Method for producing cell-containing containers and spheroids containing nervous system cells

Abstract

To provide a technique for suppressing functional variations between a plurality of neural cell-containing spheroids across a plurality of lots or within a single lot.SOLUTION: The present invention relates to a cell-containing vessel comprising a plurality of neural cell-containing spheroids for which the variation calculated by the calculation method described below is less than 20%, where the neural cell-containing spheroids contain a plurality of types of cells including neural cells. (Calculation Method) Calcium transient assays are conducted for each of a plurality of neural cell-containing spheroids, a number of spontaneous oscillations per 10 minutes is measured, an average and a standard deviation for the number of spontaneous oscillations are calculated, and a variation is calculated by the following formula (1). Variation (%)=standard deviation for number of spontaneous oscillations / average number of spontaneous oscillations×100.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a cell-containing container and a method for producing a nervous system cell-containing spheroid.

Background Art

[0002] In the research and development of drug discovery, many in vitro experimental systems using cells have been constructed. For example, in drug screening for neurological diseases and toxicity screening for the nervous system, primary cultured cells, cells differentiated from ES cells / iPS cells having undifferentiated potential, immortalized cells, cancer cells, etc. are used.

[0003] Among them, iPS cell-derived neurons can use patient-derived cells, can reflect the genetic background derived from diseases, and are attracting attention as a model that can construct a highly extrapolated model. In addition, the calcium transient assay is often used as an assay method for observing nerve activity. And the application of the calcium transient assay using spheroid-cultured neurons to toxicity evaluation and drug efficacy evaluation has been studied.

[0004] For example, Patent Document 1 (International Publication No. 2019 / 014603) describes an optical detection method comprising a spheroid composed of human cells having a single diameter, a drug, and a step of detecting a change in oscillation for the purpose of performing high-throughput functional evaluation.

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, conventional spheroids containing nerve cells have been produced by simultaneously inducing the differentiation of pluripotent stem cells such as iPS cells into astrocytes and nerve cells. Specifically, conventionally, neural progenitor cells produced from pluripotent stem cells were seeded on a spheroid production plate and cultured to form spheroids, and then differentiated into nerve cells and astrocytes using culture medium components to produce neural cell spheroids containing nerve cells and astrocytes. However, differentiation induction systems via neural progenitor cells tend to have low differentiation synchronization, making it difficult to obtain a uniform cell population.

[0006] Therefore, the proportion of cell types contained in spheroids varies depending on the degree of differentiation induction each time, such as the state of neural progenitor cells, and it is not possible to control it to be constant at all times. This results in large variability between samples and low reproducibility.

[0007] Therefore, the present invention aims to provide a technique for suppressing functional variability in spheroids containing multiple nervous system cells, either across multiple lots or within the same lot. [Means for solving the problem]

[0008] The cell-containing container according to the present invention contains multiple nerve cell-containing spheroids having a variation of less than 20% calculated according to the calculation method described below, and the nerve cell-containing spheroids contain multiple types of cells, including nerve cells. (Calculation method) For each of the spheroids containing multiple nerve cells, a calcium transient assay was performed, the number of spontaneous oscillations per 10 minutes was measured, the mean and standard deviation of the number of spontaneous oscillations were calculated, and the variability was calculated using the following formula (1). Variability (%) = Standard deviation of spontaneous oscillation count / Mean of spontaneous oscillation count × 100 …(1)

[0009] The present invention provides a method for producing spheroids containing nerve cells, which includes the step of mixing multiple types of nerve cells after cell lineage determination in predetermined proportions to form spheroids. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a technique for suppressing functional variability in spheroids containing multiple nervous system cells across multiple lots or within the same lot. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a schematic diagram illustrating a conventional method for producing spheroids containing nerve cells. [Figure 2] Figure 2 is a schematic diagram illustrating a method for producing nerve cell-containing spheroids according to one embodiment of the present invention. [Figure 3] Figure 3 is a schematic diagram illustrating a spheroid containing nerve cells according to one embodiment of the present invention. [Figure 4] Figure 4 is a schematic diagram illustrating multiple lots of nerve cell-containing spheroids obtained by a conventional manufacturing method. [Figure 5] Figure 5 is a schematic diagram illustrating multiple lots of nerve cell-containing spheroids according to one embodiment of the present invention. [Figure 6] Figure 6 is a graph showing an example of spontaneous oscillation observed in a calcium transient assay. [Figure 7] Figures 7(a) to 7(f) are graphs showing the results of calcium transient assays for three representative spheroids from test numbers 1-1 to 1-6 in Experimental Example 1, respectively. [Figure 8] Figure 8 is a graph summarizing the number of spontaneous oscillations per 10 minutes based on the results of the calcium transient assay using 24 spheroids in Experimental Example 1. [Figure 9] Figure 9 is a graph showing the diameters of spheroids from test numbers 1-1 to 1-6 in Experimental Example 1. [Figure 10] Figures 10(a) and (b) are graphs showing the results of calcium transient assays for three representative spheroids each of test numbers 2-1 and 2-2 in Experimental Example 2. [Figure 11] Figure 11 is a graph summarizing the number of spontaneous oscillations per 10 minutes based on the results of calcium transient assays for 18 spheroids in Experimental Example 2. [Figure 12] Figure 12 is a graph showing the diameters of the spheroids of test numbers 2-1 to 2-3 in Experimental Example 2. [Figure 13] Figures 13(a) and (b) are graphs showing the results of calcium transient assays for three representative spheroids each of test numbers 3-1 and 3-2 in Experimental Example 3. [Figure 14] Figure 14 is a fluorescence micrograph showing the results of immunostaining in Experimental Example 4. [Method for Producing Spheroids Containing Nervous System Cells]

