Sheet-like cell culture and method for producing same

Abstract

To provide a method for producing a sheet-like cell culture showing a three-layer structure including a cell layer of superficial cells, a cell layer of intermediate cells, and a cell layer of basal cells, in this order. Provided is a method for producing a sheet-like cell culture that includes culturing a progenitor cell population in a second compartment separated from a first compartment via a porous member to form a sheet-like cell culture in the second compartment. The sheet-like cell culture includes a cell layer of superficial cells derived from the progenitor cell population, a cell layer of intermediate cells derived from the progenitor cell population, and a cell layer of basal cells derived from the progenitor cell population, in this order. The first compartment holds an induction medium containing retinoic acid, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF).

Description

Sheet-shaped cell culture and method for producing the same

[0001] The present invention relates to the field of cell medicine. Specifically, the present invention relates to a sheet-shaped cell culture and a method for producing the same. The present invention also relates to a pharmaceutical composition comprising the sheet-shaped cell culture. The present invention also relates to matured bladder organoids and a method for producing the same.

[0002] The bladder is an organ derived from the definitive endoderm and is known to develop from the hindgut, located at the posteriormost part of the early gastrointestinal tract, through the ventral cloaca. The bladder is a sac-like organ that temporarily stores urine that is delivered from the kidneys via the ureters and excreted through the urethra. The bladder epithelial tissue of a living organism comprises, from the inside to the outside of the sac-like organ, a cell layer of superficial cells, a cell layer of intermediate cells, and a cell layer of basal cells, in this order. The urine storage or voiding function of the bladder can be reduced or lost when the bladder tissue is damaged by radiation therapy, bladder rupture, diabetes, etc.

[0003] Research into regenerative medicine is being conducted with the aim of regenerating organs that have lost function, treating intractable diseases, and making up for the chronic shortage of donors for organ transplants. In the field of regenerative medicine, research is being conducted into the use of cell cultures resembling bladder epithelial tissue, called bladder urothelial sheets, which are created in vitro from pluripotent stem cells such as ES cells or iPS cells.

[0004] Non-Patent Document 1 reports the preparation of bladder urothelial sheets from human induced pluripotent stem cells (hiPSCs). Non-Patent Document 2 reports the preparation of bladder epithelial sheets from bladder epithelial cell lines (HBLAK and HBEP) using a Transwell. Non-Patent Document 3 reports the preparation of bladder epithelial sheets from bladder epithelial cell lines (HTB-9) using a microfluidic device. Non-Patent Document 4 reports the preparation of bladder epithelial sheets from non-invasively obtainable urinary stem cells (USCs) using a Transwell.

[0005] Kotaro Suzuki et al., Sci Rep. 2019, DOI: 10.1038 / s41598-019-46848-8Harry Horsley et al., Sci Rep. 2018, DOI:10.1038 / s41598-018-19690-7Kunal Sharma et al., eLife. 2021, DOI: 10.7554 / eLife.66481Qian Wan et al., Stem Cell Res Ther. 2018, DOI: 10.1186 / s13287-018-1035-6

[0006] The sheet-shaped cell culture prepared in Non-Patent Document 1 mainly contains surface layer cells. The presence of surface layer cells was confirmed in the sheet-shaped cell culture prepared in Non-Patent Document 2. The type of bladder epithelial cells was not identified in the sheet-shaped cell culture prepared in Patent Document 3. The presence of surface layer cells was confirmed in the sheet-shaped cell culture prepared in Patent Document 4. However, the three-layer structure comprising a cell layer of surface cells, a cell layer of intermediate cells, and a cell layer of basal cells, as in bladder urothelial tissue, was not observed in the sheet-shaped cell cultures prepared in Non-Patent Documents 1 to 4.

[0007] A primary object of the present disclosure is to provide a sheet-shaped cell culture exhibiting a three-layer structure including, in this order, a cell layer of superficial cells, a cell layer of intermediate cells, and a cell layer of basal cells, and a method for producing the same. Another object of the present disclosure is to provide a sheet-shaped cell culture or mature bladder organoid exhibiting stretchability and / or urine resistance.

