Method for producing three-dimensional cell culture, method for culturing three-dimensional cell culture, three-dimensional cell culture, and method for evaluating test substance

Abstract

The present invention addresses the problem of providing: a method for producing a three-dimensional cell culture including human-derived neurons, astrocytes, and microglia that can mimic the function of a human brain, and a method for culturing for said three-dimensional cell culture; a three-dimensional cell culture including human-derived neurons, astrocytes, and microglia that can mimic the function of a human brain; and a method for evaluating a test substance using the three-dimensional cell culture. The present invention provides a method for producing a three-dimensional cell culture, the method including: a step for adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culturing container prior to co-culturing of the cells; and a step for co-culturing the astrocytes, the neurons, and the microglia in the culturing container, wherein the proportion of the astrocytes to all cells added to the culturing container is 30% or more.

Description

Method for producing three-dimensional cell culture, method for culturing three-dimensional cell culture, three-dimensional cell culture, and method for evaluating test substance

[0001] The present invention relates to a method for producing a three-dimensional cell culture using astrocytes, neurons, and microglia, a method for culturing the three-dimensional cell culture, a three-dimensional cell culture comprising astrocytes, neurons, and microglia, and a method for evaluating a test substance using the three-dimensional cell culture.

[0002] The central nervous system (CNS) is composed of neurons and glial cells (astrocytes, microglia, and oligodendrocytes), and these cells interact to maintain normal brain function (Non-Patent Document 1). For example, neurons transmit information to other neurons by releasing glutamate into the synaptic cleft, while astrocytes regulate neuronal signaling by uptake of glutamate (Non-Patent Document 2). It has also been reported that microglia become activated by aging and inflammation, affecting astrocytes, which in turn damage neurons (Non-Patent Documents 3 and 4). Therefore, co-culture systems that can evaluate the intercellular interactions between neurons and glial cells are extremely useful in CNS research.

[0003] In recent years, various three-dimensional culture systems have been reported to mimic the complex structure of the human brain (Non-Patent Document 5). Furthermore, it is known that there are species differences in neurons and glial cells (Non-Patent Documents 6 and 7), and therefore, it is extremely important to use human cells to reproduce the human brain.

[0004] Patent Literature 1 describes a three-dimensional co-culture containing astrocytes, neurons, and microglia derived from human iPS cells. Patent Literature 2 describes a three-dimensional organoid containing neurons and glia derived from human iPS cells. Patent Literature 3 describes a three-dimensional (spheroid) co-culture containing neurons and glia derived from human iPS cells.

[0005] International Publication No. WO2023 / 039567 International Publication No. WO2022 / 183015 International Publication No. WO2022 / 178395

[0006] Francesca Cerbai et al., PLoS One, Vol. 7, Article No. e45250, 2012; Jens V Andersen et al., Neuropharmacology, Vol. 14, Article No. 108719, 2021; Shane A Liddelow et al., Nature, Vol. 541, pp. 481-487, 2017; Laura E Clarke et al., Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, pp. E1896-E1905, 2018; Eduarda G Z Centeno et al., Molecular Neurodegeneration, Vol. 13, Paper No. 27, 2018; Anke M Tukker et al., Toxicological Sciences, Vol. 178, pp. 71-87, 2020; Leonid Tarassishin et al., Glia, Vol. 62, pp. 999-1013, 2014

[0007] An object of the present invention is to provide a method for producing a three-dimensional cell culture containing human-derived neurons, astrocytes, and microglia that can mimic the functions of the human brain, and a method for culturing a three-dimensional cell culture containing human-derived neurons, astrocytes, and microglia that can mimic the functions of the human brain.A further object of the present invention is to provide a three-dimensional cell culture containing human-derived neurons, astrocytes, and microglia that can mimic the functions of the human brain.A further object of the present invention is to provide a method for evaluating a test substance using the three-dimensional cell culture.

[0008] As a result of extensive research to solve the above problems, the present inventors have discovered that a three-dimensional cell culture capable of mimicking the functions of the human brain can be produced by adding human astrocytes, human neurons, and human microglia to a culture vessel prior to co-culturing the cells so that the proportion of astrocytes in the total cells added to the culture vessel is 30% or more, and then co-culturing the cells in the culture vessel.The present invention was completed based on these findings.

