Cell structure, culture device, and method for producing cell structure

Abstract

To provide a cell structure in which at least a part of a syncytiotrophoblast cell is present in a region contacting the outside, a culture device including the cell structure, and a method for manufacturing the cell structure.SOLUTION: A cell structure includes a syncytiotrophoblast cell and a cell having differentiation potency to the syncytiotrophoblast cell, where at least a part of the syncytiotrophoblast cell is present in a region contacting the outside in the cell structure. Further, a culture device includes the cell structure, culture medium, and a culture substrate. Furthermore, a method for manufacturing the cell structure is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cell structure, a culture device, and a method for producing a cell structure. [Background technology]

[0002] The human placenta is an important organ that not only produces hormones but also facilitates the exchange of nutrients, oxygen, waste products, and other substances between the mother and fetus. In the placenta, maternal and fetal blood do not mix but are separated by the blood-placental barrier, through which material exchange occurs. This blood-placental barrier is comprised of syncytiotrophoblast cells (ST cells), a type of trophoblast cell that constitutes the placental villi. Research into the morphogenesis of the blood-placental barrier and ST cells is crucial for understanding the effects of drugs or pathogens on the fetus and placental diseases. However, there is currently no suitable model for studying the human placenta, which is one of the reasons why research on the human placenta and ST cells has not progressed.

[0003] To accurately study the human placenta, a model must be constructed using human placental cells. This is because placenta differs between species, and placental cells derived from laboratory animals differ in type and function from human placental cells. However, there is a limited number of human placentas available for research, and human placental cells are generally not easy to obtain. For this reason, cancer cells derived from human choriocarcinoma (e.g., BeWo cells), which are easy to obtain and handle, have been used in human placenta research. However, these cancer cells have properties different from those of placental cells in vivo, making it difficult to create a human placenta model that mimics the living body using these cancer cells.

[0004] Recently, it has been reported that trophoblast stem cells (TS cells) have been successfully established from cytotrophoblast cells isolated from human placenta (Patent Document 1). These cells have the ability to self-renew over a long period of time and to differentiate into ST cells and extravillous trophoblast cells (EVT cells). EVT cells are responsible for reconstructing the blood vessels of the placenta.

[0005] In recent years, attempts have been made to create three-dimensional cell structures called organoids that resemble in vivo tissue structures and use them as in vivo tissue models. For the human placenta, examples have been reported in which placental organoids were created using trophoblast cells isolated from the placenta (Non-Patent Documents 1 and 2). However, the placental organoids reported so far have a structure in which ST cells are present inside the organoid, which differs from the in vivo villous structure. In the in vivo villous structure, ST cells are present on the outside. Therefore, they have not been suitable as placental models for drug discovery research or developmental research. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6400832 [Non-patent literature]

[0007] [Non-Patent Document 1] Haider, S., et al., Self-Renewing Trophoblast Organoids Recapitulate the Developmental Program of the Early Human Placenta. Stem Cell Reports 11, 537-551 (2018). [Non-patent document 2] Turco, MY, et al., Trophoblast organoids as a model for maternal-fetal interactions during human placentation. Nature 564, 263-267 (2018). Summary of the Invention [Problem to be solved by the invention]

[0008] Previously reported placental organoids have a structure in which ST cells are present inside the organoid, which differs from the placental villi structure in vivo. Therefore, they may behave differently from the placenta in vivo. To simulate placental behavior in vitro, placental organoids with ST cells present on the outside, as in vivo, are required.

[0009] Therefore, an object of the present invention is to provide a cell structure in which at least a portion of ST cells are present in an area in contact with the outside, a culture device containing the cell structure, and a method for producing the cell structure. [Means for solving the problem]

[0010] The present invention includes the following aspects. [1] Syncytiotrophoblast cells and cells capable of differentiating into syncytiotrophoblast cells, A cell structure comprising: at least a portion of the syncytiotrophoblast cells present in a region of the cell structure that is in contact with the outside. [2] The cell structure described in [1], wherein the syncytiotrophoblast cells are SDC1-positive. [3] The cell structure described in [1] or [2], wherein the cells capable of differentiating into syncytiotrophoblast cells have at least one characteristic selected from the group consisting of E-cadherin positive, GATA2 positive, GATA3 positive, TFAP2 positive, ELF5 positive, ZNF750 positive, and CDX2 negative. [4] A cell structure described in any one of [1] to [3], wherein the cell structure is a free cell aggregate and at least a portion of the syncytiotrophoblast cells are present in the outer layer of the cell aggregate. [5] The cell structure described in [4], wherein the cell aggregate comprises a core containing cells capable of differentiating into the syncytiotrophoblast cells, and a shell located on the outer layer of the core and containing the syncytiotrophoblast cells. [6] A cell structure described in any one of [1] to [3], further comprising a scaffold material, wherein the scaffold material, a cell layer containing cells capable of differentiating into the syncytiotrophoblast cells, and a cell layer containing the syncytiotrophoblast cells are present in this order. [7] A culture device comprising the cell structure according to any one of [1] to [6], a culture medium, and a culture substrate. [8] The culture device according to [7], wherein the culture substrate has a well, and the cell structure is cultured within the well. [9] The culture device according to [7], wherein the culture substrate has a flow path, and the cell structure is held within the flow path.

[10] A method for producing a cell structure described in [4] or [5], comprising a step of culturing cells capable of differentiating into syncytiotrophoblast cells in a well having a non-cell-adhesive inner wall.

[11] The method according to

[10] , wherein the diameter of the opening of the well is 100 to 1500 μm.

[12] A method for producing a cell structure described in [6], comprising the steps of: preparing a flow path device in which a first flow path and a second flow path are separated by a scaffold; and culturing cells capable of differentiating into syncytiotrophoblast cells, held in the scaffold, while perfusing a culture medium through each of the first flow path and the second flow path. [Effects of the Invention]

[0011] According to the present invention, there are provided a cell structure in which at least a portion of ST cells are present in an area in contact with the outside, a culture device containing the cell structure, and a method for producing the cell structure. [Brief explanation of the drawings]

[0012] [Figure 1A] FIG. 1 is a schematic diagram of a cell structure containing ST cells prepared by a conventional method. [Figure 1B] FIG. 1 is a schematic diagram of a cross section of a villi in vivo. [Figure 2] FIG. 1 is a diagram showing a schematic diagram of an example of a cell structure that is a free aggregate. [Figure 3] FIG. 1 is a diagram schematically illustrating an example of a cell structure including a scaffold material. [Figure 4] FIG. 1 is a diagram schematically illustrating an example of a cell structure including a scaffold material. [Figure 5] FIG. 1 is a diagram schematically illustrating an example of a cell structure including a scaffold material, which mimics in vivo villi. [Figure 6A] An example of application of a cell structure according to one embodiment of the present invention to an infection test with a pathogenic microorganism will be shown. [Figure 6B] An example of application of a cell structure according to one embodiment of the present invention to the production of a placental pathological model is shown. [Figure 6C] 1 shows an example of application of a cell structure according to one embodiment of the present invention to the production of a placental development model. [Figure 7] 1A and 1B are diagrams showing an example of a method for producing a cell structure, which is a free cell aggregate. (A) is a perspective view of a well plate. (B) is a schematic cross-sectional view of a well plate. (C) is a photograph showing an example of cells seeded in the wells of a well plate. [Figure 8A] FIG. 1 is a schematic diagram showing an example of a flow channel device used to prepare a cell structure including a scaffold material. [Figure 8B] 8B is a cross-sectional view of the flow channel device 201 of FIG. 8A taken along the line BB. [Figure 9] 8A and 8B. FIG. [Figure 10A] FIG. 2 is a diagram for schematically explaining a method of perfusion culture using a flow channel device 201. [Figure 10B] 10B is a cross-sectional view of the flow channel device 201 shown in FIG. 10A taken along the line BB at the start of perfusion culture. [Figure 11] FIG. 1 is a schematic diagram showing an example of a method for producing a flow channel device used in manufacturing a cell structure including a scaffold material. [Figure 12A] 12 shows a top view of the flow channel device 301 of FIG. [Figure 12B] 12B is an enlarged view of a portion B of the flow channel device 301 shown in FIG. 12A surrounded by a dashed line. [Figure 12C] 12B is a cross-sectional view taken along line CC. [Figure 13A] These are fluorescence microscope images of the cell structure prepared in Example 1. "SCD1" is an image stained with SCD1, a positive marker for ST cells, "E-cadherin" is an image stained with E-cadherin, a positive marker for cells differentiating into ST cells, "Nucleus" is an image stained with nuclei, and "Merged" is an image obtained by merging these images (same below). [Figure 13B] 1 shows a fluorescence microscope image of the cell structure prepared in Example 2. [Figure 14] 1 is a photograph of the microfluidic device used in Example 3. [Figure 15] 1 shows a fluorescence microscope image of the cell structure prepared in Example 3. This is the cell structure on day 8 of culture. [Figure 16] Fluorescence microscopy images of the cell constructs prepared in Example 3. These are cell constructs on day 13 of culture. The left image shows the cell constructs that were perfused with medium (perfusion (+)), and the right image shows the cell constructs that were not perfused with medium (perfusion (-)). [Figure 17] 1 shows a confocal microscope image showing a cross section of the cell structure prepared in Example 3. The arrow in the Merged image indicates the ST cell layer. DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and redundant descriptions are omitted. The dimensional ratios in each figure are exaggerated for the purpose of explanation and do not necessarily match the actual dimensional ratios.

[0014] [Cell structure] In one embodiment, the present invention provides a cell structure comprising syncytiotrophoblast cells and cells having the ability to differentiate into syncytiotrophoblast cells, wherein at least a part of the syncytiotrophoblast cells is present in a region contacting the outside in the cell structure.

[0015] The "cell structure" means a structure formed by the aggregation of multiple cells. The cell structure may be composed only of cells or may include components other than cells. The cell structure according to the present embodiment includes at least syncytiotrophoblast cells (ST cells) and cells having the ability to differentiate into ST cells (hereinafter also referred to as "ST cell-differentiating cells").

[0016] <Syncytiotrophoblast cells (ST cells)> ST cells are cells that constitute the syncytiotrophoblast. ST cells do not undergo mitosis and form the syncytiotrophoblast by fusing with each other. Examples of marker proteins of ST cells include Syndecan 1 (SCD1) and human chorionic gonadotropin (hCG). The ST cells included in the cell structure according to the present embodiment are differentiated from ST cell-differentiating cells described later. The ST cells included in the cell structure according to the present embodiment may fuse with each other to form a syncytium.

[0017] <Cells having the ability to differentiate into ST cells (ST cell-differentiating cells)> <0000The cell structure according to this embodiment includes cells capable of differentiating into ST cells (ST cell-differentiating cells). The ST cell-differentiating cells are not particularly limited as long as they have the ability to differentiate into ST cells. Examples of ST cell-differentiating cells include trophoblast stem cells (TS cells) and cytotrophoblast cells (CT cells). Marker proteins for ST cell-differentiating cells include E-cadherin, CD49f, GATA2, GATA3, TFAP2, ELF5, and ZNF750. ST cell-differentiating cells may have at least one characteristic selected from the group consisting of E-cadherin-positive, GATA2-positive, GATA3-positive, TFAP2-positive, ELF5-positive, ZNF750-positive, and CDX2-negative. "Positive" means that the protein is substantially detected. "Substantially detected" means that the protein can be detected using a conventional protein detection method (e.g., immunostaining). Alternatively, a protein may be determined to be "positive" when mRNA encoding the protein is detected by a commonly used mRNA detection means (e.g., RT-PCR). "Negative" means that the protein is not substantially detected. Cells that are not positive for a certain protein are considered to be negative for the protein.