[0012] [Method for Producing Spheroids Containing Nervous System Cells] In one embodiment, the present invention provides a method for producing a spheroid containing nervous system cells, the method including a step of mixing a plurality of types of nervous system cells after cell lineage determination at a predetermined ratio to form a spheroid.

[0013] As will be described later in the examples, the spheroids containing nervous system cells produced by the production method of the present embodiment have suppressed functional variations among multiple lots or among multiple spheroids containing nervous system cells within the same lot.

[0014] As used herein, a lot refers to products manufactured simultaneously under the same conditions. Suppression of functional variation in a plurality of nervous system cell-containing spheroids among multiple lots means that when a nervous system cell-containing spheroid representing each lot is randomly extracted from a plurality of lots of nervous system cell-containing spheroids, the functional variation of these nervous system cell-containing spheroids is small.

[0015] In addition, suppression of functional variation in a plurality of nervous system cell-containing spheroids within the same lot means that when a plurality of nervous system cell-containing spheroids are randomly extracted from one lot of nervous system cell-containing spheroids, the functional variation of these nervous system cell-containing spheroids is small.

[0016] 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 by some assay. For example, it may be the number of spontaneous oscillations per predetermined time measured by subjecting the nervous system cell-containing spheroid to a calcium transient assay. Alternatively, it may be the responsiveness to a drug. Examples of the responsiveness to a drug include, but are not particularly limited to, changes in spontaneous oscillations in the presence of the drug, changes in the expression level of a predetermined gene, and the like.

[0017] A small functional variation means, for example, that the variation calculated according to the following calculation method is less than 20%, for example less than 15%, for example less than 10%. (Calculation method) For a plurality of nervous system cell-containing spheroids among multiple lots or within the same lot, a calcium transient assay is performed on each of them, the number of spontaneous oscillations per 10 minutes is measured, the average value and standard deviation value of the number of spontaneous oscillations are calculated, and the variation is calculated by the following formula (1). Variation (%) = Standard deviation value of the number of spontaneous oscillations / Average value of the number of spontaneous oscillations × 100 …(1)

[0018] Figure 1 is a schematic diagram illustrating a conventional method for producing spheroids containing nerve cells. As shown in Figure 1, the conventional method uses neural progenitor cells before cell lineage determination to form spheroids. Therefore, differentiation induction occurs along with spheroid formation. As a result, the cells constituting the spheroid change over time, and as a result of differentiation induction, spheroids containing nerve cells and astrocytes are obtained. With this method, a uniform cell population cannot be obtained, and the resulting spheroids contain varying proportions of nerve cells and astrocytes.

[0019] Figure 2 is a schematic diagram illustrating the manufacturing method of this embodiment. As shown in Figure 2, in the manufacturing method of this embodiment, nerve cells after cell lineage determination are mixed in a predetermined proportion to form a spheroid. In the example in Figure 2, the nerve cell-containing spheroid includes differentiated nerve cells and differentiated astrocytes. Therefore, the proportion of different types of nerve cells constituting the spheroid does not change over time.

[0020] Furthermore, in the manufacturing method of this embodiment, as shown in Figure 3, by changing the proportion of nerve cells to be mixed, it is possible to produce nerve cell-containing spheroids containing multiple types of nerve cells in specific proportions.

[0021] Figure 4 is a schematic diagram illustrating multiple lots of nerve cell-containing spheroids obtained by a conventional manufacturing method. As shown in Figure 4, nerve cell-containing spheroids obtained by a conventional manufacturing method exhibit large variations in the proportion of constituent nerve cell types, both among multiple spheroids from different lots and among multiple spheroids from the same lot. Consequently, there is considerable functional variation among each nerve cell-containing spheroid.