[0009] That is, the present invention provides the following inventions: <1> A method for producing a three-dimensional cell culture, comprising the steps of adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel prior to co-culturing the cells, and co-culturing the astrocytes, neurons, and microglia in the culture vessel, wherein the astrocytes account for 30% or more of all cells added to the culture vessel. <2> The production method according to <1>, in which the astrocytes, neurons, and microglia are added to the culture vessel simultaneously. <3> The manufacturing method according to <1> or <2>, wherein the step of adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel prior to co-culturing the cells comprises a step of suspending the human-derived astrocytes, human-derived neurons, and human-derived microglia in a culture medium, and a step of simultaneously adding the culture medium containing the astrocytes, neurons, and microglia obtained above to the culture vessel. <4> The manufacturing method according to any one of <1> to <3>, wherein the astrocytes, neurons, and microglia are differentiated from human-derived pluripotent stem cells. <5> The manufacturing method according to <4>, wherein the human-derived pluripotent stem cells are human iPS cells. <6> The manufacturing method according to any one of <1> to <5>, wherein the proportion of microglia among all cells added to the culture vessel is 50% or less. <7> The manufacturing method according to any one of <1> to <6>, wherein the proportion of the neurons among all cells added to the culture vessel is 10% or more. <8> The manufacturing method according to any one of <1> to <7>, wherein the three-dimensional cell culture is in a spheroid shape. <9> A method for culturing a three-dimensional cell culture, the method comprising the steps of adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel prior to co-culturing cells, and co-culturing the astrocytes, the neurons, and the microglia in the culture vessel, wherein the proportion of the astrocytes among all cells added to the culture vessel is 30% or more.<10> The culture method according to <9>, wherein the astrocytes, the neurons, and the microglia are added to a culture vessel simultaneously. <11> The culture method according to <9> or <10>, wherein the step of adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel prior to co-culture of the cells comprises the steps of suspending the human-derived astrocytes, human-derived neurons, and human-derived microglia in a culture medium, and simultaneously adding the culture medium containing the astrocytes, neurons, and microglia obtained thereby to the culture vessel. <12> The culture method according to any one of <9> to <11>, wherein the astrocytes, neurons, and microglia are differentiated from human-derived pluripotent stem cells. <13> The culture method according to <12>, wherein the human-derived pluripotent stem cells are human iPS cells. <14> The culture method according to any one of <9> to <13>, wherein the proportion of microglia among all cells added to the culture vessel is 50% or less. <15> The culture method according to any one of <9> to <14>, wherein the proportion of neurons among all cells added to the culture vessel is 10% or more. <16> The culture method according to any one of <9> to <15>, wherein the three-dimensional cell culture is in a spheroid shape. <17> A three-dimensional cell culture comprising human-derived astrocytes, human-derived neurons, and human-derived microglia, obtained by the production method according to any one of <1> to <8>. <18> A method for evaluating a test substance, comprising contacting the three-dimensional cell culture according to <17> with a test substance. <19> A method for evaluating a test substance, comprising producing a three-dimensional cell culture by the production method according to any one of <1> to <8>, and contacting the three-dimensional cell culture with the test substance.

[0010] The three-dimensional cell culture produced by the method of the present invention is a culture system that mimics the human brain, and is useful for studying the intercellular interactions between neurons and glial cells in normal brain function and pathological mechanisms.

[0011] Figure 1 shows the results of immunostaining of the three-dimensional co-culture, and Figure 2 shows the results of calcium imaging in the three-dimensional co-culture.

[0012] Hereinafter, embodiments of the present invention will be described in detail.

[0013] The method for producing a three-dimensional cell culture of the present invention and the method for culturing a three-dimensional cell culture of the present invention are methods that include, prior to co-culturing the cells, adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel, and co-culturing the astrocytes, neurons, and microglia in the culture vessel, wherein the proportion of the astrocytes among all the cells added to the culture vessel is 30% or more.

[0014] "Prior to co-culture of cells" means that co-culture is not considered to have begun if, for example, within 1 to 24 hours after the addition of one or two types of cells. The proportion of astrocytes among all cells added to the container is substantially the same as the proportion of astrocytes in the three-dimensional cell culture. The proportion of neurons among all cells added to the container is substantially the same as the proportion of neurons in the three-dimensional cell culture. The proportion of microglia among all cells added to the container is substantially the same as the proportion of microglia in the three-dimensional cell culture.

[0015] In the present invention, preferably, astrocytes, neurons, and microglia can be added simultaneously to a culture vessel. Specifically, for example, the above three types of cells may be placed in a single vessel, suspended and mixed in a culture medium, and then added to the culture vessel. In this case, the step of adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel includes the steps of suspending the human-derived astrocytes, human-derived neurons, and human-derived microglia in a culture medium, and simultaneously adding the culture medium containing the astrocytes, neurons, and microglia obtained above to the culture vessel.

[0016] Alternatively, each of the three types of cells may be suspended in a separate culture medium and then simultaneously added to the same culture vessel. Alternatively, the three types of frozen cells that have been separately cryopreserved may be thawed and then simultaneously added to the same culture vessel. However, the manner in which astrocytes, neurons, and microglia are simultaneously added to the culture vessel is not limited to the above.

[0017] In the present invention, three types of cells (astrocytes, neurons, and microglia) can be seeded simultaneously and co-cultured at any ratio. Furthermore, culturing the three types of cells (astrocytes, neurons, and microglia) in three dimensions allows for a more accurate reflection and mimicry of the human brain than conventional culture systems, making it useful for studying the brain's inherent functions and pathogenesis mechanisms. The use of three types of cells in the present invention allows for the evaluation of intercellular interactions in brain function and pathogenesis.

[0018] Three-dimensional culture of three types of cells allows evaluation of neural activity after three weeks of culture. Since two-dimensional co-culture of three types of cells requires four weeks of culture to evaluate neural activity, it is possible that three-dimensional culture promotes the formation of neural cell networks.

[0019] Furthermore, by seeding three types of cells simultaneously, it is possible to evaluate the influence of glial cells on the process of neuronal network construction and maturation.

[0020] The astrocytes, neurons, and microglia are preferably differentiated from human-derived pluripotent stem cells.