[0008] The present disclosure provides the following inventions. [Item 1] A method for producing a sheet-shaped cell culture, comprising culturing a progenitor cell population in a second compartment separated from a first compartment by a porous member to form a sheet-shaped cell culture in the second compartment, wherein the sheet-shaped cell culture comprises, in that order, a cell layer of superficial cells derived from the progenitor cell population, a cell layer of intermediate cells derived from the progenitor cell population, and a cell layer of basal cells derived from the progenitor cell population, and the first compartment holds an induction medium containing retinoic acid, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF). [Item 2-1] The production method according to Item 1, wherein the induction medium further comprises an extracellular matrix. [Item 2-2] The production method according to Item 1, wherein the induction medium further comprises a ROCK inhibitor. [Item 2-3] The induction medium contains a calcium salt, for example, calcium chloride (CaCl ２ ), calcium carbonate (CaCO ３ ), and calcium bicarbonate (Ca(HCO ３ ) ２ Item 2-4: The method according to Item 1, wherein the induction medium further comprises an extracellular matrix and a ROCK inhibitor. Item 2-5: The induction medium further comprises an extracellular matrix and a calcium salt, for example, calcium chloride (CaCl ２ ), calcium carbonate (CaCO ３ ), and calcium bicarbonate (Ca(HCO ３ ) ２ Item 2-6: The method according to Item 1, wherein the induction medium further contains a ROCK inhibitor and a calcium salt, for example, calcium chloride (CaCl ２ ), calcium carbonate (CaCO ３ ), and calcium bicarbonate (Ca(HCO ３ ) ２Item 2-7: The method according to Item 1, wherein the induction medium further comprises an extracellular matrix, a ROCK inhibitor, and a calcium salt, such as calcium chloride (CaCl ２ ), calcium carbonate (CaCO ３ ), and calcium bicarbonate (Ca(HCO ３ ) ２Item 3: The method of claim 1, wherein the progenitor cell population comprises progenitor cells derived from a ventral hindgut organoid or a bladder organoid. [Item 4] The method of claim 1, wherein the BMP comprises at least one selected from the group consisting of BMP2, BMP4, and BMP7. [Item 5] The manufacturing method according to any one of Items 1 to 4, wherein the FGF comprises at least one selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. [Item 6] The manufacturing method according to any one of Items 1 to 5, wherein culturing the progenitor cell population comprises culturing the progenitor cell population in a second compartment substantially free of an induction medium containing retinoic acid, bone morphogenetic protein, and fibroblast growth factor. [Item 7] The manufacturing method according to any one of Items 1 to 6, wherein culturing the progenitor cell population comprises culturing the progenitor cell population in a gas phase in the second compartment. [Item 8] A culture method comprising applying a progenitor cell population capable of differentiating into superficial cells, intermediate cells, and basal cells to a second compartment separated from a first compartment by a porous member, and culturing the progenitor cell population in the second compartment, wherein the first compartment holds a culture medium containing retinoic acid, bone morphogenetic protein, and fibroblast growth factor. [Item 9-1] A sheet-shaped cell culture produced according to the production method described in any of Items 1 to 7. [Item 9-2] The sheet-shaped cell culture described in Item 9-1, wherein the sheet-shaped cell culture comprises, in this order, a cell layer of superficial cells derived from the progenitor cell population, a cell layer of intermediate cells derived from the progenitor cell population, and a cell layer of basal cells derived from the progenitor cell population.[Item 9-3] The sheet-shaped cell culture according to Item 9-2, wherein the superficial layer cells do not substantially express P63 but express uroplakin (UPK), the intermediate cells co-express P63 and UPK, and the basal cells co-express P63 and keratin 5 (KRT5). [Item 9-4] The sheet-shaped cell culture according to Item 9-2, wherein the superficial layer cells express uroplakin (UPK) and further express either CK20 and ZO1 or both, the intermediate cells co-express P63 and UPK, and the basal cells co-express P63 and keratin 5 (KRT5). [Item 9-5] The sheet-shaped cell culture according to Item 9-3, wherein the superficial layer cells further express either CK20 and ZO1 or both. [Item 9-6] The sheet-shaped cell culture according to Item 9-4, wherein the surface layer cells do not substantially express P63.

[0009] [Item 10-1] A sheet-shaped cell culture comprising, in this order, a cell layer of superficial cells, a cell layer of intermediate cells, and a cell layer of basal cells, wherein the superficial cells do not substantially express P63 but express uroplakin (UPK), the intermediate cells co-express P63 and UPK, and the basal cells co-express P63 and keratin 5 (KRT5). [Item 10-2] A sheet-shaped cell culture comprising, in this order, a cell layer of superficial cells, a cell layer of intermediate cells, and a cell layer of basal cells, wherein the superficial cells express uroplakin (UPK) and further express either CK20 or ZO1 or both, the intermediate cells co-express P63 and UPK, and the basal cells co-express P63 and keratin 5 (KRT5). [Item 11-1] The sheet-shaped cell culture according to Item 10-1, wherein the surface layer cells further express either one or both of CK20 and ZO1. [Item 11-2] The sheet-shaped cell culture according to Item 10-2, wherein the surface layer cells do not substantially express P63. [Item 12] A pharmaceutical composition comprising a sheet-shaped cell culture produced according to the production method according to any of Items 1 to 7, or the sheet-shaped cell culture according to Item 10 or 11; and a pharmaceutically acceptable carrier. [Item 13] A method for producing a non-human mammal having a sheet-shaped cell culture in the bladder or a surrounding area, the method comprising introducing the sheet-shaped cell culture produced according to the production method according to any of Items 1 to 7, or the sheet-shaped cell culture according to Item 10 or 11, into the bladder or a surrounding area. [Item 14-1] A non-human mammal having in its bladder or a surrounding area a sheet-shaped cell culture produced according to the production method of any one of Items 1 to 7, or the sheet-shaped cell culture of Item 10 or 11. [Item 14-2] A non-human mammal produced according to the method of Item 13, having in its bladder or a surrounding area a sheet-shaped cell culture.[Item 15-1] A method for evaluating drug responsiveness to a test substance, comprising: contacting a non-human mammal having, in its bladder or a surrounding area thereof, a sheet-shaped cell culture produced according to the production method of any of Items 1 to 7, or the sheet-shaped cell culture of Item 10 or 11, or the sheet-shaped cell culture of Claim 10 or 11, with the test substance; and detecting a change in the test substance or the sheet-shaped cell culture. [Item 15-2] A method for evaluating drug responsiveness to a test substance, comprising: contacting a non-human mammal having, in its bladder or a surrounding area thereof, the sheet-shaped cell culture of Item 14 with the test substance; and detecting a change in the test substance or the sheet-shaped cell culture.