[0022] Figure 5 is a schematic diagram illustrating multiple lots of nerve cell-containing spheroids obtained by the manufacturing method of this embodiment. As shown in Figure 5, since the nerve cell-containing spheroids obtained by the manufacturing method of this embodiment use nerve cells after cell lineage determination, the proportion of different types of nerve cells contained in the nerve cell-containing spheroids can be kept constant. Therefore, the variation in the proportion of different types of nerve cells is small, both for multiple nerve cell-containing spheroids across multiple lots and for multiple nerve cell-containing spheroids within the same lot. Consequently, the functional variation in each nerve cell-containing spheroid is also small.

[0023] In the manufacturing method of this embodiment, the neural cells after cell lineage determination may be differentiated neural cells or stem cells destined to differentiate into neural cells. The differentiated neural cells may be primary cells extracted from a living organism or neural cells differentiated from stem cells. Furthermore, the stem cells may be pluripotent stem cells or neural progenitor cells.

[0024] Examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells. Examples of induced pluripotent stem cells include nuclear-transplanted embryonic stem cells (ntES cells) and induced pluripotent stem cells (iPS cells). Among these, iPS cells are preferred as pluripotent stem cells.

[0025] iPS cells may be derived from healthy individuals or from patients with various neurological diseases. They may also be genetically edited, for example, cells that have been modified to possess genes that cause or are risk factors for various neurological diseases.

[0026] If iPS cells are derived from patients with various neurological disorders, they can be used to construct disease models of those disorders. Neurological disorders, while not limited to those mentioned above, include, for example, neurodegenerative diseases, autism, epilepsy, attention-deficit hyperactivity disorder (ADHD), schizophrenia, and bipolar disorder. Examples of neurodegenerative diseases include, for example, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS).

[0027] The animal species from which nerve cells originate are not particularly limited, and examples include humans, monkeys, dogs, cattle, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, and hamsters. Among these, humans are preferred.

[0028] Examples of nerve cells include nerve cells, astrocytes, oligodendrocytes, and microglia.

[0029] Nerve cells can be broadly classified into peripheral nerves and central nerves. Peripheral nerves include, for example, sensory nerves, motor nerves, and autonomic nerves. 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 be broadly classified into excitatory neurons and inhibitory neurons. Glutamate-mediated neurons are primarily responsible for excitatory transmission in the central nervous system, while GABAergic (γ-aminobutyric acid)-mediated neurons are primarily responsible for inhibitory transmission.

[0030] Other types of neurons that release neuromodulatory substances include cholinergic neurons, dopaminergic neurons, noradrenergic neurons, serotonergic neurons, and histaminergic neurons.

[0031] Furthermore, astrocytes, oligodendrocytes, microglia, etc., may be primary cultured cells or cells differentiated from stem cells.

[0032] The neural cells after cell lineage determination may be pluripotent stem cells that have undergone differentiation induction treatment into neural cells. Differentiation induction treatment may include, for example, introducing specific transcription factors into pluripotent stem cells. Specifically, cells from Elixirgen Scientific's Quick-Neuron™ series can be cited as examples of such cells. These cells are pluripotent stem cells that have undergone differentiation induction treatment into neural cells and differentiate into functionally mature neural cells in about 10 days.

[0033] The nerve cells after cell lineage determination may be cells other than nerve cells that have undergone differentiation induction treatment. The differentiation induction treatment is the same as described above. Examples of cells other than nerve cells include skeletal muscle cells, vascular endothelial cells, and blood cells. The animal species from which the cells other than nerve cells originate are not particularly limited and include, for example, humans, monkeys, dogs, cattle, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, and hamsters. Among these, humans are preferred. Furthermore, the cells other than nerve cells may be primary cultured cells or cells differentiated from stem cells.

[0034] After determining the cell lineage, multiple types of nervous system cells are mixed in predetermined proportions, then seeded and cultured in a suitable cell culture vessel such as a spheroid production plate to form spheroids. The incubation period for the cell culture vessel can be set as appropriate depending on the purpose, but it is preferable that it be at least long enough for the nervous system cells to mature to a degree that allows their function to be evaluated.

[0035] For example, when using pluripotent stem cells that have undergone differentiation induction treatment to become neural cells as neural cells after cell lineage determination, the incubation period from the time when multiple types of pluripotent stem cells that have undergone differentiation induction treatment to become neural cells are mixed and seeded 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.