[0021] Examples of human-derived pluripotent stem cells include human iPS cells (human induced pluripotent stem cells), human ES cells (human embryonic stem cells), and human mesenchymal stem cells. Human iPS cells are preferred, but are not particularly limited. Human iPS cells are iPS cells produced from human cells.

[0022] Examples of human-derived pluripotent stem cells include pluripotent stem cells derived from specimens lacking mutations in disease-related genes that cause nervous system diseases, pluripotent stem cells derived from specimens collected from healthy individuals (healthy individuals) without nervous system diseases, pluripotent stem cells derived from specimens collected from patients with diseases, and pluripotent stem cells derived from specimens with mutations in disease-related genes. "No mutations in disease-related genes" refers to the absence of mutations in disease-related genes that would cause nervous system diseases. That is, even if a gene has a mutation, if the mutation does not cause the disease, it is interpreted as the absence of a mutation in the disease-related gene. Furthermore, "mutations in disease-related genes" may refer to endogenous gene mutations inherent in the patient or exogenous gene mutations artificially introduced into the patient. It is anticipated that the three-dimensional cell culture of the present invention can be produced using pluripotent stem cells derived from specimens collected from patients with diseases or pluripotent stem cells derived from specimens with mutations in disease-related genes, thereby mimicking the pathology of disease originating from the specimen or disease-related gene mutations.

[0023] ES cells can be established, for example, by culturing pre-implantation early embryos, inner cell masses constituting the above-mentioned early embryos, single blastomeres, and the like (Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1994; Thomson, J.A. et al.: Science, 282, 1145-1147, 1998). As the early embryo, an early embryo produced by nuclear transfer of a nucleus from a somatic cell may be used (Wilmut et al.: Nature, 385, 810, 1997; Cibelli et al.: Science, 280, 1256, 1998; Akira Iritani et al.: Protein Nucleic Acid Enzymes, 44, 892, 1999; Baguisi et al.: Nature Biotechnology, 17, 456, 1999; Wakayama et al.: Nature, 394, 369, 1998; Nature Genetics, 22, 127, 1999; Proc. Natl. Acad. Sci. USA, 96, 14984, 1999; Rideout III et al. al: Nature Genetics, 24, 109, 2000, Tachibana et al: Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell, 2013, in press). As the early embryo, a parthenogenetic embryo may be used (Kim et al.: Science, 315, 482-486, 2007; Nakajima et al.: Stem Cells, 25, 983-985, 2007; Kim et al.: Cell Stem Cell, 1, 346-352, 2007; Revazova et al.: Cloning Stem Cells, 9, 432-449, 2007; Revazova et al.: Cloning Stem Cells, 10, 11-24, 2008).In addition to the above-mentioned papers, references to Strelchenko N, et al: Reprod Biomed Online. 9:623-629, 2004, Klimanskaya I, et al: Nature 444:481-485, 2006, Chung Y, et al: Cell Stem Cell 2:113-117, 2008, Zhang X, et al: Stem Cells 24:2669-2676, 2006, Wassarman, P. M. et al: Methods in Enzymology, Vol. 365, 2003, etc. are useful for the production of ES cells. Fusion ES cells obtained by cell fusion between ES cells and somatic cells are also included in the embryonic stem cells used in the method of the present invention.

[0024] Some ES cells are available from collection institutions or commercially available. For example, human ES cells are available from the Institute for Frontier Medical Sciences, Kyoto University (e.g., KhES-1, KhES-2, and KhES-3), WiCell Research Institute, ESI BIO, etc.

[0025] iPS cells refer to cells that have pluripotency (multipotency) and proliferation ability, and are produced by reprogramming somatic cells through the introduction of reprogramming factors. iPS cells exhibit properties similar to ES cells. The somatic cells used to produce iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. iPS cells can be produced by known methods. It is also naturally expected that iPS cell production methods developed in the future will also be applied.

[0026] The most basic method for generating iPS cells is to introduce four transcription factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into cells using a virus (Takahashi K, Yamanaka S: Cell 126(4), 663-676, 2006; Takahashi K, et al.: Cell 131(5), 861-72, 2007). There have been reports of the establishment of human iPS cells by introducing four factors, Oct4, Sox2, Lin28, and Nanog (Yu J, et al.: Science 318(5858), 1917-1920, 2007). It has also been reported that iPS cells can be established by introducing three factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26(1), 101-106, 2008), two factors Oct3 / 4 and Klf4 (Kim J B, et al: Nature 454(7204), 646-650, 2008), or only Oct3 / 4 (Kim J B, et al: Cell 136(3), 411-419, 2009). Furthermore, a method for introducing a protein, which is an expression product of a gene, into a cell has also been reported (Zhou H, Wu S, Joo JY, et al.: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al.: Cell Stem Cell 4, 472-476, 2009). On the other hand, it has been reported that the use of an inhibitor of histone methyltransferase G9a, such as BIX-01294, or a histone deacetylase inhibitor, such as valproic acid (VPA) or BayK8644, can improve the production efficiency and reduce the amount of factors to be introduced (Huangfu D, et al.: Nat. Biotechnol. 26(7), 795-797, 2008; Huangfu D, et al.: Nat. Biotechnol. 26(11), 1269-1275, 2008; Silva J, et al.: PLoS. Biol. 6(10), e253, 2008).Methods for gene transfer have also been investigated, and in addition to retroviruses, lentiviruses (Yu J, et al: Science 318 (5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322 (5903), 945-949, 2008), plasmids (Okita K, et al: Science 322 (5903), 949-953, 2008), transposon vectors (Woltjen K, Michael IP, Mohseni P, et al: Nature 458, 766-770, 2009; Kaji K, Norrby K, Pac a A, et al. al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat Methods 6, 363-369, 2009), or episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) have been developed for gene transfer.

