Nervous system cell-containing spheroids, method for producing nervous system cell-containing spheroids, and method for evaluating test substances

Abstract

To provide neural cell-containing spheroids with favorable drug responsiveness.SOLUTION: Provided are: a neural cell-containing spheroid, containing at least one type of neuron and other neural cells, the volume of one cell layer located at the outer edge of the spheroid accounting for 15 to 30% of the total volume of the spheroid; a method for producing the neural cell-containing spheroid; and a method for evaluating a test substance, including the steps of incubating a neural cell-containing spheroid in the presence of a test substance and evaluating the effect of the test substance on the neural cell-containing spheroid, the neural cell-containing spheroid containing at least one type of neuron and other neural cells, and the volume of one cell layer located at the outer edge of the spheroid accounting for 15 to 30% of the total volume of the spheroid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a nervous system cell-containing spheroid, a method for producing a nervous system cell-containing spheroid, and a method for evaluating a test substance. [Background technology]

[0002] In drug discovery research and development, in vitro experimental systems using cells are widely used. For example, primary cultured cells, cells differentiated from pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), immortalized cells, and cancer cells are used in drug screening for neurological disorders and toxicity screening for the neurological system.

[0003] In particular, iPS cell-derived neurons are attracting attention because they can be derived from patients, can reflect the genetic background of the disease, and can be used to construct models with high extrapolation potential.

[0004] For example, Patent Document 1 (JP 2021-185906 A) describes a neural cell-containing spheroid and a method for producing the same. It also describes that by maintaining a 1:1 ratio of neurons to astrocytes in the spheroid, the functional variability of the spheroid can be reduced. Summary of the Invention [Problem to be solved by the invention]

[0005] As described later in the Examples, the inventors have found that neural cell-containing spheroids cultured in three dimensions (3D culture) have lower drug responsiveness than neural cells cultured in two dimensions (2D culture). An object of the present invention is to provide neural cell-containing spheroids with favorable drug responsiveness. [Means for solving the problem]

[0006] The neural cell-containing spheroid of the present invention contains at least one type of neuron and neural cells other than the neuron, and the volume of one cell layer located at the outer edge of the spheroid accounts for 15 to 30% of the total volume of the spheroid. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide nervous system cell-containing spheroids with favorable drug responsiveness. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a graph showing the results of the calcium assay in Experimental Example 1. [Figure 2] FIG. 2 is a graph showing the results of the calcium assay in Experimental Example 2. [Figure 3] FIG. 3 is a graph showing the results of the calcium assay in Experimental Example 3. [Figure 4] FIG. 4 is a graph showing the results of the calcium assay in Experimental Example 4. [Figure 5] FIG. 5 is a graph showing the results of the calcium assay in Experimental Example 5. [Figure 6] FIG. 6 is a graph showing the results of the calcium assay in Experimental Example 6. [Figure 7] FIG. 7 is a graph showing the results of the calcium assay in Experimental Example 7. [Figure 8] FIG. 8 is a graph showing the results of the calcium assay in Experimental Example 8. [Figure 9] FIG. 9 is a graph showing the results of the calcium assay in Experimental Example 9. [Figure 10] FIG. 10 shows representative images of spheroids taken in Experimental Example 10. DETAILED DESCRIPTION OF THE INVENTION

[0009] [Nervous system cell-containing spheroids] In one embodiment, the present invention provides a neural cell-containing spheroid, which contains at least one type of neuron and a neural cell other than the neuron, and the volume of one cell layer located at the outer edge of the spheroid accounts for 15 to 30% of the total volume of the spheroid.

[0010] As described later in the Examples, the nervous system cell-containing spheroids of this embodiment have better drug responsiveness than conventional nervous system cell-containing spheroids. In this specification, "good drug responsiveness" may mean, for example, that drug concentration-dependent changes in signals indicating drug responsiveness can be detected with high sensitivity, that drug concentration-dependent changes in signals indicating drug responsiveness can be detected with sensitivity equal to or greater than that of 2D-cultured neurons, or that drug responsiveness is improved or high compared to conventional nervous system cell-containing spheroids (spheroids in which the volume of one cell layer located at the outer edge of the spheroid accounts for less than 15% of the total volume of the spheroid). The nervous system cell-containing spheroids of this embodiment can be said to be spheroids for evaluating drug responsiveness.

[0011] In the nervous system cell-containing spheroid of this embodiment, the ratio of the volume of one cell layer located at the outer edge of the spheroid to the total volume of the spheroid is 15 to 30%. That is, in the nervous system cell-containing spheroid of this embodiment, the ratio calculated by the following formula (1) is 15 to 30%, and may be 20 to 25%. In contrast, as described below in the Examples, in conventional nervous system cell-containing spheroids, the ratio calculated by the following formula (1) is less than 15%. The present inventors have found that when the volume of one cell layer located at the outer edge of the spheroid is 15% or more of the total volume of the spheroid, the drug responsiveness to a drug that comes into contact with the spheroid is enhanced. Furthermore, as described below in the Examples, in a nervous system cell-containing spheroid whose ratio calculated by the following formula (1) is more than 30%, the spheroid has a small volume and the number of cells constituting the spheroid is small, so the signal indicating drug responsiveness may be weak and undetectable. Based on these findings, the present inventors have discovered that the drug responsiveness of spheroids can be optimized by setting the ratio of the volume of one cell layer located on the outer edge of the spheroid to the total volume of the spheroid to be 15 to 30%, more preferably 20 to 25%.

[0012] Percentage (%) = Volume of one cell layer located at the outer edge of the spheroid / Total volume of the spheroid × 100 … (1)

[0013] Here, the volume of the entire spheroid can be calculated based on the diameter of the spheroid determined by observing the spheroid with an optical microscope or the like. The diameter of the spheroid can be determined, for example, as the diameter of a circle C having the same area as the two-dimensional projected area of ​​the spheroid. In other words, the volume of the entire spheroid can be determined as the volume of a sphere S1 having the same diameter as the circle C having the same area as the two-dimensional projected area of ​​the spheroid.

[0014] The volume of one cell layer located at the outer edge of the spheroid can be calculated by subtracting the volume of sphere S2, which has a radius 10 μm smaller than that of sphere S1, from the volume of sphere S1, assuming that the diameter of a single cell is 10 μm. Based on photographs of neural cells on the Elixirgen Scientific website, for example, it is possible to assume that the diameter of a single cell is 10 μm.