[0036] As the culture medium, a culture medium to which the necessary components have been added to the basal culture medium can be used. Examples of basal media include BrianPhys (Stem Cell Technologies), Neurobasal (Thermo Fisher Scientific), Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, D-MEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), αMEM (alpha Modified Eagle's Minimum Essential Medium, αMEM), MEM (Minimum Essential Medium), RPMI1640 (Roswell Park Memorial Institute-1640) medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, M199 medium, and High Performance Medium 199 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), StemlineII (Sigma-Aldrich), QBSF-60 (Quality Biological), 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 include Biosciences, Inc.'s NutriStem® medium (manufactured by Biological Industries, Inc.), CSTI-7 medium (manufactured by Cell Science Institute Inc.), MesenPRO RS medium (manufactured by Thermo Fisher Scientific, Inc.), MF-Medium® mesenchymal stem cell proliferation medium (manufactured by Toyobo Co., Ltd.), Sf-900II (manufactured by Thermo Fisher Scientific, Inc.), Opti-Pro (manufactured by Thermo Fisher Scientific, Inc.), etc. These may be used individually or in combination of two or more.

[0037] In addition, additives to be added to the basal culture medium include those commonly used for culturing nerve cells, 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 (ScienceCell Research), Component N (Elixirgen Scientific), Component G2 (Elixirgen Scientific), Component P (Elixirgen Scientific), N2 Supplement (Thermo Fisher Scientific), iCell Neural Supplement B (CDI), and iCell Neuvous System Supplement, B-27 plus (Thermo Fisher Scientific).

[0038] Here, we will explain the calcium transient assay. In the calcium transient assay, first, a spheroid containing nerve cells is prepared. For example, any number of nerve cells and astrocytes are mixed, seeded on a spheroid preparation plate, and cultured for 3 to 8 weeks in a medium suitable for nerve cell culture, changing the medium as needed.

[0039] Next, the spheroids are transferred to a plate suitable for optical detection, and a calcium-sensitive fluorescent dye is added to the culture medium. A calcium-sensitive fluorescent dye is a substance that is taken up by cells and emits fluorescence when it binds to calcium ions. Suitable calcium-sensitive fluorescent dyes include, for example, Cal-520, Fluo4, Calcium-6, EarlyTox Cardiotoxicity Kit (Molecular Devices), Cal-520AM (AAT Bioquest, Inc. (Former ABD Bioquest, Inc.)), etc.

[0040] Next, using a fluorescence-detecting device, set the excitation wavelength / fluorescence wavelength to match the calcium-sensitive fluorescent dye used, and perform measurements continuously for a specified period of time. Suitable devices include fluorescence plate readers, FDSS (Functional Drug Screening System, Hamamatsu Photonics), and FLIPR (Fluorometric Imaging Plate Reader, Molecular Devices). For example, measurements should be taken every 0.5 seconds for 10 minutes.

[0041] Nerve cells engaged in neural activity repeatedly take up and release calcium ions spontaneously. Therefore, when a calcium transient assay is performed using spheroids containing nerve cells, the fluorescence intensity changes periodically and is measured as spontaneous oscillation.

[0042] Figure 6 is a graph showing an example of spontaneous oscillation observed in a calcium transient assay. The horizontal axis of the graph represents time (seconds), and the vertical axis represents the ratio of the measured fluorescence intensity to the fluorescence intensity immediately after the start of the measurement.

[0043] [Spheroids containing nerve cells] In one embodiment, the present invention provides a spheroid containing multiple types of differentiated nervous system cells in a predetermined mixing ratio, wherein the variability calculated according to the calculation method described below is less than 20%. (Calculation method) For multiple spheroids containing nerve cells from different lots or from the same lot, a calcium transient assay is performed on each, 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 variability is calculated using the following formula (1). Variability (%) = Standard deviation of spontaneous oscillation count / Mean of spontaneous oscillation count × 100 …(1)

[0044] The nerve cell-containing spheroid of this embodiment can be manufactured by the manufacturing method described above. The variability calculated using the above formula (1) may be less than, for example, 15%, or less than 10%.

[0045] The spheroids containing nerve cells in this embodiment preferably have a variability of less than 15%, more preferably less than 10%, and particularly preferably less than 5%, calculated according to the calculation method described below. (Calculation method) For multiple spheroids containing nerve cells across multiple lots or within the same lot, the number of nerve cells and the number of astrocytes are measured for each. The mean and standard deviation of the ratio of nerve cells to astrocytes (number of nerve cells / number of astrocytes) are calculated, and the variability is calculated using the following formula (2). Variability (%) = Standard deviation of (Number of nerve cells / Number of astrocyte cells) / Mean value of (Number of nerve cells / Number of astrocyte cells) × 100 …(2)

[0046] In formula (2) above, the number of nerve cells and astrocyte cells can be measured, for example, by orally immunostaining spheroids to stain the nerve cells and astrocytes, respectively, and observing them with a fluorescence microscope. Examples of nerve cell markers include MAP2, Tubulin beta3, NeuN, 160kDa Neurofilament, 200kDa Neurofilament, NSE, PSD93, and PSD95. Examples of astrocyte markers include GFAP, S100β, Cx43, EAAT1, EAAT2, Glutamine synthetase, and ALDH1L1.

