simultaneously by seeding immature neurons on a substrate and culturing them. However, the long-term culture required for the maturation process can lead to problems such as detachment of the seeded neurons from the substrate or aggregation of the neurons, making it difficult to maintain the structure formed by the neural network construction.

[0005] The neuronal models described in Patent Documents 2 to 4 and Non-Patent Document 1 are intended for transplantation or for constructing a three-dimensional neuronal network, not for constructing a two-dimensional neuronal network, and are therefore not suitable for high-throughput evaluation of neurons or evaluation of drugs acting on neurons, etc. Furthermore, they cannot solve the problem of constructing and maintaining a neuronal network structure when cultured on a substrate for a long period of time.

[0006] The present invention provides a method for forming a neuronal network structure that can maintain the neuronal network structure on a substrate and stably supply neuronal models by separating the neuronal maturation process from the neuronal network structure formation process, a neuronal network structure, and a method for evaluating neurons in vitro using the neuronal network structure. [Means for solving the problem]

[0007] The method for forming a neuronal network structure includes a maturation step in which immature neurons are matured into neuronal aggregates; an arrangement step in which the neuronal aggregates obtained in the maturation step are arranged on a substrate surface so that they are spaced apart from each other in a planar view; and a network formation step in which at least two of the neuronal aggregates arranged in the arrangement step are connected to each other via neurites. [Effects of the Invention]

[0008] According to the present invention, a method for forming a neuronal network structure can be provided that prevents detachment of neurons from a substrate and aggregation of neurons that accompanies long-term culture of immature neurons in the maturation process, maintains the structure of the neuronal network on the substrate, and can stably supply neuronal models. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a plan view showing a nerve cell network structure 100 of the present invention. [Figure 2] 1 shows a microscope image of the surface of a substrate in Experimental Example 1, on which immature nerve cells have been cultured for a long period of time. [Figure 3] 1 shows microscopic images of nerve cell aggregates on days 1, 4, 8, 15, and 33 of culture in Experimental Example 2. [Figure 4] 1 shows fluorescent images of a neuronal aggregate in Experimental Example 2 at rest and during synchronous activity. [Figure 5] 10 is a graph showing changes in fluorescence intensity accompanying the activity of a nerve cell aggregate in Experimental Example 2. [Figure 6] 1 shows a microscope image of a nerve cell aggregate in Experimental Example 3 taken one day after placement on a substrate. [Figure 7] 10 is a raster plot showing action potentials one day after placing a nerve cell aggregate on a substrate in Experimental Example 3. [Figure 8] 1 shows microscopic images of nerve cell aggregates 1 hour and 2 days after placement on the substrate in Experimental Example 4. [Figure 9] 1 shows microscopic images of nerve cell aggregates 9, 13, and 26 days after placement on the substrate in Experimental Example 4. [Figure 10] 10 is a raster plot showing action potentials on days 2, 9, 13, and 26 after placing a nerve cell aggregate on a substrate in Experimental Example 5. [Figure 11] FIG. 10 shows the results of placing a nerve cell aggregate in Experimental Example 6 on a substrate containing electrodes and analyzing the connection strength between the electrodes. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, a method for forming a neuronal network structure, a neuronal network structure, and a method for evaluating neurons in vitro according to one embodiment of the present invention will be described with reference to specific embodiments and drawings as necessary. These embodiments and drawings are merely examples for facilitating understanding of the present invention and are not intended to limit the present invention. In other words, the shapes, dimensions, arrangements, etc. of the components described below may be changed or improved without departing from the spirit of the present invention, and equivalents thereof are also included in the present invention. In addition, in all drawings, similar components are given similar reference numerals, and duplicate explanations are omitted as appropriate.

[0011] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, applications, and other publications and information referenced herein are incorporated herein by reference in their entirety. In addition, in the event of any discrepancy between the publications referenced herein and the description herein, the description herein shall prevail.

[0012] <Method for forming a neuronal network structure> The method for forming a neuronal network structure of the present invention comprises a maturation step of maturing immature neurons into neuronal aggregates, an arrangement step of arranging the neuronal aggregates obtained in the maturation step on the surface of a substrate so that they are spaced apart from each other in a planar view, and a network formation step of connecting at least two of the neuronal aggregates arranged in the arrangement step via neurites.The formation method of the present invention prevents neuronal detachment from the substrate and neuronal aggregation that accompany long-term culture in the maturation step of immature neurons, maintains the structure of the neuronal network on the substrate, and enables the stable supply of neuronal models.

[0013] As used herein, a "neuronal aggregate" refers to a neuronal cell population formed by three-dimensional adhesion of two or more immature neurons or neural stem cells (hereinafter simply referred to as "immature neurons"). It may be formed from a single type of immature neuron, or may contain multiple types of immature neurons. Furthermore, a neuronal aggregate exhibits specific electrical activity characteristics as a group.

[0014] As used herein, "electrical activity characteristics" refers to properties related to electrochemical activity exhibited in cellular activity, such as the firing frequency, firing amplitude, and firing pattern of spontaneous cellular firing; the burst frequency, burst amplitude, burst pattern, and burst periodicity of synchronous bursts of neuronal aggregates; the oscillation frequency, oscillation amplitude, and oscillation phase of neural oscillations; and the intracellular cation concentrations (e.g., sodium ions, calcium ions) and their change patterns. Here, "firing" refers to a single instantaneous change in action potential, and "burst" refers to a continuous firing state, a pattern viewed more macroscopically than a single firing. Synchronized bursting refers to the overall synchronization of spontaneous firings of multiple neuronal aggregates. Furthermore, "neural oscillation" refers to a repetitive pattern of bursts and non-bursts. While a single electrical activity characteristic may be observed, in recent years, multiple characteristics (parameters) have been simultaneously measured and characterized using multivariate analysis, etc.; therefore, the "electrical activity characteristics" of this invention also encompasses characteristics resulting from a combination of such multiple characteristics. From the viewpoint of ease of measurement, electrochemical properties that can be measured using electrodes are preferred, and it is preferable to use neuronal aggregates that differ in at least one selected from the group consisting of the firing frequency, firing amplitude, and firing pattern of spontaneous firing of cells, the burst frequency, burst amplitude, burst pattern, and burst periodicity of synchronous bursts of neuronal aggregates, and the oscillation frequency, oscillation amplitude, and oscillation phase of neural oscillations. Here, "burst pattern" refers to the waveform pattern of the burst, and "burst periodicity" refers to the periodic regularity of the burst. The electrical activity characteristics of neurons or neuronal aggregates can be measured using known electrophysiological techniques. Examples of such electrophysiological techniques include, but are not limited to, measuring local field potentials using electrodes such as multi-electrode arrays, directly measuring action potentials using patch clamping techniques, measuring changes in membrane potential using voltage-sensitive dyes, and measuring cation fluctuations using calcium imaging. Examples of voltage-sensitive dyes include calcium-sensitive dyes composed of calcium chelators and fluorophores, styryl compounds, cyanine and oxonol compounds, and rhodamine derivatives. Furthermore, the phrase "exhibiting different electrical activity characteristics at the time of electrical activity measurement" means that when electrical activity characteristics are measured using the same technique in two or more neuronal aggregates, different results are obtained. This typically refers to cases where the neuronal aggregates contain different types or ratios of cells.