[0010] [Item 16] A method for producing matured bladder organoids, comprising: floating culture of immature bladder organoids using an induction medium containing retinoic acid, BMP, and FGF (e.g., FGF7 and FGF10). [Item 17] A method for producing matured bladder organoids, comprising: culturing ventral hindgut organoids using an induction medium containing retinoic acid and FGF (e.g., FGF10) to induce differentiation of immature bladder organoids; and floating culture of the immature bladder organoids using an induction medium containing retinoic acid, BMP, and FGF (e.g., FGF7 and FGF10) to induce differentiation of mature bladder organoids. [Item 18] The production method according to Item 17, wherein the induction medium used to induce the immature bladder organoids further contains BMP. [Item 19] The production method according to any one of Items 16 to 18, wherein the induction medium used to induce the matured bladder organoids further contains a calcium salt. [Item 20] Matured bladder organoids produced according to the method of any one of Items 16 to 19. [Item 21-1] Matured bladder organoids comprising a cell layer surrounding a lumen, the cell layer comprising, in this order, a cell layer of superficial cells that substantially do not express P63 but express UPK, a cell layer of intermediate cells that co-express P63 and UPK, and a cell layer of basal cells that co-express P63 and KRT5, the superficial cells facing the lumen, and the superficial cells further express CK20 and ZO1. [Item 21-2] A matured bladder organoid comprising a cell layer surrounding a lumen, wherein the cell layer comprises, in this order: a cell layer of surface cells that express UPK and further express CK20 and ZO1; a cell layer of intermediate cells that co-express P63 and UPK; and a cell layer of basal cells that co-express P63 and KRT5, wherein the surface cells face the lumen. [Item 22] A pharmaceutical composition comprising the matured bladder organoid according to Item 20 or 21, and a pharmaceutically acceptable carrier.[Item 23] A method for evaluating drug responsiveness to a test substance, comprising contacting the matured bladder organoid of Item 20 or 21 with the test substance and detecting changes in the bladder organoid in response to the test substance. [Item 24] A method for treating bladder damage or disease, comprising introducing the sheet-shaped cell culture of any of Items 9 to 11, the matured bladder organoid of Item 20 or 21, or the pharmaceutical composition of Item 12 into the bladder or a surrounding area of ​​a mammal in need thereof.

[0011] Figure 1 is a flow chart showing the protocol for preparing a bladder epithelial cell sheet. Figure 2A is a bright-field image of a cultured cell sheet containing cells induced from human iPS cells on day 26 of differentiation induction. Figure 2B is a bright-field image of a cultured cell sheet containing cells on day 42 of differentiation induction. Figure 2C is a bright-field image at low magnification of a cultured cell sheet containing cells on day 42 of differentiation induction. Figure 3A is a UPK1B fluorescent image of a bladder epithelial cell sheet composed of a cell population on day 57 of differentiation induction. Figure 3B is a P63 fluorescent image of the bladder epithelial cell sheet. Figure 3C is a KRT5 fluorescent image of the bladder epithelial cell sheet. Figure 3D is a DAPI fluorescent image of the bladder epithelial cell sheet. Figure 3E is a fluorescent image obtained by superimposing Figures 3A to 3D. Figure 4 is a series of photographs showing a bladder epithelial cell sheet on day 42 of differentiation induction being pinched with tweezers (Figure 4A), stretched to approximately 1.6 times its original width (Figure 4B), and then returned to its original size (Figure 4C). Figure 5A is a brightfield image of a bladder epithelial cell sheet on day 48 of differentiation induction (day 20 + day 28). Figure 5B is a brightfield image of a bladder epithelial cell sheet on day 48 of differentiation induction. Figure 5C is a brightfield image of a bladder epithelial cell sheet on day 48 of differentiation induction (day 20 + day 28) cultured for an additional 9 days (day 20 + day 37) without the addition of human urine (-). Figure 5D is a brightfield image of a bladder epithelial cell sheet on day 48 of differentiation induction cultured for an additional 9 days with the addition of human urine (+).