[0018] The organisms from which ST cells and ST cell-differentiating cells are derived are not particularly limited as long as they are animals with a placenta. Examples of animals from which ST cells and ST cell-differentiating cells are derived include mammals such as primates, rodents, and carnivores. Mammals are preferably primates. Examples of primates include humans, chimpanzees, rhesus monkeys, and marmosets. It is more preferable that the ST cells and ST cell-differentiating cells are human cells.

[0019] <Structure of cell structures> In the cell structure of this embodiment, at least a portion of the ST cells are present in the region of the cell structure that is in contact with the outside. "Outside" refers to the external environment of the cell structure. For example, when the cell structure is maintained in a culture medium, the external environment of the cell structure is the culture medium. The region of the cell structure that is in contact with the outside is a cell layer that separates the cell structure from the external environment, and can also be said to be the outermost layer of the cell structure. In the cell structure of this embodiment, it is preferable that the outermost layer of the cell structure is composed mainly of ST cells. For example, it is preferable that 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the cells that make up the outermost layer are composed of ST cells.

[0020] A reported method for producing cell structures containing ST cells involves culturing human trophoblast cells isolated from the placenta in Matrigel and inducing their differentiation into ST cells (Haider, S., et al., Stem Cell Reports 11, 537, 2018; Turco, M.Y., et al., Nature 564, 263, 2018). However, in the conventional Matrigel-based method, ST cells reside within the undifferentiated cell layer (see Figure 1A). In other words, ST cells are not present in the area of ​​the cell structure that contacts the outside. In vivo villous structures are structured such that ST cells reside in the outer layer (the area that contacts the outside) and cells that differentiate into ST cells, such as CT cells, reside inside (see Figure 1B). Therefore, conventional cell structures do not reproduce the in vivo villous structure.

[0021] On the other hand, in the cell structure of this embodiment, ST cells are present in the region in contact with the outside. Therefore, the structure is closer to a villus structure than conventional cell structures. In the present invention, by using the method described below, it is possible to produce such a cell structure that is closer to a villus structure.

[0022] (First embodiment) The cell structure of this embodiment may be a free cell aggregate. When the cell structure of this embodiment is a free cell structure, at least a portion of the ST cells are present in the outer layer of the cell aggregate.

[0023] "Cell aggregate" refers to a structure formed by the aggregation of multiple cells. "Free" means that the cell aggregate is not attached to a scaffold or culture substrate. A free cell aggregate can also be said to be a spheroid cell aggregate.

[0024] Figure 2 shows an example of a cell structure that is a free cell aggregate. The cell structure 100 shown in Figure 2 is composed of a cell layer L11, which is the region in contact with the outside, and a cell layer L12 inside the cell layer L11. The cell layer L11 contains ST cells 10. The cell layer L12 contains ST cell-differentiating cells 20.

[0025] The cell layer L12 containing the ST cell differentiating cells 20 can be said to be the core of the cell structure 100. The cell layer L11 containing the ST cells 10 can be said to be a shell located on the outer layer of the core. In other words, the cell structure 100 can be said to include a core containing the ST cell differentiating cells 20 and a shell located on the outer layer thereof containing the ST cells 10.

[0026] The cell layer L11 is mainly composed of ST cells 10, but may also contain other cells. Examples of other cells that the cell layer L11 may contain include ST cell-differentiating cells. Preferably, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the number of cells constituting the cell layer L11 are composed of ST cells 10. Alternatively, it is preferable that 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the volume of the cell layer L11 is composed of ST cells 10. The cell layer L11 may be a single cell layer or may be a multi-layer cell layer. The cell layer L11 may be a single cell layer composed of ST cells 10.

[0027] The cell layer L12 is mainly composed of ST cell-differentiating cells 20, but may also contain other cells. Examples of other cells that may be contained in the cell layer L12 include ST cells, fibroblasts, and umbilical vein endothelial cells. The cell layer L12 may also contain ST cells. However, it is preferable that the number of ST cells is 40% or less, 30% or less, 20% or less, or 10% or less of the number of cells that constitute the cell layer L12. Alternatively, it is preferable that the volume occupied by ST cells is 40% or less, 30% or less, 20% or less, or 10% or less of the volume of the cell layer L12.

[0028] The cell structure 100 is typically a spherical cell aggregate. The size of the cell structure 100 is not particularly limited. The size of the cell structure 100 can be appropriately set depending on the size of the wells of the well plate used in the production method described below. The maximum diameter of the cell structure 100 may be, for example, approximately 50 to 3000 μm. The maximum diameter of the cell structure 100 can be, for example, 100 μm or more, 150 μm or more, 200 μm or more, 250 μm or more, or 300 μm or more. The maximum diameter of the cell structure 100 can be, for example, 2000 μm or less, 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, or 500 μm or less.

[0029] (Second embodiment) The cell structure of this embodiment may include a scaffold. When the cell structure of this embodiment includes a scaffold, the cell structure contains the scaffold, a cell layer containing ST cell differentiating cells, and a cell layer containing ST cells, in this order.

[0030] The term "scaffold" refers to a substance that serves as a base for cells to adhere and form cell aggregates. The scaffold is not particularly limited as long as it can serve as a base for cells to form cell aggregates. Preferably, the scaffold has cell adhesive properties and low cytotoxicity. Examples of scaffolds include, but are not limited to, extracellular matrices such as collagen (type I, type II, type III, type V, type XI, etc.), gelatin, elastin, proteoglycan, glycosaminoglycan, fibronectin, vitronectin, laminin, pectin, hyaluronic acid, chitin, and chitosan; cell-adhesive polysaccharides such as alginic acid and starch; and cell-adhesive amino acid polymers such as polylysine and polyarginine. Alternatively, commercially available scaffolds such as Matrigel® (Corning) may be used. The scaffold may be a gel of the above-mentioned materials or their derivatives. The scaffold material may be a three-dimensional structure formed from the scaffold material, or the surface of a culture substrate or the like may be coated with the scaffold material, or a sheet-like scaffold material may be used, or a sheet-like scaffold material may be fixed to a support or the like.

[0031] FIG. 3 shows an example of a cell structure including a scaffold material. The cell structure 200 shown in FIG. 3 is composed of a scaffold material 250, a cell layer L22 containing ST cell-differentiating cells 20, and a cell layer L21 containing ST cells 10. The cell layer L22 is located adjacent to the scaffold material 250, and the cell layer L21 is located adjacent to the cell layer L22. The cell layer L22 contains ST cell-differentiating cells 20. The cell structure 200 can also be said to have a structure in which the cell layer L22 is located between the scaffold material 250 and the cell layer L21. When the scaffold material 250 is placed on the bottom side, the cell layer L21 is located in the uppermost layer of the cell structure 200.

[0032] The scaffold 250 has a sheet-like shape in the cell structure 200. Examples of such a scaffold 250 include gel films of the substances listed above as examples of the scaffold. The gel film of the scaffold 250 may be fixed to a support or the like. The material of the support for fixing the gel film of the scaffold 250 is not particularly limited, but is preferably one with low cytotoxicity. Examples of materials for the support include, but are not limited to, polyimide, polyethylene terephthalate (PET), polystyrene, polyethylene, polypropylene, nylon, polytetrafluoroethylene (PTFE), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-hexafluoropropylene copolymer (FEP), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), polydimethylsiloxane (PDMS), and the like. The support may be, for example, a resin film formed from the above-mentioned materials. The method for fixing the gel film of the scaffold 250 to the support is not particularly limited. For example, the gel film of the scaffold 250 can be fixed to the support using a fixing material such as adhesive tape. The fixing material used for fixing is not particularly limited, but is preferably one with low cytotoxicity. Examples of the material for the fixing material include the same materials as those used for the support. In the example of the cell structure 200, the scaffold 250 is in a sheet shape, but the shape of the scaffold 250 is not limited to a sheet shape. The scaffold 250 may have, for example, any three-dimensional structure.

[0033] The cell layer L22 is a cell layer containing ST cell differentiating cells 20. The cell layer L22 is mainly composed of ST cell differentiating cells 20, but may also contain other cells. Examples of other cells that may be contained in the cell layer L22 include ST cells, fibroblasts, and umbilical vein endothelial cells. Preferably, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the number of cells constituting the cell layer L22 are ST cell differentiating cells 20. Alternatively, preferably, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the volume of the cell layer L22 is composed of ST cell differentiating cells 20. The cell layer L22 may contain ST cells. However, it is preferable that ST cells account for 40% or less, 30% or less, 20% or less, or 10% or less of the number of cells constituting the cell layer L22. Alternatively, the volume occupied by ST cells is preferably 40% or less, 30% or less, 20% or less, or 10% or less of the volume of the cell layer L22. 3, the cell layer L22 is a single cell layer, but the cell layer L22 may include multiple cell layers. Furthermore, the cell layer L22 may form any three-dimensional structure.

[0034] The cell layer L21 is a cell layer containing ST cells 10. The cell layer L21 is mainly composed of ST cells 10, but may also contain other cells. Examples of other cells that the cell layer L21 may contain include ST cell-differentiating cells. The cell layer L21 is preferably composed of ST cells 10, with 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the number of cells constituting the cell layer L21 being ST cells 10. Alternatively, it is preferable that 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the volume of the cell layer L21 be composed of ST cells 10. The cell layer L21 may be a single cell layer or may be a multi-layer cell layer. The cell layer L21 is preferably a single cell layer composed of ST cells 10, for example.

[0035] The size of the cell structure 200 is not particularly limited. The size of the cell structure 200 can be appropriately set depending on the size of the channel of the channel device used in the fabrication method described below. The width of the cell structure 200 may be, for example, approximately 100 to 5000 μm. The width of the cell structure 200 can be, for example, 150 μm or more, 200 μm or more, 250 μm or more, or 300 μm or more. The width of the cell structure 200 can be, for example, 4000 μm or less, 3000 μm or less, 2000 μm or less, 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, or 500 μm or less. The thickness of the cell structure 200 may be, for example, approximately 100 to 5000 μm. The thickness of the cell structure 200 can be, for example, 150 μm or more, 200 μm or more, 250 μm or more, or 300 μm or more. The thickness of the cell structure 200 can be, for example, 4000 μm or less, 3000 μm or less, 2000 μm or less, 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, or 500 μm or less.

[0036] <<Variation 1>> Fig. 4 shows a modified example of a cell structure including a scaffold. The cell structure 300 shown in Fig. 4 is present in a channel 311 formed in a first substrate 310. In the cell structure 300, a scaffold 350 is present in a central channel compartment 311C defined by microposts 312 and 313. On one side of the scaffold 350, the scaffold 350, a cell layer L32 containing ST cell differentiating cells 20, and a cell layer L31 containing ST cells 10 are present in this order. Furthermore, on the other side of the scaffold 350, a cell layer L33 containing cells 30 is present.