[0015] The nerve cell network structure of the present invention can be formed by a method comprising the steps described below.

[0016] (maturation process) Neurons can be broadly divided into peripheral and central nerve cells. Peripheral nerve cells include, for example, sensory nerve cells, motor nerve cells, and autonomic nerve cells. Central nerve cells include, for example, interneurons and projection neurons. Projection neurons include, for example, cortical neurons, hippocampal neurons, and amygdala neurons. Central nerve cells can also be broadly divided into excitatory neurons and inhibitory neurons. Examples include glutamatergic neurons, which are primarily responsible for excitatory transmission in the central nervous system, and GABAergic (γ-aminobutyric acid) neurons, which are primarily responsible for inhibitory transmission. Other neurons that release neuromodulators include cholinergic neurons, dopaminergic neurons, noradrenergic neurons, serotonergic neurons, and histaminergic neurons. The immature nerve cells used in the present invention are not particularly limited as long as they are cells that can differentiate into nerve cells, and may be neural stem cells.

[0017] The immature neurons used to prepare a neuronal aggregate are not particularly limited as long as they are immature neurons. They may be primary cultured cells, passaged cells, established cell lines, immortalized cells, or cells that have undergone various gene editing processes. Furthermore, from the viewpoint of facilitating the production of cell aggregates containing a large number of desired cells, cells induced by differentiation from stem cells are preferred. Examples of stem cells include embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, umbilical cord blood-derived stem cells, and neural stem cells. Examples of induced pluripotent stem cells include nuclear transfer embryonic stem cells (ntES cells) and induced pluripotent stem cells (iPS cells). Examples of mesenchymal stem cells include bone marrow mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells. Among these, iPS cells are preferred as stem cells. iPS cells may be derived from healthy individuals or from patients with various nervous system disorders. Furthermore, they may be subjected to various gene editing processes, for example, to carry genes that are causative or risk factors for various nervous system disorders. iPS cells derived from patients with various nervous system disorders can be used to construct models of those disorders. Examples of nervous system disorders include, but are not limited to, neurodegenerative disorders, autism, epilepsy, attention-deficit hyperactivity disorder (ADHD), schizophrenia, and bipolar disorder. Examples of neurodegenerative disorders include Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

[0018] Animal species from which nerve cells are derived include, but are not limited to, humans, monkeys, dogs, cattle, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, hamsters, etc. Among these, mammals are preferred, and humans are particularly preferred.

[0019] The immature neurons that form the neuronal aggregates may be collected from a living organism, established and cultured, or differentiated from stem cells, as described above. The immature neurons that form the neuronal aggregates may be cells obtained as described above, or may be formed by mixing them with other cells. From the viewpoint of easily obtaining neuronal aggregates with desired properties, it is preferable that the immature neurons are derived from stem cells, i.e., immature neurons obtained by inducing differentiation of stem cells. When used in the evaluation method of this embodiment, it is preferable that all immature neurons that form the neuronal aggregates are derived from stem cells, from the viewpoint of easily constructing a desired evaluation system. Furthermore, when forming neuronal aggregates, the immature neurons may be mixed with extracellular matrix (ECM) or glial cells such as astrocytes.

[0020] When the immature neurons that form the neuronal aggregates are derived from iPS cells, the iPS cells can be differentiated into immature neurons using known methods and then matured into neuronal aggregates. To induce differentiation of iPS cells into immature neurons, commercially available differentiation induction kits, such as those manufactured by Elixirgen Scientific (specifically, a kit for inducing differentiation into a mixed culture of dopaminergic neurons, cholinergic neurons, glutamatergic neurons, and serotonergic neurons; a kit for inducing differentiation into cholinergic neurons; a kit for inducing differentiation into dopaminergic neurons; and a kit for inducing differentiation into GABAergic neurons), can be used. Alternatively, commercially available iPS cell-derived differentiated neurons may also be used.

[0021] In the maturation process, first, neuronal aggregates are prepared from immature neurons. To prepare neuronal aggregates, a suspension of immature neurons is added to a substrate such as a U-bottom microwell plate. The number of cells can be adjusted as needed. On the substrate, the immature neurons spontaneously form neuronal aggregates.

[0022] The substrate for producing neural cell aggregates is not limited to U-bottom microwell plates, and is not particularly limited as long as it can produce neural cell aggregates. The shape of the neural cell aggregates is not limited to spherical, and any shape may be used. In addition, neural cell aggregates that have undergone complex differentiation, such as organoids, can also be used.

[0023] Next, the maturity of the formed neuronal aggregates is confirmed. Methods for confirming the maturity of neuronal aggregates include, for example, Ca imaging and patch clamp. Random tests can be performed as needed to confirm the maturity of neuronal aggregates using general cell evaluations such as cell viability, ATP, and LDH, as well as the expression of proteins that serve as maturation indicators using immunostaining and mRNA levels using PCR. This allows for confirmation of the maturity of neuronal aggregates, which is optimal for the neuronal network formation process described below.

[0024] For example, by detecting biomarkers specific to neuronal cell types, it is possible to confirm whether a neuronal aggregate is a mature neuronal aggregate that has been induced to differentiate into a desired neuronal cell. For example, a mature neuronal aggregate can be identified based on the expression of at least one marker selected from the group consisting of Dcx, MAP-2, synapsin 1, TuJ1, NSE, Map2a, Gap43, NF, CD24, CDH2 / CD325, synaptophysin, and CD56 / NCAM.