[0047] Alternatively, the number of nerve cells and astrocytes in the spheroid may not be measured as a cell count, but the variability may be calculated by considering the sum of the fluorescence intensities of each stained cell as the value corresponding to the cell count. Alternatively, the variability may be calculated by considering the area or volume occupied by each stained cell in the microscopic image as the value corresponding to the cell count.

[0048] In this case, the variation may be calculated using the following formula (3) instead of the above formula (2). Variability (%) = Standard deviation of (Value corresponding to the number of nerve cells / Value corresponding to the number of astrocyte cells) / Mean value of (Value corresponding to the number of nerve cells / Value corresponding to the number of astrocyte cells) × 100 …(3)

[0049] In the nerve cell-containing spheroid of this embodiment, the ratio of the number of nerve cells to the number of astrocyte cells (value corresponding to the number of nerve cells / value corresponding to the number of astrocyte cells) may be approximately 1.

[0050] As described later in the examples, when the ratio of nerve cell count to astrocyte count is 1, that is, when the ratio of nerve cell count to astrocyte count is approximately 1:1, the functional variability of nerve cell-containing spheroids may be lower compared to when the ratio is approximately 2:1 or approximately 4:1.

[0051] The ratio of nerve cell count to astrocyte cell count does not need to be exactly 1; for example, it could be between 0.8 and 1.2, or between 0.9 and 1.1. Furthermore, the proportion of each cell type that results in low functional variability in nerve cell-containing spheroids may vary depending on the nerve cells and astrocytes used.

[0052] [Cell-containing container] In one embodiment, the present invention provides a cell-containing container comprising the above-described nerve cell-containing spheroid. The form of the container is not particularly limited and includes, for example, a tube, a multiwell plate, etc. Examples of multiwell plates include 24-well, 48-well, 96-well, 384-well, and 1,536-well plates.

[0053] There are no particular restrictions on the shape, volume, material, color, etc., of the wells in a multiwell plate; they can be appropriately selected according to the purpose.

[0054] The shape of the well is not particularly limited as long as it can accommodate the spheroid, and can be appropriately selected according to the purpose. Examples include flat bottom, round bottom, U-bottom, and V-bottom shapes.

[0055] The volume of the wells is not particularly limited and can be appropriately selected depending on the purpose. For example, considering the amount of reagents used in a typical evaluation method, it may be 5 to 1000 μL, 30 to 300 μL, or 50 to 200 μL.

[0056] The color of the multiwell plate can be transparent, translucent, colored, or completely light-shielding. Furthermore, when evaluating the optical system, a container with a transparent bottom and colored sides is preferable from the viewpoint of suppressing interference between adjacent wells.

[0057] The material of the multiwell plate can be appropriately selected according to the purpose. Examples include 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, acrylic materials such as urethane acrylate, organic materials such as cellulose and polydimethylsiloxane (PDMS), and inorganic materials such as glass and ceramics.

[0058] To prevent spheroids from adhering to the bottom or sides of the multiwell plate, it is preferable that the surface of the multiwell plate be treated with an ultra-low adsorption treatment. Examples include coating with a polymer having a phosphorylcholine group, covalent treatment with a electrically neutral hydrophilic gel, and coating with a synthetic polymer having the same structure as the polar group of phosphatidylcholine. Any treatment that suppresses cell adhesion can be selected as appropriate, and is not limited to these examples.

[0059] The container in this embodiment may be a multi-well plate, with one spheroid containing neural cells in each well. Such a container is convenient because it can be used directly in various assays.

[0060] Because the container of this embodiment contains spheroids with reduced functional variability, when used for efficacy evaluation or toxicity evaluation, it yields data with less variability and high reproducibility.

[0061] [Method for evaluating test substances] In one embodiment, the present invention provides a method for evaluating the efficacy or toxicity of a test substance, comprising the step of evaluating the function of a nerve cell-containing spheroid in the presence of the test substance, wherein the nerve cell-containing spheroid contains a predetermined mixture ratio of differentiated nerve cells, and the variability calculated according to the calculation method described below is less than 20%. (Calculation method) For multiple spheroids containing nerve cells from different lots or from the same lot, a calcium transient assay is performed on each, 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 variability is calculated using the following formula (1). Variability (%) = Standard deviation of spontaneous oscillation count / Mean of spontaneous oscillation count × 100 …(1)

[0062] The evaluation method of this embodiment can also be described as a drug screening method. The test substance is not particularly limited and can be, for example, a natural compound library, a synthetic compound library, an existing drug library, a metabolite library, etc. [Examples]

[0063] 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.