[0037] Microposts 312 and 313 are arranged in the flow channel 311 in which the cell structure 300 exists. The microposts 312 and 313 have the function of dividing the flow channel 311 into three flow channel compartments: flow channel compartment 311L, flow channel compartment 311C, and flow channel compartment 311R. The microposts 312 and 313 are arranged in the flow channel 311 at intervals that prevent the scaffold material 350 in the flow channel compartment 311C from flowing out of the compartment and allow the cells of the cell layer L31 and the cell layer L33 to adhere to the scaffold material 350.

[0038] The scaffold 350 is present in the flow channel section 311C. Examples of the scaffold 350 include the same scaffolds as those listed above. For example, an extracellular matrix gel can be used as the scaffold 350. A specific example of the extracellular matrix gel is collagen gel.

[0039] The cell layer L32 is a cell layer containing ST cell differentiating cells 20. The cell layer L32 is located adjacent to one side of the scaffold 350. The cell layer L32 is mainly composed of ST cell differentiating cells 20 but may contain other cells. Examples of other cells that may be contained in the cell layer L32 include ST cells, fibroblasts, and umbilical vein endothelial cells. Preferably, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the number of cells constituting the cell layer L32 are composed of ST cell differentiating cells 20. Alternatively, preferably, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the volume of the cell layer L32 is composed of ST cell differentiating cells 20. The cell layer L32 may contain ST cells. However, it is preferable that ST cells account for 40% or less, 30% or less, 20% or less, or 10% or less of the number of cells constituting the cell layer L32. Alternatively, it is preferable that the volume occupied by ST cells is 40% or less, 30% or less, 20% or less, or 10% or less of the volume of the cell layer L32. 5, the cell layer L32 is a single cell layer, but the cell layer L32 may include multiple cell layers. Furthermore, the cell layer L32 may form any three-dimensional structure.

[0040] The cell layer L31 is a cell layer containing ST cells 10. The cell layer L31 is located adjacent to the cell layer L32. The cell layer L31 is mainly composed of ST cells 10, but may also contain other cells. Examples of other cells that the cell layer L31 may contain include ST cell-differentiating cells. The cell layer L31 is preferably composed of 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the number of cells constituting the cell layer L31, which are ST cells 10. Alternatively, it is preferable that 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the volume of the cell layer L31 is composed of ST cells 10. The cell layer L31 may be a single cell layer or may be a multi-layer cell layer. The cell layer L31 is, for example, a single cell layer composed of ST cells 10.

[0041] The cell layer L33 is a cell layer containing cells 30. The cell layer L33 is a cell layer located adjacent to the other side of the scaffold 350. Any cells can be used as the cells 30 depending on the intended use of the cell structure 300. For example, placenta-derived cells can be used as the cells 30. Examples of such cells include umbilical vein endothelial cells. The cells 30 are preferably derived from the same animal as the animal from which the ST cells 10 and ST cell-differentiating cells 20 are derived. The cells 30 may be one type of cell or a mixture of two or more types of cells. 4, the cell layer L33 is a single cell layer, but the cell layer L33 may include multiple cell layers. In addition, the cell layer L33 may form any three-dimensional structure.

[0042] The cell structure 300 is a cell structure in which a cell layer L33, a scaffold 350, a cell layer L32, and a cell layer L31 exist in this order. The scaffold 350 exists between the cell layer L33 and the cell layer L32. The cell layer L32 exists between the scaffold 350 and the cell layer L31. The cell layer L31 and the cell layer L32 exist in the flow channel section 311R, and the cell layer L33 exists in the flow channel section 311L.

[0043] The channel section 311R, in which the cell layer L31 and the cell layer L32 are present, is filled with culture medium M1. The channel section 311L, in which the cell layer L33 is present, is filled with culture medium M2. The culture medium M1 and the culture medium M2 constitute the external environment of the cell structure 300. In the cell structure 300, the cell layer L31 and the cell layer L33 are in contact with the external environment, that is, the culture medium M1 and the culture medium M2, respectively.

[0044] The culture medium M1 and the culture medium M2 may be the same medium or different media. Examples of culture medium M1 that can be used include an ST cell induction medium and an ST cell maintenance medium. Examples of ST cell induction medium include a medium prepared by adding a ROCK inhibitor (Y27632, etc.), a GSK3β inhibitor (CHIR99021, etc.), a p38 MAPK inhibitor (SB202190, etc.), and a growth factor (EGF, BMP4, bFGF, etc.) to a basal medium for animal cells (e.g., the TS basal medium used in the Examples). Specific examples of such media include the 3D-1 medium, 3D-2 medium, and TS perfusion medium used in the Examples. The culture medium M2 can be appropriately selected depending on the type of cells 30. The culture medium M2 may be any medium capable of maintaining the cells 30. For example, the culture medium M2 may be a basal medium for animal culture supplemented with growth factors (FGFs, BGFs, EGF, etc.), enzyme inhibitors (GSK3 inhibitors, ROCK inhibitors, MAPK inhibitors, etc.), signal transduction inhibitors (Wnt inhibitors, etc.), etc.

[0045] <<Variation 2>> Another modified example of a cell structure including a scaffold material is shown in Figure 5. In the cell structure 300' shown in Figure 5, the scaffold material 350 in the cell structure 300 includes the cells 30 and 40.

[0046] The cells 30 are, for example, umbilical vein endothelial cells, and the cells 30 present in the scaffold material 350 form a three-dimensional structure. The cells 40 are cells present in the scaffold 350, and any cells can be used depending on the intended use of the cell structure 300'. An example of the cells 40 is a fibroblast. As shown in Figure 1B, villi in vivo have a structure in which an extracellular matrix containing blood vessels and fibroblasts is covered with a cell layer containing undifferentiated cells such as CT cells, and an outer cell layer containing ST cells is present. Therefore, by using umbilical vein endothelial cells as cells 30 and fibroblasts as cells 40, a cell structure that mimics villi in vivo can be created.

[0047] <Application example> Compared to conventional cell constructs containing ST cells, the cell construct of this embodiment has a structure that is closer to the structure of villi in vivo, and therefore can be used in a variety of studies, such as placental development research, functional research, drug response evaluation, etc. Figures 6A to 6C show application examples of the cell construct of this embodiment.

[0048] FIG. 6A shows an example of application to an infection test with a pathogenic microorganism. The infection test can be performed by exposing the cell structure of this embodiment to a pathogenic microorganism. Examples of pathogenic microorganisms include microorganisms that cause stillbirth, such as Toxoplasma gondii. Either the cell structure of the first or second embodiment may be used in the infection test.

[0049] 6B shows an example of application to the creation of a placenta pathology model. For example, by creating the cell structure of this embodiment using TS cells derived from a diseased placenta such as hydatidiform mole, a pathology model of the diseased placenta can be created. The cell structure used in the pathology model may be the cell structure of either the first or second embodiment.

[0050] FIG. 6C shows an example of application to the creation of a placental development model. For example, a model of placental development can be created by allowing the cell structure of this embodiment to interact with cells that arise during placental development. The cell structure of this embodiment can be contacted with a cell aggregate containing decidual fibroblasts, for example, to evaluate its interaction with decidual fibroblasts. The cell structure used in the placental development model may be the cell structure of either the first or second embodiment. In vivo, as pregnancy progresses and villi grow, the villi come into contact with the maternal decidua. This contact causes ST cell-differentiating cells, such as CT cells, to emerge from the villi and migrate onto and / or into the decidua, where they come into contact with the decidua. The ST cell-differentiating cells that come into contact with the decidua differentiate into extravillous trophoblast cells (EVT cells) and reconstruct maternal blood vessels within the decidua. As a result of this vascular remodeling, more blood flows into the placenta, allowing more nutrients to be provided to the fetus. Therefore, by allowing the cell structure of this embodiment to interact with a cell aggregate containing decidual fibroblasts that mimics the decidua, a model of placental development can be constructed.

[0051] [Method for producing cell structures] In one embodiment, the present invention provides a method for producing a cell structure according to the above embodiment.

[0052] <Method for producing cell structure of first embodiment> The method for producing the cell structure of the first embodiment includes a step of culturing cells capable of differentiating into syncytiotrophoblast cells in a well having a non-cell-adhesive inner wall.

[0053] Figure 7 is a diagram schematically illustrating an example of a method for producing a cell structure according to the first embodiment. Figure 7(A) shows a well plate 110, which is an example of a culture substrate used in the production method according to this embodiment. Figure 7(B) shows a cross-sectional view of the well plate 110. The well plate 110 has a well 111. The well plate 110 is only required to have at least one well 111, and the number of wells 111 is not particularly limited. Figure 7(C) is a photograph taken from the bottom of the well 111, showing cells seeded in the well 111.

[0054] (well plate) The well 111 has a non-cell-adhesive inner wall. The term "non-cell-adhesive inner wall" refers to an inner wall in which, when cells come into contact with the inner wall, they do not adhere at all, or even if they temporarily adhere, they naturally detach. The entire inner wall (inner surface) of the well 111, including the inner side and inner bottom surfaces, is preferably non-cell-adhesive. The well 111 may be formed of a non-cell-adhesive material, or the inner wall may be coated with a non-cell-adhesive material. Examples of non-cell-adhesive materials include non-cell-adhesive hydrogels. Examples of raw materials for non-cell-adhesive hydrogels include, but are not limited to, non-cell-adhesive polysaccharides such as agarose and cellulose; polyethylene glycol and its derivatives; monomers and polymers thereof, such as 2-methacryloyloxyethyl phosphorylcholine (MPC) and its derivatives, hydroxyethyl methacrylate (HEMA) and its derivatives; segmented polyurethane (SPC) and its derivatives; and non-cell-adhesive proteins such as albumin. Among these, non-cell-adhesive polysaccharides are preferred, and agarose is more preferred, due to their high biocompatibility and high substance permeability.

[0055] The well plate 110 may contain a cell-non-adhesive substance only on the inner walls of the wells 111, or the entire well plate 110 may be formed from a cell-non-adhesive substance. A commercially available cell culture well plate having a cell-non-adhesive inner wall may be used as the well plate 110. Alternatively, the well plate 110 may be produced by gelling a cell-non-adhesive substance such as agarose using a commercially available well plate mold (e.g., MicroTissues 3D petri dish micro-mold; SIGMA).

[0056] The shape of well 111 is not particularly limited, but the shape of the bottom surface is preferably round, U-shaped, or conical. The shape of the opening and cross section of well 111 is preferably circular or elliptical. By forming well 111 in such a shape, spherical cell structures are more likely to be formed.

[0057] The size of the well 111 is not particularly limited. By adjusting the size of the well 111, the size of the cell structure to be formed can be adjusted. The diameter of the opening of the well 111 can be, for example, approximately 50 to 3000 μm. The maximum diameter of the opening of the well 111 can be, for example, 2000 μm or less, 1500 μm or less, 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, or 500 μm or less. When the maximum diameter of the opening of the well 111 is equal to or less than the above upper limit, spherical cell structures are more likely to be formed. The lower limit of the maximum diameter of the opening of the well 111 is not particularly limited as long as it is a size that allows the formation of cell structures. The lower limit of the maximum diameter of the opening of the well 111 can be, for example, 100 μm or more, 150 μm or more, 200 μm or more, 250 μm or more, or 300 μm or more. The maximum diameter of the opening of the well 111 is, for example, preferably 100 to 1500 μm, and more preferably 200 to 1000 μm.

[0058] The depth of well 111 is not particularly limited as long as it is a depth that allows the formation of a cell structure. The depth of well 111 can be, for example, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, or 500 μm or more. The upper limit of the depth of well 111 is not particularly limited, but can be, for example, 10,000 μm or less, 9,000 μm or less, 8,000 μm or less, 7,000 μm or less, 6,000 μm or less, or 5,000 μm or less.