[0012] Figure 6A is a UPK1B fluorescent image of a bladder epithelial cell sheet containing cells on day 57 of differentiation induction, which includes a 9-day culture period after the addition of urine. Figure 6B is a P63 fluorescent image of the bladder epithelial cell sheet. Figure 6C is a KRT5 fluorescent image of the bladder epithelial cell sheet. Figure 6D is a DAPI fluorescent image of the bladder epithelial cell sheet. Figure 6E is a fluorescent image obtained by superimposing Figures 6A to 6D. Figure 7A is a CK20 fluorescent image of a bladder epithelial cell sheet composed of a cell population on day 57 of differentiation induction without the addition of urine. Figure 7B is a ZO1 fluorescent image of the bladder epithelial cell sheet. Figure 7C is a DAPI fluorescent image of the bladder epithelial cell sheet. Figure 7D is a fluorescent image obtained by superimposing Figures 7A to 7C. Figure 7E is a CK20 fluorescent image of a bladder epithelial cell sheet composed of a cell population on day 57 of differentiation induction, which includes a 9-day culture period after the addition of urine. Figure 7F is a ZO1 fluorescent image of the bladder epithelial cell sheet. Figure 7G is a DAPI fluorescent image of the bladder epithelial cell sheet. Figure 7H is a fluorescent image obtained by superimposing Figures 7E to 7G. Figure 8A is a fluorescent image of matured bladder organoids on day 67 of differentiation induction, including the culture period in a whole embryo culture system. Figure 8B is a CK20 and DAPI fluorescent image (left) and a ZO1 and DAPI fluorescent image (right) of the matured bladder organoids. Figure 9A is a photograph showing the results of injecting a green dye solution into the lumen of matured bladder organoids. Figure 9B is a series of fluorescent images taken after injecting FITC dye into the lumen of matured bladder organoids. Figure 9C is a line graph showing the change over time in fluorescence intensity from the matured bladder organoids after FITC injection.

[0013] The term "sheet-shaped cell culture" refers to a membrane-like cell aggregate formed in vitro. A sheet-shaped cell culture can be formed, for example, by contact culturing cells to connect them to each other. A sheet-shaped cell culture can have, for example, a thin membrane-like form. A sheet-shaped cell culture can have, for example, a first main surface and a second main surface opposite the first main surface. A sheet-shaped cell culture has, for example, a thickness that is small relative to the maximum length (e.g., major axis) of its surface. A sheet-shaped cell culture has, for example, a surface area according to its intended use. A sheet-shaped cell culture having a predetermined surface area can be obtained, for example, by contact culturing cells in a culture compartment having a culture area corresponding to the predetermined surface area.

[0014] The sheet-shaped cell culture comprises, for example, a cell layer of superficial cells, a cell layer of intermediate cells, and a cell layer of basal cells, in this order, similar to the bladder urothelium in a living bladder. The sheet-shaped cell culture is also referred to as a bladder epithelial cell sheet. The "superficial cells" express uroplakin and further express either CK20 or ZO1 or both, and / or do not substantially express P63. The "intermediate cells" co-express P63 and uroplakin. The "basal cells" co-express P63 and keratin 5. The bladder epithelial cell sheet can be produced by contact culturing a progenitor cell population that can differentiate into superficial cells, intermediate cells, and basal cells.

[0015] For example, the surface layer cells do not substantially express P63 but express uroplakin. For example, the surface layer cells express uroplakin and further express either CK20 and ZO1, or both. For example, the surface layer cells do not substantially express P63 but express uroplakin and further express either CK20 and ZO1, or both. The surface layer cells preferably do not substantially express P63 but express uroplakin and further express both CK20 and ZO1. The surface layer cells express uroplakin and further express CK20. The surface layer cells express uroplakin and further express ZO1. The surface layer cells preferably express uroplakin and further express CK20 and ZO1.

[0016] The term "progenitor cell" refers to a cell that is in the process of differentiating from a stem cell to a functional cell and has the ability to differentiate into said functional cell. The term "cell population" refers to a collection of multiple cells. A cell population, for example, contains at least two of one cell type. A cell population, for example, contains at least two cell types, with at least one of each cell type. A progenitor cell population according to the present disclosure contains at least two progenitor cells that have the ability to differentiate into one or more of superficial cells, intermediate cells, and basal cells. A progenitor cell population that can differentiate into superficial cells, intermediate cells, and basal cells, for example, contains multiple progenitor cells that have the ability to differentiate into one or more of superficial cells, intermediate cells, and basal cells.