[0025] Furthermore, whether a nerve cell aggregate has matured can be confirmed by whether it has an axon and dendrites and whether it generates action potentials.

[0026] Maturation into specific types of neurons can be confirmed based on whether they express phenotypic markers characteristic of dopaminergic neurons (at least one marker selected from the group consisting of TH, AaDC, Dat, Otx-2, FoxA2, LMX1A, and VMAT2), cholinergic neurons (at least one marker selected from the group consisting of NGF and ChAT), GABAergic neurons (at least one marker selected from the group consisting of GAD67 and vGAT), glutamatergic neurons (vGLUT1), serotonergic neurons, motor neurons (at least one marker selected from the group consisting of HB9, SMN, ChAT, and NKX6), sensory neurons (at least one marker selected from the group consisting of POU4F1 and peripherin), astrocytes (at least one marker selected from the group consisting of GFAP and Tapal), and oligodendrocytes (at least one marker selected from the group consisting of O1, O4, CNPase, and MBP).

[0027] Maturation of neurons sensitive to specific neurotransmitters can be confirmed by the presence of receptors and enzymes involved in neurotransmitter biosynthesis, release, and reuptake, as well as ion channels involved in depolarization and repolarization events associated with synaptic transmission. Synapse formation can be confirmed by staining for synaptophysin. Sensitivity to specific neurotransmitters can be confirmed by detecting receptors for, for example, gamma-aminobutyric acid (GABA), glutamate, dopamine, 3,4-dihydroxyphenylalanine (DOPA), noradrenaline, acetylcholine, and serotonin.

[0028] The formation method of the present invention may include a processing step of processing the neuronal aggregate mass obtained in the maturation step. This processing step can result in neuronal aggregate masses with different properties, which can then be subjected to the placement step described below. For example, neuronal aggregate masses with different electrical activity characteristics can be obtained. For example, neuronal aggregate masses with different electrical activity characteristics can be obtained by adding an agent that affects the electrical activity characteristics (e.g., an NMDA receptor inhibitor), coating with a material that affects the electrical activity characteristics, or connecting with another neuronal aggregate mass via neurites, thereby changing the electrical activity characteristics. Such changes in electrical activity characteristics need only be achieved by the time the electrical activity of at least two neuronal aggregate masses is measured. The agent that affects the electrical activity characteristics can be a single compound or a combination of multiple compounds.

[0029] Processing of the nerve cell aggregate may involve adjusting the size of the nerve cell aggregate by adding or cutting the nerve cell aggregate.

[0030] (Placement process) Next, the multiple nerve cell aggregates obtained in the maturation step are placed on the surface of the substrate so that they are spaced apart from each other in a plan view. In the placement step, first, a substrate for placing the neuronal aggregate is prepared. The substrate for placing the neuronal aggregate is not particularly limited as long as it is capable of placing the neuronal aggregate and forming a neuronal network structure, and may or may not have electrodes on its surface. However, a substrate with electrodes on its surface is preferred because it allows measurement of the electrical activity of the placed neuronal aggregate and the subsequently constructed neuronal network structure.

[0031] As used herein, "measuring electrical activity" means measuring the time-dependent changes in the above-mentioned electrical activity characteristics associated with neuronal activity. By measuring the electrical activity of at least two or more neuronal aggregates connected to each other via neurites, it is possible to evaluate how these neuronal aggregates exchange signals, and ultimately how each neuronal aggregate is organized and connected. The measurement of electrical activity may be carried out for as long as it is possible to evaluate the electrical activity, and may be carried out multiple times. In one embodiment of the present invention, the electrical activity is continuously measured after the neuronal aggregate is placed on the surface of a substrate.

[0032] Specific examples of substrates having electrodes on their surfaces include multi-electrode arrays (MEAs). Furthermore, the substrate is preferably configured to hold a culture medium for forming a neuronal network structure, and examples thereof include a culture vessel in which an MEA is placed within a well. The shape of the culture vessel may be a dish-type having one well, or a multi-well plate-type having multiple wells.

[0033] In one embodiment of the present invention, electrical activity is measured using electrodes. Electrodes are preferred because they allow direct and simple measurement of electrical activity. Examples of such electrodes include multi-electrode arrays (MEAs) that can measure local field potentials, as well as micro-glass electrodes that can directly measure cellular action potentials. The use of such electrodes will be described in detail in the section on neuronal network structures, along with specific examples.

[0034] The substrate may be made of any material that is not toxic to cells, but is preferably made of an elastic material or a metal material such as glass, ceramic, or stainless steel. Examples of elastic materials include synthetic resins such as cycloolefin, polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, polyester (e.g., polyethylene terephthalate), polyurethane, polysulfone, polyacrylate, polymethacrylate (e.g., polymethyl methacrylate (PMMA)), and polyvinyl; silicone resins such as PDMS (Poly-Dimethylsiloxane); synthetic rubbers such as EPDM (Ethylene Propylene Diene Monomer); and natural rubber. The substrate may be made of one of these materials alone or in combination of two or more of them.

[0035] The substrate only needs to have cell adhesive properties at least in the portion where the neuronal aggregate is to be placed. To allow the neuronal aggregate to adhere to the surface, at least the surface of the portion where the neuronal aggregate is to be placed may be coated with a coating agent or the like. The coating agent is not particularly limited, but may be a chemical material such as poly-D-lysine or poly-L-ornithine, or a biomaterial such as collagen or laminin, either singly or in combination.

[0036] To prevent unintended structural formation due to floating of the neuronal aggregates until they adhere to the substrate, it is preferable to place a partition member between the areas where multiple neuronal aggregates are placed on the surface of the substrate. In this case, a frame member may be placed to surround each neuronal aggregate to keep each neuronal aggregate within a specific area on the substrate. The materials constituting the partition member and frame member may be any material that is not toxic to neurons, including those exemplified as materials for the substrate. For example, a PDMS precursor can be poured into a mold and cured to create a transferred microscopic three-dimensional structure, which can then be attached to the substrate to form a partition member on the substrate. It is also possible to control the neural network structure that is constructed by patterning cell adhesive and non-adhesive regions on a substrate using cell adhesive proteins, polymers, hydrogels, etc.