[0059] (Cells with the ability to differentiate into ST cells (ST cell differentiating cells)) In the production method of this embodiment, cells capable of differentiating into ST cells are cultured in well 111. Examples of cells capable of differentiating into ST cells include TS cells and CT cells. TS cells may be induced from blastocysts, CT cells, or pluripotent stem cells. TS cells can be induced from placental cells by known methods. For example, cells can be isolated from placental tissue by appropriate mechanical and / or enzymatic treatment, and then cultured in a medium that induces TS cells. TS cells can then be established using the expression of TS cell markers (e.g., GATA2-positive, GATA3-positive, TFAP2-positive, ELF5-positive, ZNF750-positive, CDX2-negative) as an indicator. As a method for inducing TS cells from CT cells, for example, the method described in Japanese Patent No. 6400832 can be used. Methods for inducing TS cells from pluripotent stem cells include, for example, methods described in International Publication No. 2020 / 250438. CT cells can be isolated from the placenta, for example. CT cells can be isolated from the placenta by, for example, appropriately treating placental tissue mechanically and / or enzymatically to separate the cells, and then isolating the CT cells using the expression of CT cell markers (CD49f positive, E-cadherin positive, etc.) as an indicator (Japanese Patent No. 6400832, Haider, S., et al., Stem Cell Reports 11, 537-551 (2018)).

[0060] TS cells can be maintained and cultured in a TS cell maintenance medium. A known TS cell maintenance medium can be used. Examples of TS cell maintenance media include those prepared by adding a ROCK inhibitor (Y27632, etc.), a growth factor (EGF, etc.), an HDAC inhibitor (valproic acid: VPA, etc.), an ALK5 inhibitor (A83-01, etc.), a GSK3β inhibitor (CHIR99021, etc.), or the like to a basal medium for animal cell culture. Specific examples of TS cell maintenance media include the TS medium used in the Examples described below.

[0061] (Culture medium) The culture medium used to culture cells capable of differentiating into ST cells can be the same medium used to induce ST cells from TS cells (ST cell induction medium). For example, a medium typically used for culturing animal cells can be used as the basal medium, supplemented with a ROCK inhibitor. The culture medium may further contain growth factors, ROCK inhibitors, GSK3β inhibitors, p38 MAPK inhibitors, etc.

[0062] <Basal medium> Examples of basal media include Doulbecco's modified Eagle's Medium (DMEM), DMEM / F12, IMDM, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Ham's F12, RPMI 1640, Fischer's, and mixtures thereof. A preferred basal medium is DMEM / F12.

[0063] The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or serum substitutes, as needed. Examples of serum substitutes include albumin, transferrin, sodium selenite, ITS-X (Invitrogen), Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The basal medium may also contain components, such as lipids, amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth factors, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts, as needed. These components can be used in appropriate combinations.

[0064] The basal medium may be, for example, a medium obtained by adding bovine serum albumin (BSA), ITS-X, L-ascorbic acid, and antibiotics (penicillin, streptomycin, etc.) to the above-mentioned basal medium (e.g., DMEM / F12). A specific example of the basal medium is the TS basal medium used in the Examples described below.

[0065] ≪Growth factors≫ Growth factors are not particularly limited, but examples thereof include epidermal growth factor (EGF), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP).

[0066] EGF binds to EGF receptors (EGFs) present on the cell surface to induce EGF signal transduction and act as a mitogen. There are no particular limitations on the organism from which EGF is derived, but human EGF is preferred. Human FGF may be a recombinant form produced by non-human cells. Commercially available EGF can be used. There are no particular limitations on the concentration of EGF in the medium, but it can be, for example, 5 to 200 ng / mL. The concentration of EGF in the medium is preferably 10 to 150 ng / mL, more preferably 10 to 100 ng / mL, even more preferably 10 to 80 ng / mL, and particularly preferably 10 to 60 ng / mL.

[0067] FGF has a high affinity for heparan sulfate proteoglycans and forms complexes with FGF receptors (FGFRs) on cell surfaces together with heparan sulfate proteoglycans to induce FGF signaling and act as a mitogen. The organism from which FGF is derived is not particularly limited, but human FGF is preferred. Human FGF may also be a recombinant form produced by non-human cells. FGF2 (also known as bFGF) is preferred as FGF. Commercially available FGFs can be used. The concentration of FGF (e.g., FGF2) in the medium is not particularly limited, but can be, for example, 5 to 200 ng / mL. The concentration of FGF (e.g., FGF2) in the medium is preferably 10 to 150 ng / mL, more preferably 20 to 100 ng / mL, even more preferably 30 to 80 ng / mL, and particularly preferably 40 to 60 ng / mL.

[0068] When the medium contains FGF, it may also contain heparin. Heparin has the effect of promoting the activity of FGF. Heparin is preferably in the form of a salt. Examples of heparin salts include salts with alkali metals such as lithium, sodium, and potassium; salts with alkaline earth metals such as calcium, barium, and magnesium; salts with metals such as aluminum, zinc, copper, and iron; ammonium salts; salts with organic bases; and salts with amino acids. Commercially available heparin can be used. The concentration of heparin in the medium is not particularly limited, but can be, for example, 0.001 to 10 μg / mL. The concentration of heparin in the medium is preferably 0.005 to 5 μg / mL, more preferably 0.01 to 3 μg / mL, even more preferably 0.05 to 1 μg / mL, and particularly preferably 0.07 to 0.5 μg / mL.

[0069] BMP is a protein belonging to the transforming growth factor β (TGFβ) superfamily, and controls cell death induction, cell differentiation, etc. The organism from which BMP is derived is not particularly limited, but human BMP is preferred. Human BMP may also be a recombinant form produced by non-human cells. BMP4 is preferred as the BMP. Commercially available BMPs can be used. The concentration of BMP (e.g., BMP4) in the medium is not particularly limited, but can be, for example, 5 to 200 ng / mL. The concentration of BMP (e.g., BMP4) in the medium is preferably 10 to 150 ng / mL, more preferably 20 to 100 ng / mL, even more preferably 30 to 80 ng / mL, and particularly preferably 40 to 60 ng / mL.

[0070] <ROCK inhibitors> ROCK (Rho-associated coiled-coil containing protein kinase: Rho-associated kinase) inhibitors are substances that inhibit the function of Rho-associated kinase. Examples of ROCK inhibitors include trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 1-(5-isoquinolinylsulfonyl)homopiperazine, and salts thereof. Other examples include small molecule inhibitors such as Fasudil / HA1077, H-1152, and Wf-536, as well as derivatives thereof. ROCK inhibitors may be antisense nucleic acids, siRNAs, dominant-negative mutants, or expression vectors thereof against ROCK. Commercially available trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide or a salt thereof includes Y27632 ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl·HO). ROCK inhibitors may be used singly or in combination of two or more. The ROCK inhibitor used is preferably Y27632. The concentration of the ROCK inhibitor in the medium is not particularly limited, but can be, for example, 0.1 to 50 μM, preferably 1 to 20 μM, more preferably 1 to 10 μM, and even more preferably 3 to 8 μM.

[0071] <GSK3β inhibitor> GSK (Glycogen Synthase Kinase) 3β inhibitors are substances that inhibit the functions of GSK3β, such as its kinase activity (for example, its ability to phosphorylate β-catenin). Examples of GSK3β inhibitors include 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile, Kenpaullone, 1-Azakenpaullone, CHIR98014, AR-A014418, CT99021, CT20026, SB216763, AR-A014418, lithium, SB415286, TDZD-8, BIO, BIO-acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopentadienylruthenium complex, and TDZD-8. 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT, alpha-4-dibromoacetophenone, AR-AO 144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione; TWSl 19 pyrrolopyrimidine compound, L803 Examples of suitable GSK-3β inhibitors include H-KEAPPAPPQSpP-NH2 or its myristoylated form; and small molecule inhibitors such as 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, SB216763, and SB415286. GSK-3β inhibitors may be antisense nucleic acids, siRNAs, dominant-negative mutants, and expression vectors thereof against GSK-3β. Commercially available 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile includes CHIR99021. GSK-3β inhibitors may be used singly or in combination of two or more. It is preferable to use CHIR99021 as the GSK3β inhibitor. The concentration of the GSK3β inhibitor in the medium is not particularly limited, but can be, for example, 0.1 to 20 μM, preferably 0.2 to 10 μM, more preferably 0.5 to 5 μM, and even more preferably 0.5 to 3 μM.

[0072] <p38 MAPK inhibitor> p38 MAPK inhibitors are substances that inhibit the function of p38 MAPK (P38 mitogen-activated protein kinase). Examples of p38 MAPK inhibitors include SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole), SB203580 (4-[4-(4-fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-1H-imidazol-5-yl]pyridine), VX702 (6-(N-carbamoyl-2,6-difluoroanilino)-2-(2,4-difluorophenyl)pyridine-3-carboxyamide), VX745 (5-(2,6-dichlorophenyl)-2-[2,4-difluorophenyl)thio]-6H-pyrimidin[ 1,6-b]pyridazine-6-one), PD169316 (4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole), RO4402257 (6-(2,4-difluorophenoxy)-2-{[3-hydroxy-1-(2-hydroxyethyl)propyl]amino}-8-methylpyrido[2,3-D]pyrimidin-7(8h)-one), BIRB796 (1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea), etc. The p38 MAPK inhibitor may be an antisense nucleic acid, siRNA, dominant-negative mutant, or expression vector thereof against p38 MAPK. The p38 MAPK inhibitor may be used alone or in combination of two or more. The p38 MAPK inhibitor used is preferably SB202190. The concentration of the p38 MAPK inhibitor in the medium is not particularly limited, but can be, for example, 0.1 to 20 μM, preferably 0.2 to 10 μM, more preferably 0.5 to 5 μM, and even more preferably 0.5 to 3 μM.

[0073] In addition to the above components, the medium may contain other components as needed.

[0074] Examples of media used for culturing cells differentiated into ST cells include a medium obtained by adding a ROCK inhibitor, EGF, a GSK3β inhibitor, and a p38 MAPK inhibitor to a basal medium, and a medium obtained by adding a ROCK inhibitor, EGF, a GSK3β inhibitor, a p38 MAPK inhibitor, BMP4, FGF2, and heparin to a basal medium. Specific examples of media used for culturing cells differentiated into ST cells include 3D-1 medium, 3D-2 medium, and TS perfusion medium used in the Examples described below.

[0075] (Culture method) As shown in FIG. 7(B), the ST cell differentiating cells are cultured in wells 111 of a well plate 110. For example, the ST cell differentiating cells 20 can be suspended in a medium to form a cell suspension, and the suspension can be seeded in the wells 111. The amount of the cell suspension added to the wells 111 can be adjusted appropriately depending on the size of the wells 111. The cell concentration of the cell suspension is not particularly limited, but may be, for example, 10 4 ~10 8 The medium used to prepare the cell suspension is not particularly limited, but for example, a basal medium or the above-mentioned culture medium can be used.

[0076] After seeding the ST cell differentiating cells 20 in the wells 111, culture medium can be added to the wells 111 to start culturing. The culturing may be performed by placing the well plate 110 in a culture vessel having a size that can accommodate the well plate 110. In this case, the wells 111 can be filled with the culture medium by adding the culture medium to the culture vessel. When the culture medium needs to be replaced during culturing, the culture medium in the culture vessel can be replaced.