[0015] In one embodiment, the percentage calculated by the above formula (1) can be adjusted by adjusting the total number of cells constituting the spheroid. As described below, the percentage calculated by the above formula (1) may vary depending on the type of cells constituting the spheroid (volume per cell). As described below in the Examples, a spheroid with a percentage calculated by the above formula (1) of 15 to 30% can be said to have a total cell count of more than 2,000 but less than 16,000 (a spheroid with more than 2,000 but less than 16,000 cells constituting the spheroid). Therefore, when defined by the number of cells constituting the spheroid, the nervous system cell-containing spheroid according to this embodiment has a total cell count of more than 2,000 but less than 16,000, preferably 3,000 to 12,000, and more preferably 4,000 to 8,000.

[0016] In one embodiment, spheroids with a ratio calculated by the above formula (1) of 15 to 30% can be said to have a diameter of 150 to 450 μm. Therefore, when defined by the diameter of the spheroid, the neural cell-containing spheroid according to this embodiment has a diameter of 150 to 450 μm, preferably 192 to 394 μm, and more preferably 225 to 325 μm.

[0017] As will be described later in the Examples, spheroids with a cell density of 15 to 30% calculated by the above formula (1) have a cell density of 0.35 to 1.1 cells / 10 3 μm 3Therefore, when defined by the cell density of the spheroid, the neural cell-containing spheroid of this embodiment has a cell density of 0.35 to 1.1 cells / 10 3 μm 3 and 0.38 to 0.81 pieces / 10 3 μm 3 It is preferable that the number of pieces is 0.45 to 0.67 pieces / 10 3 μm 3 Here, the cell density of the spheroid can be calculated, for example, by dividing the number of cells constituting the spheroid by the volume of the sphere S1.

[0018] When the number of cells constituting a spheroid is small, gaps tend to form between the cells, whereas when the number of cells constituting a spheroid is large, the gaps between the cells tend to become smaller. Even if the number of cells constituting a spheroid is the same, when the culture period after seeding the cells is 2 to 3 days, the gaps between the cells tend to become large, and when the culture period is 7 days or longer, the gaps between the cells tend to become small.

[0019] The spheroids of this embodiment preferably have small intercellular spaces. In one embodiment, spheroids cultured for a period of, for example, about 30 to 50 days after seeding with cells can be suitably used for evaluating drug responsiveness.

[0020] As used herein, "nervous system cells" refers to cells that constitute the nervous system, preferably cells that constitute the central nervous system of a vertebrate. Examples of nervous system cells include neurons as well as glial cells such as astrocytes, oligodendrocytes, and microglia.

[0021] The neural cell-containing spheroid of this embodiment contains at least one type of neuron and neural cells other than neurons. The neural cells other than neurons preferably include astrocytes. The neural cell-containing spheroid of this embodiment may further contain neural cells other than neurons and astrocytes.

[0022] In the nervous system cell-containing spheroid of this embodiment, the ratio of the number of neurons to the number of astrocytes (number of neurons / number of astrocytes) is preferably 1. In this specification, a ratio of the number of neurons to the number of astrocytes of 1 includes cases where the ratio varies by about ±20%. In other words, the value of the number of neurons / number of astrocytes does not need to be exactly 1, and may be, for example, 0.8 to 1.2, or 0.9 to 1.1.

[0023] The inventors previously demonstrated that spheroids with a neuron:astrocyte ratio of approximately 1:1 tend to have less functional variability than spheroids with a neuron:astrocyte ratio of approximately 2:1 or approximately 4:1.

[0024] The function of a nervous system cell-containing spheroid refers to a value measured by evaluating the activity of the nervous system cell-containing spheroid using some kind of assay. For example, the function may be the number of spontaneous oscillations per predetermined time measured by subjecting the nervous system cell-containing spheroid to a calcium assay. Calcium assays are described below. Alternatively, the function may be responsiveness to a drug. Examples of responsiveness to a drug include, but are not limited to, changes in spontaneous oscillations in the presence of a drug and changes in the expression level of a specific gene.

[0025] A small functional variation may mean, for example, that the variation calculated according to the calculation method below is less than 20%, such as less than 15%, for example less than 10%. (Calculation method) A calcium assay is performed on multiple neural cell-containing spheroids across multiple lots or within the same lot, and the number of spontaneous oscillations per 10 minutes is measured. The mean and standard deviation of the number of spontaneous oscillations are calculated, and the variance is calculated using the following formula (2). Variability (%) = Standard deviation of spontaneous oscillation count / Mean of spontaneous oscillation count × 100 … (2)

[0026] The number of neurons and astrocytes contained in the spheroids may be considered to be equal to the number of neurons and astrocytes mixed in the process of producing neural cell-containing spheroids.

[0027] Alternatively, for example, spheroids may be immunofluorescently stained to stain neurons and astrocytes, and then measured by observing them under a fluorescence microscope. Examples of neuronal markers include MAP2, tubulin beta3, NeuN, 160 kDa neurofilament, 200 kDa neurofilament, NSE, PSD93, and PSD95. Examples of astrocyte markers include GFAP, S100β, Cx43, EAAT1, EAAT2, glutamine synthetase, and ALDH1L1.

[0028] Alternatively, the number of neurons and astrocytes in the spheroid may not be measured as a cell count, but the sum of the fluorescence intensities of the stained cells may be considered as a value corresponding to the cell count, and the ratio of the number of neurons to the number of astrocytes may be calculated.

[0029] Alternatively, the area or volume on the microscopic image occupied by each stained cell may be regarded as a value corresponding to the number of cells, and the ratio of the number of neurons to the number of astrocytes may be calculated.

[0030] The neural cell-containing spheroids of this embodiment are usually contained in a container. The shape of the container is not particularly limited, and examples thereof include tubes and multi-well plates. Examples of multi-well plates include well plates with 24 wells, 48 ​​wells, 96 wells, 384 wells, and 1,536 wells.

[0031] There are no particular limitations on the shape, volume, material, color, etc. of the wells of the multi-well plate, and they can be selected appropriately depending on the purpose.

[0032] The shape of the well is not particularly limited as long as it can accommodate spheroids, and can be appropriately selected depending on the purpose. Examples of shapes include flat, round, U-bottom, and V-bottom.

[0033] The volume of the well is not particularly limited and can be selected appropriately depending on the purpose. For example, taking into consideration the amount of reagent used in a typical evaluation method, the volume may be 5 to 1000 μL, 30 to 300 μL, or 50 to 200 μL.

