Method for producing brain organoids, brain organoids and applications thereof

Abstract

The present invention provides a method for producing a brain organoid, a brain organoid and its application. A method for producing a brain organoid comprises: culturing a neuroectoderm marker-positive cell aggregate in a culture medium containing an extracellular matrix at a concentration exceeding 10% by volume.

Description

[0001] This application claims priority based on Japanese Patent Application No. 2018-214930 filed in Japan on November 15, 2018, the contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to a method for producing brain organoids. More specifically, the present invention relates to a method for producing brain organoids, brain organoids, a kit for evaluating the efficacy of a test substance, a method for evaluating the efficacy of a test substance, a therapeutic drug for a disease caused by a disorder of a nervous system cell or nervous tissue, and a pharmaceutical composition for treating a disease caused by a disorder of a nervous system cell or nervous tissue. Background Art

[0003] The cerebral cortex of mammals has a multi-layer structure (layers I-VI), which is slowly formed from the formation period of the cerebral cortex of the fetus. The cerebral cortex is born from the neuroepithelium of the dorsal telencephalon (cerebral cortex), and forms hemispherical brain cells on both sides while slowly expanding outward. The dorsal and caudal sides of the cerebral cortex are adjacent to the cortical margin, while the ventral and rostral sides are adjacent to the lateral ganglionic eminence (LGE (Lateral Ganglionic Eminence), striatal primordium) or septum through the septal procortex. In the multi-layer structure of the cerebral cortex of an adult, there is the most superficial layer I (the primordium during the viviparous period is called the marginal zone), and most of the most superficial layer I is derived from adjacent tissues such as the cortical margin or septum and is mainly formed by Cajal-Retzius cells that are positive for reelin (in the case of the human cerebral cortex, a part of the reelin-positive cells can also be directly born from the cerebral cortical neuroepithelium). The remaining layers of the cortical plate have a characteristic pattern in which nerve cells are regularly born and arranged in time and space. This is called the inside-out model, where neurons in deeper layers are generated earlier from neural progenitor cells.

[0004] Unlike the generation of the cerebral cortex of mice, from which a large amount of information can be obtained, the generation of the human cerebral cortex cannot be understood in detail due to the limited use of human fetal brain tissue. So far, a three-dimensional culture method (serum-free floating culture of embryoid body-like aggregates with quickreaggregation, SFEBq) method) using mouse embryonic stem cells (ES cells) has been established, showing that the aggregate reproduces the initial process of the generation of the cerebral cortex. It is reported that the method can also be adapted to human induced pluripotent stem cells (iPS cells: induced pluripotent stem cells) (Patent Document 1).

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2015 / 076388 Summary of the invention

[0008] Problems to be solved by the invention

[0009] The object of the present invention is to provide a method for producing telencephalon marker-positive brain organoids.

[0010] Technical means of solving problems

[0011] The present invention includes the following embodiments.

[0012] [1] A method for producing brain organoids, comprising: culturing a neuroectoderm marker-positive cell aggregate in a culture medium containing an extracellular matrix at a concentration exceeding 10% by volume.

[0013] [2] The method for producing a brain organoid according to [1], wherein the concentration of the extracellular matrix in the culture medium is 20% to 50% by volume.

[0014] [3] The method for producing a brain organoid according to [1] or [2], wherein the culture medium further contains a Wnt signaling enhancing substance.

[0015] [4] The method for producing a brain organoid according to any one of [1] to [3], wherein the culture medium further contains a transforming growth factor β family signaling pathway inhibitory substance.

[0016] [5] A method for producing a brain organoid according to any one of [1] to [4], wherein the step is a step of culturing cell aggregates positive for neural ectoderm markers in a state where they are dispersed in a culture medium containing an extracellular matrix at a concentration exceeding 10% by volume without being inserted into an extracellular matrix.

[0017] [6] The method for producing a brain organoid according to any one of [1] to [5], further comprising a step of culturing in a culture medium substantially free of extracellular matrix after the step.

[0018] [7] The method for producing brain organoids according to [6], wherein the step of culturing in a culture medium substantially free of extracellular matrix is ​​performed by suspension culture.

[0019] [8] The method for producing a brain organoid according to [6] or [7], wherein the step of culturing in a culture medium substantially free of extracellular matrix is ​​carried out under high oxygen partial pressure conditions.

[0020] [9] The method for producing a brain organoid according to any one of [6] to [8], wherein the step of culturing in a culture medium substantially free of extracellular matrix is ​​performed while stirring.

[0021]

[10] The method for producing a brain organoid according to any one of [1] to [9], wherein the neuroectoderm marker-positive cell aggregates are derived from humans.

[0022]

[11] A brain organoid produced by the production method according to any one of [1] to

[10] .

[0023]

[12] The brain organoid according to

[11] , wherein the brain organoid comprises at least cells positive for telencephalon markers.

[0024]

[13] The brain organoid according to

[12] , wherein the brain organoid further comprises cells positive for telencephalic tissue markers.

[0025]

[14] The brain organoid according to

[13] , wherein the cells positive for the telencephalic tissue marker are at least one selected from the group consisting of cerebral cortex, basal ganglia, hippocampus and choroid plexus.

[0026]

[15] A kit for evaluating the efficacy of a test substance, comprising the brain organoid according to any one of

[11] to

[14] .

[0027]

[16] A method for evaluating the efficacy of a test substance, comprising: a step of bringing the test substance into contact with a brain organoid according to any one of

[11] to

[14] ; and a step of detecting the effect of the test substance on the brain organoid.

[0028]

[17] A therapeutic drug for a disease caused by a disorder of nervous system cells or nervous tissue, comprising the brain organoid according to any one of

[11] to

[14] .

[0029]

[18] A pharmaceutical composition for treating a disease caused by a disorder of nervous system cells or nervous tissue, comprising the brain organoid according to any one of

[11] to

[14] as an active ingredient.

[0030] The present invention may also include the following embodiments.

[0031] [P1] A method for producing a brain organoid, comprising culturing a neuroectoderm marker-positive cell aggregate in a first culture medium containing more than 10% by volume of an extracellular matrix component, thereby forming a brain organoid.

[0032] [P2] The method for producing a brain organoid according to [P1], wherein the concentration of the extracellular matrix component in the first culture medium is 30% to 50% by volume.

[0033] [P3] The method for producing a brain organoid according to [P1] or [P2], wherein the first culture medium further contains a Wnt signaling enhancing substance.

[0034] [P4] The method for producing a brain organoid according to any one of [P1] to [P3], wherein the first culture medium further contains a transforming growth factor (TGF)-β family signal transduction pathway inhibitor.

[0035] [P5] The method for producing a brain organoid according to any one of [P1] to [P4], further comprising culturing the brain organoid in suspension in a second culture medium.

[0036] [P6] The method for producing a brain organoid according to [P5], wherein the second culture medium does not substantially contain extracellular matrix components.

[0037] [P7] The method for producing a brain organoid according to [P5] or [P6], wherein the suspension culture is carried out under high oxygen partial pressure conditions.

[0038] [P8] The method for producing a brain organoid according to any one of [P5] to [P7], wherein the suspension culture is performed by stirring culture.

[0039] [P9] The method for producing a brain organoid according to any one of [P1] to [P8], wherein the neuroectoderm marker-positive cell aggregates are derived from humans.

[0040] [P10] The method for producing a brain organoid according to any one of [P1] to [P9], wherein the brain organoid comprises a forebrain, telencephalon, or telencephalon partial-like tissue.

[0041] [P11] The method for producing a brain organoid according to [P10], wherein the telencephalon-like tissue is a cerebral cortex, basal ganglia, hippocampus or choroid plexus.

[0042] [P12] A brain organoid produced by the production method according to any one of [P1] to [P11].

[0043] [P13] A kit for evaluating the toxicity and efficacy of a test substance, comprising the brain organoid described in [P12].

[0044] [P14] A method for evaluating the toxicity and efficacy of a test substance, comprising: bringing the test substance into contact with the brain organoid described in [P12]; and examining the effect of the test substance on the brain organoid.

[0045] [P15] A therapeutic drug for a disease caused by a disorder of nervous system cells or nervous tissue, comprising the brain organoid described in [P12].

[0046] [P16] A pharmaceutical composition for treating a disease caused by a disorder of nervous system cells or nervous tissue, comprising the brain organoid described in [P12] as an active ingredient.

[0047] Effects of the Invention

[0048] According to the present invention, a method for producing telencephalon marker-positive cerebral organoids can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a microscope photograph showing the results of Experimental Example 1.

[0050] Figure 2 This is a microscope photograph showing the results of Experimental Example 1.

[0051] Figure 3 This is a microscopic photograph showing the results of bright field observation of cell aggregates on the 14th day of culture in Experimental Example 3.

[0052] Figure 4 This is a fluorescence microscope photograph showing the results of immunostaining in Experimental Example 3.

[0053] Figure 5 This is a fluorescence microscope photograph showing the results of immunostaining in Experimental Example 3.

[0054] Figure 6 These are representative microscopic photographs showing the results of bright field observation of cell aggregates containing different basal culture media in Experimental Examples 3 and 4.

[0055] Figure 7 Yes Figure 6 The results are numerically graphed.