[0077] Culture conditions that are commonly used for culturing animal cells can be used. The culture temperature can be, for example, 32 to 40°C, preferably 35 to 38°C. Generally, it can be 37°C. The CO2 concentration can be, for example, about 2 to 5%, generally 5% CO2 concentration. The culture can be static culture.

[0078] To maintain and grow the cells during culture, it is preferable to change the culture medium every 1 to 5 days. The medium change may be performed with the same culture medium or with a different culture medium. For example, the cells may first be cultured in a culture medium (first culture medium) containing a high concentration of a GSK3β inhibitor, and then cultured in a culture medium (second culture medium) containing a lower concentration of a GSK3β inhibitor than the first culture medium. That is, the "step of culturing ST cell differentiating cells in a well having a non-cell-adhesive inner wall" can include the steps of (i) culturing ST cell differentiating cells in a first culture medium containing a GSK3β inhibitor, and (ii) culturing ST cell differentiating cells in a second culture medium containing a GSK3β inhibitor, the second culture medium having a lower concentration of the GSK3β inhibitor than the first culture medium.

[0079] The concentration of the GSK3β inhibitor in the first culture medium can be, for example, 0.8 μM or more, 1.0 μM or more, 1.2 μM, or 1.5 μM or more. The upper limit of the concentration of the GSK3β inhibitor in the first culture medium is not particularly limited, but can be, for example, 10 μM or less, 7 μM or less, 5 μM or less, 4 μM or less, or 3 μM or less. The concentration of the GSK3β inhibitor in the second culture medium can be set according to the concentration of the GSK3β inhibitor in the first culture medium. The concentration of the GSK3β inhibitor in the second culture medium can be, for example, 1.0 μM or less, 0.8 μM or less, 0.7 μM or less, or 0.6 μM or less. The lower limit of the concentration of the GSK3β inhibitor in the second culture medium is not particularly limited, and can be, for example, 0.01 μM or more, 0.05 μM or more, 0.1 μM or more, 0.2 μM or more, or 0.3 μM or more.

[0080] The first culture medium and the second culture medium may contain, in addition to the GSK3β inhibitor, a ROCK inhibitor, growth factors (EGF, FGF2, BMP4, etc.), a p38 MAPK inhibitor, etc. The first culture medium and the second culture medium are, for example, media prepared by adding a GSK3β inhibitor, a ROCK inhibitor, EGF, FGF2, heparin, BMP4, and a p38 MAPK inhibitor to a basal medium (e.g., TS basal medium). A specific example of the first culture medium is the 3D-2 medium used in the Examples described below. A specific example of the second culture medium is the 3D-1 medium used in the Examples described below.

[0081] The culture period in the first culture medium can be, for example, 1 to 5 days from the start of culture. The culture period in the second culture medium can be, for example, 3 days or more after changing the culture medium from the first culture medium to the second culture medium. The culture period in the second culture medium is preferably 5 days or more, more preferably 6 days or more. The upper limit of the culture period in the second culture medium is not particularly limited, and culture can be continued until the cell structure reaches the desired size. For example, the culture period in the second culture medium can be 30 days or less, 25 days or less, 20 days or less, 15 days or less, 12 days or less, 10 days or less, or 8 days or less. It is preferable to change the culture medium appropriately during both the culture period in the first culture medium and the culture period in the second culture medium. The interval for changing the culture medium can be, for example, every 1 to 5 days or every 2 to 4 days.

[0082] As described above, by culturing the ST cell-differentiating cells 20 in the well 111, some of the ST cell-differentiating cells 20 differentiate into ST cells, forming a cell structure with ST cells present in the outer layer. Because the inner wall of the well 111 is non-cell-adhesive, the cell structure does not adhere to the inner wall of the well 111 and forms a free cell aggregate. Therefore, the production method according to this embodiment makes it possible to produce the cell structure of the first embodiment, which is a free cell aggregate.

[0083] <Method for producing cell structure according to the second embodiment> The method for producing the cell structure of the second embodiment includes the steps of preparing a flow path device in which a first flow path and a second flow path are separated by a scaffold material, and culturing cells capable of differentiating into syncytiotrophoblast cells held in the scaffold material while perfusing a culture medium through each of the first flow path and the second flow path.

[0084] (Step of Preparing a Flow Channel Device) Fig. 8A is a perspective view of a flow channel device 201 used to fabricate a cell structure according to the second embodiment. Fig. 8B is a cross-sectional view of the flow channel device 201 taken along line BB shown in Fig. 8A. Fig. 9 is a diagram for schematically explaining a method for fabricating the flow channel device 201. The flow path device 201 is composed of a first substrate 210, a second substrate 220, a scaffold 250 fixed to a support 251 by a fixing material 252, and tubes 231, 232, 233, and 234. A first flow path 211 is formed in the first substrate 210, and a second flow path 221 is formed in the second substrate 220. The first flow path 211 and the second flow path 221 are separated by the scaffold 250. "Separated by the scaffold" means that at least a portion of the first flow path 211 and the second flow path 221 is separated by the scaffold 250.

[0085] The first substrate 210 and the second substrate 220 have approximately the same size and shape. The first flow path 211 and the second flow path 221 are formed in each substrate such that at least a portion of the flow paths overlap when the first substrate 210 and the second substrate 220 are stacked. The first flow path 211 and the second flow path 221 are preferably formed such that, when the first substrate 210 and the second substrate 220 are stacked, ends of each flow path are located at different positions. In the flow path device 201, the first substrate 210 and the second substrate 220 are stacked such that the surface on which the first flow path 211 is formed and the surface on which the second flow path 221 is formed face each other.

[0086] The material of the first substrate 210 and the second substrate 220 is not particularly limited, but a material that is highly biocompatible and has high oxygen permeability is preferable. "Oxygen permeability" means the property of passing molecular oxygen. By using an oxygen-permeable material for the first substrate 210 and the second substrate 220, oxygen can reach the inside of each flow channel. The oxygen permeability is, for example, about 100 to 5000 cm 3 / m 2 24h atm. The oxygen permeability is approximately 1100 to 3000 cm 3 / m 2 24h atm, or approximately 1250-2750cm 3 / m 2 ·24h·atm. "cm 3 / m 2 24h atm is the amount of oxygen that passes through 1m3 of the material in 24 hours under an atmosphere of 1 atmosphere. 2 Capacity per (cm 3 ) Examples of oxygen-permeable materials include oxygen-permeable polymers. Examples of oxygen-permeable polymers include fluororesins and silicones (such as PDMS). Among these, PDMS is preferred as the oxygen-permeable polymer.

[0087] The sizes of the first channel 211 and the second channel 221 are not particularly limited as long as they allow the formation of a multi-layered cell structure. The channel width of first channel 211 and second channel 221 can be, for example, 50 to 5000 μm. The channel width of first channel 211 and second channel 221 can be, for example, 50 μm or more, 100 μm or more, or 150 μm or more. The upper limit of the channel width of first channel 211 and second channel 221 can be, for example, 5000 μm or less, 3000 μm or less, 1000 μm or less, 800 μm or less, 600 μm or less, 500 μm or less, or 300 μm or less. The depth of the first flow path 211 and the second flow path 221 can be, for example, 50 to 5000 μm. The depth of the first flow path 211 and the second flow path 221 can be, for example, 50 μm or more, 100 μm or more, or 150 μm or more. The upper limit of the depth of the first flow path 211 and the second flow path 221 can be, for example, 5000 μm or less, 3000 μm or less, 1000 μm or less, 800 μm or less, 600 μm or less, 500 μm or less, or 300 μm or less.

[0088] The scaffold 250 is fixed to the support 251 by a fixing material 252 so that at least a portion of the scaffold 250 is exposed. In the flow path device 201, the support 251 to which the scaffold 250 is fixed is sandwiched and fixed between the first substrate 210 and the second substrate 220. At this time, the exposed scaffold 250 is arranged so as to separate the first flow path 211 and the second flow path 221. That is, when the first substrate 210 and the second substrate 220 are stacked, the exposed scaffold 250 is arranged at a position where the first flow path 211 and the second flow path 221 overlap. It is sufficient that at least a portion of the overlapping portion of the first flow path 211 and the second flow path 221 is separated by the scaffold 250. It is preferable that the remaining overlapping portion of the first flow path 211 and the second flow path 221 is separated by the support 251. In the flow channel device 201, exposed portions of the scaffold 250 are provided on both surfaces of the support 251, and form part of the wall surfaces of the first flow channel 211 and the second flow channel 221. For example, by forming a through-hole in the support 251 and fixing the scaffold 250 so that the through-hole is covered with the scaffold 250, the scaffold 250 can be exposed on both surfaces of the support 251. The exposed portion of the scaffold 250 may be provided on only one surface of the support 251. When the exposed portion of the scaffold 250 is provided on only one surface of the support 251, the exposed portion of the scaffold 250 is disposed on the side of the first substrate 210 and forms part of the wall surface of the first flow channel 211. Examples of the scaffold 250, the fixing material 252, and the support 251 include those similar to those listed in the above section [Cell Structure].

[0089] A tube 231 and a tube 232 are connected to both ends of the first flow path 211. A tube 233 and a tube 234 are connected to both ends of the second flow path 221. The material of the tubes 231, 232, 233, and 234 is not particularly limited, and may be, for example, a synthetic resin such as silicone, Teflon (registered trademark), polyethylene, polypropylene, or polysiloxane.

[0090] The flow channel device 201 can be fabricated by, for example, the method shown in FIG. 9. First, a scaffold 250 is fixed to a support 251 using a fixing material 252. Next, a laminate is formed by stacking a first substrate 210, a scaffold 250, a support 251, and a second substrate 220 in this order (FIGS. 9(A) and 9(B)). At this time, the surface on which the first flow channel 211 is formed and the surface on which the second flow channel 221 is formed face each other. In addition, the exposed scaffold 250 is positioned between the first flow channel 211 and the second substrate 220. Next, a tube 231 and a tube 232 are connected to both ends of the first flow path 211. A tube 233 and a tube 234 are connected to both ends of the second flow path 221 (FIG. 9(C)). In this manner, the flow path device 201 can be prepared.

[0091] (Step of culturing ST cell-differentiating cells) Fig. 10A is a schematic diagram illustrating a method for culturing ST cell differentiating cells 20 using a flow channel device 201. Fig. 10B is a cross-sectional view taken along line BB of the flow channel device 201 shown in Fig. 10A at the start of culturing.

[0092] With the ST cell differentiating cells 20 held on the scaffold 250 included in the flow path device 201, the cells are cultured while a culture medium is perfused through each of the first flow path 211 and the second flow path 221. For example, as shown in Fig. 9A, the culture medium M1 can be circulated through the first flow path 211 by flowing the culture medium M1 through a tube 231 connected to one end of the first flow path 211 and discharging it through a tube 232 connected to the other end. The culture medium M2 can be perfused through the second flow path 221 by flowing the culture medium M2 through a tube 233 connected to one end of the second flow path 221 and discharging it through a tube 234 connected to the other end.