[0064] [Experimental Example 1] (Spheroid production using human iPSC-derived nerve cells and human primary cultured astrocytes) Human iPSC-derived nerve cells (Mixed Neurons, Elixirgen Scientific) and human primary cultured astrocytes (Thermo Fisher Scientific) were mixed so that the number of cells per well matched the cell counts shown in Table 1 below, numbered 1-1 to 1-6. Subsequently, each mixed cell was seeded onto a spheroid preparation plate and cultured to produce spheroids. BrianPhys (Stem Cell Technologies), supplemented with SM1 supplement (Stem Cell Technologies), N2 supplement A (Stem Cell Technologies), and 10% astrocyte culture supernatant (ScienceCell Research), was used as the culture medium.

[0065] Next, a calcium transient assay was performed on each spheroid that had been cultured for at least four weeks. Specifically, each spheroid was transferred to a measurement plate, and a calcium-sensitive fluorescent dye (EarlyTox Cardiotoxicity Kit, Molecular Devices Inc.) was added to the culture medium and incubated. Subsequently, fluorescence was measured using a fluorescence plate reader at 0.6-second intervals for 10 minutes.

[0066] Figures 7(a) to 7(f) are graphs showing the measurement results for three representative spheroids from test numbers 1-1 to 1-6, respectively. In Figures 7(a) to 7(f), the horizontal axis of the graphs represents time (seconds). The vertical axis represents the ratio of the measured fluorescence intensity to the fluorescence intensity immediately after the start of measurement. Figure 8 is a graph summarizing the number of spontaneous oscillations per 10 minutes based on the results of the calcium transient assay for 24 spheroids. In Figure 8, the values ​​in the graphs represent the mean ± standard deviation.

[0067] Table 1 below shows the results of calculating the variability using the following formula (1) based on the mean and standard deviation of the number of spontaneous oscillations. Variability (%) = Standard deviation of spontaneous oscillation count / Mean of spontaneous oscillation count × 100 …(1)

[0068] [Table 1]

[0069] As a result, it was found that a variability of less than 20% (reproducibility of 80% or more) can be achieved when the ratio of cell numbers (neurons: astrocytes) is 1:1.

[0070] Next, images of each spheroid cultured for 8 weeks were taken using a phase-contrast microscope and analyzed using software (named "ImageJ," https: / / imagej.nih.gov / ij / ) to calculate the diameter of the spheroids. Figure 9 is a graph showing the diameters of the spheroids produced under each condition. In Figure 9, the values ​​on the graph represent the mean ± standard deviation.

[0071] [Experimental Example 2] (Spheroid production using human iPSC-derived nerve cells and human iPSC-derived astrocytes) Human iPSC-derived nerve cells (Mixed Neurons, Elixirgen Scientific) and human iPSC-derived astrocytes (Human iPS-derived Astrocytes (Mature), Xcell Science) were mixed so that the number of cells per well was as shown in Table 2 below, with test numbers 2-1 and 2-2. Subsequently, each mixed cell was seeded onto a spheroid production plate and cultured to produce spheroids. BrianPhys (Stem Cell Technologies), supplemented with SM1 supplement (Stem Cell Technologies) and 10% astrocyte culture supernatant (ScienceCell Research), was used as the culture medium.

[0072] Next, a calcium transient assay was performed on each spheroid that had been cultured for at least four weeks. Specifically, each spheroid was transferred to a measurement plate, and a calcium-sensitive fluorescent dye (EarlyTox Cardiotoxicity Kit, Molecular Devices Inc.) was added to the culture medium and incubated. Subsequently, fluorescence was measured using a fluorescence plate reader at 0.6-second intervals for 10 minutes.

[0073] Figures 10(a) and (b) are graphs showing the measurement results for three representative spheroids from test numbers 2-1 and 2-2, respectively. In Figures 10(a) and (b), the horizontal axis of the graphs represents time (seconds). The vertical axis represents the ratio of the measured fluorescence intensity to the fluorescence intensity immediately after the start of measurement. Figure 11 is a graph summarizing the number of spontaneous oscillations per 10 minutes based on the results of the calcium transient assay for 18 spheroids. In Figure 11, the values ​​in the graphs represent the mean ± standard deviation.

[0074] Table 2 below shows the results of calculating the variability using the following formula (1) based on the mean and standard deviation of the number of spontaneous oscillations. Variability (%) = Standard deviation of spontaneous oscillation count / Mean of spontaneous oscillation count × 100 …(1)

[0075] [Table 2]

[0076] As a result, it was found that a variability of less than 20% (reproducibility of 80% or more) can be achieved when the ratio of cell numbers (neurons: astrocytes) is 1:1.

[0077] Next, images of each spheroid cultured for 7 weeks were taken using a phase-contrast microscope and analyzed using software (named "ImageJ," https: / / imagej.nih.gov / ij / ) to calculate the diameter of the spheroids. Figure 12 is a graph showing the diameters of the spheroids produced under each condition. In Figure 12, the values ​​on the graph represent the mean ± standard deviation.