[0056] Figure 8 This is a microscopic photograph showing the results of bright field observation of cell aggregates on the 40th day of culture in Experimental Example 5.

[0057] Fig. 9 This is a fluorescence micrograph showing the results of immunostaining of brain organoids cultured on day 40 in Experimental Example 5 (Matrigel concentration: 50% by volume).

[0058] Fig.10 This is a fluorescence micrograph showing the results of immunostaining of brain organoids cultured on day 40 in Experimental Example 5 (Matrigel concentration: 30% by volume).

[0059] Fig.11 This is a fluorescence micrograph showing the results of immunostaining of brain organoids cultured on day 40 in Experimental Example 5 (Matrigel concentration: 10% by volume).

[0060] Fig.12 This is a fluorescence micrograph showing the results of immunostaining of brain organoids cultured on day 40 in Experimental Example 5 (Matrigel concentration: 2 vol%).

[0061] Fig.13 This is a fluorescence micrograph showing the results of immunostaining of brain organoids cultured on day 70 in Experimental Example 5 (Matrigel concentration: 50% by volume).

[0062] Fig.14 This is a fluorescence micrograph showing the results of immunostaining of brain organoids cultured on day 70 in Experimental Example 5 (Matrigel concentration: 30% by volume).

[0063] Fig.15 This is a fluorescence micrograph showing the results of immunostaining of brain organoids cultured on day 70 in Experimental Example 5 (Matrigel concentration: 10% by volume). DETAILED DESCRIPTION

[0064] Hereinafter, the present invention will be described in more detail with reference to embodiments, but the present invention is not limited to the following embodiments at all.

[0065] [Expression of gene name and protein name]

[0066] In this specification, human genes and human proteins are represented by capital letters. In addition, mouse genes are represented by capital letters representing the initial characters and lowercase letters representing the characters after the initial characters. In addition, mouse proteins are represented by capital letters. However, depending on the situation, human genes, mouse genes, genes of other types, human proteins, mouse proteins, and proteins of other types are sometimes described without strict distinction.

[0067] In this specification, the expression showing a numerical range such as "A to B" has the same meaning as "A or more and B or less", and it is assumed that A and B are included in the numerical range.

[0068] Unless otherwise specified, each substance exemplified in the present specification, for example, a substance contained in a culture medium or a substance used in each step may be used alone or in combination of two or more.

[0069] In the present specification, the term "medium containing substance X" or "in the presence of substance X" refers to a medium to which an exogenous substance X is added, a medium containing an exogenous substance X, or in the presence of an exogenous substance X. That is, when cells or tissues present in the medium express, secrete, or produce the substance X endogenously, the endogenous substance X is distinguished from the exogenous substance X, and a medium that does not contain an exogenous substance X does not fall within the scope of "medium containing substance X" even if it contains endogenous substance X.

[0070] In this specification, the so-called "cell aggregate" refers to a mass of cells adhered to each other. Cell aggregates, embryoid bodies, spheres, spheroids and brain organoids are also included in cell aggregates. The cells of the cell aggregates adhere to each other on their surfaces. So-called surface adhesion refers to the ratio of the surface area of ​​a certain cell to the surface adhesion of another cell, for example, more than 1%, preferably more than 3%, and more preferably more than 5%. The surface of the cell can be observed by staining caused by a reagent (such as DiI) for membrane staining or immunostaining of cell adhesion factors (for example, E-cadherin or N-cadherin). In addition, the adhesion area occupied in the membrane can also be quantified. There is a situation in which the cells form cell-cell junctions or adhesion junctions and other cell adhesions in part or all of the cell aggregates.

[0071] [Method for producing brain organoids]

[0072] Regarding the manufacture of brain organoids, it is widely known that a cell aggregate positive for neuroectoderm markers is inserted into a gelatinous matrix gel particle for suspension culture. However, the gelatinous matrix gel particles are easily broken when inserted, so in order not to break, skilled techniques are required, and there is a problem that it is not suitable for mass production using automated devices. In addition, in the case where it is impossible to insert into the center of the gelatinous matrix gel particles, in the suspension culture, the cell aggregates are detached from the gelatinous matrix gel particles and brain organoids cannot be formed. In addition, since the formation of solid matrix gel particles or the insertion of the cell aggregates takes time, a method for making brain organoids more easily is sought. Therefore, the present embodiment provides a method for producing brain organoids, which includes a step of culturing neuroectodermal marker-positive cell aggregates in a culture medium (also referred to as a "first culture medium") containing an extracellular matrix at a concentration exceeding 10% by volume, preferably a step of culturing neuroectodermal marker-positive cell aggregates in a state of being dispersed in a first culture medium containing an extracellular matrix at a concentration exceeding 10% by volume without being inserted into the extracellular matrix, and as a result, brain organoids can be formed. As described later in the examples, according to the production method of the present embodiment, brain organoids can be easily produced.

[0073] In the present embodiment, the brain organoid refers to a cell aggregate containing at least cells that are positive for telencephalon markers. As telencephalon markers, FOXG1 (also called BF1), SIX3, etc. can be listed, but are not limited to these. Furthermore, FOXG1 and SIX3 are also forebrain markers. The brain organoid of the present embodiment contains cells that express at least one telencephalon marker. In a preferred form, the brain organoid is a cell aggregate containing FOXG1-positive cells. Slices of brain organoids can also be made, and immunostaining using 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole, DAPI) and anti-FOXG1 antibodies can be used to calculate the proportion of FOXG1-positive cells in the brain organoids. Preferably, the brain organoid is more than 50%, preferably more than 70%, and more preferably more than 90% of the number of cells contained in the brain organoid that are positive for telencephalon markers.

[0074] The brain organoids manufactured by the manufacturing method of the present embodiment preferably contain at least one selected from cells positive for forebrain markers and cells positive for telencephalic part-like tissue markers in addition to cells positive for telencephalic markers. Cells positive for telencephalic part-like tissue markers include cells positive for telencephalic part-like tissue markers such as the cerebral cortex, basal ganglia, hippocampus and choroid plexus. In addition to cells positive for telencephalic markers, brain organoids can also be morphologically determined to determine whether they contain cells positive for forebrain markers and cells positive for telencephalic part-like tissue markers. Alternatively, the expression of characteristic marker genes or marker proteins can be measured for each cell to determine the expression.

[0075] As forebrain markers, for example, FOXG1, SIX3, etc. can be cited. In addition, as cerebral cortex markers, for example, PAX6, a neural stem cell-neural precursor cell marker, CTIP2, a layer V marker of the cerebral cortex, SATB2, etc. can be cited. In addition, as basal ganglia markers, for example, NKX2.1, GSH2, etc. can be cited. In addition, as hippocampal markers, for example, KA1, ZBTB2, etc. can be cited. In addition, as choroid plexus markers, for example, TTR, LMX1A, etc. can be cited.

[0076] The neuroectoderm marker-positive cell aggregates are preferably induced from stem cells, and the stem cells are preferably pluripotent stem cells or stem cells induced from pluripotent stem cells.

[0077] In this embodiment, the so-called "iPS cell" refers to a cell in which pluripotency is induced by reprogramming somatic cells using existing methods, etc. Specifically, it can be mentioned that somatic cells differentiated from fibroblasts, peripheral blood mononuclear cells, or lymphocytes are reprogrammed by expressing any one of a combination of multiple genes selected from a reprogramming gene group including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, Lin28, Esrrb, etc., thereby inducing pluripotent cells.

[0078] Preferred combinations of initialization gene groups include, for example: (1) a combination of Oct3 / 4, Sox2, Klf4 and Myc (c-Myc or L-Myc), or (2) a combination of Oct3 / 4, Sox2, Klf4, Lin28 and L-Myc (see Okita K., et al., An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells, Stem Cells, 31(3), 458-466, 2013).

[0079] iPS cells were established by Yamanaka et al. using mouse cells in 2006 (Takahashi K. and Yamanaka S., Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell, 126(4), 663-676, 2006). In 2007, iPS cells were established using human fibroblasts, which have pluripotency and self-replication ability like ES cells (for example, see K. Takahashi K., et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell, 131(5), 861-872, 2007; J. Yu J., et al., Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells, Science, 318(5858), 1917-1920, 2007; M. Nakagawa M., et al., Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts). fibroblasts), Nature Biotechnology (Nat Biotechnol), 26(1), 101-106, 2008, etc.).

[0080] In addition to methods for producing iPS cells from somatic cells by initialization using gene expression, iPS cells can also be induced from somatic cells by adding compounds, etc. (Hou P., et al., Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds, Science, 341(6146), 651-654, 2013).

[0081] In this embodiment, iPS cells can also be used after straining. For example, 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, 1231A3 cells and other iPS cell lines established by Kyoto University can be obtained from Kyoto University and iPS Academia Japan Co., Ltd. Furthermore, as iPS cells after straining, for example, PCPhiPS771 sold by ReproCELL Co., Ltd. can be listed. In addition, as iPS cells after straining, XFiPS-F44-3F-2 established by the Institute of Health Carlos III can be listed.

[0082] Examples of somatic cells used in producing iPS cells include tissue-derived fibroblasts, blood cell lineage cells (eg, peripheral blood mononuclear cells or T cells), hepatocytes, pancreatic cells, intestinal epithelial cells, and smooth muscle cells.