[0027] Cells that have been transformed into iPS cells, i.e., reprogrammed, can be selected using the expression of pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct3 / 4, Fgf-4, Esg-1, and Cript as indicators. The selected cells can be recovered as iPS cells.

[0028] In addition to direct reprogramming through gene expression, iPS cells can also be produced by inducing them from somatic cells through the addition of compounds (Hou P, et al: Science 341(6146), 651-654, 2013).

[0029] iPS cells can also be obtained from, for example, FUJIFILM Cellular Dynamics, Inc. (FCDI), Kyoto University, or the RIKEN BioResource Center.

[0030] Examples of human-derived astrocytes include astrocytes induced to differentiate from astrocyte precursor cells (e.g., XCell Science, XCS-AP-001-1V), iCell (registered trademark) astrocytes (FUJIFILM Cellular Dynamics, C1037), etc., with astrocytes induced to differentiate from astrocyte precursor cells being preferred. When inducing differentiation from astrocyte precursor cells into astrocytes, it is preferable to induce differentiation in the absence of serum.

[0031] Methods for obtaining human-derived nerve cells include, for example, deriving them from somatic cells collected from healthy individuals (healthy subjects) who do not have a mutation in a disease-related gene that causes a nervous system disease or a nervous system disease, or from human iPS cells produced from somatic cells collected from a patient with a nervous system disease, or from an established human iPS cell line.

[0032] The nerve cells induced to differentiate from human-derived pluripotent stem cells are not particularly limited, but are preferably motor nerve cells, cerebral cortical excitatory nerve cells, or substantia nigra nerve cells.

[0033] Methods for inducing differentiation of human-derived pluripotent stem cells into neurons include, but are not limited to, a method in which neural stem cells are produced from pluripotent stem cells using treatment with low molecular weight compounds, etc., and then the differentiation into neurons is induced directly by gene expression, etc.

[0034] Methods for inducing differentiation of pluripotent stem cells into neural cells include, for example, (1) a method of culturing in a serum-free medium to form embryoid bodies (cell masses containing neural precursor cells) and then differentiating them (SFEB method: Watanabe K, et al., Nat. Neurosci., 8: 288-296, 2005; SFEBq method: Wataya T, et al., Proc. Natl. Acad. Sci. USA., 105: 11796-11801, 2008); (2) a method of culturing on stromal cells to differentiate them (SDIA method: Kawasaki H, et al., Neuron, 28: 31-40, 2000); (3) a method of culturing on drug-added matrigel to differentiate them (Chambers S.M, et al., al, Nat. Biotechnol., 27:275-280, 2009); (4) a method of differentiation by culturing in a medium containing a low molecular weight compound as a substitute for cytokines (U.S. Pat. No. 5,843,780); (5) a method of differentiation by introducing a neural induction factor (such as a gene encoding Neurogenin2 (NGN2) protein) into pluripotent stem cells and expressing it (WO2014 / 148646; and Zhang Y., et al, Neuron, 78:785-98, 2013); (6) a method of differentiation by introducing miR-9 / 9*-124 into pluripotent stem cells and expressing it; and combinations of these methods.

[0035] Of the above, (5) the method of introducing a gene encoding the NGN2 protein into pluripotent stem cells and expressing it is preferred because it allows mature neurons to be obtained in a short period of time and with high efficiency.

[0036] Examples of human-derived neurons include induced neurons differentiated from iPS cells by forced expression of NGN2, iCell (registered trademark) glutamatergic neurons (FUJIFILM Cellular Dynamics, C1033), iCell (registered trademark) GABAergic neurons (FUJIFILM Cellular Dynamics, C1008), iCell (registered trademark) dopaminergic neurons (FUJIFILM Cellular Dynamics, C1028), and iCell (registered trademark) motor neurons (FUJIFILM Cellular Dynamics, C1048), of which induced neurons differentiated from iPS cells by forced expression of NGN2 and iCell (registered trademark) glutamatergic neurons are preferred.

[0037] Examples of human-derived microglia include iCell (registered trademark) microglia (FUJIFILM Cellular Dynamics, C1110) and microglia (Axol Bioscience, AX0664), with iCell (registered trademark) microglia being preferred.

[0038] Astrocytes are cells that express at least one astrocyte-specific marker gene, which includes GFAP (glial fibrillary acidic protein), S100β (S100 calcium binding protein B), KCNJ10 (potassium inwardly rectifying channel subfamily J member 10), AQP4 (aquaporin-4), and SLC1A3 (solute carrier family 1 member 3).

[0039] Neurons are preferably cells that express at least one or more neuron-specific marker genes, including β-III tubulin, NeuN, N-CAM (neural cell adhesion molecule), and MAP2 (microtubule-associated protein 2), and have β-III tubulin-positive processes (hereinafter referred to as neurites).