[0078] [Experimental Example 3] (Study of drug response using spheroids) Spheroids were prepared using human iPSC-derived neurons and human iPSC-derived astrocytes, and their drug responsiveness was investigated. First, human iPSC-derived neurons (Mixed Neurons, Elixirgen Scientific) and human iPSC-derived astrocytes (Human iPSC-derived Astrocytes (Mature), Xcell Science) were mixed so that the number of cells per well was as shown in Table 3 below, with test numbers 3-1 and 3-2. Next, each mixed cell was seeded onto a spheroid preparation plate and cultured to produce spheroids. BrianPhys (Stem Cell Technologies), supplemented with SM1 supplement (Stem Cell Technologies) and 10% astrocyte culture supernatant (ScienceCell Research), was used as the culture medium.

[0079] [Table 3]

[0080] Next, calcium transient assays were performed on each spheroid cultured for 7 weeks in the presence of a drug. The drug used was 4-aminopyridine (4-AP), a potassium channel blocker. It is known that 4-AP induces hyperexcitability in nerve cells. In calcium transient assays, this hyperexcitability is detected as an increase in oscillation frequency or an elevation of the baseline.

[0081] Specifically, each spheroid was first transferred to a measurement plate, and a calcium-sensitive fluorescent dye (EarlyTox Cardiotoxicity Kit, Molecular Devices Inc.) was added to the culture medium and incubated. Subsequently, 4-AP was added to the culture medium to a final concentration of 30 μM, and fluorescence measurements were taken at 0.6-second intervals for 10 minutes immediately following the addition.

[0082] Figures 13(a) and (b) are graphs showing the measurement results for three representative spheroids from test numbers 3-1 and 3-2, respectively. In Figures 13(a) and (b), the horizontal axis of the graphs represents time (seconds). The vertical axis represents the ratio of the measured fluorescence intensity to the fluorescence intensity immediately after the start of measurement.

[0083] As a result, when the cell ratio (neurons:astrocytes) was 1:1, the expected changes in the oscillation waveform were observed, whereas when the cell ratio (neurons:astrocytes) was 4:1, the expected changes in the oscillation waveform were not observed. From these results, it became clear that the cell ratio (neurons:astrocytes) affects not only spontaneous oscillations but also the response to drugs.

[0084] [Experimental Example 4] (Immunostaining of nerve cells and astrocytes in spheroids) Human iPSC-derived nerve cells (Mixed Neurons, Elixirgen Scientific) and human iPSC-derived astrocytes (Human iPSC-derived Astrocytes (Mature), Xcell Science) were used, with each well containing 8 × 10⁶ cells. 3 Each was mixed individually to form a single unit.

[0085] Next, the mixed cells were seeded onto spheroid production plates and cultured to produce spheroids. BrianPhys (Stem Cell Technologies), supplemented with SM1 supplement (Stem Cell Technologies) and 10% astrocyte culture supernatant (ScienceCell Research), was used as the culture medium.

[0086] Next, immunohistochemical staining of spheroids was performed at 5 and 7 weeks after the start of culture. After washing the spheroids with phosphate-buffered saline (PBS), they were fixed with 4% paraformaldehyde and blocked with fetal bovine serum (BSA).

[0087] Next, the cells were incubated with primary antibodies. As primary antibodies, we used an anti-MAP2 antibody (610460, BD Transduction Laboratories) that recognizes MAP2, a marker for nerve cells, and an anti-GFAP antibody (Z0334, Daco) that recognizes GFAP, a marker for astrocytes.

[0088] Next, the samples were washed and incubated with a secondary antibody. Depending on the type of antibody used, the secondary antibody used was either an anti-mouse secondary antibody (Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, A11001, Thermo Fisher Scientific) or an anti-rabbit secondary antibody (Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594, A11037, Thermo Fisher Scientific).

[0089] Next, the cells were washed, and the nuclei were stained with Hoechst33342 (Thermo Fisher Scientific) and observed under a fluorescence microscope. Figure 14 shows fluorescence micrographs of the immunohistochemical staining results. Each image obtained was analyzed using software (named "ImageJ", https: / / imagej.nih.gov / ij / ) to calculate the number of MAP2-positive cells and GFAP-positive cells, and to determine the ratio of neurons to astrocytes. Figure 14 also shows the percentage of MAP2-positive cells (%), GFAP-positive cells (%), and Hoechst33342-positive cells (%).

[0090] As a result, the cell ratio (neurons:astrocytes) was approximately 1:1 in both spheroids at 5 weeks and 7 weeks after the start of culture.