[0037] Next, the plurality of nerve cell aggregates are arranged on the surface of the substrate so as to be spaced apart from one another in a plan view, sandwiching the partition member therebetween. Alternatively, when a substrate on which a frame member is further arranged is used, the nerve cell aggregates are arranged so as to be contained within the area enclosed by the frame member.

[0038] The nerve cell aggregates are arranged in a manner that involves mixing the nerve cell aggregates with a medium or buffer solution to prepare a nerve cell aggregate suspension, and then seeding the suspension using a micropipette. 2 In the case of a narrow area such as the area below, the suspension may be seeded using an inkjet bioprinter. Seeding using a high-precision bioprinter is particularly preferable because it allows the nerve cell aggregates to be spaced apart at a narrow distance even without a partition member or the like. The placement method may be a physical method such as tweezers, an optical method such as optical tweezers, or an electrical method such as dielectrophoresis.

[0039] Before placing the neuronal aggregates on the surface of the substrate, a medium is added to the substrate. If the substrate has micropores such as microwells, it is preferable to degas the substrate as needed to prevent air bubbles from being trapped in the micropores. If the neuronal aggregates are added directly to the substrate, it is not necessarily necessary to add a medium to the substrate beforehand.

[0040] The medium may be any basal medium containing components necessary for the survival of neuronal aggregates (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, etc.), and can be appropriately selected depending on the type of neuronal cell. Examples include, but are not limited to, basal media such as Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), RPMI-1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle's Medium:Nutrient Mixture F-12 (DMEM / F-12), and Glasgow Minimum Essential Medium (Glasgow MEM) to which necessary components have been added. Commercially available media for culturing neuronal cells may also be used, such as BrainPhys (Stemcell Technologies), Neurobasal, and Neurobasal Plus (both Thermo Fisher Scientific).

[0041] The distance between the neuronal aggregates is not particularly limited as long as connection via neurites is achieved, but is preferably 100 μm or more. By ensuring that the distance is equal to or greater than the lower limit, the action potentials of the neuronal aggregates are not confused with each other, allowing for more accurate measurement. Meanwhile, the upper limit of the distance can be set to, for example, about 3 cm, taking into account the distance that neurites can extend. Typically, a range of 100 μm to 1 cm is preferred.

[0042] (Network formation process) After placing each neuronal aggregate on the substrate, it is cultured until each neuronal aggregate adheres to the substrate. The culture conditions can be, for example, for a fixed period of about 10 to 48 hours, under normal cell culture conditions such as 37°C and 5% CO2. The culture medium used during culture can also be appropriately selected depending on the type of cell, and includes the same media as those exemplified in the above-mentioned placement step.

[0043] After confirming that the neuronal aggregate has adhered to the substrate, the partition member is removed and the cells are cultured. Removing the partition member prevents the outgrowth of neurites derived from the neuronal aggregate. Note that, since the partition member can be provided with a microchannel or the like, allowing neurites to progress through the channel, removing the partition member is not necessarily required if neurite outgrowth is not to be impeded or if the direction of the neurites needs to be controlled.

[0044] The culture conditions are typically for 5 to 60 days, for example, 18 to 28 days, under typical cell culture conditions such as 37°C and 5% CO. Culturing for this period allows a neurite-mediated network to be formed between at least two of the multiple neuronal aggregates arranged on the substrate.

[0045] When the substrate has electrodes on its surface, it is possible to confirm whether a network has been formed between at least two of the multiple neuronal aggregates arranged on the substrate via neurites by measuring the electrical activity of the neuronal aggregates. The electrical activity characteristics of the neuronal aggregates arranged on the substrate can be confirmed by measuring the action potential of the neuronal aggregates, the synchronized bursts exhibited by the neuronal aggregates, etc. using known electrophysiological techniques. Specifically, as shown in the Examples described below, the neuronal aggregates can be confirmed by placing them on a substrate equipped with an MEA and measuring the action potential for about 5 minutes using an MEA system such as the MED64 system (manufactured by SCREEN Holdings Co., Ltd.), Maestro MEA (manufactured by Axion BioSystems), or MEA systems (manufactured by Multichannel Systems).

[0046] When measuring the electrical activity of neuronal aggregates to confirm the formation of a neuronal network, the electrical activity is measured for at least two neuronal aggregates, and may be measured for, for example, two, three, four, five, or more neuronal aggregates. These neuronal aggregates are arranged spaced apart from each other in a planar view. The at least two neuronal aggregates whose electrical activity is measured are connected to each other via neurites (axons). Here, "connected to each other via neurites" means that a signal is transmitted from one neuronal aggregate to another via neurites, and synchronous firing or bursts are typically observed. When the electrical activity of three or more neuronal aggregates is measured, the connection via neurites is not particularly limited as long as a signal generated in one neuronal aggregate is transmitted to all neuronal aggregates, and may be serial or parallel. For example, each neuronal aggregate may be connected to each other independently in parallel, or all neuronal aggregates may be connected in series, or a mixture of these may be used.

[0047] <Neuronal network structure> The neuronal network structure of the present invention is a neuronal network structure used for evaluating neurons in vitro, and comprises a substrate and a plurality of neuronal aggregates arranged on the surface of the substrate at a distance from each other in a planar view, the neuronal aggregates being adhered to the surface of the substrate, at least two of the plurality of neuronal aggregates being connected to each other via neurites, and at least two of the plurality of neuronal aggregates being electrically synchronized.

[0048] The neuronal network structure of the present invention can be produced by the above-mentioned method for forming a neuronal network structure. The neuronal network structure of the present invention can be used for evaluating neurons in vitro as described below. The neuronal network structure of the present invention will be described in detail below using specific examples of embodiments.

[0049] Fig. 1 is a plan view showing a neuronal network structure 100 of the present invention. The neuronal network structure 100 shown in Fig. 1 includes four neuronal aggregates, but the number of neuronal aggregates may be two or more. By including multiple neuronal aggregates, more complex connections can be established via neurites, and electrophysiological interactions in each neuronal aggregate can be evaluated.