[0040] Microglia are cells that express at least one of the microglia-specific marker genes, including IBA1 (ionized calcium-binding adapter molecule 1), CD33, CD45, TREM2 (triggering receptor expressed on myeloid cells 2), P2RY12 (purinergic receptor P2Y, G-protein coupled, 12), TMEM119 (transmembrane protein 119), and CX3CR1 (CX3C-chemokine receptor 1).

[0041] The expression level of a marker gene can usually be analyzed by the amount of transcription product corresponding to the gene, or the amount or activity of its translation product. The expression level can be measured by measuring mRNA, which is a transcription product of the gene, or protein, which is a translation product of the gene, but is preferably measured by measuring mRNA or cDNA, which is a reverse transcription product of the gene. The expression of the translation product (protein) can be detected or measured by immunocytostaining, which uses an antibody to detect intracellular proteins.

[0042] In the present invention, the proportion of astrocytes among all cells added to the culture vessel is 30% or more, preferably 35% or more, more preferably 40% or more, and even more preferably 45% or more. There is no upper limit to the proportion of astrocytes among all cells added to the culture vessel, but it is generally 80% or less, preferably 70% or less.

[0043] By ensuring that the proportion of astrocytes among all cells added to the culture vessel is 30% or more, the maturation of neurons is promoted, and calcium oscillations, which are indicators of neuronal maturation and network construction, can be stably and clearly detected when evaluating neural activity using calcium imaging, etc.

[0044] The proportion of microglia in all cells added to the culture vessel is preferably 50% or less, more preferably 40% or less, and even more preferably 30% or less. There is no particular lower limit to the proportion of microglia in all cells added to the culture vessel, but it is generally 0.1% or more, preferably 1% or more, and even more preferably 10% or more.

[0045] By setting the proportion of microglia to 0.1% or higher, it has been confirmed that microglia activated by neuroinflammation affect other cells, making it possible to evaluate cell-to-cell interactions during neuroinflammation.

[0046] The proportion of neurons among all cells added to the culture vessel is preferably 10% or more, more preferably 15% or more, even more preferably 20% or more, and particularly preferably 25% or more. There is no upper limit to the proportion of neurons, but it is generally 65% ​​or less, preferably 60% or less.

[0047] By increasing the proportion of neurons to 10% or more, calcium oscillations can be clearly detected by calcium imaging, which is useful for evaluating neural activity. Furthermore, in this invention, it has been confirmed by immunostaining that neurons, astrocytes, and microglia can be cultured for a long period of time, allowing the influence of glial cells on neural activity to be evaluated.

[0048] In the present invention, astrocytes, neurons, and microglia are co-cultured in a culture vessel. The culture vessel may be a plate, dish, cell culture insert, or cell culture flask with wells, with wells being preferred. Examples include PrimeSurface® plates (Sumitomo Bakelite) and Cell Repellent plates (Greiner Bio One), with PrimeSurface plates (Sumitomo Bakelite) being preferred.

[0049] The shape of the culture vessel is not particularly limited, but may be a round bottom, a U-bottom, or a V-bottom.

[0050] The surface in the container that comes into contact with the medium is preferably non-cell-adhesive. This allows the cells to be cultured in a suspended state, facilitating the formation of spheroids. Alternatively, only certain areas of the surface inside the container may be cell-adhesive, while the remaining areas may be non-cell-adhesive. In this case, cells will gather at the cell-adhesive areas, allowing spheroids to form.

[0051] The lower limit of the number of cells (the number of cells is the total number of three types of cells; the same applies below) when seeded onto a 96-well plate is not particularly limited, but is, for example, 0.7 × 10 ４ Preferably, the number of cells / well is 1.0 x 10 ４ More preferably, 1.2 x 10 cells / well or more ４ More preferably, 1.4 x 10 cells / well or more ４ Even more preferably, 1.6 x 10 cells / well or more ４ More than 1.8 x 10 cells / well is particularly preferred. ４ The upper limit of the cell density at the time of seeding into the culture vessel is not particularly limited, but is, for example, 8.0 × 10 ４ cells / well or less, 8.0 x 10 ４ Preferably less than 5.0 x 10 cells / well ４ cells / well or less, more preferably 3.0 x 10 ４ More preferably, 2.5 x 10 cells / well or less ４ Even more preferably, 2.3 x 10 cells / well or less ４ Cells / well or less is particularly preferred.

[0052] The number of cells seeded onto a 96-well plate is preferably 1.0 x 10 ４ Cells / well > 8.0 x 10 ４ cells / well or less, more preferably 1.2 x 10 ４ 5.0 x 10 cells / well or more ４ cells / well or less, more preferably 1.4 x 10 ４ Cells / well > 3.0 x 10 ４cells / well or less, and even more preferably 1.6 x 10 ４ 2.5 x 10 cells / well or more ４ cells / well or less, and particularly preferably 1.8 x 10 ４ Cells / well > 2.3 x 10 ４ cells / well or less.

[0053] The medium can be selected from known or commercially available media. The medium used for culture can be a basal medium to which additives have been added. Here, the basal medium may be, for example, DMEM, DMEM (Dulbecco's Modified Eagle Medium) / F12, BrainPhys (registered trademark) Neuronal Medium, Neurobasal (trademark) Medium-A, Neurobasal (trademark) Medium, Neural Progenitor Basal Medium, NS-A Basal Medium, Basal Medium Eagle (BME), BGJb Medium, CMRL 1066 Medium, Glasgow Minimum Essential Medium (MEM), Improved MEM Zinc Examples of the medium include Option, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle MEM, αMEM, Ham's F12 Medium, RPMI 1640 Medium, and Fischer's Medium. A single medium may be used, or two or more types of medium may be used in combination.