[0034] The color of the multiwell plate can be, for example, transparent, translucent, colored, completely light-shielding, etc. When optical evaluation is performed, a container with a transparent bottom and colored sides is preferred from the viewpoint of suppressing interference between adjacent wells.

[0035] The material of the multiwell plate can be selected appropriately depending on the purpose, and examples include acrylic materials such as polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetylcellulose), polyimide (PI), nylon (Ny), low-density polyethylene (LDPE), medium-density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, and urethane acrylate; organic materials such as cellulose and polydimethylsiloxane (PDMS); and inorganic materials such as glass and ceramics.

[0036] To prevent spheroids from adhering to the bottom or sides of the multiwell plate, the surface of the multiwell plate is preferably subjected to a low-adsorption treatment. Examples include coating with a polymer containing phosphorylcholine groups, covalent bonding with a neutrally charged hydrophilic gel, and coating with a synthetic polymer having the same structure as the polar group of phosphatidylcholine. Treatments that suppress cell adhesion are not limited to these and can be selected as appropriate.

[0037] The neural cell-containing spheroids of this embodiment are contained in a container, which may be in the form of a multiwell plate, with one neural cell-containing spheroid in each well. Such a container is convenient because it can be used directly in various assays.

[0038] [Method for producing neural cell-containing spheroids] In one embodiment, the present invention provides a method for producing neural cell-containing spheroids. The production method of this embodiment includes a step of mixing at least one type of lineage-committed neuronal cell and a neural cell other than the neuronal cell at a predetermined ratio to form a spheroid, and in this step, the ratio of the volume of one cell layer located at the outer edge of the spheroid to the total volume of the spheroid is adjusted to 15 to 30%.

[0039] As used herein, "lineage-committed cells" refer to cells whose terminally differentiated cell type has been determined when undifferentiated cells are differentiated, and refer to cells that have differentiation potential but have been determined to differentiate into a specific cell type, or cells that have been terminally differentiated. Therefore, "lineage-committed neural cells" may be differentiated neural cells or stem cells that are destined to differentiate into neural cells. Differentiated neural cells may be primary cells extracted from a living organism, or neural cells induced to differentiate from stem cells. Furthermore, stem cells may be pluripotent stem cells or neural progenitor cells.

[0040] Examples of pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), nuclear transfer embryonic stem cells (ntES cells), etc. Among these, iPS cells are preferred as pluripotent stem cells.

[0041] iPS cells may be derived from healthy individuals or from patients with various nervous system disorders. They may also be cells that have undergone various gene editing processes, for example, cells that have been engineered by gene editing to contain genes that are causative or risk factors for various nervous system disorders.

[0042] 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.

[0043] The animal species from which the nerve cells are derived is not particularly limited, and examples thereof include primates such as humans and monkeys, mammals such as rabbits and dogs, ungulates such as pigs, sheep, horses and cows, and rodents such as mice, rats, guinea pigs and hamsters. Of these, humans are preferred.

[0044] As described above, nervous system cells include not only neurons but also glial cells such as astrocytes, oligodendrocytes, and microglia.

[0045] Neurons can be broadly divided into, for example, peripheral nerves and central nerves. Peripheral nerves include, for example, sensory nerves, motor nerves, and autonomic nerve cells. Central nerves 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.

[0046] Other neurons that release neuromodulators include cholinergic neurons, dopaminergic neurons, noradrenergic neurons, serotonergic neurons, and histaminergic neurons.

[0047] Furthermore, astrocytes, oligodendrocytes, microglia, etc. may be primary cultured cells or cells induced to differentiate from stem cells. When cells induced to differentiate from stem cells are used, it is preferable to use neural cells after their lineage has been determined, i.e., neural cells after it has been determined that they will differentiate into astrocytes, oligodendrocytes, microglia, etc.

[0048] The neural cells after lineage commitment may be pluripotent stem cells that have been subjected to differentiation induction treatment into neural cells. For example, the differentiation induction treatment may involve introducing a specific transcription factor into the pluripotent stem cells. Specific examples of such cells include Quick-Neuron (Eluixirgen Scientific) and other pluripotent stem cells. TM These cells are pluripotent stem cells that have been induced to differentiate into neural cells, and differentiate into functionally mature neural cells in approximately 10 days.

[0049] After lineage determination, multiple types of neural cells are mixed at a predetermined ratio and then seeded into an appropriate cell culture vessel such as a spheroid-producing plate and cultured to form spheroids. The incubation period for the cell culture vessel can be appropriately determined depending on the purpose, but it is preferably at least long enough for the neural cells to mature to a level at which their functions can be evaluated.

[0050] For example, when using pluripotent stem cells that have been induced to differentiate into neural cells as the neural cells after lineage commitment, the incubation period can be, for example, 20 days or more, for example, 30 days or more, for example, 40 days or more, for example, 50 days or more, for example, 60 days or more, or for example, 70 days or more from the time when pluripotent stem cells that have been induced to differentiate into multiple types of neural cells are mixed and seeded. In one embodiment, spheroids that have been aged for, for example, about 30 to 50 days from the time when pluripotent stem cells that have been induced to differentiate into multiple types of neural cells are mixed and seeded can be suitably used for evaluating drug responsiveness.

[0051] The medium may be a basal medium supplemented with necessary ingredients. Examples of basal media include BrianPhys (Stem Cell Technologies), Neurobasal (Thermo Fisher Scientific), Neurobasal Plus (Thermo Fisher Scientific), Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, D-MEM / F12, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Minimum Essential Medium (MEM), RPMI 1640 (Roswell Park Memorial Institute-1640), and Iscove's Modified Dulbecco's Medium (Iscove's Modified Dulbecco's Medium). Medium, IMDM), MCDB131 medium, Williams' medium E, IPL41 medium, Fischer's medium, M199 medium, High Performance Medium 199, StemPro34 (Thermo Fisher Scientific), X-VIVO 10 (Chembrex), X-VIVO 15 (Chembrex), HPGM (Chembrex), StemSpan H3000 (Stem Cell Technologies), StemSpanSFEM (Stem Cell Technologies), Stemline II (Sigma-Aldrich), QBSF-60 (Quality Biologics), StemProhESCSFM (Thermo Fisher Scientific), Essential8® Medium (Thermo Fisher Scientific), mTeSR1 or mTeSR2 Medium (Stem Cell Technologies), ReproFF or ReproFF2 (ReproCell), PSGro hESC / iPSC Medium (SystemExamples of suitable medium include NutriStem (registered trademark) medium (Biological Industries, Inc.), CSTI-7 medium (Cell Science Institute, Inc.), MesenPRO RS medium (Thermo Fisher Scientific, Inc.), MF-Medium (registered trademark) mesenchymal stem cell growth medium (Toyobo Co., Ltd.), Sf-900II (Thermo Fisher Scientific, Inc.), and Opti-Pro (Thermo Fisher Scientific, Inc.). These may be used alone or in combination of two or more.