[0083] In the case of initialization by expression of several genes when manufacturing iPS cells, the method for expressing genes is not particularly limited. As the method, there can be cited: infection method using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors and adeno-associated virus vectors), gene introduction method using plasmid vectors (e.g., plasmid vectors and episomal vectors) (e.g., calcium phosphate method, lipofection method, recombinant human fibrin fragment (RetroNectin) method and electroporation method), gene introduction method using ribonucleic acid (RNA) vectors (e.g., calcium phosphate method, lipofection method and electroporation method), and direct injection method of protein or mRNA, etc.

[0084] The pluripotent stem cells used in the production method of the present embodiment are preferably iPS cells.

[0085] Genetically modified pluripotent stem cells can be easily prepared by transfecting artificial nucleases such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) into pluripotent stem cells. Artificial nucleases introduce double-stranded deoxyribonucleic acid (DNA) breaks (DSB) into target genes, and introduce indel mutations through non-homologous end joining (NHEJ), which is one of the double-strand break (DSB) repair mechanisms, thereby preparing a cell line in which the target gene is destroyed and the gene is modified, that is, a gene knockout cell line. Compared with previous gene modification technologies, gene knockout cell lines can be produced in a short time, at a low cost and efficiently, and therefore it is widely used as a technology for producing gene-modified cells (for example, see Hsu PD et al., Development and Applications of CRISPR-Cas9 for Genome Engineering, Cell, 157(6), 1262-1278, 2014, etc.).

[0086] Attempts have also been made to knock in genes such as green fluorescent protein (GFP) into target genomic regions (or genes) using artificial nucleases. A donor plasmid with about 500bp-1k bp of the same sequence in the target genomic region at both ends of the gene knock-in sequence of GFP, etc. is used. By introducing the donor plasmid into pluripotent stem cells together with the artificial nuclease, the artificial nuclease introduces DSB into the target sequence, using the homologous sequence of the donor plasmid, and knocking genes such as GFP into the target sequence through homologous recombination (HR: homologous recombination), which is another DSB repair mechanism.

[0087] In addition, as a method that does not use a donor plasmid, by using single-stranded DNA (ssODN: single-stranded oligodeoxynucleotides), a single base replacement of the target gene or the introduction of a short DNA sequence of less than tens of bp such as His tag or LoxP can be achieved. By artificially synthesizing single-stranded oligodeoxynucleotides (ssODN) containing 40bp-60bp homologous sequences at both ends by clamping the base sequence to be introduced, and introducing them into fertilized eggs together with artificial nucleases, using single-strand annealing (SSA) as a highly efficient DSB repair mechanism, gene knock-in cell lines can be simply and efficiently produced.

[0088] When utilizing donor plasmid to carry out gene knock-in, it is necessary to attach the homologous sequence of the target genome region to the plasmid containing the gene to be knocked-in. Utilize polymerase chain reaction (Polymerase Chain Reaction, PCR) etc. to increase homologous sequence, connect and utilize Escherichia coli to clone and make donor plasmid. In addition, as the screening method of gene knock-in cell strain, methods such as positive selection, promoter selection, negative selection, polyadenylic acid (polyA) selection can be used. As the method for selecting target homologous recombinant from the cell strain screened, Southern hybridization method (Southern hybridization method) or PCR method etc. for genomic DNA can be enumerated.

[0089] The pluripotent stem cells are preferably pluripotent stem cells derived from mammals, and the mammals are preferably rodents and primates, more preferably primates, and the primates are preferably humans.

[0090] “Mammals” include rodents, ungulates, carnivores, primates, etc. Rodents include mice, rats, hamsters, guinea pigs, etc. Ungulates include pigs, cattle, goats, horses, sheep, etc. Carnivores include dogs, cats, etc. “Primates” refers to mammals belonging to the order Primates. Examples of primates include the suborder Prosimians such as lemurs, slow lorises, and tree shrews; and the suborder Anthropoids such as monkeys, apes, and humans.

[0091] Proliferation of pluripotent stem cells

[0092] In the method for producing brain organoids of the present embodiment, pluripotent stem cells may be proliferated for use. When proliferating pluripotent stem cells, the proliferating may be performed in the presence of feeder cells or in the absence of feeder cells (feeder-free).

[0093] When iPS cells are propagated in the presence of feeder cells, iPS cells can be propagated in the presence of undifferentiated maintenance factors using existing methods. As a proliferation medium used when iPS cells are propagated in the absence of feeder cells, for example, an existing iPS cell maintenance medium or a medium for establishing iPS cells in a feeder-free layer can be used. As a feeder-free medium for establishing iPS cells in a feeder-free layer, Essential 8 medium or TeSR medium, mTeSR medium and mTeSR-E8 medium (all manufactured by STEMCELL Technologies) and StemFit medium (manufactured by Ajinomoto Co., Ltd.) can be listed.

[0094] If the culture vessel used for the proliferation of pluripotent stem cells is a culture vessel capable of "adhesion culture", the culture vessel can be appropriately selected according to the scale, culture conditions and culture period of the culture. Examples of such culture vessels include flasks, tissue culture flasks, culture dishes (plates), tissue culture plates, multilayer plates, microplates, microwell plates, multilayer plates, multi-well plates, chamber slides, bowls, tubes, trays, culture bags, microcarriers, beads, stacked plates, spinner bottles or roller bottles.

[0095] In order to be able to carry out adhesion culture, these culture vessels are preferably cell-adhesive. As a cell-adhesive culture vessel, a culture vessel in which the surface of the culture vessel is artificially treated for the purpose of improving adhesion to the cells can be cited. Specifically, a culture vessel with surface processing or a culture vessel coated with a coating agent inside can be cited. As a coating agent, for example, laminin (including laminin α5β1γ1 (hereinafter, sometimes referred to as "laminin 511"), laminin α1β1γ1 (hereinafter, sometimes referred to as "laminin 111"), laminin fragments (laminin 511E8, etc.), entactin, collagen, gelatin, vitronectin (Vitronectin), Synthemax (Corning), extracellular matrix such as matrigel, etc.; polymers such as polylysine and polyornithine, etc. As a surface-processed culture vessel, a culture container with surface processing such as positive charge treatment can be cited.

[0096] The proliferation of pluripotent stem cells is preferably carried out by adding a Rho-associated coiled-coil forming kinase (ROCK: Rho-associated coiled-coil forming kinase / Rho binding kinase) inhibitor to the culture medium for culturing. By adding a ROCK inhibitor, cell death can be suppressed when the pluripotent stem cells, especially human iPS cells, are dispersed. In the case of peeling cells, it is preferred to add a ROCK inhibitor appropriately after peeling and culture. In the case of adding a ROCK inhibitor, it is sufficient to culture for at least 1 day after adding it, and it is preferably removed after culturing. In addition, the proliferation of pluripotent stem cells can also be cultured in a culture medium containing a ROCK inhibitor from more than 1 day before the pluripotent stem cells are peeled from the culture medium, preferably 1 day before.

[0097] The ROCK inhibitor is not particularly limited as long as it is an inhibitor that can inhibit the function of Rho kinase (ROCK), and examples thereof include: Y-27632 (for example, see T. Ishizaki T., et al., Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases, Molecular Pharmacology, Mol Pharmacol, 57(5), 976-983, 2000; S. Narumiya S., et al., Use and properties of ROCK-specific inhibitor Y-27632, Methods in Enzymology, Methods Enzymol) 325, 273-284, 2000, etc.), Fasudil / HA1077 (for example, refer to M. Uehata M., et al., Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension, Nature, 389(6654), 990-994, 1997), H-1152 (for example, refer to Y. Sasaki Y., et al., The novel and specific Rho kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probe molecule for Rho kinase-related pathways inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathway), Pharmacology & Therapeutics, Pharmacol Ther., 93(2-3), 225-232, 2002), Wf-536 (for example, refer to M.Nakajima M., et al., Effect of Wf-536, a novel ROCK inhibitor, against metastasis of B16 melanoma, Cancer Chemotherapy and Pharmacology, 52(4), 319-324, 2003) and inducers thereof, as well as antisense nucleic acids, RNA interference-inducing nucleic acids (e.g., siRNA), dominant negative mutants and expression vectors thereof for ROCK. .

[0098] 《Cell aggregates positive for neuroectoderm markers》

[0099] Then, the proliferated multifunctional stem cells are cultured to produce neuroectoderm marker-positive cell aggregates.

[0100] The medium for culturing neuroectoderm marker-positive cell aggregates (hereinafter, sometimes referred to as "aggregation medium") is described below.

[0101] As the basal medium for the aggregation medium, a medium commonly used for culturing animal cells can be used. As the basal medium, for example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, or mixed mediums thereof, etc., can be used for culturing animal cells.

[0102] In the culture of neuroectoderm marker-positive cell aggregates, serum-free medium can also be used. So-called "serum-free medium" refers to a medium that does not contain unadjusted or unpurified serum. As long as it does not contain unadjusted or unpurified serum, a medium mixed with purified blood-derived components or animal tissue-derived components (e.g., growth factors) is also included in the serum-free medium. Serum-free medium can also contain serum substitutes. As serum substitutes, for example, serum substitutes that are suitable for containing albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or thioglycerol, equivalents of these, etc. can be listed.