[0017] The term "cell layer" refers to an overlap of cell populations that are distinguishable from one another. A cell layer of a predetermined cell type is, for example, a cell layer of basal cells, a cell layer of intermediate cells, or a cell layer of superficial cells. A cell layer of a predetermined cell type is, for example, a cell population in which the predetermined cell type is present at 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. A predetermined cell can be identified by detecting a cell marker (e.g., either or both of protein expression and gene transcription) corresponding to the predetermined cell type.

[0018] The basal cell layer is, for example, a cell population in which 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of cells co-express P63 and keratin 5. The intermediate cell layer is, for example, a cell population in which 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of cells co-express P63 and uroplakin. The superficial cell layer is, for example, a cell population in which 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of cells express uroplakin but do not substantially express P63. The superficial cell layer is, for example, a cell population in which 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of cells express uroplakin and either CK20 or ZO1 or both (preferably both)

[0019] The cell layer of a sheet-shaped cell culture can be identified by analyzing and measuring cells within a circle with a radius of 100 μm from the center of the circumscribing circle of the cell culture when the cell culture is viewed perpendicular to its main surface. If a sheet-shaped cell culture contains tears or holes, the cell layer of the sheet-shaped cell culture can be identified by analyzing and measuring cells within a circle with a radius of 100 μm from the center of the circumscribing circle of a substantially flat area with a substantially uniform thickness and no tears or holes. The proportion of a given cell type in the cell layer can be measured by observing a sheet-shaped cell culture in which a cell marker corresponding to the cell type has been visualized under a microscope, counting the number of the given cell type and other cell types present within the cell monolayer within the given circle, and calculating the ratio of the number of the given cell type to the total number of cells. The proportion of a given cell type in the cell layer of basal cells may be counted, for example, in the cell monolayer facing the exterior of the sheet-shaped cell culture. The proportion of a given cell type in the cell layer of superficial cells may be counted, for example, in the cell monolayer facing the exterior of the sheet-shaped cell culture. The proportion of a given cell type in the intermediate cell layer may be counted, for example, in a cell monolayer located intermediate between the basal cell layer and the superficial cell layer.

[0020] In the context of a sheet-shaped cell culture, "not substantially expressing" a given marker means that the expression level of the given marker is so low that the cells cannot be characterized by the marker. In a sheet-shaped cell culture according to one embodiment of the present disclosure, cells that "not substantially express" P63 have an expression level of P63 that is reduced to at least 20%, 10%, 5%, 2.5%, or 1% of the expression level in cells that express P63 and keratin 5 in the sheet-shaped cell culture.

[0021] The expression level of a marker can be measured, for example, by quantitative PCR or immunostaining. The expression level of a marker measured by quantitative PCR may be, for example, the expression level of mRNA encoding a predetermined marker. The expression level of a marker measured by immunostaining may be, for example, the intensity of a signal (e.g., fluorescence intensity) derived from a substance (e.g., a fluorescent substance) that can generate a signal when directly or indirectly bound to an antibody that binds to a predetermined marker. A substance that can generate a signal when indirectly bound to an antibody (primary antibody) that binds to a predetermined marker may be, for example, a substance that can generate a signal when directly bound to a secondary antibody that can bind to the primary antibody.

[0022] The term "porous material" refers to a material having a large number of pores that can permeate a polymer compound and that allows the cultivation of anchorage-dependent cells. The polymer compound may be, for example, retinoic acid, BMP, or FGF. The porous material may also be permeable to, for example, calcium ions. The porous material may have voids that are not in communication with the outside space.

[0023] The porous member may be, for example, a filter paper, an ultrafiltration membrane, a nonwoven fabric, a mesh (e.g., a gauze-like mesh), a porous membrane, a membrane filter, or a semipermeable membrane. The porous member may be, for example, formed of silicone, nylon, polyvinylidene fluoride, acetyl cellulose, nitrocellulose, polyethylene terephthalate, polycarbonate, polyester (PET), or polytetrafluoroethylene (PTFE). The porous member is preferably a mesh, a porous membrane, or a membrane filter. The porous member is preferably a mesh, a porous membrane, or a membrane filter made of polycarbonate, polyester (PET), or polytetrafluoroethylene (PTFE). The porous member is preferably a porous membrane or a membrane filter made of polycarbonate, polyester (PET), or polytetrafluoroethylene (PTFE).

[0024] The pores have a size that allows permeation of, for example, polymer compounds (e.g., retinoic acid, BMP, and FGF) and enables the cultivation of anchorage-dependent cells. A size that allows the cultivation of anchorage-dependent cells is, for example, a size that does not allow cells to pass through. The size of the pores is, for example, a major axis of 0.01 to 100 μm, preferably 0.02 to 50 μm, and more preferably 0.02 to 25 μm, 0.1 to 50 μm, 0.1 to 25 μm, 0.1 to 10 μm, 0.2 to 50 μm, 0.2 to 25 μm, 0.2 to 10 μm, 0.4 to 50 μm, 0.4 to 25 μm, or 0.4 to 10 μm. The size of the pores is, for example, a major axis of 0.4 μm.