[0052] In addition, additives to be added to the basal medium include those commonly used in culturing neurons, such as SM1 Supplement (Stem Cell Technologies), N2 Supplement A (Stem Cell Technologies), rat astrocyte culture supernatant (Fujifilm Wako Pure Chemical Industries), human astrocyte culture supernatant (Sciencell Research), Component N (Elixirgen Scientific), Component G2 (Elixirgen Scientific), Component P (Elixirgen Scientific), N2 Supplement (Thermo Fisher Scientific), iCell Neural Supplement B (CDI), iCell Neuvous System Supplement, B-27 plus (Thermo Fisher Scientific), etc.

[0053] In the process of forming spheroids, adjusting the ratio of the volume of one cell layer located on the outer edge of the spheroid to the total volume of the spheroid to 15 to 30% means adjusting the ratio calculated by the above formula (1) to 15 to 30%, more preferably 20 to 25%, and specifically, this can be achieved by adjusting the number of neural cells to be mixed.

[0054] As described later in the Examples, spheroids with a ratio calculated by the above formula (1) of 15 to 30% can be said to be spheroids with a total cell count of more than 2,000 but less than 16,000 (spheroids with a cell count of more than 2,000 but less than 16,000). Therefore, by forming spheroids with the number of neural cells mixed in being more than 2,000 but less than 16,000, preferably 3,000 to 12,000, and more preferably 4,000 to 8,000, it is possible to produce neural cell-containing spheroids with a ratio calculated by the above formula (1) of 15 to 30%.

[0055] As will be described later in the Examples, spheroids with a cell density of 15 to 30% calculated by the above formula (1) have a cell density of 0.35 to 1.1 cells / 10 3 μm 3 Therefore, in the production method of this embodiment, the cell density is 0.35 to 1.1 cells / 10 3 μm 3 The cell density is 0.38 to 0.81 cells / 10 3 μm 3 It is preferable that the number of pieces is 0.45 to 0.67 pieces / 10 3 μm 3 It is more preferable that:

[0056] Different cell types may produce different spheroid sizes even with the same cell number. Therefore, it is necessary to identify the appropriate cell number (range) for each cell type used, which will result in a cell layer volume ratio of 15-30%.

[0057] Specifically, first, multiple spheroids are formed using varying numbers of cells (for example, total cell numbers of 20,000, 16,000, 12,000, 8,000, 4,000, 2,000, etc.). Then, the "volume ratio of one cell layer to the total volume" for each cell number is calculated based on the above formula (1). The cell number (range) that gives this value of 15-30% is then determined. Thereafter, spheroids are produced using the previously determined cell number depending on the type of cell used. This makes it possible to produce spheroids with favorable drug sensitivity (drug responsiveness).

[0058] In the production method of this embodiment, the neural cells other than neurons preferably include astrocytes. The neural cells may further include neural cells other than neurons and astrocytes.

[0059] In the manufacturing method of this embodiment, the ratio of the number of neurons to the number of astrocytes (number of neurons / number of astrocytes) is preferably 1. The ratio of the number of neurons to the number of astrocytes being 1 is the same as described above.

[0060] [Test substance evaluation method] In one embodiment, the present invention provides a method for evaluating a test substance, comprising the steps of incubating a nervous system cell-containing spheroid in the presence of a test substance and evaluating the effect of the test substance on the nervous system cell-containing spheroid, wherein the nervous system cell-containing spheroid contains at least one type of neuron and nervous system cells other than the neuron, and the volume of one cell layer located at the outer edge of the spheroid accounts for 15 to 30% of the total volume of the spheroid.

[0061] The evaluation method of this embodiment is a method for evaluating a test substance using the above-mentioned nervous system cell-containing spheroids. The above-mentioned nervous system cell-containing spheroids have better drug responsiveness than conventional nervous system cell-containing spheroids, so the evaluation method of this embodiment can appropriately evaluate the test substance.

[0062] The evaluation method of this embodiment can also be said to be a drug screening method. The test substance is not particularly limited, and examples thereof include a natural compound library, a synthetic compound library, an existing drug library, a metabolite library, etc.

[0063] The method for evaluating the effect of a test substance on the neural cell-containing spheroid is not particularly limited, and examples include calcium assay, RNA-seq, immunostaining of the entire spheroid, immunostaining of thin sections of the spheroid, and microelectrode array (MEA) analysis.

[0064] Here, we will explain the calcium assay. In the calcium assay, neural cell-containing spheroids are first prepared. For example, neurons and astrocytes are mixed in any number and seeded on a spheroid-producing plate. The mixture is then cultured for 3 to 8 weeks in a medium suitable for neuronal culture, with medium changes as needed.

[0065] Next, the spheroids are transferred to a plate suitable for optical detection, and a calcium-sensitive fluorescent dye is added to the medium. The calcium-sensitive fluorescent dye is a substance that is taken up into cells and emits fluorescence when bound to calcium ions. Examples of calcium-sensitive fluorescent dyes that can be used include Cal-520 (AAT Bioquest), Cal-520AM (AAT Bioquest), Fluo4 (AAT Bioquest), Calcium-6 (Molecular Devices), and EarlyTox Cardiotoxicity Kit (Molecular Devices).

[0066] Next, using a fluorescence detection device, set the excitation wavelength / emission wavelength appropriate for the calcium-sensitive fluorescent dye used and perform continuous measurement for a desired period of time. Usable devices include a fluorescence plate reader, FDSS (Functional Drug Screening System, Hamamatsu Photonics), and FLIPR (Fluorometric Imaging Plate Reader, Molecular Devices). For example, measurements should be taken every 0.6 seconds for 20 minutes.

[0067] Neurons undergoing neural activity spontaneously take up and release calcium ions. Therefore, when performing a calcium assay using neural cell-containing spheroids, the fluorescence intensity changes periodically, which can be measured as spontaneous oscillations.