[0054] Specific examples of additives that can be added to the culture medium include serum, retinoic acid, Wnt, bone morphogenetic protein (BMP) (e.g., BMP-4), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH), insulin-like growth factor 1 (IGF-1), Activin A, Heregulin β-1, interleukins, 8-Br-cAMP, heparin, heparan sulfate, laminin, collagen, fibronectin, progesterone, selenite, B-27 (registered trademark) supplement, N2 supplement with transferrin (Apo), N2 Examples of such supplements include, but are not limited to, Supplement with Transferrin, GlutaMAX™, L(+)-ascorbic acid, ITS-Supplement, MEM Non-Essential Amino Acid, GlutaMAX™ Supplement, etc. Furthermore, antibiotics (e.g., Penicillin, Streptomycin) may also be added.

[0055] The culture conditions may be selected from those for general cell culture: 37°C, 5% CO ２ During the culture, the medium may be changed at appropriate intervals (preferably once every 1 to 7 days, more preferably once every 2 to 3 days), but it is not necessary to change the medium during the culture period.

[0056] The three-dimensional cell cultures produced in the present invention are preferably spheroids. Spheroids can be formed by co-culturing astrocytes, neurons, and microglia in a culture vessel. Alternatively, three-dimensional cell cultures can be produced by co-culturing astrocytes, neurons, and microglia in a gelling culture medium in a culture vessel, and then gelling the medium after co-culturing.

[0057] According to the present invention, there is provided a three-dimensional cell culture comprising human-derived astrocytes, human-derived neurons, and human-derived microglia, which is obtained by the above-described method for producing a three-dimensional cell culture of the present invention. In the present invention, human-derived astrocytes, human-derived neurons, and human-derived microglia are added to a culture vessel prior to co-culture of the cells. Therefore, it is expected that in the three-dimensional cell culture of the present invention, astrocytes, neurons, and microglia are distributed throughout the core and surface of the three-dimensional cell culture.

[0058] The three-dimensional cell culture of the present invention is a three-dimensional culture of three types of cells (astrocytes, neurons, and microglia), and can therefore reflect and mimic the human brain, making it useful for screening new drugs useful for treating neurodegenerative diseases, such as Alzheimer's disease (AD), spinocerebellar degeneration, frontotemporal lobar degeneration (FTLD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), Lewy body disease, Huntington's disease, and Niemann-Pick disease.

[0059] That is, the present invention provides a method for evaluating a test substance, which comprises contacting the above-described three-dimensional cell culture of the present invention with the test substance.The present invention also provides a method for evaluating a test substance, which comprises producing a three-dimensional cell culture by the production method of the present invention and contacting the three-dimensional cell culture with the test substance.

[0060] More specifically, the present invention further provides a drug screening method, comprising: (1) contacting the three-dimensional cell culture of the present invention with a test substance; (2) culturing the three-dimensional cell culture contacted with the test substance in step (1) and a control three-dimensional cell culture of the present invention that has not been contacted with the test substance; (3) measuring a function of the three-dimensional cell culture (e.g., glutamine uptake, calcium oscillation, etc.); and (4) selecting, as a candidate drug, a test substance that changes the function compared to a control that has not been contacted with the test substance.

[0061] Examples of test substances include proteins, peptides, antibodies, nucleic acids (gene expression vectors, siRNA, miRNA, antisense oligonucleotides, mRNA, etc.), viral vectors (AAV, lentivirus, adenovirus, etc.), non-peptide compounds, synthetic compounds, synthetic low molecular weight compounds, natural compounds, cell extracts, extracellular vesicles, plant extracts, animal tissue extracts, plasma, extracts derived from marine organisms, cell culture supernatants, and microbial fermentation products.

[0062] Test substances can also be obtained using any of the many approaches to combinatorial library technology known in the art, including (1) biological library technology, (2) synthetic library technology using deconvolution, (3) one-bead one-compound library technology, and (4) synthetic library technology using affinity chromatography selection. While the biological library technology using affinity chromatography selection is limited to peptide libraries, other approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12:145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al. (1994) J. Med. Chem. 37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et. al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al. (1994) J. Med. Chem. 37:1233-51).Compound libraries can be stored in solution (see Houghten (1992) Bio / Techniques 13:412-21) or on beads (Lam (1991) Nature 354:82-4), chips (Fodor (1993) Nature 364:555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-9), or phage (Scott and Smith (1990) Science 249:386-90; Devlin (1990) Science 249:404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-82; Felici (1991) J. Mol. Biol. 222:301-10; U.S. Patent Application Publication No. 2002 / 0103360).

[0063] The contact between the three-dimensional cell culture and the test substance may be carried out by adding the test substance to the culture medium of the three-dimensional cell culture. The contact time is not particularly limited as long as a change in the indicator can be confirmed, but may be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. The concentration of the test substance to be added can be appropriately adjusted depending on the properties of the compound (solubility, toxicity, etc.).

[0064] The culture medium for the three-dimensional cell culture used when contacting the three-dimensional cell culture with the test substance is not particularly limited as long as it is a medium capable of culturing the three-dimensional cell culture.