[0103] Serum substitutes can be prepared, for example, by the method described in WO98 / 30679. As serum substitutes, commercial products can also be used. As commercially available serum substitutes, for example, Knockout (TM) Serum Replacement (manufactured by Thermo Fisher Scientific: sometimes referred to as "KSR"), Chemically-defined Lipid Concentrated (manufactured by Thermo Fisher Scientific), Glutamax (TM) (manufactured by Thermo Fisher Scientific), B27 (manufactured by Thermo Fisher Scientific), N2 supplement (manufactured by Thermo Fisher Scientific), 1× non-essential amino acids (manufactured by Thermo Fisher Scientific), etc.

[0104] The aggregation medium preferably contains a bone morphogenetic protein (BMP) signal transduction pathway inhibitor or a transforming growth factor-β (TGF-β) family signal transduction pathway inhibitor.

[0105] The BMP signal transduction pathway inhibitory substance is not particularly limited as long as it is a substance that inhibits the signal transduction pathway caused by BMP, and may be any of nucleic acids, proteins, and low molecular weight organic compounds. Here, examples of BMP include BMP2, BMP4, BMP7, and Growth Differentiation Factor (GDF) 7.

[0106] As BMP signal transduction pathway inhibitors, for example, substances that directly act on BMP (Noggin protein, antibodies, aptamers, etc.), substances that inhibit the expression of genes encoding BMP (antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of BMP receptors (Bone Morphogenetic Protein Receptor, BMPR) and BMP, and substances that inhibit physiological activity caused by signal transduction based on BMP receptors. As BMPR, ALK2 and ALK3 can be listed.

[0107] As BMP signal transduction pathway inhibitors, LDN193189 and dorsomorphin can be cited. LDN193189 (4-[6-(4-piperazine-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) is a BMPR (ALK2 / 3) inhibitor (hereinafter referred to as a BMPR inhibitor), which is usually sold in the form of hydrochloride. In addition, proteins known as BMP signal transduction pathway inhibitors (such as chordin or noggin) can also be used.

[0108] The BMP signal transduction pathway inhibitory substance is preferably dorsomorphin.

[0109] The final concentration of the BMP signaling pathway inhibitor contained in the aggregation medium is preferably 0.5 μM to 10 μM, more preferably 0.75 μM to 5 μM, and even more preferably 1 μM to 3 μM. When it is within the above numerical range, the neurons forming the layer structure are spatially and regularly arranged in the brain organoid, which is preferred.

[0110] The TGF-β family signal transduction pathway inhibitory substance refers to a substance that inhibits the TGF-β family signal transduction pathway, that is, the signal transduction pathway mediated by the Smad family, and specific examples thereof include TGF-β signal transduction pathway inhibitory substances and Nodal / Activin signal transduction pathway inhibitory substances.

[0111] The TGF-β signal transduction pathway inhibitory substance is not particularly limited as long as it is a substance that inhibits the signal transduction pathway caused by TGF-β, and may be any of nucleic acid, protein, and low molecular weight organic compound.

[0112] Examples of TGF-β signal transduction pathway inhibitory substances include substances that directly act on TGF-β (e.g., proteins, antibodies, aptamers, etc.), substances that inhibit the expression of genes encoding TGF-β (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of TGF-β receptors to TGF-β, and substances that inhibit physiological activity caused by signal transduction based on TGF-β receptors (e.g., TGF-β receptor inhibitors, Smad inhibitors, etc.).

[0113] As for proteins known as TGF-β signal transduction pathway inhibitors, Lefty and the like can be cited. As TGF-β signal transduction pathway inhibitors, compounds known to those skilled in the art can be used, and specifically, SB431542, LY-364947, SB-505124, and A-83-01 and the like can be cited. Here, SB431542 (4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide) and A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-thiocarboxamide) are compounds known as inhibitors of TGF-β receptor (ALK5) and activin receptor (ALK4 / 7) (i.e., TGF-βR inhibitors). The TGF-β signaling pathway inhibitory substance is preferably SB431542 or A-83-01. A-83-01 is preferred because it facilitates the advancement of differentiation into the initial telencephalon.

[0114] The final concentration of the TGF-β signaling pathway inhibitor contained in the aggregation medium is preferably 0.5 μM to 10 μM, more preferably 0.75 μM to 5 μM, and even more preferably 1 μM to 3 μM.

[0115] Regarding the aggregation medium, when the BMP signal transduction pathway inhibitor is dorsomorphin and the TGF-β signal transduction pathway inhibitor is A-83-01, the induction period from pluripotent stem cells to neuroectoderm marker-positive cell aggregates is preferably 3 to 14 days, more preferably 4 to 12 days, and further preferably 5 to 10 days. When it is within the range of the numerical value, the induction efficiency of brain cells containing neuroepithelial cells is improved, so it is preferred.

[0116] As a culture method utilizing aggregation culture medium, suspension culture is preferably performed while stirring aggregation culture medium. Furthermore, so-called suspension culture refers to a method for cultivating and performing the culture while maintaining a state in which a cell aggregate is suspended in a culture fluid. Suspension culture is performed under the condition that cells or cell aggregates are not adhered to culture equipment, etc., and the culture (adhesion culture or adhesion culture method) performed under the condition that adheres to culture equipment, etc. is not included in the scope of suspension culture. In the case described, so-called cell adhesion refers to a firm cell-substratum junction that can be formed between cells or cell aggregates and culture equipment. In more detail, so-called suspension culture refers to a culture under the condition that a firm cell-substratum junction is not formed between cells or cell aggregates and culture equipment, etc., and so-called adhesion culture refers to a culture under the condition that a firm cell-substratum junction is formed between cells or cell aggregates and culture equipment, etc.

[0117] When culturing pluripotent stem cells using aggregation medium, pluripotent stem cells can be dispersed in advance. By dispersing pluripotent stem cells, cell aggregates positive for neuroectoderm markers with excellent uniformity can be obtained. So-called dispersion refers to separation of cells or tissues into small cell clusters (more than 2 cells, less than 100 cells, preferably less than 50 cells) or single cells by dispersion treatments such as enzyme treatment or physical treatment. So-called a certain number of dispersed cells refers to a collection of a certain number of cell clusters or single cells. As a method for dispersing pluripotent stem cells, for example, mechanical dispersion treatment, cell dispersion liquid treatment and cell protective agent addition treatment can be listed. These treatments can also be combined for carrying out. It is preferred to carry out cell dispersion liquid treatment, followed by mechanical dispersion treatment. As a method for mechanical dispersion treatment, pipetting treatment or scraping operation using a scraper can be listed.

[0118] As the cell dispersion liquid used in the cell dispersion liquid treatment, for example, there can be mentioned: a solution containing any of enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNA enzyme and papain, or a chelating agent such as ethylenediaminetetraacetic acid. Commercially available cell dispersion liquids, such as TrypLE Select (manufactured by Thermo Fisher Scientific) or TrypLE Express (manufactured by Thermo Fisher Scientific) can also be used.

[0119] When dispersing pluripotent stem cells, cell death of pluripotent stem cells can be suppressed by treating them with a cytoprotectant. Examples of the cytoprotectant used in the cytoprotectant treatment include substances acting on the fibroblast growth factor (FGF) signal transduction pathway, heparin, substances acting on the insulin-like growth factor (IGF) signal transduction pathway, serum, and serum substitutes.

[0120] In order to inhibit cell death induced by dispersion (especially cell death of human pluripotent stem cells), ROCK inhibitors or myosin inhibitors can be added during dispersion. As ROCK inhibitors, Y-27632, Fasudil (HA1077), H-1152, etc. can be listed. As myosin inhibitors, Blebbistatin can be listed. As preferred cell protectants, ROCK inhibitors can be listed.

[0121] In the culture of neuroectoderm marker-positive cell aggregates, it is preferred that the dispersed pluripotent stem cells are quickly taken to a narrow space for culture. If cultured in the manner described above, an epithelial-like structure can be reproducibly formed in the brain organoids induced by differentiation of the formed cell aggregates. As a culture plate with a narrow space, for example, a plate with a narrow hole (for example, the bottom area of ​​the hole is 0.1 cm in terms of flat bottom conversion) can be cited. 2 ~2.0cm 2 Plates on the left and right), microwells and small centrifuge tubes.

[0122] Examples of plates having narrow holes include a 24-well plate (area 1.88 cm in terms of flat bottom conversion). 2 ), 48-well plate (the area is calculated as 1.0 cm based on the flat bottom conversion 2 ), 96-well plate (the area is 0.3cm when converted to a flat bottom 2 About 6 mm to 8 mm in inner diameter), 384-well plate. Of these, 96-well plate is preferred.

[0123] Regarding the shape of the plate with narrow holes, as the shape of the bottom surface when the hole is observed from above, polygons, rectangles, ellipses, and perfect circles can be listed, and preferably perfect circles can be listed. Regarding the shape of the plate with narrow holes, as the shape of the bottom surface when the hole is observed from the side, it can be a flat bottom structure, or it can be a concave structure with a high periphery and a low inner concave portion. As the shape of the bottom surface, for example, a U-shaped bottom, a V-shaped bottom, and an M-shaped bottom can be listed, preferably a U-shaped bottom or a V-shaped bottom can be listed, and further preferably a V-shaped bottom can be listed. As a plate with narrow holes, a plate having a concave or concave bottom surface on a cell culture dish (for example, a 60mm to 150mm dish, a culture bottle) can also be used. As the bottom surface of a plate with narrow holes, it is preferred to use a non-cell-adhesive bottom surface, preferably a non-cell-adhesive coated bottom surface.