[0068] In the evaluation method of this embodiment, the neural cells other than neurons that constitute the neural cell-containing spheroid preferably include astrocytes. The neural cells may further include neural cells other than neurons and astrocytes.

[0069] In the evaluation method of this embodiment, the ratio of the number of neurons to the number of astrocytes constituting the nervous system cell-containing spheroid (number of neurons / number of astrocytes) is preferably 1. The ratio of the number of neurons to the number of astrocytes being 1 is the same as described above. [Example]

[0070] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0071] [Experimental Example 1] (Investigation of drug responsiveness in 2D-cultured and 3D-spheroid-cultured neurons) 2D culture of neurons Human iPSC-derived neurons (Excitatory Neurons, Elixirgen Scientific) and primary human astrocytes (ScienCell Research Laboratories) were seeded into 384-well plates at 10,000 neurons and 2,500 astrocytes per well. Neurobasal Plus (Thermo Fisher Scientific) medium supplemented with B-27 Plus Supplement (Thermo Fisher Scientific), Glutamax (Thermo Fisher Scientific), 200 μM ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10% Neuron Culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. The cells were cultured for 6 weeks, and then a calcium assay was performed.

[0072] 《3D spheroid culture of neural cells》 Human iPSC-derived neurons (Excitatory Neurons, Elixirgen Scientific) and primary human astrocytes (ScienCell Research Laboratories) were mixed at 8,000 neurons and 8,000 astrocytes per well and seeded onto spheroid plates. Neurobasal Plus (Thermo Fisher Scientific) medium supplemented with B-27 Plus Supplement (Thermo Fisher Scientific), Glutamax (Thermo Fisher Scientific), 200 μM ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10% Neuron Culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. The cells were then cultured for 6 weeks, and a calcium assay was performed as described below.

[0073] Calcium Assay We performed calcium assays in the presence of drugs to examine the drug responsiveness of 2D-cultured and 3D-cultured spheroid neurons. The drug used was chlorpromazine.

[0074] For 2D-cultured neurons, calcium-sensitive fluorescent dye (Cal-520AM, AAT Bioquest) and drugs were added to the culture medium, and calcium spikes were measured using a fluorescent plate reader at 0.5-second intervals for 20 minutes.

[0075] For 3D spheroid-cultured neurons, each spheroid was transferred to a measurement plate, and calcium-sensitive fluorescent dye (Cal-520AM, (AAT Bioquest)), Pluronic F-127 (AAT Bioquest) and drugs were added to the medium. Fluorescence was measured at 0.5-second intervals for 20 minutes using a fluorescence plate reader to count calcium spikes.

[0076] "result" Figure 1 is a graph showing the measurement results. The values ​​shown in the graph represent the mean ± standard deviation. The horizontal axis of the graph represents the concentration of chlorpromazine in the medium, and the vertical axis of the graph represents the percentage change in the number of calcium spikes. Additionally, "2D" represents the results for 2D-cultured neurons, and "3D" represents the results for 3D-spheroid-cultured neurons.

[0077] The percentage change in the number of calcium spikes when dimethyl sulfoxide (DMSO), used as the drug solvent, was added was set to 0%, and a positive value was shown if the number of calcium spikes increased due to the addition of the drug, and a negative value was shown if the number of calcium spikes decreased due to the addition of the drug.

[0078] As a result, in 2D-cultured neurons, the number of calcium spikes decreased in a drug-dose-dependent manner, and calcium spikes disappeared at a concentration of 3 μM. In contrast, in 3D-cultured neurons, no change in the number of calcium spikes was observed at any drug concentration.

[0079] These results indicate that neurons cultured in 3D spheroids are less drug-responsive than neurons cultured in 2D.

[0080] [Experimental Example 2] (Study of drug responsiveness of 3D spheroid-cultured neurons 1) Using spheroids with different cell numbers, calcium assays were performed in the presence of drugs to examine the drug responsiveness of neurons.

[0081] Human iPSC-derived neurons (Excitatory Neurons, Elixirgen Scientific) and primary human astrocytes (ScienCell Research Laboratories) were mixed and seeded onto spheroid plates at cell counts of 8,000 neurons and 8,000 astrocytes, 4,000 neurons and 4,000 astrocytes, or 2,000 neurons and 2,000 astrocytes per well. The culture medium used was Neurobasal Plus (Thermo Fisher Scientific) supplemented with B-27 Plus Supplement (Thermo Fisher Scientific), Glutamax (Thermo Fisher Scientific), 200 μM ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10% Neuron Culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.). Subsequently, the cells were cultured for 5 weeks, and a calcium assay was performed in the same manner as in Experimental Example 1. Chlorpromazine was used as the drug. The expected drug response is a decrease in the number of calcium spikes when chlorpromazine is applied.

[0082] Figure 2 is a graph showing the measurement results. The values ​​on the graph represent the mean ± standard deviation. The horizontal axis of the graph represents the concentration of chlorpromazine in the medium, and the vertical axis of the graph represents the relative value, with the number of calcium spikes when dimethyl sulfoxide (DMSO), used as the drug solvent, was added, taken as 100%. If the number of calcium spikes increased with the addition of the drug, the value is greater than 100%, and if the number of calcium spikes decreased, the value is less than 100%. 0% represents a state in which calcium spikes completely disappeared.

[0083] In Figure 2, "16,000 cells" indicates the results for spheroids prepared by mixing 8,000 neurons and 8,000 astrocytes, "8,000 cells" indicates the results for spheroids prepared by mixing 4,000 neurons and 4,000 astrocytes, and "4,000 cells" indicates the results for spheroids prepared by mixing 2,000 neurons and 2,000 astrocytes.

[0084] As a result, when drug responsiveness to 1 μM and 3 μM chlorpromazine was evaluated, no concentration-dependent response was observed at 16,000 cells and 8,000 cells, whereas a concentration-dependent response was observed at 4,000 cells.

[0085] These results indicate that drug responsiveness varies depending on the number of cells constituting the spheroid, and that drug responsiveness tends to be more favorable when the cell number is lower.

[0086] [Experimental Example 3] (Study of drug responsiveness of 3D spheroid-cultured neurons 2) Except for changing the drug to bicuculline, calcium assays were performed using spheroids with different cell numbers in the same manner as in Experimental Example 2 to examine the drug responsiveness of neurons. The expected drug response is an increase in the number of calcium spikes when bicuculline is applied.