[0025] The porous member can be permeable to, for example, polymer compounds of about 1,000 kDa or less, for example, about 500 kDa or less, about 250 kDa or less, about 200 kDa or less, about 100 kDa or less, about 80 kDa or less, about 60 kDa or less, and about 50 kDa or less.

[0026] The porous member is permeable to, for example, retinoic acid, BMP, and FGF, enables the cultivation of anchorage-dependent cells, and is permeable to polymeric compounds of about 1,000 kDa or less. For example, the porous member has pores with a maximum length (e.g., major axis) of 0.01 to 100 μm, and is permeable to polymeric compounds of about 1,000 kDa or less. For example, when the pore size is 0.01 μm or more and 100 μm or less (preferably 50 μm or less, or 25 μm or less), the porous member is permeable to polymeric compounds of about 1,000 kDa or less, about 500 kDa or less, about 250 kDa or less, about 200 kDa or less, about 100 kDa or less, about 80 kDa or less, about 60 kDa or less, and about 50 kDa or less. For example, when the pore size of the porous member is 0.02 μm or more and 50 μm or less (preferably 25 μm or less), it can be permeable to polymer compounds of about 1,000 kDa or less, about 500 kDa or less, about 250 kDa or less, about 200 kDa or less, about 100 kDa or less, about 80 kDa or less, about 60 kDa or less, and about 50 kDa or less. For example, when the pore size of the porous member is 0.1 μm or more (preferably 0.2 μm or more or 0.4 μm or more) and 50 μm or less (preferably 25 μm or less or 10 μm or less), it can be permeable to polymer compounds of about 1,000 kDa or less, about 500 kDa or less, about 250 kDa or less, about 200 kDa or less, about 100 kDa or less, about 80 kDa or less, about 60 kDa or less, and about 50 kDa or less.

[0027] The porous member may be subjected to a treatment to promote cell adhesion, for example. The treatment may be, for example, applying a coating agent to the porous member or treating the surface by a physical method. The coating agent may be, for example, a basement membrane preparation, laminin, entactin, collagen, gelatin, an extracellular matrix such as Matrigel (trademark), or a polymer such as polylysine or polyornithine. The physical method may be, for example, a positive charge treatment. If the porous member functions sufficiently as a scaffold for cells, treatment to promote cell adhesion is not necessarily required.

[0028] The term "compartment" refers to a space separated by a member. In the present disclosure, a first compartment is separated from a second compartment by a porous member. The first compartment and the second compartment can each hold, for example, a culture medium. The first compartment and the second compartment can hold, for example, a cell suspension containing cells. The first compartment and / or the second compartment can, for example, not hold a liquid but hold a gas (be in a gas phase). Holding a liquid or a gas means that the liquid or gas can be present for a certain period of time.

[0029] The term "induction medium" refers to a medium that can be used to induce differentiation or produce desired cells, spheroids, or organoids. The induction medium can be prepared, for example, by adding an additive or differentiation inducer (solid or liquid) according to the present disclosure to a basal medium (liquid). The induction medium used to produce a sheet-shaped cell culture according to the present disclosure is also referred to as a "sheet maturation medium." The sheet maturation medium contains a basal medium and an additive containing retinoic acid, bone morphogenetic protein, and fibroblast growth factor.

[0030] The term "retinoic acid (RA)" generally refers to a compound of the chemical formula C ２０ H ２８ O ２ and is a compound registered under CAS number 302-79-4. Retinoic acid includes, for example, all-trans retinoic acid (ATRA), 9-cis-retinoic acid, 11-cis-retinoic acid, and 13-cis-retinoic acid. The retinoic acid is preferably ATRA or 9-cis-retinoic acid, and more preferably ATRA.

[0031] The term "bone morphogenetic protein (BMP)" refers to a protein that belongs to the transforming growth factor β superfamily and induces ectopic bone formation. The BMP may be, for example, BMP2, BMP4, or BMP7, or a combination thereof. The BMP is preferably BMP4.

[0032] The term "fibroblast growth factor (FGF)" refers to a protein with a molecular weight of 16,000 to 20,000 that promotes the proliferation of fibroblasts or endothelial cells. The FGF may be, for example, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23, or a combination thereof. The FGF is preferably at least one selected from the group consisting of FGF4, FGF7, and FGF10. The FGF is preferably either FGF4 or FGF10, or a combination thereof. The FGF is preferably either FGF4 or FGF7, or a combination thereof. The FGF is preferably either FGF7 or FGF10, or a combination thereof. The FGF is preferably FGF4. The FGF is preferably FGF7. The FGF is preferably FGF10.