[0050] The cell substrate 100 comprises a substrate 5 having a plurality of detection units 3, and a first neuronal aggregate 10, a second neuronal aggregate 20, a second neuronal aggregate 30, and a fourth neuronal aggregate arranged on different detection units 3 at a distance from each other in a planar view. A culture medium 4 is filled on the substrate 5. The first neuronal aggregate 10, the second neuronal aggregate 20, the third neuronal aggregate 30, and the fourth neuronal aggregate 40 may be neuronal aggregates having the same properties or different properties. The first neuronal aggregate 10 and the second neuronal aggregate 20 form a connection via neurites 1a.

[0051] Each neuronal aggregate may be placed on one or more detection units 3, but it is preferable to place it on two or more detection units 3. This allows the field potentials generated by changes in the action potentials of multiple cells contained in each neuronal aggregate to be detected by each detection unit 3, and by combining the field potentials obtained from the multiple electrodes, more accurate field potential data can be obtained.

[0052] (first nerve cell aggregate) The first neuronal aggregate 10 is a neuronal aggregate including one or more neurons 1. As long as the first neuronal aggregate 10 exhibits specific electrical activity characteristics as a group, it may be a neuronal aggregate consisting of only neurons 1, or a neuronal aggregate consisting of a mixture of neurons 1 and cells other than the neurons 1. Furthermore, when the first neuronal aggregate 10 is a neuronal aggregate consisting of only neurons 1, it may be a neuronal aggregate consisting of one type of neurons 1, or a neuronal aggregate consisting of a mixture of two or more types of neurons 1.

[0053] As the nerve cell 1, the nerve cell described above can be used.

[0054] The animal species from which the cells contained in the first nerve cell aggregate 10 are derived may be any of the animal species described above.

[0055] (secondary neuronal aggregate) The second neuronal aggregate 20 is a neuronal aggregate including one or more cells 2 (hereinafter sometimes simply referred to as "cell 2") that transmit electrical signals to and from neuron 1. The second neuronal aggregate 20 may be a neuronal aggregate consisting of the same cells as the first neuronal aggregate 10, or a neuronal aggregate consisting of different cells. As long as the second neuronal aggregate mass 20 exhibits synchronous action potentials spontaneously or in response to an external stimulus, it may be a neuronal aggregate mass consisting of only cells 2, or a neuronal aggregate mass consisting of a mixture of cells 2 and cells other than the cells 2. Furthermore, when the second neuronal aggregate mass 20 is a neuronal aggregate mass consisting of only cells 2, it may be a neuronal aggregate mass consisting of one type of cell 2, or a neuronal aggregate mass consisting of a mixture of two or more types of cells 2.

[0056] The cells contained in the second nerve cell aggregate mass 20 may be the same as those exemplified for the first nerve cell aggregate mass 10 above.

[0057] Examples of animal species from which the cells contained in the second nerve cell aggregate 20 are derived include those similar to those exemplified for the first nerve cell aggregate 10 above.

[0058] (Third and fourth neuronal aggregates) The third neuronal aggregate 30 and the fourth neuronal aggregate 40 are arranged on a detection unit 3 other than the detection unit 3 on which the first neuronal aggregate 10 and the second neuronal aggregate 20 are arranged, and are spaced apart from the first neuronal aggregate 10 and the second neuronal aggregate 20 in a planar view. The third neuronal aggregate mass 30 and the fourth neuronal aggregate mass 40 are neuronal aggregates that exhibit synchronous action potentials spontaneously or in response to external stimuli. The third neuronal aggregate mass 30 and the fourth neuronal aggregate mass 40 may be neuronal aggregate masses composed of the same cells as the first neuronal aggregate mass 10 or the second neuronal aggregate mass 20, or may be neuronal aggregate masses composed of cells different from either the first neuronal aggregate mass 10 or the second neuronal aggregate mass 20. The third neuronal aggregate mass 30 and the fourth neuronal aggregate mass 40 can form a connection with at least one of the first neuronal aggregate mass 10 and the second neuronal aggregate mass 20 via neurites. As shown in FIG. 1 , the first neuronal aggregate mass 10 and the third neuronal aggregate mass 30 may form a connection via neurites 1a and 2b, and the second neuronal aggregate mass 20 and the fourth neuronal aggregate mass 40 may form a connection via neurites 2a and 2c. Furthermore, the third nerve cell aggregate mass 30 and the fourth nerve cell aggregate mass 40 may form a connection via the neurites 2b and 2c.

[0059] Cells contained in the third nerve cell aggregate mass 30 and the fourth nerve cell aggregate mass 40 include the same cells as those exemplified in the first nerve cell aggregate mass 10 and the second nerve cell aggregate mass 20 above.

[0060] The cell substrate is not limited to that shown in Figure 1, and may be one in which some of the components shown in Figure 1 have been modified or removed, or one in which other components have been added to those described above, as long as the effects of the present invention are not impaired.

[0061] For example, in the nerve cell network structure 100 shown in Fig. 1, the second nerve cell aggregate 20 may be a nerve cell aggregate containing one or more nerve cells of a type different from nerve cell 1. Examples of nerve cells contained in the second nerve cell aggregate 20 include the same types as those exemplified in the first nerve cell aggregate 10 above.

[0062] (Detection unit) The detection unit 3 is configured to detect field potentials generated by changes in the action potentials of each cell contained in the first neuronal aggregate mass 10, the second neuronal aggregate mass 20, the third neuronal aggregate mass 30, and the fourth neuronal aggregate mass 40. Note that "detecting" here encompasses detecting the generation of a field potential, as well as detecting the specific frequency, amplitude, and phase of changes in the field potential. The detection unit 3 may be embedded in the substrate 5 so that its surface is exposed and in contact with each neuronal aggregate mass, or may be disposed on the substrate 5.

[0063] The number of detectors 3 may be two or more, and may be, for example, four, eight, sixteen, thirty-two, sixty-four, or the like.

[0064] Specific examples of the detection unit 3 include electrodes. When the detection unit 3 is an electrode, it is possible to detect changes over time in the frequency, amplitude, and phase of the field potential generated by a nerve cell aggregate.

[0065] <Evaluation method using neuronal network structures> The above-mentioned nerve cell network structure can be used as a system for evaluating nerve cells in vitro (hereinafter, sometimes simply referred to as "evaluation system").