[0065] The culture temperature when contacting the test substance with the three-dimensional cell culture is, but not limited to, about 30 to 40°C, preferably about 37°C, and CO ２ The culture was carried out under an atmosphere of CO ２ The concentration is preferably about 2-5%.

[0066] The present invention will be specifically explained by the following examples, but the present invention is not limited to the scope of the examples.

[0067] <Preparation of three-dimensional co-culture> Human iPS cell-derived astrocyte precursor cells (XCell Science, XCS-AP-001-1V) were seeded onto a flask coated with Matrigel basement membrane matrix (Corning, 354234) and cultured for 5 days (37°C, 5% CO) using differentiation-inducing medium. ２ ) to induce differentiation into astrocytes.

[0068]

[0069] The differentiated astrocytes were detached using TrypLE™ Select (Thermo Fisher Scientific, 12563-029) and centrifuged at 600 × g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and the cells were suspended in 1 mL of co-culture medium and counted.

[0070] Neurons generated by forced expression of the Ngn2 gene from iPS cells (WO2014 / 148646; and Zhang Y, et al., Neuron, 78:785-98, 2013) were thawed in a 37°C water bath. After thawing, the cells were added to co-culture medium and centrifuged at 600 x g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and the cells were suspended in 1 mL of co-culture medium and counted.

[0071] iPS cell-derived microglia (iCell® Microglia, FCDI, C1110) were thawed in a 37°C water bath. After thawing, the cells were added to co-culture medium and centrifuged at 600 × g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and the cells were suspended in 1 mL of co-culture medium and counted.

[0072] Astrocytes, neurons, and microglia were mixed at a cell number ratio of 10:6:3 (53%:32%:16%), and 1.9 × 10 ４ The cells were seeded onto a 96-well plate (PrimeSurface (registered trademark) Plate 96U, Sumitomo Bakelite, MS-9096U) at 37°C and 5% CO ２ The cells were cultured under these conditions, and half of the medium was replaced three times a week.

[0073]

[0074] <Cell fixation and immunostaining> Three-week-cultured three-dimensional co-cultures were fixed by treatment with 4% paraformaldehyde / phosphate buffer (Fujifilm Wako Pure Chemical Industries, Ltd., 161-20141) for 30 minutes. After washing with PBS(-), permeabilization was performed by treatment with Triton® X-100 solution (BioVision, 2104-100) diluted to 0.4% with PBS(-) for 15 minutes. After permeabilization, blocking was performed by treatment with BSA solution (Sigma Aldrich, A416) adjusted to a concentration of 4% with PBS(-) for 30 minutes. Subsequently, as the primary antibody reaction, the sections were treated with 600-fold diluted anti-MAP2 antibody (Novus, NB300-213), 600-fold diluted anti-IBA1 antibody (Fujifilm Wako Pure Chemical Industries, 019-19741), and 3000-fold diluted anti-GFAP antibody (Merck, MAB3402), and left to stand overnight at 4°C.

[0075] The next day, after washing with PBS(-), secondary antibody reactions were performed using 1000-fold diluted Goat anti-rabbit Alexa Fluor® 488 (Thermo Fisher Scientific, A11008), 1000-fold diluted Goat anti-Chicken Alexa Fluor® 594 (Thermo Fisher Scientific, A11042), 1000-fold diluted Goat anti-Mouse Alexa Fluor® 647 (Thermo Fisher Scientific, A32728), and 1000-fold diluted Hoechst® 33342. The cells were treated with PBS solution (Dojindo, H342) and left to stand at room temperature for 60 minutes. After washing with PBS(-), images were captured using a confocal quantitative image cytometer CQ1 (Yokogawa Electric Corporation, CellVoyager® CQ1). Figure 1 shows the captured images. (A) shows nuclei (Hoechst), (B) shows microglia (IBA1), (C) shows neurons (MAP2), and (D) shows astrocytes (GFAP). Signals were detected in all cells. Furthermore, astrocytes, neurons, and microglia were confirmed to be distributed throughout the core and surface of the 3D co-culture.

[0076] <Evaluation of neural activity> A working solution and a recording solution were prepared as follows.

[0077]

[0078]

[0079] Three-dimensional co-cultures prepared with a ratio of neurons, astrocytes, and microglia of 5:85:10, 80:10:10, or 60:30:10 were cultured for three weeks, and then an equal volume of working solution was added to the culture medium and incubated at 37°C for one hour. The cells were then washed with PBS(-), and an equal volume of recording solution was added to the culture medium. Green fluorescent images of Calibryte™ 520 AM were then acquired using a CQ1 confocal quantitative image cytometer (3 images / second, 2 minutes). Fluorescence intensity was then quantified over time using ImageJ (NIH, version 1.50i). The results are shown in Figure 2. In three-dimensional co-cultures with a 5% neuronal ratio (Figure 2A), calcium oscillations (repeated, synchronized influx of calcium ions), an indicator of neural activity, were weak and unclear. On the other hand, in a 3D co-culture with 80% neurons and 10% astrocytes (Figure 2B), clear calcium oscillations were observed, but at irregular intervals. In a 3D co-culture with 60% neurons and 30% astrocytes (Figure 2C), regular and clear calcium oscillations were observed. These results demonstrate that 3D co-cultures with a neuronal ratio of 10% or more and an astrocyte ratio of 30% or more are suitable for evaluating the intensity and frequency of calcium oscillations.