[0124] The formation of cell aggregates and the uniformity of cell aggregates can be judged based on the size and number of cells in the cell aggregates, the macroscopic morphology, the microscopic morphology obtained based on tissue staining analysis and its uniformity, etc. In addition, the formation of epithelial-like structures in cell aggregates and the uniformity of epithelial-like structures can be judged based on the macroscopic morphology of cell aggregates, the microscopic morphology obtained based on tissue staining analysis and its uniformity, the expression of differentiation and undifferentiation markers and their uniformity, the expression control of differentiation markers and their synchronization, the reproducibility of differentiation efficiency between cell aggregates, etc.

[0125] The formation of a neuroectoderm marker-positive cell aggregate can be determined by the neuroectoderm marker being positive. Examples of neuroectoderm markers include nestin and βIII-tubulin.

[0126] 《Cultivation of neuroectoderm marker-positive cell aggregates》

[0127] The manufacturing method of this embodiment includes a step of culturing a neuroectodermal marker-positive cell aggregate in a first culture medium containing an extracellular matrix at a concentration exceeding 10% by volume. Preferably, the step includes culturing a neuroectodermal marker-positive cell aggregate in a state of being dispersed in a first culture medium containing an extracellular matrix at a concentration exceeding 10% by volume without being inserted into the extracellular matrix. Brain organoids can be formed by culturing neuroectodermal marker-positive cell aggregates in a first culture medium.

[0128] The so-called "extracellular matrix component" refers to various components usually found in the extracellular matrix. In the present embodiment, it is preferred to use a basement membrane component. As a basement membrane component, for example, it can be listed: type IV collagen, laminin, heparan sulfate proteoglycan, entactin and nidogen. As an extracellular matrix component added to the culture medium, commercial products can be used, for example, matrigel (manufactured by Corning), human laminin (manufactured by Sigma), etc. Matrigel is a basement membrane preparation derived from Engelbreth Holm Swarm (EHS) mouse sarcoma. The main components of matrigel are type IV collagen, laminin, heparan sulfate proteoglycan, entactin and nidogen, in addition to these, it also contains TGF-β, FGF, tissue plasminogen activator, and growth factors naturally produced by EHS tumors. Regarding the growth factor reduced products of Matrigel, the concentration of growth factors is lower than that of ordinary Matrigel, and its standard concentration is Epidermal Growth Factor (EGF) < 0.5ng / mL, Nerve Growth Factor (NGF) < 0.2ng / mL, Platelet-Derived Growth Factor (PDGF) < 5pg / mL, IGF-1 ≤ 5ng / mL, TGF-β ≤ 1.7ng / mL. As the extracellular matrix component, it is preferred to use the growth factor reduced products of Matrigel.

[0129] The concentration of the extracellular matrix component in the first culture medium contributes to the formation of a neural tube-like structure. In addition, in the construction of a cerebral cortex-like layered structure in long-term culture, it also affects the timing of the formation of upper layer neural cells or the thickness of the upper layer.

[0130] When Matrigel is used as an extracellular matrix component, the Matrigel and the first culture medium are mixed by pipetting on an ice bath. When the pipetting is performed more than 15 times, the Matrigel is dispersed in the culture medium. Dispersion refers to a state in which no clumps of the extracellular matrix can be visually observed in the culture medium. When the Matrigel cannot be dispersed in the culture medium, aggregation between the obtained brain organoids occurs.

[0131] The volume obtained by adding the first culture medium and the extracellular matrix is ​​set to 100 volume %, and the upper limit of the concentration of the extracellular matrix is ​​preferably 70 volume %, more preferably 60 volume %, and further preferably 50 volume %. On the other hand, the lower limit of the concentration of the extracellular matrix is ​​preferably 10 volume %, more preferably 20 volume %, and further preferably 30 volume %. When it is below 10 volume %, in the case of immunostaining of the cerebral organoid slices of the 70th day of cultivation, the expression of the II / III layer marker of the cerebral cortex, i.e. SATB2, is not substantially observed. In addition, when it is within the range of 30 volume % to 50 volume %, in the cerebral organoid slices of the 70th day of cultivation, the expression of the II / III layer marker of the cerebral cortex, i.e. SATB2, can be clearly observed. In addition, when it is within the range of 30 volume % to 50 volume %, the period until the expression of SATB2 is short, so it is preferred.

[0132] The first culture medium can be prepared by using a culture medium for culturing animal cells as a basal culture medium. As the basal culture medium, for example, BME culture medium, BGJb culture medium, CMRL 1066 culture medium, Glasgow MEM culture medium, Improved MEM Zinc Option culture medium, IMDM culture medium, Medium 199 culture medium, Eagle MEM culture medium, αMEM culture medium, DMEM culture medium, Ham's medium, Ham's F-12 culture medium, RPMI 1640 culture medium, Fischer's medium, mixed culture medium thereof, and other culture mediums that can be used for culturing animal cells are not particularly limited.

[0133] The first culture medium may contain N2 supplement (product name of Thermo Fisher Scientific Inc.), chemically defined lipid concentrate, serum, serum substitute, heparin, etc. The serum substitute is the same as the serum substitute described above.

[0134] The manufacturing method of this embodiment only needs to include the step of culturing the neuroectoderm marker-positive cell aggregates in the first culture medium, and may also include other steps that do not use the first culture medium. For example, after the step of culturing the neuroectoderm marker-positive cell aggregates using a culture medium other than the first culture medium (a culture medium containing N2 supplements and chemically defined lipid concentrates (Chemically Defined Lipid Concentrate) and not containing serum, heparin, and extracellular matrix components), the step of switching to the first culture medium from midway (for example, after the stage of forming a hemispherical neural tube-like structure with a ventricle-like cavity in the Foxg1-positive cell aggregates) and culturing in the first culture medium is performed.

[0135] The first culture medium may also contain a Wnt signal enhancer. Examples of Wnt signal enhancers include Wnt proteins, glycogen synthase kinase-3β (GSK-3β) inhibitors, Wnt agonists such as R-Spondin, and Dkk (inhibitor of Wnt inhibitory proteins). Among these, Wnt signal enhancers are preferably Wnt proteins and GSK-3β inhibitors.

[0136] Examples of GSK-3β inhibitors include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), Kenpaullone, and 6-Bromoindirubin-3'-oxime (6-Bromoindirubin-3'-oxime, BIO). Among these, CHIR99021 is preferred as a GSK-3β inhibitor.

[0137] As Wnt proteins, Wnt proteins derived from various organisms can be used. Among the Wnt proteins derived from organisms, Wnt proteins derived from mammals are preferred. Examples of Wnt proteins from mammals include: Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. Among them, Wnt3a is preferred, and Wnt3a is more preferably a complex with α-albumin.

[0138] When CHIR99021 is used as a Wnt signal enhancer, the final concentration of CHIR99021 contained in the culture medium is preferably 0.1 μM to 30 μM, more preferably 0.5 μM to 10 μM, and even more preferably 1 μM to 5 μM. When it is within the above numerical range, the induction efficiency of brain cells including neuroepithelial cells is improved, which is preferred.

[0139] When Wnt3a is used as a Wnt signal enhancer, the final concentration of Wnt3a contained in the culture medium is preferably 0.1 ng / mL to 20 ng / mL, more preferably 0.5 ng / mL to 15 ng / mL, and even more preferably 1 ng / mL to 10 ng / mL. When it is within the above numerical range, the induction efficiency of brain cells including neuroepithelial cells is improved, which is preferred.

[0140] The first culture medium may further contain a TGF-β family signal transduction pathway inhibitory substance. The details of the TGF-β family signal transduction pathway inhibitory substance are the same as those of the TGF-β family signal transduction pathway inhibitory substance. When SB431542 is used as the TGF-β family signal transduction pathway inhibitory substance, the final concentration in the culture medium is preferably 0.5 μM to 10 μM, more preferably 0.75 μM to 5 μM, and further preferably 1 μM to 3 μM.

[0141] The culturing of the neuroectoderm marker-positive cell aggregates in the first culture medium is performed for a period required to obtain telencephalon marker-positive cerebral organoids. The telencephalon marker is the same as the telencephalon marker described above.

[0142] For example, when the first culture medium contains a Wnt signal enhancer and a TGF-β family signal transduction pathway inhibitor, the Wnt signal enhancer is Wnt3a and CHIR99021, and the TGF-β family signal transduction pathway inhibitor is SB431542, the culture period of the neuroectoderm marker-positive cell aggregates in the first culture medium is preferably 3 to 14 days, more preferably 4 to 12 days, further preferably 5 to 10 days, and particularly preferably 6 to 8 days.

[0143] "Step of culturing in a medium substantially free of extracellular matrix components"

[0144] The manufacturing method of the present embodiment may include a process of culturing in a culture medium (hereinafter, also referred to as "the second culture medium") that is substantially free of extracellular matrix components. Specifically, it is preferred to further include the following process: the cell aggregates obtained by culturing the cell aggregates positive for neuroectoderm markers in the first culture medium are cultured in a culture medium that is substantially free of extracellular matrix components. By including the process, cell aggregates (brain organoids) containing cells positive for telencephalic markers, cells positive for forebrain markers, and cells positive for telencephalic part-like tissue markers can be obtained. The culture is preferably carried out by suspension culture.