[0087] Figure 3 is a graph showing the measurement results. The values ​​on the graph represent the mean ± standard deviation. The horizontal axis of the graph represents the concentration of bicucullin in the medium, and the vertical axis of the graph represents the relative value, with the number of calcium spikes when dimethyl sulfoxide (DMSO), used as the drug solvent, was added, taken as 100%. If the number of calcium spikes increased with the addition of the drug, the value is greater than 100%, and if the number of calcium spikes decreased, the value is less than 100%. 0% represents a state in which calcium spikes completely disappeared.

[0088] In Figure 3, "16,000 cells" indicates the results for spheroids prepared by mixing 8,000 neurons and 8,000 astrocytes, "8,000 cells" indicates the results for spheroids prepared by mixing 4,000 neurons and 4,000 astrocytes, and "4,000 cells" indicates the results for spheroids prepared by mixing 2,000 neurons and 2,000 astrocytes.

[0089] The results showed that the number of calcium spikes increased in a concentration-dependent manner in 4,000 cells. The response in 8,000 cells was not as pronounced as in 4,000 cells, but there was a tendency for the number of calcium spikes to increase at 30 μM. In 16,000 cells, no increase in the number of calcium spikes was observed at any concentration.

[0090] This result further supports the idea that drug responsiveness varies depending on the number of cells constituting the spheroid, and that drug responsiveness tends to be more favorable when the cell number is low.

[0091] [Experimental Example 4] (Study of drug responsiveness of 3D spheroid-cultured neurons 3) A calcium assay was performed in the same manner as in Experimental Example 2, except that the drug was changed to GABA and the number of cells constituting the spheroids was changed to 16,000 or 4,000, and drug responsiveness of the neurons was examined. The expected drug response is a decrease in the number of calcium spikes when GABA is applied.

[0092] Human iPSC-derived neurons (Excitatory Neurons, Elixirgen Scientific) and primary human astrocytes (ScienCell Research Laboratories) were mixed at 4,000 neurons and 4,000 astrocytes or 2,000 neurons and 2,000 astrocytes per well and seeded onto spheroid plates. Neurobasal Plus (Thermo Fisher Scientific) medium supplemented with B-27 Plus Supplement (Thermo Fisher Scientific), Glutamax (Thermo Fisher Scientific), 200 μM ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10% Neuron Culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. The cells were cultured for 5 weeks, and calcium assays were performed as in Experimental Example 2. GABA was used as the drug. When GABA is administered, the expected drug response is a decrease in the number of calcium spikes.

[0093] Figure 4 is a graph showing the measurement results. The values ​​shown on the graph represent the mean ± standard deviation. The horizontal axis of the graph represents the concentration of GABA in the medium, and the vertical axis of the graph represents the relative change in the number of calcium spikes (log2). The response observed upon addition of DMSO, the drug solvent, is represented as 0 (no change). A positive value indicates an increase in the number of calcium spikes due to drug addition, and a negative value indicates a decrease in the number of calcium spikes.

[0094] In Figure 4, "16,000 cells" indicates the results for spheroids prepared by mixing 8,000 neurons and 8,000 astrocytes, and "4,000 cells" indicates the results for spheroids prepared by mixing 2,000 neurons and 2,000 astrocytes.

[0095] As a result, it was shown that the drug showed a stronger effect at lower concentrations in a concentration-dependent manner at 4,000 cells compared to 16,000 cells, and favorable drug sensitivity was obtained.

[0096] This result further supports the idea that drug responsiveness varies depending on the number of cells constituting the spheroid, and that drug responsiveness tends to be more favorable when the cell number is low.

[0097] [Experimental Example 5] (Study of drug responsiveness of 3D spheroid-cultured neurons 4) Except for changing the drug to bicucullin, a calcium assay was performed in the same manner as in Experimental Example 4 to examine the drug responsiveness of neurons. The expected drug response is an increase in the number of calcium spikes when bicucullin is applied.

[0098] Figure 5 is a graph showing the measurement results. The values ​​shown on the graph represent the mean ± standard deviation. The horizontal axis of the graph represents the concentration of bicucullin in the medium, and the vertical axis of the graph represents the relative change in the number of calcium spikes (log2). The response observed upon addition of DMSO, the drug solvent, is represented as 0 (no change). A positive value indicates an increase in the number of calcium spikes due to drug addition, and a negative value indicates a decrease in the number of calcium spikes.

[0099] In Figure 5, "16,000 cells" indicates the results for spheroids prepared by mixing 8,000 neurons and 8,000 astrocytes, and "4,000 cells" indicates the results for spheroids prepared by mixing 2,000 neurons and 2,000 astrocytes.

[0100] As a result, no drug response was observed at 16,000 cells, whereas a concentration-dependent drug response was observed at 4,000 cells.

[0101] This result further supports the idea that drug responsiveness varies depending on the number of cells constituting the spheroid, and that drug responsiveness tends to be more favorable when the cell number is low.

[0102] [Experimental Example 6] (Study of drug responsiveness of 3D spheroid-cultured neurons 5) A calcium assay was performed in the same manner as in Experimental Example 4, except that the drug was changed to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and the drug responsiveness of neurons was examined. The expected drug response is a decrease in the number of calcium spikes when CNQX is applied.

[0103] Figure 6 is a graph showing the measurement results. The values ​​on the graph represent the mean ± standard deviation. The horizontal axis of the graph represents the concentration of CNQX in the medium, and the vertical axis of the graph represents the relative change in the number of calcium spikes (log2). The response observed upon addition of DMSO, the drug solvent, is represented as 0 (no change). A positive value indicates an increase in the number of calcium spikes due to drug addition, and a negative value indicates a decrease in the number of calcium spikes.

[0104] In Figure 6, "16,000 cells" indicates the results for spheroids prepared by mixing 8,000 neurons and 8,000 astrocytes, and "4,000 cells" indicates the results for spheroids prepared by mixing 2,000 neurons and 2,000 astrocytes.

[0105] As a result, it was shown that the drug showed a stronger effect at lower concentrations in a concentration-dependent manner at 4,000 cells compared to 16,000 cells, and favorable drug sensitivity was obtained.

[0106] This result further supports the idea that drug responsiveness varies depending on the number of cells constituting the spheroid, and that drug responsiveness tends to be more favorable when the cell number is low.