[0080] <Cellular interactions during neuroinflammation> Three-dimensional co-cultures cultured for 3 weeks were treated with lipopolysaccharide (LPS) from E. coli O127 (Fujifilm Wako Pure Chemical Industries, Ltd., 124-05151) to a final concentration of 100 ng / mL, and fixed by treatment with 4% paraformaldehyde phosphate buffer (Fujifilm Wako Pure Chemical Industries, Ltd., 161-20141) for 30 minutes after 0, 0.5, 1, 3, 6, and 24 hours. After washing with PBS(-), permeabilization was performed by treatment with Triton (registered trademark) X-100 solution (BioVision, 2104-100) diluted to 0.4% with PBS(-) for 15 minutes. After permeabilization, the sections were blocked by treating them with a 4% BSA solution (Sigma Aldrich, A416) in PBS(-) for 30 minutes. Then, as the primary antibody reaction, they were treated with a 1000-fold diluted anti-IBA1 antibody (Fujifilm Wako Pure Chemical Industries, Ltd., 011-27991) and an 800-fold diluted anti-NF-κB p65 antibody (Cell Signaling, 8242), and left overnight at 4°C. The next day, after washing with PBS(-), the sections were treated with 1000-fold diluted Donkey anti-goat Alexa Fluor (registered trademark) 488 (Thermo Fisher Scientific, A11055), 1000-fold diluted Donkey anti-rabbit Alexa Fluor (registered trademark) 594 (Thermo Fisher Scientific, A32754), and 1000-fold diluted Hoechst (registered trademark) 33342 solution (Dojindo, H342) as a secondary antibody reaction, and allowed to stand at room temperature for 60 minutes. After washing with PBS(-), images are taken using a confocal quantitative image cytometer CQ1 (Yokogawa Electric Corporation, CellVoyager (registered trademark) CQ1) to obtain images.

[0081] By observing changes in the nuclear localization of NF-κB after LPS treatment, it can be confirmed that activation of microglia affects other cells, that is, that cell-cell interactions occur.

Claims

1. A method for producing a three-dimensional cell culture, comprising the steps of adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel prior to co-culturing cells, and co-culturing the astrocytes, the neurons, and the microglia in the culture vessel, wherein the proportion of the astrocytes among the total cells added to the culture vessel is 30% or more.

2. The manufacturing method according to claim 1, wherein the astrocytes, the nerve cells, and the microglia are added to a culture vessel simultaneously.

3. The manufacturing method according to claim 1, wherein the step of adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel prior to co-culturing the cells comprises the steps of suspending the human-derived astrocytes, human-derived neurons, and human-derived microglia in a culture medium, and simultaneously adding the culture medium containing the astrocytes, neurons, and microglia obtained as described above to the culture vessel.

4. The manufacturing method described in claim 1, wherein the astrocytes, neurons, and microglia are differentiated from human-derived pluripotent stem cells.

5. The manufacturing method described in claim 4, wherein the human-derived pluripotent stem cells are human iPS cells.

6. A manufacturing method described in any one of claims 1 to 5, wherein the proportion of said microglia among all cells added to the culture vessel is 50% or less.

7. A manufacturing method described in any one of claims 1 to 5, wherein the proportion of said nerve cells among all cells added to the culture vessel is 10% or more.

8. The method according to any one of claims 1 to 5, wherein the three-dimensional cell culture is in the form of a spheroid.

9. A method for culturing a three-dimensional cell culture, comprising the steps of adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel prior to co-culturing the cells, and co-culturing the astrocytes, the neurons, and the microglia in the culture vessel, wherein the proportion of the astrocytes among the total cells added to the culture vessel is 30% or more.

10. The culture method according to claim 9, wherein the astrocytes, the nerve cells, and the microglia are added to a culture vessel simultaneously.

11. The culture method according to claim 9, wherein the step of adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel prior to co-culture of the cells comprises the steps of suspending the human-derived astrocytes, human-derived neurons, and human-derived microglia in a culture medium, and simultaneously adding the culture medium containing the astrocytes, neurons, and microglia obtained as described above to the culture vessel.

12. The culture method according to claim 9, wherein the astrocytes, neurons, and microglia are differentiated from human-derived pluripotent stem cells.

13. The culture method according to claim 12, wherein the human-derived pluripotent stem cells are human iPS cells.

14. A culture method according to any one of claims 9 to 13, wherein the proportion of said microglia among all cells added to the culture vessel is 50% or less.

15. A culture method according to any one of claims 9 to 13, wherein the proportion of said nerve cells among all cells added to the culture vessel is 10% or more.

16. The culture method according to any one of claims 9 to 13, wherein the three-dimensional cell culture is in the form of a spheroid.

17. A three-dimensional cell culture comprising human astrocytes, human neurons, and human microglia, obtained by the manufacturing method according to any one of claims 1 to 5.

18. A method for evaluating a test substance, comprising contacting the three-dimensional cell culture according to claim 17 with the test substance.

19. A method for evaluating a test substance, comprising producing a three-dimensional cell culture by the production method described in any one of claims 1 to 5, and contacting the three-dimensional cell culture with the test substance.

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