[0093] <Culture medium> Culture medium M1 and culture medium M2 may be the same or different, but are preferably the same. Culture medium M1 and culture medium M2 can be a medium used to induce ST cells from TS cells (ST cell induction medium). Culture medium M1 and culture medium M2 can be the same as the culture media exemplified in the section "Method for Producing a Cell Structure of the First Embodiment." Culture medium M1 and culture medium M2 can be, for example, a basal medium supplemented with a ROCK inhibitor. The culture medium may further contain growth factors, ROCK inhibitors, GSK3β inhibitors, p38 MAPK inhibitors, etc.

[0094] Examples of culture medium M1 and culture medium M2 include media prepared by adding a ROCK inhibitor, a growth factor (EGF, etc.), a GSK3β inhibitor, a p38 MAPK inhibitor, etc. to a basal medium (e.g., TS basal medium). The concentrations of the components in culture medium M1 or culture medium M2 include the same concentrations as those exemplified in the section <Method for producing a cell structure according to the first embodiment>. The concentration of the GSK3β inhibitor in culture medium M1 or culture medium M2 can be 0.01 to 2.0 μM. The concentration of the GSK3β inhibitor in culture medium M1 or culture medium M2 can be, for example, 2.0 μM or less, 1.0 μM or less, 0.8 μM or less, 0.7 μM or less, or 0.6 μM or less. The lower limit of the concentration of the GSK3β inhibitor is not particularly limited, but can be, for example, 0.01 μM or more, 0.05 μM or more, 0.1 μM or more, 0.2 μM or more, or 0.3 μM or more. Specific examples of the culture medium M1 and the culture medium M2 include the TS perfusion medium used in the Examples described below.

[0095] ≪ST cell differentiated cell≫ The same cells as those mentioned in the section <Method for Producing a Cell Structure of the First Embodiment> can be used as the ST cell differentiating cells 20. The ST cell differentiating cells 20 can be retained in the scaffold 250 by introducing a cell suspension in which the ST cell differentiating cells 20 are suspended into the first flow channel 211 and allowing the cells to stand for a certain period of time. To prepare the cell suspension, an ST cell induction medium or a buffer solution such as PBS can be used. To prepare the cell suspension, it is preferable to use an ST cell induction medium. As the ST cell induction medium, it is preferable to use the culture medium M1 used in perfusion culture. The cell concentration of the cell suspension to prepare the cell suspension is not particularly limited, but for example, 10 4 ~10 8 cells / mL, or 10 5 ~10 7 It can be expressed as cells / mL. The period for static culture after the cell suspension of ST cell differentiating cells 20 is introduced into the first flow channel 211 is not particularly limited, but can be, for example, 1 to 10 days. From the viewpoint of stably retaining the ST cell differentiating cells 20 in the scaffold 250, the period for static culture is preferably, for example, 1 day or more, 2 days or more, or 3 days or more. From the viewpoint of starting the perfusion culture as early as possible and supplying sufficient nutrients to the ST cell differentiating cells 20, the period for static culture is preferably, for example, 10 days or less, 7 days or less, 5 days or less, or 4 days or less. Culture conditions for static culture can be those generally used for culturing animal cells. For example, the culture temperature can be 32 to 40°C (preferably 35 to 38°C) and the CO2 concentration can be 2 to 5% (preferably 5%).

[0096] During static culture, a culture medium M2 may be introduced into the second flow path 221. The culture medium M2 may be the same as the solution used to prepare the cell suspension. For example, if the culture medium M1 is used to prepare the cell suspension, the culture medium M2 may be a medium with the same composition as the culture medium M1. During the static culture period, it is preferable to replace the culture medium in the first flow path 211 and the second flow path 221 as appropriate. The interval for replacing the culture medium can be, for example, every 1 to 3 days, every 1 to 2 days, or every day. It is preferable to use culture medium M1 for replacing the culture medium in the first flow path 211, and it is preferable to use culture medium M2 for replacing the culture medium in the second flow path 221.

[0097] Before introducing the cell suspension of the ST cell differentiating cells 20 into the first flow channel 211, the first flow channel 211 may be treated with a solution containing a cell adhesive substance. As the cell adhesive substance, for example, a cell adhesive extracellular matrix can be used. For example, the first flow channel 211 may be treated with a buffer solution (such as PBS) containing Matrigel (registered trademark) or the like. Treatment with the cell adhesive substance improves the stability of the retention of the ST cell differentiating cells 20 on the scaffold material 250. After treatment with the solution containing the cell adhesive substance, the first flow channel 211 and the second flow channel 221 may be washed. As the washing liquid, for example, a buffer solution such as PBS, or a basal medium, etc. can be used.

[0098] The flow channel device 201 may be degassed before the cell suspension of ST cell differentiating cells 20 is introduced into the first flow channel 211. The degassing may be performed using, for example, a desiccator. When treatment with a cell adhesive substance is performed, the degassing may be performed before or after the treatment with the cell adhesive substance.

[0099] The ST cell differentiating cells 20 may be pre-cultured before being introduced into the first flow path 211. For example, an ST cell induction medium can be used for the pre-culture. As the ST cell induction medium, it is preferable to use the culture medium M1 used in perfusion culture. The pre-culture period is not particularly limited, but can be, for example, 1 to 15 days. From the viewpoint of preventing excessive differentiation of the ST cell differentiating cells 20, the pre-culture period can be, for example, 15 days or less, 12 days or less, 10 days or less, or 8 days or less. From the viewpoint of acclimatizing the cells to the ST cell induction medium, the pre-culture period can be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more.

[0100] ≪Perfusion culture≫ Perfusion culture can be performed by perfusing culture medium M1 through first flow path 211 and culture medium M2 through second flow path 221. The perfusion rate is not particularly limited and can be appropriately set depending on the sizes of first flow path 211 and second flow path 221. The perfusion rate can be, for example, 10 to 1000 μL / h. To prevent detachment of cells held on scaffold 250, the perfusion rate can be, for example, 1000 μL / h or less, 800 μL / h or less, 500 μL / h or less, 400 μL / h or less, or 350 μL / h or less. To promote the formation of a cell structure, the perfusion rate can be, for example, 10 μL / h or more, 50 μL / h or more, 100 μL / h or more, 150 μL / h or more, 200 μL / h or more, or 250 μL / h or more. Perfusion can be performed using a pump or the like, and the perfusion rate can be adjusted by adjusting the pumping rate.

[0101] The culture conditions for perfusion culture can be those generally used for culturing animal cells, such as a culture temperature of 32 to 40°C (preferably 35 to 38°C) and a CO2 concentration of 2 to 5% (preferably 5%).

[0102] The period of perfusion culture is not particularly limited. The period of perfusion culture can be, for example, 5 to 20 days. From the viewpoint of sufficiently promoting the formation of cell structures, the period of perfusion culture can be, for example, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, or 10 days or more. The upper limit of the period of perfusion culture is not particularly limited, and culture can be continued until the cell structures reach the desired size. For example, the period of perfusion culture can be 20 days or less, 15 days or less, or 12 days or less.

[0103] As described above, by perfusion culturing the ST cell differentiating cells 20 using the flow channel device 201, some of the ST cell differentiating cells 20 differentiate into ST cells, forming a cell structure in which ST cells exist in the outer layer (the region in contact with the external environment). Therefore, the production method according to this embodiment can produce the cell structure of the second embodiment, which is a cell structure in which the scaffold 250, a cell layer containing the ST cell differentiating cells 20, and a cell layer containing the ST cells exist in this order.

[0104] (Method for producing cell structure according to variant 1) <Step of preparing a flow channel device> Fig. 11 is a schematic diagram showing an example of a method for producing a flow channel device 301 used to produce a cell structure 300 (see Fig. 4) of Modification 1. Fig. 12A is a top view of the flow channel device 301. Fig. 12B is an enlarged view of a portion B of the flow channel device 301 surrounded by a dashed line in Fig. 12A. Fig. 12C is a cross-sectional view taken along line CC of the portion shown in Fig. 12B.

[0105] The flow channel device 301 is composed of a first substrate 310 and a second substrate 320. A flow channel 311, ports P1 to P4, and flow channels p1 to p4 are formed in the first substrate 310. The ports P1 to P4 are connected to the flow channel 311 by flow channels p1 to p4, respectively. Flow path 311 and flow paths p1 to p4 are open on one side of first substrate 310, and are closed on the other side. Second substrate 320 is stacked on first substrate 310 so as to close the openings of flow path 311 and flow paths p1 to p4. Therefore, second substrate 320 forms the bottoms of flow path 311 and flow paths p1 to p4.

[0106] Two rows of microposts 312 and 313 are arranged in the flow channel 311 along the flow channel direction. The flow channel 311 is partitioned by the microposts 312 and 313 into flow channel compartments 311R, 311C, and 311L. The flow channel compartment 311C is filled with a scaffold 350. The scaffold 350 can be filled into the flow channel compartment 311C before the flow channel 311 is covered with the second substrate 320. Alternatively, after the flow channel 311 is covered with the second substrate 320, a solution containing the scaffold 350 may be introduced into the flow channel compartment 311C from the injection port P311a of the flow channel 311 and gelled, thereby filling the flow channel compartment 311C with the scaffold 350.

[0107] The material of the first substrate 310 is not particularly limited, but is preferably a material that is highly biocompatible and highly oxygen permeable, such as the same materials as those listed as the materials for the first substrate 210 and the second substrate 220. The material of the first substrate 310 is preferably PDMS. The material of the second substrate 320 is not particularly limited, but is preferably a material that has high biocompatibility and low cell adhesiveness. Examples of materials for the second substrate 320 include glass, various synthetic resins, and metals.

[0108] The size of the flow channel 311 is not particularly limited as long as it allows the formation of cell aggregates in the flow channel compartment 311R and the flow channel compartment 311L and allows the filling of the scaffold material 350 in the flow channel compartment 311C. The width and depth of each flow channel compartment may be, for example, the same as those exemplified for the first flow channel 211 and the second flow channel 221.

[0109] <Step of culturing ST cell-differentiating cells> With the ST cell differentiating cells 20 held in the scaffold 350 filled in the channel compartment 311C, culture is performed while perfusing a culture medium through each of the channel compartments 311R and 311L. The channel compartments 311R and 311L correspond to a first channel and a second channel, respectively, separated by the scaffold 350. As shown in Fig. 11(A), the culture medium M1 can be perfused into the flow path compartment 311R by injecting the culture medium M1 through port P1 and discharging it through port P2. As shown in Fig. 11(A), the culture medium M2 can be perfused into the flow path compartment 311L by injecting the culture medium M2 through port P3 and discharging it through port P4.

[0110] The ST cell differentiating cells 20 can be retained in the scaffold 350 by introducing a cell suspension containing the ST cell differentiating cells 20 from port P1 into the flow channel compartment 311R and allowing the suspension to stand for a certain period of time. In this case, the ST cell differentiating cells 20 retained in the scaffold 350 are present in the flow channel compartment 311R. Alternatively, a cell suspension of cells 30 may be introduced into the flow channel compartment 311L from port P3 and statically cultured for a certain period of time, thereby allowing the cells 30 held in the scaffold 350 to reside in the flow channel compartment 311L. The static culture of the ST cell differentiating cells 20 and the static culture of the cells 30 may be carried out simultaneously or separately. The static culture period for the ST cell differentiating cells 20 may be the same as those listed above. The static culture period for the cells 30 may be set appropriately depending on the type of the cells 30. For example, the static culture period may be the same as that for the ST cell differentiating cells 20.