[0066] That is, the method for evaluating neurons in vitro of the present invention is a method for evaluating neurons in vitro using the above-mentioned neuronal network structure after forming the neuronal network structure, and includes a maturation step of maturing immature neurons into neuronal aggregates, an arrangement step of arranging multiple neuronal aggregates obtained in the maturation step on a substrate surface so that they are spaced apart from each other in a planar view, a network formation step of connecting at least two of the multiple neuronal aggregates arranged in the arrangement step via neurites, and a measurement step of measuring the electrical activity of at least two of the neuronal aggregates in the neuronal network structure obtained in the network formation step.

[0067] In the evaluation method of the present invention, the maturation step and the network formation step are the same as those described in the above-mentioned method for forming a neuronal network structure.

[0068] In the evaluation method of the present invention, the measurement step of measuring the electrical activity of at least two neuronal aggregates in the neuronal network structure obtained in the network formation step includes, for example, a step of evaluating the properties of each neuronal aggregate and the correlation between neuronal aggregates by confirming synchronized bursts in the formed neuronal network structure. For example, when observing the bursts of arbitrary neuronal aggregates A and B connected via neurites, if a burst synchronized with the spontaneous burst of neuronal aggregate B is confirmed in addition to a spontaneous burst in neuronal aggregate A, it can be evaluated that neuronal aggregate A is receiving a transmission signal from neuronal aggregate B. Furthermore, in this case, if a burst synchronized with the spontaneous burst of neuronal aggregate A is not confirmed in neuronal aggregate B, it can be evaluated that neuronal aggregate B is a neuronal aggregate upstream of neuronal aggregate A and is not receiving feedback projection.

[0069] In a preferred embodiment of the evaluation method of the present invention, an agent suspected of acting on neurons (referred to as a "candidate agent" in this disclosure) is added to a neuronal aggregate whose electrical activity is being measured. If any change in electrical activity occurs as a result of the addition of the candidate agent, the candidate agent can be determined to act on neurons. This makes it possible to efficiently select candidate agents that act on neurons. Therefore, in one embodiment of the present invention, the effect of the candidate agent on a neuronal aggregate (or the neurons present therein) is evaluated, and in a further embodiment, screening for effective candidate agents based on such evaluation is included. In other words, the present invention includes a method for screening candidate agents using the evaluation method of the present invention. The evaluation method of the present invention will be described in detail below.

[0070] In one embodiment of the present invention, the method comprises measuring electrical activity in at least two neuronal aggregates, followed by severing at least one of the neurites connecting the neuronal aggregates. Because reconnection does not occur once a neurite is severed, electrical activity must be measured at least once before severance. By measuring changes in electrical activity in the neuronal aggregates whose electrical activity was measured after severance, it is possible to evaluate how communication via such neurites affected the neuronal aggregates. Furthermore, by confirming the effects of the candidate agent in an evaluation system in which the neurites have been severed, the candidate agent can be evaluated as a model for traumatic brain injury, etc.

[0071] [How to use] The method of using the evaluation system of this embodiment will be described in detail below. For example, when a neuronal network structure comprising two neuronal aggregates is used, if the firing patterns of some of the action potentials of the first neuronal aggregate 10 and the second neuronal aggregate 20 are synchronized, it can be determined that there is electrophysiological interaction between the first neuronal aggregate 10 and the second neuronal aggregate 20 due to connection via neurites.

[0072] Furthermore, the formed connections can be artificially cut using a medical scalpel or the like, and the action potentials of the first neuronal aggregate 10 and the second neuronal aggregate 20 can be measured after the cut, thereby evaluating the electrical activity of each neuronal aggregate. For example, if the electrical activity characteristics of the first neuronal aggregate 10 and the second neuronal aggregate 20 after cutting return to different electrical activity characteristics, similar to those before the formation of a connection via a neurite, it can be determined that the synchronization of the firing patterns of some of the action potentials is based on the formation of that connection.

[0073] For example, when using a neuronal network structure 100 having four neuronal aggregates, the action potentials of the first neuronal aggregate 10, the second neuronal aggregate 20, the third neuronal aggregate 30, and the fourth neuronal aggregate 40 are detected by the detection unit 3, and the action potential data is transmitted from the detection unit 3 via wiring to the measurement unit for analysis. This makes it possible to determine which neuronal aggregates each individual neuronal aggregate affects or influences, or whether it is not affected or does not influence, within a complex cell substrate.

[0074] Furthermore, the electrical activity of each neuronal aggregate can be evaluated by severing one or more of the formed connections and measuring the action potential of each neuronal aggregate after severing. In this case, all connections between each neuronal aggregate can be severed, or only connections between desired neuronal aggregates can be partially severed, or connections between neuronal aggregates can be sequentially severed in a desired order.

[0075] The evaluation method and evaluation system of this embodiment are extremely useful in basic cellular and molecular research into neural development and disorders, such as axon guidance, neurodegenerative disorders, neuronal plasticity, and neuronal learning and memory.

[0076] The evaluation method and evaluation system of this embodiment can also be used to evaluate any compound. Specifically, a compound is added to one or more of a plurality of neuronal aggregates, and then the action potential of each neuronal aggregate is measured. This allows the effect of any compound on each neuronal aggregate to be evaluated, specifically, the ability of any compound to activate a neuronal network or alter synchronous firing, as well as the toxicity of the compound. Furthermore, based on this evaluation, it can be determined whether or not any compound may be useful in treating a specific disease.

[0077] For example, when a compound is exposed to a first neuronal aggregate containing many excitatory neurons and a second neuronal aggregate containing many inhibitory neurons, which are connected via neurites, and the firing pattern of the action potential of the first neuronal aggregate is completely synchronized with that of the second neuronal aggregate, the compound can be determined to have the ability to suppress synchronous firing of neurons. Based on this determination, the compound can be determined to be potentially useful for treating diseases in which synchronous firing is excessive (e.g., epilepsy, autism, schizophrenia, etc.). Thus, the evaluation method and evaluation system of this embodiment are suitable for use in compound screening.