[0145] In the cell aggregates in suspension culture, cells adhere to the cell surface. In the cell aggregates in suspension culture, no cell-matrix indirect bonding is formed between the culture equipment, etc., or even if it is formed, its contribution is small. In a part of the form, in the cell aggregates in suspension culture, endogenous cell-matrix indirect bonding is present in the inside of the aggregate, but no cell-matrix indirect bonding is formed between the culture equipment, etc., or even if it is formed, its contribution is small.

[0146] The culture vessel used when performing suspension culture is not particularly limited if it is a culture vessel that can perform "suspension culture", and can be appropriately determined by those skilled in the art. Examples of such culture vessels include flasks, flasks for tissue culture, culture dishes (plates), petri dishes, plates for tissue culture, multilayer plates, microplates, microwell plates, microporous plates, multilayer plates, multi-well plates, chamber slides, bowls, tubes, trays, culture bags, spinner flasks, conical flasks, roller bottles, etc.

[0147] In order to be able to carry out suspension culture, these culture vessels are preferably non-cell adhesive. As a non-cell adhesive culture vessel, it is possible to use a culture vessel that is not artificially treated for the purpose of improving the adhesiveness with cells (for example, coating treatment or surface processing such as positive charge treatment using extracellular matrix such as basement membrane preparations, laminin, nestin (entactin), collagen, gelatin, etc. or high molecules such as polylysine, polyornithine, etc.). As a non-cell adhesive culture vessel, it is possible to use a culture vessel that is artificially treated for the purpose of reducing the adhesiveness with cells (for example, super-hydrophilic treatment such as 2-methacryloyloxy ethyl phosphorylcholine (2-Methacryloyloxy ethyl phosphorylcholine, MPC) polymer, protein low adsorption treatment, etc.). A rotating flask, a roller bottle, a small bioreactor with a stirring blade, etc. can be used to stir. The culture surface of the culture vessel can be flat or have concave-convex.

[0148] Suspension culture is preferably carried out under high oxygen partial pressure conditions. By carrying out suspension culture under high oxygen partial pressure conditions, long-term maintenance culture of the ventricular zone contained in the brain organoid can be achieved, and cell aggregates can be formed that have not only cells positive for telencephalon markers but also cells positive for at least one type of cells selected from cells positive for forebrain markers and cells positive for telencephalon part-like tissue markers.

[0149] The high oxygen partial pressure condition refers to an oxygen partial pressure condition higher than the oxygen partial pressure in air (20%). As the high oxygen partial pressure condition, culturing can also be performed at 30% to 60%.

[0150] The second culture medium can be prepared by using a culture medium for culturing animal cells as a basal culture medium. As the basal culture medium, for example, BME culture medium, BGJb culture medium, CMRL 1066 culture medium, Glasgow MEM culture medium, Improved MEM Zinc Option culture medium, IMDM culture medium, Medium 199 culture medium, Eagle MEM culture medium, αMEM culture medium, DMEM culture medium, Ham's medium, Ham's F-12 culture medium, RPMI 1640 culture medium, Fischer's medium, mixed culture medium thereof, and other culture mediums that can be used for culturing animal cells are not particularly limited.

[0151] The second culture medium may also further contain N2 supplement, chemically defined lipid concentrate, serum, serum substitute, heparin, etc. The serum substitute is the same as the serum substitute.

[0152] The second culture medium is substantially free of extracellular matrix components. Here, the so-called "substantially free of extracellular matrix components" refers to the intentional addition of extracellular matrix components to the second culture, allowing a portion or all of the extracellular matrix components added to the first culture medium to be mixed into the second culture medium; and the second culture medium contains extracellular matrix components produced by the brain organoids themselves, etc. In one form, the volume obtained by adding the second culture medium and the matrigel is set to 100 volume%, and the concentration of the extracellular matrix components in the second culture medium is preferably less than 1 volume%, more preferably less than 0.5 volume%, and further preferably less than 0.1 volume%.

[0153] Since the second culture medium does not substantially contain an extracellular matrix component, a cell aggregate containing not only cells positive for the telencephalon marker but also cells positive for at least one or more of cells positive for the forebrain marker and cells positive for a telencephalic part-like tissue marker can be produced.

[0154] The culture period in the second culture medium is preferably 7 to 154 days, more preferably 14 to 147 days, and still more preferably 21 to 140 days. When cultured in the second culture medium for 77 days or more, brain organoids with a clear hierarchical structure (inside-out pattern) of the cerebral cortex tend to be easily obtained.

[0155] [Brain Organoids]

[0156] In one embodiment, the present invention provides a cerebral organoid manufactured by the method for manufacturing a cerebral organoid of the present embodiment. The cerebral organoid of the present embodiment and a cerebral organoid manufactured by a manufacturing method different from the method for manufacturing the cerebral organoid of the present embodiment may differ in gene expression patterns and the like. However, whether such a difference exists is uncertain, and in order to identify such a difference and to identify the cerebral organoid of the present embodiment by gene expression patterns and the like, a lot of trial and error is required, and it is essentially impossible to achieve. Therefore, it can be said that what is actually happening is that the cerebral organoid of the present embodiment is specified by the case where it is manufactured using the manufacturing method.

[0157] As described in the examples later, the brain organoids of this embodiment confirmed the inside-out pattern when immunostained for neural stem cell-neural precursor cell marker PAX6, cerebral cortex layer V marker CTIP2, and cerebral cortex layer II / III marker SATB2. In addition, by performing long-term culture and immunostaining, it can be confirmed that the brain organoids of this embodiment have astrocyte markers S100β or glial fibrillary acidic protein (GFAP) positive cells.

[0158] [Toxicity-Efficacy Evaluation Method and Kit of Test Substances]

[0159] In one embodiment, the present invention provides a method for evaluating the toxicity-efficacy of a test substance, comprising: contacting the test substance with a brain organoid of the present embodiment; and examining the effect of the test substance on the brain organoid.

[0160] The method of this embodiment can evaluate the efficacy of a test substance on the brain with good reproducibility. In addition, by using human-derived brain organoids, the efficacy of a test substance in humans can be accurately evaluated.

[0161] Examples of test substances include natural compound libraries, synthetic compound libraries, and existing drug libraries.

[0162] Methods for examining the effects of test substances on brain organoids include measurement of diameter in the presence and absence of test substances, analysis of morphology, analysis of cell survival rate, analysis of gene expression patterns, immunostaining of sections, and measurement of neural activity.

[0163] [Therapeutic drugs and pharmaceutical compositions for diseases caused by disorders of nervous system cells or nervous tissues]

[0164] The brain organoid of this embodiment can be used as a therapeutic drug for treating diseases by being transplanted into the brain of a patient suffering from a disease caused by a disorder of nervous system cells or nervous tissues, such as Alzheimer's disease, microcephaly, and autism.

[0165] In addition, the therapeutic drug of this embodiment can also be prepared in the form of a therapeutic pharmaceutical composition, which contains the brain organoid of this embodiment as an active ingredient, and further includes a buffer for suspending the brain organoid, an existing therapeutic drug for a disease caused by a disorder of nervous system cells or nervous tissue, etc. Here, "containing as an active ingredient" means containing an amount of brain organoids to the extent that a therapeutic effect can be obtained on a disease caused by a disorder of nervous system cells or nervous tissue, and the content is not particularly limited.

[0166] Example

[0167] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0168] [Experimental Example 1]

[0169] Human iPS cells (PChiPS771 strain, Lot. A01QM28, manufactured by ReproCELL) were cultured without feeder layer according to the method described in "M. Nakagawa M., et al., A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells, Scientific Reports, 4, 3594, 2014". As a feeder-free medium, StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) was used or iMatrix-511 (manufactured by Nippi Co., Ltd.) was used in a feeder-free scaffold.

[0170] As a specific expansion culture operation, first, human iPS cells (PChiPS771 strain, Lot.A01QM28, manufactured by ReproCELL) with a confluence of 60% to 80% (60% to 80% of the culture area is covered by cells) are washed with phosphate buffered saline (hereinafter referred to as "PBS"), and then dispersed into single cells using TrypLE Select (manufactured by Thermo Fisher Scientific). Then, the human iPS cells dispersed into single cells are inoculated in a plastic culture dish coated with iMatrix-511 (manufactured by Nippi) and cultured without a feeder layer in StemFit AK02N medium supplemented with Y27632 (ROCK inhibitor, final concentration 10 μM). As the plastic culture dish, a 60 mm dish (manufactured by Iwaki, for cell culture) was used, and the number of cells seeded with the human iPS cells dispersed into single cells was set to 3×10 4 indivual.

[0171] One day after the cells were seeded, the medium was replaced with StemFit AK02N medium not containing Y27632. Thereafter, the medium was replaced with StemFit AK02N medium not containing Y27632 every 1 to 2 days. Then, 6 days after the cells were seeded, the cells reached 80% confluence. Figure 1 The figure shows a microscope photograph of iPS cells observed using a bright field microscope 7 days after the cells were seeded.