[0111] Before introducing the cell suspension of ST cell differentiating cells 20 into the flow channel device 301, similar to the above-described flow channel device 201, treatment with a cell adhesive substance, washing treatment, degassing treatment, etc. may be performed. Furthermore, the ST cell differentiating cells 20 and the cells 30 may be pre-cultured as appropriate.

[0112] The culture medium M1 perfused through the flow channel section 311R can be the same as the culture medium M1 in the flow channel device 201. The culture medium M2 perfused through the flow channel section 311L can be selected appropriately depending on the type of cells 30.

[0113] The perfusion rate is not particularly limited and can be set appropriately depending on the sizes of the flow channel sections 311 R and 311 L. Examples of the perfusion rate include those similar to those listed for the flow channel device 201 above.

[0114] The period of perfusion culture can be set appropriately depending on the application of the cell structure. Examples of the period of perfusion culture include the same periods as those mentioned for the flow channel device 201 above.

[0115] As described above, by perfusion culturing the ST cell differentiating cells 20 and the cells 30 using the flow channel device 301, the cell structure 300 of Modification 1 (see FIG. 4) can be produced.

[0116] (Method for producing cell structure according to modified example 2) The cell structure 300' of Modification 2 (see FIG. 5) can be produced using a flow channel device 301. In the cell structure 300' of Modification 2, cells 30 and cells 40 are present in addition to the scaffold material 350 in the flow channel section 311C. Therefore, a flow channel device 301 in which the scaffold material 350 containing the cells 30 and cells 40 is filled in the flow channel section 311C may be used. The cell structure 300' of Modification 2 can be produced in the same manner as in Modification 1, except that the flow channel 311 is filled with the scaffold material 350 containing the cells 30 and cells 40. A flow path device 301 in which a scaffold material 350 containing cells 30 and cells 40 is filled in a flow path section 311C can be produced by containing cells 30 and cells 40 in a solution containing scaffold material 350, introducing the solution into the flow path section 311C, and causing it to gel.

[0117] [Culture device] In one embodiment, the present invention provides a culture device comprising the cell structure of the above embodiment, a culture medium, and a culture substrate. "Culture substrate" refers to an apparatus used for culturing cells (culture vessel, culture tank, well plate, fluidic device, etc.). A culture substrate is an apparatus that accommodates cells and a culture medium during culture and provides a culture environment for the cells.

[0118] <Culture device containing the cell structure of the first embodiment> The culture device containing the cell structure of the first embodiment can include, as the culture substrate, the culture substrate used for producing the cell structure of the first embodiment. Examples of such a culture substrate include the well plate mentioned in the above section "Method for producing a cell structure" (e.g., well plate 110 shown in FIG. 7). Examples of culture media include the media listed in the section "Method for Producing a Cell Structure." Examples of culture media include the first culture medium or the second culture medium listed in the section "Method for Producing a Cell Structure." The culture medium is preferably the second culture medium.

[0119] <Culture device containing the cell structure of the second embodiment> The culture device containing the cell structure of the second embodiment can include, as the culture substrate, the culture substrate used for producing the cell structure of the second embodiment. Examples of such culture substrates include the fluidic devices listed in the above section "Method for producing a cell structure" (e.g., the flow channel device 201 shown in FIG. 8 and the flow channel device 301 shown in FIG. 11). Examples of the culture medium include the media listed in the section [Method for producing a cell structure], such as culture medium M1 and culture medium M2 listed in the section [Method for producing a cell structure].

[0120] The culture device of this embodiment can be used for maintaining and managing cell structures, and for various tests using the cell structures. [Example]

[0121] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0122] <Culture medium> The method for preparing the medium used in the test is described below.

[0123] (TS basal medium) TS basal medium was prepared by adding the following components to DMEM / F12 medium (FUJIFILM Wako). The concentrations shown below are the final concentrations of each component in the TS basal medium. Bovine serum albumin (BSA) (FUJIFILM Wako) 0.15% Penicillin 5,000 units / mL Streptomycin (Thermo Fisher Scientific) 5,000 μg / mL ITS-X (FUJIFILM Wako) 1% KSR(Thermo Fisher Scientific) 1% L-ascorbic acid (FUJIFILM Wako) 0.2mM

[0124] (TS medium) TS medium was prepared by adding the following components to TS basal medium. The concentrations shown below are the final concentrations of each component in TS medium. Y27632 (FUJIFILM Wako) 2.5 μM EGF (FUJIFILM Wako) 25ng / mL VPA (FUJIFILM Wako) 0.8mM A83-01(FUJIFILM Wako) 5μM CHIR99021 (FUJIFILM Wako) 2 μM

[0125] (3D-1 medium) 3D-1 medium was prepared by adding the following components to TS basal medium. The concentrations shown below are the final concentrations of each component in 3D-1 medium. Y27632 (FUJIFILM Wako) 2.5 μM EGF (FUJIFILM Wako) 25ng / mL BMP4 (FUJIFILM Wako) 20ng / mL bFGF(Thermo Fisher Scientific) 50ng / mL Heparin (Sigma) 0.1 μg / mL CHIR99021(FUJIFILM Wako) 0.5μM SB202190(FUJIFILM Wako) 2μM

[0126] (3D-2 medium) 3D-2 medium was prepared by adding the following components to TS basal medium. The concentrations shown below are the final concentrations of each component in 3D-2 medium. Y27632 (FUJIFILM Wako) 2.5 μM EGF (FUJIFILM Wako) 25ng / mL BMP4 (FUJIFILM Wako) 20ng / mL bFGF(Thermo Fisher Scientific) 50ng / mL Heparin (Sigma) 0.1 μg / mL CHIR99021 (FUJIFILM Wako) 2 μM SB202190(FUJIFILM Wako) 2μM

[0127] (TS perfusion medium) The following components were added to TS basal medium to prepare TS perfusion medium. The concentrations shown below are the final concentrations of each component in the TS perfusion medium. Y27632 (FUJIFILM Wako) 2.5 μM EGF (FUJIFILM Wako) 50ng / mL CHIR99021(FUJIFILM Wako) 0.5μM SB202190(FUJIFILM Wako) 2μM

[0128] [Example 1] Creation of cell structures, which are free cell aggregates (1)

[0129] <Method> (Preparation of 256-well agarose microwell plates) Agarose powder (SeaKem GTG Agarose, Lonza) was dissolved in 0.9% NaCl solution to prepare a 2% agarose solution. The agarose solution was poured into a MicroTissues 3D petri dish micro-mold (16 x 16 spheroids / mold) (product number Z764000-6EA, Sigma). The agarose solution was allowed to gel at 4°C to form an agarose microwell plate. The agarose microwell plate was then removed from the mold and transferred into a well of a 12-well plate. 2 mL of phosphate buffered saline (PBS) (Fujifilm Wako) was added to the well containing the agarose microwell plate. After removing bubbles from the microwells of the agarose microwell plate, the PBS(-) in the wells was replaced with DMEM / F12 medium (FUJIFILM Wako) and stored in a 37°C incubator until use.

[0130] Hereinafter, the wells of the 12-well plate containing the agarose microwell plates will be referred to as "wells containing microwell plates."

[0131] (cell culture) TS cells were maintained and managed in TS medium. TS cells were induced from CT cells and established as TS cells (Patent No. 6400832, Okae et al. Cell Stem Cell 22 50-68 (2018)). TS cells were collected from the maintenance culture dish and suspended in TS basal medium to prepare a cell suspension. The 12-well plate was removed from the 37°C incubator, and the DMEM / F12 medium in the wells containing the microwell plate was removed. 40 μL of the cell suspension (1 x 10 6 cells / mL) were seeded onto agarose microwell plates (4x10 4cells / agarose plate). 2 mL of 3D-2 medium was placed in each well of the microwell plate, and culturing was initiated at 37°C and 5% CO2. Three days after the start of culturing, the medium was replaced with 2 mL of 3D-2 medium. On the fourth day of culturing, the medium was replaced with 2 mL of 3D-1 medium, and on the sixth and eighth days of culturing, the medium was replaced with 3D-1 medium. The culturing was terminated on the tenth day of culturing.

[0132] (Preparation of frozen section samples) The 3D-1 medium in the wells of the microwell plate was removed, and 4% paraformaldehyde (PFA) (FUJIFILM Wako) was added to the wells. The cell aggregates were fixed in paraformaldehyde for 40 minutes. The paraformaldehyde solution in the wells of the microwell plate was then replaced with 1% neutral buffered formalin (FUJIFILM Wako) and stored at 4°C until further treatment.

[0133] To permeabilize the cells, they were treated with 0.3% Triton X-100 (FUJIFILM Wako) diluted in PBS(-) for 1 hour at room temperature. The cells were then washed with PBS(-) twice for 5 minutes. The cells were then treated with 15% sucrose / PBS(-) solution for 2 hours at 4°C. The 15% sucrose / PBS(-) solution was then replaced with 30% sucrose / PBS(-) solution and treated overnight at 4°C. The cells were removed from the 30% sucrose / PBS(-) solution and transferred into a cryomold (No. 1) (Sakura Finetek Japan). After removing the 30% sucrose / PBS(-) solution from the cryomold, Tissue-Tek OCT Compound (Sakura Finetek Japan) was placed into the cryomold and the cell aggregates and OCT Compound were frozen on dry ice. The frozen cell aggregate / OCT compound was stored at -20°C until further processing. The frozen cell aggregate sample was cut using a cryostat (LEICA CM1950) to prepare frozen section samples with a thickness of approximately 12 μm.

[0134] (Immune cell staining) The frozen section samples were washed with PBS(-) to remove the OCT compound. A primary antibody solution was prepared by adding PE-conjugated anti-SDC1 (Miltenyi Biotec) (1 / 500 dilution) and E-Cadherin (24E10) Rabbit mAb (Cell Signaling) (1 / 500 dilution) to antibody diluent (PBS(-) containing 0.1% Tween 20 and 2% FBS). 100–200 μL of the primary antibody solution was applied to the frozen section samples, and the samples were left overnight at 4°C, taking care not to allow them to dry out.

[0135] The primary antibody solution was then removed from the frozen section samples, and the sections were washed with 200 μL of PBS(-) (3 minutes, three times). Next, 100–200 μL of secondary antibody solution was applied to the frozen section samples and allowed to stand at room temperature for 1 hour, protected from light. The secondary antibody solution was prepared by adding anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate) (1 / 400 dilution), anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor 555 Conjugate) (1 / 400 dilution), and Hoechst 33258 (Dojindo) (1 / 1250 dilution) to the antibody diluent. The secondary antibody solution was then removed from the frozen section samples, and the sections were washed with 200 μL of PBS(-) (3 minutes, three times). Next, VECTASHIELD Mounting Medium (Vector Laboratories) containing DAPI was dropped onto the frozen section sample, and a cover glass was placed over the section to mount it.

[0136] (Fluorescence microscopy analysis) The frozen section samples were analyzed using an all-in-one fluorescence microscope BZ-X800 (Keyence).

[0137] <Result> Figure 13A shows the results of immunocytochemistry. "SCD1" is an image stained for SCD1, a positive marker for ST cells; "E-cadherin" is an image stained for E-cadherin, a positive marker for ST cell-differentiating cells; "Nucleus" is an image stained with nuclei; and "Merged" is an image obtained by merging these images (same below). The results of immunocytochemistry confirmed that SDC1-expressing cells covered the surface of the cell aggregate in a single layer. Furthermore, E-cadherin-expressing cells were present inside the cell aggregate. The results of immunocytochemistry demonstrated that this method can produce cell aggregates in which ST cells reside in the outer layer of the cell aggregate and undifferentiated trophoblast cells reside inside the cell aggregate.