[0078] For example, the present invention includes the following aspects. (1) A method for forming a neuronal network structure, comprising: a maturation step of maturing immature neurons into neuronal aggregates; an arrangement step of arranging the neuronal aggregates obtained in the maturation step on a substrate surface so that they are spaced apart from each other in a planar view; and a network formation step of connecting at least two of the neuronal aggregates arranged in the arrangement step via neurites. (2) The method according to (1), wherein the substrate has an electrode on its surface. (3) The formation method described in (1) or (2), which includes a processing step of processing the nerve cell aggregate obtained in the maturation step to obtain nerve cell aggregates having different characteristics. (4) The method according to any one of (1) to (3), wherein in the placing step, the nerve cell aggregate is placed on the surface of the base material using a partition member. (5) The method according to any one of (1) to (4), wherein the immature nerve cells are differentiated from stem cells. (6) The method according to (5), wherein the stem cells are induced pluripotent stem cells. (7) A neuronal network structure used to evaluate neurons in vitro, comprising a substrate and a plurality of neuronal aggregates arranged on the surface of the substrate at a distance from each other in a planar view, the neuronal aggregates being adhered to the surface of the substrate, at least two of the plurality of neuronal aggregates being connected to each other via neurites, and at least two of the plurality of neuronal aggregates being electrically synchronized. (8) The structure according to (7), wherein the surface of the substrate has an electrode. (9) A method for evaluating neurons in vitro, comprising: a maturation step of maturing immature neurons into neuronal aggregates; an arrangement step of arranging the neuronal aggregates obtained in the maturation step on a substrate surface so that they are spaced apart from each other in a planar view; a network formation step of connecting at least two of the neuronal aggregates arranged in the arrangement step via neurites; and a measurement step of measuring the electrical activity of at least two of the neuronal aggregates in the neuronal network structure obtained in the network formation step. (10) The evaluation method according to (9), wherein the surface of the substrate has an electrode. (11) The evaluation method according to (9) or (10), comprising a processing step of processing the neuronal aggregate obtained in the maturation step to obtain neuronal aggregates having different characteristics. (12) The evaluation method according to any one of (9) to (11), wherein in the placing step, the nerve cell aggregate is placed on the surface of the substrate using a partition member. (13) The evaluation method according to any one of (9) to (12), wherein the immature nerve cells are differentiated from stem cells. (14) The evaluation method according to (13), wherein the stem cells are induced pluripotent stem cells. (15) The evaluation method according to any one of (9) to (14), further comprising adding an arbitrary agent to at least one of the at least two nerve cell aggregates. (16) The evaluation method according to (15), further comprising evaluating the effect of the candidate agent on the nerve cell aggregate based on the measurement results of the electrical activity. (17) The evaluation method according to any one of (9) to (16), further comprising cutting the neurite. [Example]

[0079] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0080] [Experimental Example 1] (Seeding of immature neural cells onto the substrate) Frozen iPS cell-derived immature neurons (iCell® GABAergic neurons) commercially available from Cellular Dynamics International were suspended in culture medium and seeded onto a substrate containing electrodes of a multielectrode array (MEA; SCREEN Holdings Co., Ltd.) that had been pre-coated with 0.05% polyethyleneimine (PEI; Sigma) and 28 μg / mL of Matrigel (Growth Factor Reduced Matrigel; Corning Incorporated).

[0081] After seeding, immature neurons were cultured for 30 days while observing the state of the cells on the substrate under a microscope. Figure 2 shows microscopic images taken on days 1, 7, 14, 22, and 30 after seeding. As shown in Figure 2, immature neurons were uniformly spread across the substrate on day 1 after seeding. However, as the number of culture days increased, partial cell aggregation and detachment from the substrate occurred. This revealed that when immature neurons are cultured directly on a substrate, even if the desired neural network pattern is formed in advance, the structure collapses as the culture progresses. In particular, when evaluating neurons using electrodes attached to the substrate, the degree of adhesion between the neurons and the electrode substrate is important for obtaining electrical signals, so detachment and structural collapse make electrical evaluation of neurons difficult.

[0082] [Experimental Example 2] (Creation of neural cell aggregates and evaluation of their maturation) Frozen iPS cell-derived neurons (GABAergic neurons from healthy donors) commercially available from Elixirgen Scientific were suspended in culture medium, and the cell suspension was added to a V-bottom microwell plate for culture. Figure 3 shows microscopic images taken on days 1, 4, 8, 15, and 33 of culture. From the microscopic images, it was possible to select neuronal aggregates that appeared to be clearly abnormal. Next, we evaluated the function of the neuronal aggregates. Because calcium ions flow into neurons when action potentials are generated, loading cells with a calcium-labeled fluorescent reagent allows us to observe changes in fluorescence intensity associated with neuronal activity. Therefore, we evaluated the function of the neuronal aggregates by calcium imaging on day 22 of culture. Figure 4 shows a fluorescent image of the neuronal aggregate on day 22 of culture, and Figure 5 shows a graph of the change in fluorescence intensity associated with neuronal activity. As shown in Figure 4, synchronous activity was observed throughout the entire neuronal aggregate on day 22 of culture. Furthermore, as shown in Figure 5, we confirmed that the fluorescence intensity during synchronous activity increased compared to the fluorescence intensity at rest, and that synchronous activity occurred periodically. These results indicate that calcium imaging can be used as an indicator of maturation.

[0083] [Experimental Example 3] (Measurement of electrical activity in neuronal aggregates) Frozen iPS cell-derived neurons (GABAergic neurons from healthy donors) commercially available from Elixirgen Scientific were cultured in V-bottom microwell plates for 50 days. Neuronal aggregates were then placed in microwells fabricated using PDMS on a multielectrode array (MEA; SCREEN Holdings Co., Ltd.) substrate containing electrodes, which had been pre-coated with 0.05% polyethyleneimine (PEI; Sigma) and 28 μg / mL growth factor-reduced Matrigel (Corning). Figure 6 shows a microscopic image of the neuronal aggregates 1 day after placement on the MEA substrate. As shown in Figure 6, the PDMS microwells confirmed that the neuronal aggregates were positioned and contained near the electrodes. Furthermore, neurite extension was observed 1 day after placement, confirming adhesion of the neuronal aggregates to the substrate. Next, action potential measurements were performed using a multi-electrode measurement device, MED64-Basic (MEA; SCREEN Holdings Co., Ltd.). The results are shown in Figure 7. As shown in Figure 7, numerous neural firings were observed at the electrodes near the neuronal aggregate, and synchronized bursts, in which neural firing was detected simultaneously across multiple electrodes, were detected. These synchronized bursts are considered to be one indicator of neural maturation, and these results demonstrate that synchronized bursts can be used to confirm the maturation of neuronal aggregates.