[0172] [Experimental Example 2]

[0173] Human iPS cells (PChiPS771 strain, Lot. A01QM28, manufactured by ReproCELL) at 80% confluence were treated in the presence of Y27632 (ROCK inhibitor, 10 μM) for 2 hours to obtain human iPS cells.

[0174] The human iPS cells treated for 2 hours were treated as single cells using a cell dispersion (product name "TrypLE Select", manufactured by Thermo Fisher Scientific) and pipetting. The human iPS cells made into single cells were suspended in 100 μL of aggregation medium at 37°C and in the presence of 5% CO2 by volume in the container, so that each well of a non-cell-adhesive 96-well culture plate (product name "PrimeSurface 96V bottom plate", manufactured by Sumitomo Bakelite) was 2×10 4 indivual.

[0175] As the aggregation medium, a medium prepared by adding non-essential amino acids (manufactured by Thermo Fisher Scientific, diluted to 200-fold concentration), penicillin / streptomycin (manufactured by Nacalai Tesque, diluted to 100-fold concentration), Glutamax (manufactured by Thermo Fisher Scientific, diluted to 100-fold concentration), 1×2-mercaptoethanol (manufactured by Thermo Fisher Scientific, diluted to 1000-fold concentration), Dorsomorphin (manufactured by Sigma, final concentration 2 μM), and A-83-01 (final concentration 2 μM) to StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) was used.

[0176] At the start of suspension culture (culture day 0, hereinafter, unless otherwise specified, the number of culture days is expressed as the number of culture days from the start of suspension culture), Y27632 (final concentration 30 μM) was added to the aggregation medium. In addition, on the first day after the start of suspension culture, 150 μL of a medium containing Y27632 (final concentration 10 μM) was added to a medium having the same composition as the aggregation medium, and suspension culture was continued without medium replacement until the 7th day after the start of suspension culture. Figure 2 The micrographs show the results of bright field observation of cell aggregates on day 7 after the start of suspension culture using an inverted microscope (Olympus).

[0177] The cell aggregates on the 7th day after the suspension culture was started were immunostained to study the expression of neuroectoderm markers. Specifically, the cell aggregates were fixed using a 4 mass % paraformaldehyde aqueous solution, and then replaced using a sucrose solution, and frozen sections were made using a Leica CM3050 S (manufactured by Leica). Then, for these frozen sections, neuroectoderm markers were immunostained.

[0178] βIII-TUBULIN was investigated as a neuroectoderm marker, and it was found that cell aggregates on day 7 after the start of suspension culture were positive for neuroectoderm markers.

[0179] [Experimental Example 3]

[0180] On the 7th day after the start of suspension culture in Experimental Example 2, 230 μL of the aggregation medium was removed from each well. Then, the first medium was added at 150 μL / well, mixed on an ice bath so that the extracellular matrix and other components were evenly dispersed, and suspension culture was performed at 37°C in the presence of 5% by volume CO2 in the container without stirring.

[0181] As the first culture medium, various Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (manufactured by Thermo Fisher Scientific) supplemented with 1×N2 supplement (manufactured by Thermo Fisher Scientific, diluted to 200 times), heparin sodium salt (manufactured by Sigma, final concentration 10 μg / mL), non-essential amino acids (manufactured by Thermo Fisher Scientific, diluted to 200 times), penicillin / streptomycin (manufactured by Nacalais) were prepared. The first culture medium contains different concentrations of the extracellular matrix, including 1% (human, recombinant, manufactured by R&D Systems, final concentration 4 ng / mL), CHIR99021 (manufactured by Axon, final concentration 1 μM) and SB-431542 (manufactured by Sigma, final concentration 1 μM), and further contains 2% by volume, 10% by volume, 30% by volume or 50% by volume of Matrigel (manufactured by Corning Incorporated).

[0182] Figure 3These are microscopic photographs showing the results of bright field observation of each cell aggregate in Experimental Example 3 on the 14th day after the start of the suspension culture.

[0183] Next, for each cell aggregate on the 14th day after the start of suspension culture in Experimental Example 3, frozen sections were prepared in the same manner as in Experimental Example 2, and immunostaining was performed to evaluate brain markers.

[0184] Specifically, the expression of forebrain and telencephalon marker FOXG1 (anti-FOXG1 antibody, ABCAM, rabbit), immature neural cell marker βIII-TUBULIN (anti-βIII-TUBULIN antibody, SIGMA, mouse), neural stem cell and neural progenitor cell marker PAX6 (anti-PAX6 antibody, MBL, rabbit) was studied. Fluorescence microscopy observation was performed on each sample after immunostaining using a confocal microscope (manufactured by Zeiss).

[0185] Figure 4 and Figure 5 The fluorescence micrographs show the results of immunostaining of cell aggregates cultured at a Matrigel concentration of 30% by volume. Figure 4 In the figure, “DAPI” represents the result of staining the frozen sections of cell aggregates using DAPI, “βIII-TUBULIN” represents the result of staining the frozen sections of cell aggregates using anti-βIII-TUBULIN antibody, “PAX6” represents the result of staining the frozen sections of cell aggregates using PAX6 antibody, and “MERGE” represents the result obtained by synthesizing the above results.

[0186] Figure 5 In the table, “DAPI” and “MERGE” represent Figure 4 These have the same meaning, “SOX2” indicates the result of staining a frozen section of a cell aggregate with an anti-SOX2 antibody, and “FOXG1” indicates the result of staining a frozen section of a cell aggregate with an anti-FOXG1 antibody.

[0187] The results showed that the cell aggregates cultured at a Matrigel concentration of 30% by volume (14 days after the start of suspension culture in Experimental Example 3) were positive for FOXG1, βIII-TUBULIN, and PAX6, and were brain organoids positive for at least forebrain and telencephalon markers.

[0188] [Experimental Example 4]

[0189] In the first culture medium of Experimental Example 3, StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) was used instead of Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (manufactured by Thermo Fisher Scientific Inc.). In addition, a culture medium containing matrigel at a concentration of 50 volume % (the first culture medium of Experimental Example 4) was prepared in the same manner as in Experimental Example 3, and the cell aggregates on the 7th day after the start of the suspension culture of Experimental Example 2 were cultured.

[0190] Figure 6 The micrographs show the results of bright field observation of each cell aggregate on the 7th day, the 9th day, the 11th day, and the 14th day after the start of suspension culture. Figure 7 It means according to Figure 6 The graphs are the results of measuring the two-dimensional projection area of ​​each cell aggregate based on the photographs.

[0191] Figure 6 and Figure 7 In the table, “DMEM F12” indicates the result of using the first culture medium of Experimental Example 3 with a Matrigel concentration of 50% by volume, and “AK02N” indicates the result of using the first culture medium of Experimental Example 4.

[0192] For the cell aggregates cultured in the first culture medium of Experimental Example 4 in the same manner as in Experimental Example 3, frozen sections were prepared and immunostained to evaluate brain markers in the same manner as in Experimental Example 3. As a result, it was clear that the cell aggregates cultured in the first culture medium of Experimental Example 4 were also positive for FOXG1, βIII-TUBULIN, and PAX6, and were brain organoids that were positive for at least forebrain and telencephalon markers.

[0193] [Experimental Example 5]

[0194] In Experimental Example 3, each cell aggregate cultured on the 14th day in various first culture media containing different concentrations of the extracellular matrix was recovered from each well and transferred to a 50 mL Falcon (registered trademark) conical tube (manufactured by Corning Incorporated) containing 10 mL of PBS. Then, the tube was inverted and mixed 5 times, and the supernatant was removed to remove the matrigel (extracellular matrix).

[0195] Then, each cell aggregate was recovered from a 50 mL Falcon (registered trademark) conical tube and transferred to a 30 mL single-use bioreactor. Then, 20 mL of the second culture medium was added and suspension culture was started while stirring. The stirring speed was set to 50 rpm, and the culture medium was replaced once every 3 to 4 days.

[0196] As the second culture medium, a culture medium containing no extracellular matrix was used. Specifically, as the second culture medium, a medium obtained by adding 1×N2 supplement (manufactured by Thermo Fisher Scientific, diluted to 200 times), B-27 supplement (manufactured by Thermo Fisher Scientific, diluted to 100 times), 1×Non-essential Amino Acids (manufactured by Thermo Fisher Scientific, diluted to 200 times), 1×Penicillin / Streptomycin (manufactured by Nacalai Tesque, diluted to 100 times), 1×2-mercaptoethanol (manufactured by Thermo Fisher Scientific, diluted to 1000 times) and insulin solution (human, recombinant, manufactured by Wako Pure Chemical Industries, Ltd., 2.5 μg / mL) to Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (manufactured by Thermo Fisher Scientific) was used.

[0197] For each cell aggregate on the 40th and 70th day after the start of suspension culture, frozen sections were prepared in the same manner as in Experimental Example 3, and immunostained and brain markers were evaluated. Specifically, immunostained neural stem cell-neural precursor cell marker PAX6 (anti-PAX6 antibody, sheep), cerebral cortex layer V marker CTIP2 (anti-CTIP2 antibody, ABCAM, rat), cerebral cortex layer II / III marker SATB2 (anti-SATB2 antibody, mouse) were immunostained and observed by fluorescence microscopy using a confocal microscope (manufactured by Zeiss).