[0138] [Example 2] Creation of cell structures, which are free cell aggregates (2)

[0139] <Method> (cell culture) Cell culture was carried out in the same manner as in Example 1, except that the seeding amount of TS cells and the timing of medium replacement were changed. TS cells were cultured in 10 μL of cell suspension (1 × 10 6 cells / mL) were seeded onto agarose microwell plates (1x10 4 Cells / agarose plate). 2 mL of 3D-2 medium was added to the wells to initiate culture, and 3 days after the start of culture, this was replaced with 2 mL of 3D-1 medium. Thereafter, the medium was replaced with 3D-1 medium every two days, and culture was terminated on the 10th day of culture.

[0140] (Preparation of frozen section samples, immunocytochemical staining, and fluorescence microscopy analysis) In the same manner as in Example 1, frozen section samples were prepared, immunostained, and analyzed under a fluorescent microscope.

[0141] <Result> The results of immunofluorescence staining are shown in Figure 13B. Similar to the results of immunocytostaining in Example 1, it was confirmed that SDC1-expressing cells covered the surface of the cell aggregates in a monolayer, and E-cadherin-expressing cells were present inside the cell aggregates. The results of immunocytostaining showed that even when the cell seeding amount and the timing of medium change were changed, it was possible to produce cell aggregates in which ST cells were present in the outer layer of the cell aggregates and ST cell-differentiating cells were present inside the cell aggregates.

[0142] [Example 3] Fabrication of cell-based constructs containing scaffolds

[0143] <Method> (3D printing materials) The flow channel device was fabricated by 3D printing. 1% (w / w) photoinitiator (Omnirad 819 (former Irgacure 819, IGM Resins BV) and 1% (w / w) photosensitizer (2-isopropylthioxanthone, Tokyo Chemical Industry Co., Ltd.) were dissolved in polyethylene glycol diacrylate (PEGDA) (Mn: 250) (Sigma) to prepare the 3D printing material.

[0144] (Fabrication of flow channel device) A channel mold was created using a 3D printer (QiDi Tech Shadow 5.5s, QIDI Technology Co., Ltd.). The 3D printed object was treated with 99.5% ethanol (FUJIFILM Wako) for 1-2 minutes and then air-dried. The front and back surfaces of the 3D printed object were irradiated with ultraviolet light and treated at 80°C overnight to completely harden the 3D printed object.

[0145] The flow channel device was fabricated using polydimethylsiloxane (PDMS) (SILPOT 184, Toray Dow Corning). A PDMS solution, made by mixing the base resin and curing agent at a ratio of 10:1, was poured into a mold and heated to 65-80°C overnight to harden. Two of these were fabricated, and these were used as the first and second substrates of the flow channel device.

[0146] A flow channel device was fabricated as shown in Figure 9. A collagen vitrigel (registered trademark) membrane was used as the scaffolding material. A polyimide film was used as the support for the collagen membrane. Kapton (registered trademark) tape was used as the fixing material for fixing the collagen membrane to the polyimide film.

[0147] To incorporate a collagen membrane into a flow channel device, a collagen-coated sheet was prepared. First, a 4 mm diameter hole was drilled in Kapton tape (0.069 mm thick, Nitto Denko America, #5-5018-02, P-221) using a biopsy trephine (Kai Industries). A collagen vitrigel membrane (ad-MED Vitrigel® 2, Kanto Chemical) was pressed onto the adhesive surface of the Kapton tape to cover the hole. A 7.5 μm thick polyimide film (Toray-DuPont, 63-2884-80, 30EN) with a 4 mm diameter hole was placed on the adhesive side of the Kapton tape, sandwiching the collagen vitrigel membrane. The prepared polyimide film with collagen vitrigel membrane was sandwiched between the first and second substrates.

[0148] Four tubes (inner diameter 0.5 mm, outer diameter 1 mm) were connected to the channel device fabricated as described above. A thicker tube (inner diameter 1 mm, outer diameter 2 mm) was attached to the tip of each tube. To secure the tubes to the channel device, PDMS solution was used as an adhesive, and the PDMS was solidified by heat treatment. The channel device fabricated is shown in Figure 14.

[0149] (cell culture) TS cells were maintained and managed using TS medium. 1 mL of PBS(-) containing 3 μg / well of collagen IV (Corning) was placed in the wells of a 6-well plate. After standing at 37°C for 30 minutes or more, the solution in the wells was removed and the plate was washed twice with PBS(-). 0.3x10 cells were added to the collagen IV-coated wells. 5 Cells were placed at 1000 cells / well and pre-cultured in TS perfusion medium. On days 3 and 5 of culture, the medium was replaced with 2 mL of TS perfusion medium. On day 7 of culture, cells were seeded into the microfluidic device using the method described below.

[0150] (Seeding cells into the flow channel device) The flow channel device was sterilized by UV irradiation for 15 minutes. The flow channel device was then placed in a desiccator and degassed for 15 minutes. PBS(-) containing 0.5 mg / mL Matrigel (Corning) was poured into the first flow channel of the flow channel device. PBS(-) was poured into the second flow channel. The device was then left to stand at 37°C for 1 hour. It was then washed twice with TS basal medium and degassed again in a desiccator.

[0151] Cells pre-cultured in TS perfusion medium for 7 days were detached from the wells and suspended in TS perfusion medium at 2 x 10 6 A cell suspension of 100 cells / mL was prepared. 40 μL of the cell suspension was poured into the first channel, and TS perfusion medium was poured into the second channel (day 0 of culture). The medium was changed daily (40 μL each for the first and second channels) until day 3 of culture. From day 3 of culture, TS perfusion medium was perfused into the first and second channels at a flow rate of 30 μL / h. Culture was terminated on day 8 of culture (day 5 of perfusion) or day 13 of culture (day 10 of perfusion). For comparison, a sample was prepared that had been cultured in the same manner but without perfusion from day 3 of culture (non-perfused sample). For the non-perfused sample, the medium was manually changed once a day (40 μL each for the first and second channels) until day 13 of culture. In both samples, cells were fixed with 4% PFA after culture was terminated.

[0152] (immunostaining) For permeabilization, 0.3% Triton X 100 / PBS(-) was poured into the first and second flow paths and left to stand at room temperature for 1 hour to treat the cells. After washing the cells twice with PBS(-), 40 μL of primary antibody solution was added to each of the first and second flow paths. The mixture was then left to stand overnight at 4°C. The primary antibody solution used was the same as in Example 1.

[0153] The primary antibody solution was then removed, and the first and second flow paths were washed with PBS(-). A secondary antibody solution was poured into the first and second flow paths, and the devices were left standing at room temperature for 1 hour in the dark. The same secondary antibody solution as in Example 1 was used. The secondary antibody solution was then removed from the first and second flow paths, and PBS(-) was poured into the first and second flow paths to wash the cells. The film with the cell / collagen vitrigel membrane was peeled off from the flow path device and placed on a glass-bottom dish together with VECTASHIELD Mounting Medium (Vector Laboratories) containing DAPI.

[0154] (Fluorescence microscopy analysis) The cell structure samples were observed using an all-in-one fluorescence microscope BZ-X800 (Keyence) and a confocal microscope (LSM 700, Carl Zeiss).

[0155] <Result> Figure 15 shows immunocytochemical fluorescence microscopy images of the cell structure on day 8 of culture (day 5 of perfusion). SDC1 expression was observed, and cell fusion, a characteristic of ST cells, was observed. SDC1 was expressed on the surface of the fused cells.

[0156] Figure 16 shows fluorescent microscopic images of immunocytochemical staining of cell constructs on day 13 of culture (day 10 of perfusion). The image on the left shows a cell construct that underwent perfusion culture (perfusion (+)), and the image on the right shows a control cell construct that did not undergo perfusion culture (perfusion (-)). Perfusion culture increased the number of cells and elevated SDC1 expression.

[0157] Figure 17 shows a confocal microscopic image of a cross section of the cell structure on day 8 of culture (day 5 of perfusion). It was confirmed that the cell structure partially had a two-layer structure, with the upper layer cells expressing SDC1 and the lower layer cells expressing low levels of SDC1.

[0158] These results demonstrate that this method can produce cell aggregates in which ST cells reside in the upper layer (the area in contact with the outside) and cells with the potential to differentiate into ST cells reside in the lower layer. Furthermore, it is suggested that the cell structures produced by this method have a structure similar to that of the villi in vivo. [Industrial Applicability]

[0159] According to the present invention, there are provided a cell structure in which at least a portion of ST cells are present in an area in contact with the outside, a culture device containing the cell structure, and a method for producing the cell structure. [Explanation of symbols]

[0160] 10...ST cells, 20...ST cell differentiating cells, 30,40...cells, 100,200,300,300'...cell structure, 110...well plate, 111...well, 210,310...first substrate, 220,320...second substrate, 211...first flow channel, 221...second flow channel, 231,232,233,234...tube, 250,350...scaffold material, 251...support, 252...fixation material, 311...flow channel, 312,313...micropost.

Claims

1. Syncytiotrophoblast cells; cells capable of differentiating into syncytiotrophoblast cells; A cell structure comprising: At least a portion of the syncytiotrophoblast cells are present in a region of the cell structure that is in contact with the outside, The cell structure is a free spherical cell aggregate, The cell aggregates a core having cells capable of differentiating into the syncytiotrophoblast cells; a shell located on the outer layer of the core and containing the syncytiotrophoblast cells; Including, The cell structure is a placental organoid. Cell structure.

2. Syncytiotrophoblast cells, cells capable of differentiating into syncytiotrophoblast cells; A cell structure comprising a scaffold material, At least a portion of the syncytiotrophoblast cells are present in a region of the cell structure that is in contact with the outside, the scaffold, a cell layer containing cells capable of differentiating into the syncytiotrophoblast cells, and a cell layer containing the syncytiotrophoblast cells are present in this order; The cell structure is a placental organoid. Cell structure.

3. The cell structure according to claim 1 or 2, wherein the syncytiotrophoblast cells are SDC1 positive.

4. The cell structure according to any one of claims 1 to 3, wherein the cells capable of differentiating into syncytiotrophoblast cells have at least one characteristic selected from the group consisting of E-cadherin-positive, GATA2-positive, GATA3-positive, TFAP2-positive, ELF5-positive, ZNF750-positive, and CDX2-negative.

5. The cell structure according to any one of claims 1 to 4, a culture medium; A culture substrate; A culture device comprising:

6. the culture substrate comprises a well; The cell structure is cultured in the well. The culture device of claim 5 .

7. The culture substrate has a flow path, The cell structure is held within the channel. The culture device of claim 5 .

8. A method for producing a cell structure according to any one of claims 1 to 4, comprising a step of culturing cells capable of differentiating into syncytiotrophoblast cells in a well having a non-cell-adhesive inner wall.

9. 9. The method according to claim 8, wherein the diameter of the opening of the well is 100 to 1500 μm.

10. preparing a flow channel device in which a first flow channel and a second flow channel are separated by a scaffold; Culturing cells retained in the scaffold and capable of differentiating into syncytiotrophoblast cells while perfusing a culture medium through each of the first flow path and the second flow path; The method for producing the cell structure according to claim 2, comprising:

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