[0084] [Experimental Example 4] (Adhesion of neural cell aggregates after placement on a substrate) Frozen iPSC-derived neurons (Mixed-Human iPSC-derived Neurons) commercially available from Elixirgen Scientific were cultured in U-bottom microwell plates for 36 days. Four neuronal aggregates were then placed in microwells fabricated using PDMS on a multielectrode array (MEA; SCREEN Holdings Co., Ltd.) substrate containing electrodes pre-coated with 0.05% polyethyleneimine (PEI; Sigma) and 28 μg / mL growth factor-reduced Matrigel (Corning). Figure 8 shows microscopic images of the neuronal aggregates on the substrate, taken 1 hour after placement and on day 2, while Figure 9 shows microscopic images on days 9, 13, and 26. The PDMS microwells were removed 2 days after placement of the neuronal aggregates. As shown in Figure 8, the neuronal aggregates remained fixed in place even after removal of the PDMS microwells. Furthermore, as shown in Figure 9, the positions of the neuronal aggregates remained intact even on days 9, 13, and 26 after placement on the substrate, and it was observed that the neuronal aggregates were connected to each other due to the extension of neurites and cell migration.

[0085] [Experimental Example 5] (Network formation of neuronal aggregates) As in Experimental Example 4, four neuronal aggregates were placed on a substrate containing MEA electrodes, and neuronal connectivity was analyzed using action potential measurements with a MED64-Basic (MEA; SCREEN Holdings Co., Ltd.) multi-electrode measurement device to measure neuronal firing synchronization. The results are shown in Figure 10. As shown in Figure 10, two days after placement of the neuronal aggregates on the substrate containing electrodes, each of the four neuronal aggregates repeatedly fired sporadically, whereas 13 and 26 days after placement on the substrate, synchronous neuronal firing activity was observed. These results confirmed that continued culture of neuronal aggregates on the substrate containing electrodes led to the formation of connections between the neuronal aggregates and the formation of a network.

[0086] [Experimental Example 6] (Evaluation of network formation in neuronal aggregates) As in Experiment 4, four neuronal aggregates were placed on a substrate containing MEA electrodes. Using the timing of burst occurrence as a timestamp, the cross-correlation function of the timestamps at each electrode was calculated to analyze the connection strength between the electrodes using the free software MEAnalyzer. Figure 11 shows the visualization results for cross-correlation functions between electrodes that exceeded a threshold. Note that in Figure 11, the thicker the lines connecting the electrodes, the stronger the correlation. As shown in Figure 11, when the cross-correlation function between electrodes exceeded a threshold, functional connectivity as measured by bursts was not observed on the second day after placing the neuronal aggregates on the substrate, but connectivity was confirmed at the electrodes centered on each cell aggregate on the ninth day. Partial connectivity between cell aggregates was observed on the 13th day, and complete connectivity was confirmed on the 26th day. These results enable noninvasive determination of the timing at which cell aggregates become functionally connected, which can be used as an indicator for the timing of drug screening. These results also suggested that the speed of connection varies depending on the distance between neuronal aggregates, and that the timing of connection between neuronal aggregates can be controlled by controlling the distance at which the neuronal aggregates are placed on the substrate. [Explanation of symbols]

[0087] 1a, 2a, 2b, 2c: neurites 3: Detection unit (electrode) 4: Culture medium 5: Base material 10: First neuronal aggregate (neuronal aggregate A) 20: Second neuronal aggregate (neuronal aggregate B) 30: Third nerve cell aggregate 40: Fourth neuron aggregate 100: Neuronal network structure [Prior art documents] [Patent documents]

[0088] [Patent Document 1] Special Publication No. 2017-528127 [Patent Document 2] Patent No. 6074765 [Patent Document 3] International Publication No. 2019 / 113080 [Patent Document 4] Japanese Patent Application Publication No. 2019-37245 [Non-patent literature]

[0089] [Non-Patent Document 1] Advanced Healthcare Materials, 6(15), 1-7.

Claims

1. A method for forming a neuronal network structure, comprising: a maturation step of maturing immature neurons into neuronal aggregates; an arrangement step of arranging the plurality of nerve cell aggregates obtained in the maturation step on a substrate surface so as to be spaced apart from each other in a plan view; a network forming step of connecting at least two of the nerve cell aggregates arranged in the arrangement step via neurites; A method for forming a neuronal network structure, comprising:

2. The method according to claim 1 , wherein the substrate has an electrode on its surface.

3. The method according to claim 1 or 2, further comprising a processing step of processing the nerve cell aggregate obtained in the maturation step to obtain nerve cell aggregates having different characteristics.

4. The method according to any one of claims 1 to 3, wherein in the placing step, the nerve cell aggregate is placed on the surface of the substrate using a partition member.

5. The method according to any one of claims 1 to 4, wherein the immature nerve cells are differentiated from stem cells.

6. The method of claim 5 , wherein the stem cells are induced pluripotent stem cells.

7. 1. A method for assessing neural cells in vitro, comprising: a maturation step of maturing immature neurons into neuronal aggregates; an arrangement step of arranging the plurality of nerve cell aggregates obtained in the maturation step on a substrate surface so that the nerve cell aggregates are spaced apart from each other in a planar view; a network forming step of connecting at least two of the nerve cell aggregates arranged in the arrangement step via neurites; a measuring step of measuring electrical activities of at least two of the neuronal aggregates in the neuronal network structure obtained in the network forming step; Evaluation methods, including:

8. The evaluation method according to claim 7 , wherein the surface of the substrate has an electrode.

9. The evaluation method according to claim 7 or 8, further comprising a processing step of processing the nerve cell aggregate obtained in the maturation step to obtain nerve cell aggregates having different characteristics.

10. The evaluation method according to any one of claims 7 to 9, wherein in the placing step, the nerve cell aggregate is placed on the surface of the substrate using a partition member.

11. The evaluation method according to any one of claims 7 to 10, wherein the immature nerve cells are differentiated from stem cells.

12. The evaluation method according to claim 11 , wherein the stem cells are induced pluripotent stem cells.

13. The evaluation method according to any one of claims 7 to 12, further comprising adding an arbitrary agent to at least one of the at least two nerve cell aggregate masses.

14. The evaluation method according to claim 13, further comprising evaluating the effect of a candidate agent on a nerve cell aggregate based on the measurement results of the electrical activity.

15. The evaluation method according to any one of claims 7 to 14, further comprising cutting the neurite.

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