[0107] [Experimental Example 7] (Study of drug responsiveness of 3D spheroid-cultured neurons 6) Except for changing the drug to chlorpromazine, a calcium assay was performed in the same manner as in Experimental Example 4 to examine the drug responsiveness of neurons. The expected drug response is a decrease in the number of calcium spikes when chlorpromazine is applied.

[0108] Figure 7 is a graph showing the measurement results. The values ​​on the graph represent the mean ± standard deviation. The horizontal axis of the graph represents the concentration of chlorpromazine in the medium, and the vertical axis of the graph represents the relative change in the number of calcium spikes (log2). The response observed upon addition of DMSO, the drug solvent, is represented as 0 (no change). A positive value indicates an increase in the number of calcium spikes due to drug addition, and a negative value indicates a decrease in the number of calcium spikes.

[0109] In Figure 7, "16,000 cells" indicates the results for spheroids prepared by mixing 8,000 neurons and 8,000 astrocytes, and "4,000 cells" indicates the results for spheroids prepared by mixing 2,000 neurons and 2,000 astrocytes.

[0110] As a result, it was shown that the drug showed a stronger effect at lower concentrations in a concentration-dependent manner at 4,000 cells compared to 16,000 cells, and favorable drug sensitivity was obtained.

[0111] This result further supports the idea that drug responsiveness varies depending on the number of cells constituting the spheroid, and that drug responsiveness tends to be more favorable when the cell number is low.

[0112] [Experimental Example 8] (Study of drug responsiveness of 3D spheroid-cultured neurons 7) A calcium assay was performed in the same manner as in Experimental Example 4, except that the drug was changed to dopamine, and the drug responsiveness of neurons was examined. The expected drug response is a decrease in the number of calcium spikes when dopamine is administered.

[0113] Figure 8 is a graph showing the measurement results. The values ​​on the graph represent the mean ± standard deviation. The horizontal axis of the graph represents the concentration of dopamine in the medium, and the vertical axis of the graph represents the relative change in the number of calcium spikes (log2). The response observed upon addition of DMSO, the drug solvent, is represented as 0 (no change). A positive value indicates an increase in the number of calcium spikes due to drug addition, and a negative value indicates a decrease in the number of calcium spikes.

[0114] In Figure 8, "16,000 cells" indicates the results for spheroids prepared by mixing 8,000 neurons and 8,000 astrocytes, and "4,000 cells" indicates the results for spheroids prepared by mixing 2,000 neurons and 2,000 astrocytes.

[0115] As a result, it was shown that the drug showed a stronger effect at lower concentrations in a concentration-dependent manner at 4,000 cells compared to 16,000 cells, and favorable drug sensitivity was obtained.

[0116] This result further supports the idea that drug responsiveness varies depending on the number of cells constituting the spheroid, and that drug responsiveness tends to be more favorable when the cell number is low.

[0117] [Experimental Example 9] (Study of drug responsiveness of 3D spheroid-cultured neurons8) A calcium assay was performed in the same manner as in Experimental Example 4, except that the drug was changed to carbamazepine, and the drug responsiveness of neurons was examined. The expected drug response is a decrease in the number of calcium spikes when carbamazepine is administered.

[0118] Figure 9 is a graph showing the measurement results. The values ​​on the graph represent the mean ± standard deviation. The horizontal axis of the graph represents the concentration of carbamazepine in the medium, and the vertical axis of the graph represents the relative change in the number of calcium spikes (log2). The response observed upon addition of DMSO, the drug solvent, is represented as 0 (no change). A positive value indicates an increase in the number of calcium spikes due to drug addition, and a negative value indicates a decrease in the number of calcium spikes.

[0119] In Figure 9, "16,000 cells" indicates the results for spheroids prepared by mixing 8,000 neurons and 8,000 astrocytes, and "4,000 cells" indicates the results for spheroids prepared by mixing 2,000 neurons and 2,000 astrocytes.

[0120] As a result, it was shown that the drug showed a stronger effect at lower concentrations in a concentration-dependent manner at 4,000 cells compared to 16,000 cells, and favorable drug sensitivity was obtained.

[0121] This result further supports the idea that drug responsiveness varies depending on the number of cells constituting the spheroid, and that drug responsiveness tends to be more favorable when the cell number is low.

[0122] [Experimental Example 10] (Study of 3D spheroid-cultured neurons) Spheroids with different cell numbers were prepared, their diameters were measured, and various parameters shown in Table 1 below were calculated.

[0123] Human iPSC-derived neurons (Excitatory Neurons, Elixirgen Scientific) and primary human astrocytes (ScienCell Research Laboratories) were mixed and seeded onto spheroid plates at the following cell counts per well: 8,000 neurons and 8,000 astrocytes, 4,000 neurons and 4,000 astrocytes, 2,000 neurons and 2,000 astrocytes, 1,000 neurons and 1,000 astrocytes, 500 neurons and 500 astrocytes, and 250 neurons and 250 astrocytes. The culture medium used was Neurobasal Plus (Thermo Fisher Scientific) supplemented with B-27 Plus Supplement (Thermo Fisher Scientific), Glutamax (Thermo Fisher Scientific), 200 μM ascorbic acid (Fujifilm Wako Pure Chemical Industries), and 10% Neuron Culture medium (Fujifilm Wako Pure Chemical Industries). The cells were cultured for 5 weeks and then photographed using a phase-contrast microscope.

[0124] Representative images of the spheroids are shown in Figure 10. In Figure 10, "16,000 cells" indicates a spheroid created by mixing 8,000 neurons and 8,000 astrocytes, "8,000 cells" indicates a spheroid created by mixing 4,000 neurons and 4,000 astrocytes, "4,000 cells" indicates a spheroid created by mixing 2,000 neurons and 2,000 astrocytes, "2,000 cells" indicates a spheroid created by mixing 1,000 neurons and 1,000 astrocytes, "1,000 cells" indicates a spheroid created by mixing 500 neurons and 500 astrocytes, and "500 cells" indicates a spheroid created by mixing 250 neurons and 250 astrocytes.