[0091] The present invention includes the following embodiments. [1] A cell-containing container comprising multiple nerve cell-containing spheroids having a variation of less than 20% calculated according to the calculation method below, wherein the nerve cell-containing spheroids contain multiple types of cells, including nerve cells. (Calculation method) For each of the spheroids containing multiple nerve cells, a calcium transient assay was performed, the number of spontaneous oscillations per 10 minutes was measured, the mean and standard deviation of the number of spontaneous oscillations were calculated, and the variability was calculated using the following formula (1). Variability (%) = Standard deviation of spontaneous oscillation count / Mean of spontaneous oscillation count × 100 …(1) [2] The cell-containing container according to [1], wherein the nerve cell-containing spheroid contains nerve cells and astrocytes, and the variability calculated according to the calculation method below is less than 15%. (Calculation method) For multiple spheroids containing nerve cells, the number of nerve cells and astrocytes are measured for each. The mean and standard deviation of the ratio of nerve cells to astrocytes (number of nerve cells / number of astrocytes) are calculated, and the variability is calculated using the following formula (2). Variability (%) = Standard deviation of (Number of nerve cells / Number of astrocyte cells) / Mean value of (Number of nerve cells / Number of astrocyte cells) × 100 …(2) [3] The cell-containing container described in [2], wherein the variability calculated according to the calculation method below is less than 5%. (Calculation method) For multiple spheroids containing nerve cells, the number of nerve cells and astrocytes are measured for each. The mean and standard deviation of the ratio of nerve cells to astrocytes (number of nerve cells / number of astrocytes) are calculated, and the variability is calculated using the following formula (2). Variability (%) = Standard deviation of (Number of nerve cells / Number of astrocyte cells) / Mean value of (Number of nerve cells / Number of astrocyte cells) × 100 …(2) [4] The cell-containing container according to [2] or [3], wherein the spheroid containing nerve cells has a ratio of the number of nerve cells to the number of astrocyte cells (number of nerve cells / number of astrocyte cells) of approximately 1. [5] A multiwell plate comprising one nerve cell-containing spheroid in each well, the cell-containing container according to any one of [1] to [4]. [6] A method for producing a spheroid containing nerve cells, comprising the step of mixing several types of nerve cells after cell lineage determination in predetermined proportions to form a spheroid.

[0092] The present invention can also be said to include the following embodiments. [P1] A method for producing a spheroid containing nerve cells, comprising the step of mixing multiple types of nerve cells after cell lineage determination in predetermined proportions to form a spheroid. [P2] A spheroid containing multiple types of differentiated nervous system cells in a predetermined mixing ratio, with a variability of less than 20% calculated according to the calculation method described below. (Calculation method) For multiple spheroids containing nerve cells from different lots or from the same lot, a calcium transient assay is performed on each, 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 variability is calculated using the following formula (1). Variability (%) = Standard deviation of spontaneous oscillation count / Mean of spontaneous oscillation count × 100 …(1) [P3] Spheroids containing nerve cells as described in [P2], wherein the variability calculated according to the calculation method below is less than 15%. (Calculation method) For multiple spheroids containing nerve cells across multiple lots or within the same lot, the number of nerve cells and the number of astrocytes are measured for each. The mean and standard deviation of the ratio of nerve cells to astrocytes (number of nerve cells / number of astrocytes) are calculated, and the variability is calculated using the following formula (2). Variability (%) = Standard deviation of (Number of nerve cells / Number of astrocyte cells) / Mean value of (Number of nerve cells / Number of astrocyte cells) × 100 …(2) [P4] Spheroids containing nerve cells as described in [P2], wherein the variability calculated according to the calculation method below is less than 5%. (Calculation method) For multiple spheroids containing nerve cells across multiple lots or within the same lot, the number of nerve cells and the number of astrocytes are measured for each. The mean and standard deviation of the ratio of nerve cells to astrocytes (number of nerve cells / number of astrocytes) are calculated, and the variability is calculated using the following formula (2). Variability (%) = Standard deviation of (Number of nerve cells / Number of astrocyte cells) / Mean value of (Number of nerve cells / Number of astrocyte cells) × 100 …(2) [P5] A spheroid containing nerve cells as described in any of [P2] to [P4], wherein the ratio of nerve cell numbers to astrocyte cell numbers (number of nerve cells / number of astrocyte cells) is 1. A container containing a nerve cell-containing spheroid as described in any of [P6], [P2], or [P5]. [P7] A multiwell plate, the container according to [P6], wherein one spheroid containing the nerve cells is contained in each well. [Prior art documents] [Patent Documents]

[0093] [Patent Document 1] International Publication No. 2019 / 014603

Claims

1. The process includes mixing multiple types of nerve cells after cell lineage determination in predetermined proportions to form spheroids. The aforementioned multiple types of nerve cells include nerve cells and astrocytes, The predetermined ratio is the ratio of the number of nerve cells to the number of astrocyte cells (number of nerve cells / number of astrocyte cells), and this ratio is between 0.8 and 1.

2. A method for producing spheroids containing nerve cells.

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