[0033] The term "extracellular matrix (ECM)" refers to a collection of extracellular molecules that can provide structural and biochemical support to surrounding cells. The structural and biochemical support can provide anchorage-dependent cells with a scaffold for cell growth. The structural and biochemical support can be in the form of, for example, a gel or a viscous liquid. The ECM includes, for example, water, polysaccharides, elastin, integrins, and glycoproteins. Glycoproteins include, for example, collagen, entactin (nidogen), fibronectin, and laminin.

[0034] ECM can be prepared, for example, by culturing ECM-producing cells (e.g., epithelial cells, endothelial cells, parietal endoderm-like cells, or fibroblasts) in vitro and then removing the ECM-producing cells. The ECM-producing cells may be, for example, chondrocytes, which primarily produce collagen and proteoglycans; fibroblasts, which primarily produce type IV collagen, laminin, interstitial procollagen, and fibronectin; and colonic myofibroblasts, which primarily produce collagen (types I, III, and V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tenascin-C.

[0035] ECM is commercially available. Commercially available extracellular matrices may be, for example, extracellular matrix proteins (Invitrogen), basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex® Basement Membrane Extract (Trevigen, Inc.), or Matrigel® (Corning)). The ECM may be a synthetic extracellular matrix (e.g., ProNectin (Sigma Z378666)). The extracellular matrix may be one type or a mixture of two or more types. In one embodiment, the ECM is Matrigel.

[0036] The term "ROCK inhibitor" refers to a compound that inhibits Rho-associated, coiled-coil containing protein kinase (ROCK). Examples of ROCK inhibitors include N-(4-pyridinyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (Y-27632), Fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepine (H-1152), 4β-[(1R)-1-aminoethyl]-N-(4-pyridyl)benzene-1αcarboxamide (Wf-536), and N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4β-[(R)-1-aminoethyl]cyclohexane-1α. carboxamide (Y-30141), N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide (GSK269962A), or N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A), or a combination thereof. The ROCK inhibitor may be, for example, Y-27632 alone or in combination with another ROCK inhibitor. The ROCK inhibitor is preferably Y-27632.

[0037] ROCK is a serine-threonine protein kinase. ROCK phosphorylates, for example, myosin-binding subunit 1 (MYPT1) of myosin light chain phosphatase (MLCP) to inhibit its enzymatic activity. The ROCK inhibitory effect can be measured, for example, based on the amount of phosphorylation of MYPT1 by ROCK in the presence of a compound capable of inhibiting the phosphorylation effect. In one example, the ROCK inhibitory effect can be measured by measuring the amount of phosphorylation of MYPT1 by ROCK, measuring the amount of phosphorylation of MYPT1 by ROCK in the presence of a compound capable of inhibiting the phosphorylation effect, and comparing the amounts of phosphorylation. The ROCK inhibitory activity can be measured using a commercially available kit (for example, a 96-well ROCK Activity Assay Kit (CELL BIOLABS, INC., Catalog No.: STA-416) or a ROCK Activity Immunoblot Kit (STA-416, CELL BIOLABS, INC., Catalog No.: STA-415)).

[0038] "Induction medium A" comprises a basal medium and an additive containing activin A and a GSK3β inhibitor. Induction medium A can be used, for example, to induce differentiation of pluripotent stem cells (e.g., human iPS cells) into definitive endoderm cells.

[0039] "Induction medium B" comprises a basal medium and an additive containing fibroblast growth factor (FGF) and a GSK3β inhibitor, and may further contain bone morphogenetic protein (BMP). Induction medium B can be used, for example, to induce differentiation of ventral hindgut organoids from definitive endoderm cells.

[0040] Induction medium B can be used, for example, to induce differentiation of hindgut organoids from definitive endoderm cells, and then induce differentiation of ventral hindgut organoids from the hindgut organoids. When inducing differentiation of hindgut organoids from definitive endoderm cells, induction medium B is characterized by being substantially free of BMP. The induction medium B is also referred to as "hindgut induction medium B." Hindgut induction medium B contains a basal medium and an additive containing FGF and a GSK3β inhibitor, and is substantially free of BMP.

[0041] When inducing differentiation of ventral hindgut organoids from hindgut organoids, the induction medium B is characterized by further containing BMP. The induction medium B is also referred to as "ventralization induction medium B." Ventralization induction medium B contains a basal medium and an additive containing FGF, a GSK3β inhibitor, and BMP.

[0042] When inducing differentiation of hindgut organoids from definitive endoderm cells without changing the combination of additives, induction medium B is characterized by containing BMP. Induction medium B is also referred to as "ventral hindgut induction medium B." Ventral hindgut induction medium B contains a basal medium and additives containing FGF, a GSK3β inhibitor, and BMP.