[0125] Table 1 below shows various parameters for each spheroid. Some of the parameters were calculated based on the diameter of the spheroid measured using the image in Figure 10. In Table 1 below, "surface area" refers to the surface area of ​​the spheroid, "volume" refers to the volume of the spheroid, and "cell density" refers to the cell density of the spheroid. "10 μm inner radius" refers to the radius of the spheroid when one cell layer is removed from the outer edge of the spheroid, assuming that the diameter of a single cell is 10 μm. "10 μm inner volume" refers to the volume of the spheroid when one cell layer is removed from the outer edge of the spheroid, assuming that the diameter of a single cell is 10 μm. "Volume within 10 μm of the surface" refers to the volume of one cell layer removed from the outer edge of the spheroid, assuming that the diameter of a single cell is 10 μm. "The percentage (%) of the volume of the surface 10 μm to the total volume" refers to the percentage (%) of the volume of one cell layer located on the outer edge of the spheroid, assuming a diameter of one cell of 10 μm. "The number of cells present in a volume of the surface 10 μm" refers to the number of cells in one cell layer located on the outer edge of the spheroid, assuming a diameter of one cell of 10 μm.

[0126] [Table 1]

[0127] The results of Experimental Examples 2 to 10 showed that spheroids with a total cell count of 4,000 tended to exhibit the most favorable drug responsiveness, followed by spheroids with a total cell count of 8,000 and spheroids with a total cell count of 16,000. Although spheroids with a total cell count of 2,000 or less are considered to exhibit drug responsiveness, it was presumed that this was below the detection sensitivity of the device used in the calcium assay and therefore could not be detected, resulting in no drug response being observed.

[0128] In this experiment, the total cell count was 4,000 vs. 8,000, and furthermore, the total cell count was 8,000 vs. 16,000, resulting in a relative decrease in drug sensitivity. This is thought to be due to the large number of cells and the large size of the spheroids, or because the drug only penetrated the surface of the spheroids, resulting in fewer cells that came into contact with the drug and showed a drug response compared to cells that did not come into contact with the drug and showed no drug response (the drug response that should have been observed was hidden).

[0129] Therefore, to observe drug responses with high sensitivity, it is preferable to have a high proportion of cells in the spheroid that can come into contact with the drug and show a drug response. Therefore, the inventors focused on the surface of the spheroid and investigated the "volume ratio of one cell layer" that is suitable for expressing the proportion of cells present on the surface, and found the optimal conditions for spheroids with excellent drug responsiveness.

[0130] Spheroid size may vary depending on the cell type, even with the same cell number. Therefore, it is necessary to identify the appropriate cell number (range) for each cell type, which will result in a cell layer volume ratio of 15-30%.

[0131] Specifically, first, multiple spheroids are formed using varying numbers of cells (for example, total cell numbers of 20,000, 16,000, 12,000, 8,000, 4,000, 2,000, etc.). Then, the "volume ratio of one cell layer to the total volume" for each cell number is calculated based on the above formula (1). The cell number (range) that gives this value of 15-30% is then determined. Thereafter, spheroids are produced using the previously determined cell number depending on the type of cell used. This makes it possible to produce spheroids with favorable drug sensitivity (drug responsiveness).

[0132] The present invention includes the following aspects. [1] A nervous system cell-containing spheroid, comprising at least one type of neuron and other nervous system cells, wherein the volume of one cell layer located at the outer edge of the spheroid accounts for 15 to 30% of the total volume of the spheroid. [2] The neural cell-containing spheroid according to [1], wherein the neural cells other than neurons include astrocytes. [3] A neural cell-containing spheroid according to [2], in which the ratio of the number of neurons to the number of astrocytes (number of neurons / number of astrocytes) is 1. [4] A method for producing spheroids containing neural cells, comprising a step of mixing at least one type of neural cell after lineage determination and neural cells other than the neural cells in a predetermined ratio to form a spheroid, wherein in the step, the ratio of the volume of one layer of cells located at the outer edge of the spheroid to the total volume of the spheroid is adjusted to 15 to 30%. [5] The manufacturing method described in [4], wherein the nervous system cells other than neurons include astrocytes. [6] The manufacturing method described in [5], wherein the ratio of the number of neurons to the number of astrocytes (number of neurons / number of astrocytes) is 1. [7] A method for evaluating a test substance, comprising the steps of incubating a nervous system cell-containing spheroid in the presence of the test substance and evaluating the effect of the test substance on the nervous system cell-containing spheroid, wherein the nervous system cell-containing spheroid contains at least one type of neuron and nervous system cells other than the neuron, and the volume of one layer of cells located at the outer edge of the spheroid accounts for 15 to 30% of the total volume of the spheroid. [8] The evaluation method according to [7], wherein the non-neuronal nervous system cells include astrocytes. [9] The evaluation method described in [8], wherein the ratio of the number of neurons to the number of astrocytes (number of neurons / number of astrocytes) is 1. [Prior art documents] [Patent documents]

[0133] [Patent Document 1] Patent Publication No. 2021-185906

Claims

1. A neural cell-containing spheroid, At least one type of nerve cell and a nervous system cell other than the nerve cell, The ratio of the volume of one cell layer located at the outer edge of the spheroid to the total volume of the spheroid is 15 to 30%. Neural cell-containing spheroids.

2. The neural cell-containing spheroid according to claim 1 , wherein the neural cells other than neurons include astrocytes.

3. The nervous system cell-containing spheroid according to claim 2, wherein the ratio of the number of neurons to the number of astrocytes (number of neurons / number of astrocytes) is 1.

4. A method for producing neural cell-containing spheroids, comprising: a step of mixing at least one type of lineage-committed neuronal cell and a neural cell other than the neuronal cell at a predetermined ratio to form a spheroid, In the step, the ratio of the volume of one cell layer located on the outer edge of the spheroid to the total volume of the spheroid is adjusted to 15 to 30%.

5. The method according to claim 4 , wherein the non-neuronal neural cells include astrocytes.

6. The method of claim 5, wherein the ratio of the number of neurons to the number of astrocytes (number of neurons / number of astrocytes) is 1.

7. A method for evaluating a test substance, comprising: Incubating the neural cell-containing spheroids in the presence of a test substance; and evaluating the effect of the test substance on the nervous system cell-containing spheroids, The evaluation method, wherein the nervous system cell-containing spheroid contains at least one type of nerve cell and a nervous system cell other than the nerve cell, and the ratio of the volume of one cell layer located on the outer edge of the spheroid to the total volume of the spheroid is 15 to 30%.

8. The evaluation method according to claim 7 , wherein the non-neuronal nervous system cells include astrocytes.

9. The evaluation method according to claim 8, wherein the ratio of the number of neurons to the number of astrocytes (number of neurons / number of astrocytes) is 1.

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