[0198] Figure 8 It is a microscope photo showing the result of observing each cell aggregate with different matrigel concentration in the bright field on the 40th day after suspension culture started. As a result, it is clear that the size of the cell aggregate becomes larger as the concentration of matrigel increases. In addition, it is clear that when the matrigel concentration is more than 10 volume %, an aggregate with a structure of a neural tube sample can be formed.

[0199] Fig. 9It is a fluorescence microscope photograph showing the following results: on the 40th day after the start of suspension culture, frozen sections were made from the cell aggregates obtained by using the first culture medium with a matrix gel concentration of 50% by volume, and the results obtained by immunostaining and evaluating brain markers were obtained. As a result, it is clear that the cell aggregates obtained on the 40th day after the start of suspension culture have a neural tube-like structure, and there are areas with a high density of cells positive for neural stem cells-neural precursor cells, namely PAX6. In addition, the outer side of the area with a high density of cells positive for PAX6 is configured with the Vth layer marker of the cerebral cortex, namely CTIP2 positive cells, but the II / III layer marker of the cerebral cortex, namely SATB2 positive cells, are not observed. According to the above results, it is clear that the cell aggregates obtained are at least neural stem cells-neural precursor cells and brain organoids positive for the Vth layer marker of the cerebral cortex.

[0200] Furthermore, Fig. 9 In the table, "DAPI" indicates the result of staining with DAPI, "SATB2" indicates the result of staining with anti-SATB2 antibody, "PAX6" indicates the result of staining with anti-PAX6 antibody, "CTIP2" indicates the result of staining with anti-CTIP2 antibody, and "MERGE" indicates the result of synthesizing the above results. Figure 10 to Figure 15 , the same is true.

[0201] Fig.10 It is a fluorescence microscope photograph showing the following results: on the 40th day after the start of suspension culture, frozen sections were made from the cell aggregates obtained by using the first culture medium with a matrix gel concentration of 30% by volume, and the results obtained by immunostaining and evaluating brain markers were obtained. As a result, it is clear that the cell aggregates obtained on the 40th day after the start of suspension culture have a neural tube-like structure, and there are areas with a high density of cells positive for neural stem cells-neural precursor cells, namely PAX6. In addition, the outer side of the area with a high density of cells positive for PAX6 is configured with the Vth layer marker of the cerebral cortex, namely CTIP2 positive cells, but the II / III layer marker of the cerebral cortex, namely SATB2 positive cells, are not observed. According to the above results, it is clear that the cell aggregates obtained are at least neural stem cells-neural precursor cells and brain organoids positive for the Vth layer marker of the cerebral cortex.

[0202] Fig.11It is a fluorescence microscope photograph showing the following results: on the 40th day after the start of suspension culture, frozen sections were made from the cell aggregates obtained by using the first culture medium with a matrix gel concentration of 10 volume %, and the results obtained by immunostaining and evaluating brain markers were obtained. As a result, it is clear that the cell aggregates obtained on the 40th day after the start of suspension culture have a neural tube-like structure, and there are areas with high cell density of neural stem cells-neural precursor cells, i.e., PAX6-positive cells. In addition, the outer side of the area with high cell density of PAX6-positive cells is configured with the Vth layer marker of the cerebral cortex, i.e., CTIP2-positive cells, but the II / III layer marker of the cerebral cortex, i.e., SATB2-positive cells, are not observed. According to the above results, it is clear that the cell aggregates obtained are at least neural stem cells-neural precursor cells and brain organoids with positive Vth layer markers of the cerebral cortex.

[0203] Fig.12 It is a fluorescence microscope photograph showing the following results: On the 40th day after the start of suspension culture, frozen sections were made from cell aggregates obtained using the first culture medium with a matrix gel concentration of 2 volume%, and the results obtained by immunostaining and evaluating brain markers. As a result, the cell aggregates obtained on the 40th day after the start of suspension culture had fewer cells positive for the neural stem cell-neural precursor cell marker, namely PAX6, compared with the cell aggregates with a matrix gel concentration of 10 volume % to 50 volume %. In addition, no CTIP2-positive cells, which are the markers of layer V of the cerebral cortex, and SATB2-positive cells, which are the markers of layer II / III of the cerebral cortex, were observed. Based on the above results, it is clear that the obtained cell aggregates are at least brain organoids positive for neural stem cells-neural precursor cells and layer V markers of the cerebral cortex.

[0204] Fig.13 It is a fluorescence microscope photograph showing the following results: on the 70th day after the start of suspension culture, frozen sections were made from the cell aggregates obtained by using the first culture medium with a matrigel concentration of 50 volume %, and the results obtained by immunostaining and evaluating brain markers were obtained. As a result, it is clear that the cell aggregates obtained on the 70th day after the start of suspension culture have a neural tube-like structure, and there is a high cell density area of ​​neural stem cell-neural precursor cell marker, i.e., PAX6 positive. In addition, it is shown that there is a layer V marker of the cerebral cortex, i.e., a CTIP2 positive cell layer, outside the region with a high cell density of PAX6 positive, and then the layer II / III marker of the cerebral cortex, i.e., SATB2 positive cells, are configured outside it, thereby forming a layered structure of the cerebral cortex sample. According to the above results, it is clear that the cell aggregates obtained are at least neural stem cells-neural precursor cells and the layer V of the cerebral cortex and the layer II / III marker of the cerebral cortex, which are positive for brain organoids.

[0205] Fig.14It is a fluorescence microscope photograph showing the following results: on the 70th day after the start of suspension culture, frozen sections were made from the cell aggregates obtained by using the first culture medium with a matrigel concentration of 30% by volume, and the results obtained by immunostaining and evaluating brain markers were obtained. As a result, it is clear that the cell aggregates obtained on the 70th day after the start of suspension culture have a neural tube-like structure, and there is a high cell density area of ​​neural stem cell-neural precursor cell marker, i.e., PAX6 positive. In addition, it is shown that there is a layer V marker of the cerebral cortex, i.e., a CTIP2 positive cell layer, outside the region with a high cell density of PAX6 positive, and then the layer II / III marker of the cerebral cortex, i.e., SATB2 positive cells, are configured outside it, thereby forming a layered structure of the cerebral cortex sample. According to the above results, it is clear that the cell aggregates obtained are at least neural stem cells-neural precursor cells and the layer V of the cerebral cortex and the layer II / III marker of the cerebral cortex, which are positive for brain organoids.

[0206] Fig.15 It is a fluorescence microscope photo showing the following results: on the 70th day after the start of suspension culture, frozen sections were made from the cell aggregates obtained using the first culture medium with a matrix gel concentration of 10 volume %, and the results obtained by immunostaining and evaluating brain markers were obtained. As a result, it is clear that the cell aggregates obtained on the 70th day after the start of suspension culture have a neural tube-like structure, and there are areas with a high density of cells positive for neural stem cell-neural precursor cell markers, namely PAX6. In addition, the Vth layer marker of the cerebral cortex, namely CTIP2 positive cells, are configured outside the area with a high density of cells positive for PAX6. On the other hand, the II / III layer marker of the cerebral cortex, namely SATB2 positive cells, are not substantially observed. According to the above results, it is clear that the cell aggregates obtained are at least neural stem cells-neural precursor cells and cerebral cortical Vth layer marker positive brain organoids.

[0207] Industrial Applicability

[0208] According to the present invention, a method for producing telencephalon marker-positive cerebral organoids can be provided.

Claims

1. A method for producing a brain organoid, comprising: The step of culturing the human induced pluripotent stem cells in suspension in an aggregation medium for more than 7 days to obtain neuroectoderm marker-positive cell aggregates; A step of suspending and culturing the neuroectodermal marker-positive cell aggregates in a first culture medium containing an extracellular matrix at a concentration of 20% to 50% by volume for 7 days or more, wherein the step is a step of culturing the neuroectodermal marker-positive cell aggregates in a state of being dispersed in the first culture medium without being inserted into the extracellular matrix; and After the above step, a step of performing suspension culture in a second culture medium substantially free of extracellular matrix; Wherein, the extracellular matrix is ​​matrigel; The aggregation medium comprises StemFit AK02N, non-essential amino acids at a final concentration of 200 times dilution, glutamine at a final concentration of 100 times dilution, 1×2-mercaptoethanol at a final concentration of 1000 times dilution, doxorubicin at a final concentration of 2 μM, and A-83-01 at a final concentration of 2 μM; The first culture medium comprises Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 or StemFit AK02N, a final concentration of 1×N2 supplement diluted 200 times, a final concentration of 4 ng / mL Wnt-3a, a final concentration of 1 μM CHIR99021, and a final concentration of 1 μM SB-431542; The second culture medium comprises Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, 1×N2 supplement diluted 200 times at a final concentration, B-27 supplement diluted 100 times at a final concentration, 1×2-mercaptoethanol diluted 1000 times at a final concentration, and insulin solution at a final concentration of 2.5 μg / mL.

2. The method for producing a brain organoid according to claim 1, wherein: The step of culturing in the second culture medium substantially free of extracellular matrix is ​​performed under high oxygen partial pressure conditions.

3. The method for producing a brain organoid according to claim 1 or 2, wherein: The step of culturing in the second culture medium substantially free of extracellular matrix is ​​performed with stirring.

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