[0043] "Induction medium C" comprises a basal medium and an additive containing retinoic acid and a fibroblast growth factor. Induction medium C can be used, for example, to induce differentiation of a sheet-shaped cell culture (bladder epithelial cell sheet) from the progenitor cell population according to the present disclosure. The induction medium C is also referred to as "sheet maturation medium C." Sheet maturation medium C is characterized by, for example, containing a relatively low concentration (e.g., 1 nM) of retinoic acid and further containing a bone morphogenetic protein. Sheet maturation medium C comprises, for example, a basal medium and an additive containing a relatively low concentration of retinoic acid, a fibroblast growth factor, and a bone morphogenetic protein.

[0044] Induction medium C can be used, for example, to induce bladder epithelial cells in ventral hindgut organoids and induce differentiation into bladder organoids. Induction medium C can be used, for example, to induce differentiation into immature bladder organoids from ventral hindgut organoids, and then to induce differentiation into mature bladder organoids from the immature bladder organoids.

[0045] Induction medium C can be used, for example, to induce differentiation of immature bladder organoids from ventral hindgut organoids. The induction medium C is also referred to as "immature organoid induction medium C." Immature organoid induction medium C is characterized by, for example, containing a relatively high concentration (e.g., 1 μM) of retinoic acid. Immature organoid induction medium C, for example, contains a basal medium and an additive containing a relatively high concentration of retinoic acid and fibroblast growth factor.

[0046] Induction medium C can be used, for example, to induce differentiation of immature bladder organoids into mature bladder organoids. The induction medium C is also referred to as "organoid maturation medium C." Organoid maturation medium C is characterized by, for example, containing a relatively low concentration (e.g., 1 nM) of retinoic acid and further containing a bone morphogenetic protein. Organoid maturation medium C, for example, contains a basal medium and an additive containing a relatively low concentration of retinoic acid, a fibroblast growth factor, and a bone morphogenetic protein.

[0047] Induction medium C can be used, for example, to induce differentiation of bladder organoids from ventral hindgut organoids. The induction medium C is also referred to as "bladder organoid induction medium C." Bladder organoid induction medium C is characterized by, for example, containing a relatively medium concentration (e.g., 100 nM) of retinoic acid and further containing bone morphogenetic protein. Bladder organoid induction medium C, for example, contains a basal medium and an additive containing a relatively medium concentration of retinoic acid, a fibroblast growth factor, and a bone morphogenetic protein.

[0048] The term "organoid" refers to a three-dimensional cell aggregate formed in vitro that resembles living tissue. The cells that make up organoids are, for example, mostly cells with differentiation and proliferation capabilities. As used herein, an organoid expressing a specific marker refers to an organoid that contains a specific proportion of cells that express the specific marker. The specific proportion may be, for example, 3% or more, 5% or more, 10% or more, or 15% or more of the cells that make up the organoid.

[0049] "Hindgut organoids" can be formed, for example, by culturing definitive endoderm cells using an induction medium (hindgut induction medium B) containing fibroblast growth factor and a GSK3β inhibitor. Hindgut organoids can be formed, for example, by culturing definitive endoderm cells using hindgut induction medium B, and then three-dimensionally culturing them in an extracellular matrix gel using hindgut induction medium B. Hindgut organoids in the extracellular matrix gel can be recovered according to known methods or using commercially available reagents. Hindgut organoids in the extracellular matrix gel can be recovered, for example, by gently breaking down the extracellular matrix gel using metalloprotease.

[0050] The term "ventral hindgut organoid" refers to an organoid that expresses at least one ventral hindgut marker (P63, ΔNp63, GATA3, ISL1, and SATB2) and does not substantially express at least one dorsal hindgut marker (SOX2, CDX2, and T), or if expressed, the expression level is lower than that in hindgut organoids. Ventral hindgut organoids can be produced, for example, by ventralizing hindgut organoids. Ventralization involves culturing hindgut organoids using ventralization induction medium B in the presence of an extracellular matrix.

[0051] Ventral hindgut organoids can be induced to differentiate from definitive endoderm-differentiated cells using induction medium B. Ventral hindgut organoids can be formed, for example, by culturing definitive endoderm-differentiated cells using induction medium B to form floating spheroids, culturing the floating spheroids in the presence of an extracellular matrix using hindgut induction medium B to form hindgut organoids, and then culturing the hindgut organoids using ventralization induction medium B. Ventral hindgut organoids formed by the differentiation induction method are also referred to as ventral hindgut spheroids or cloacal spheroids.

[0052] The term " bladder organoid " refers to an organoid having at least two cell layers. The at least two cell layers include a first cell layer comprising cells that do not substantially express P63 but express UPK1B and / or UPK2; and a second cell layer that is located outward relative to the first cell layer and comprises cells that co-express P63 and UPK1B and / or UPK2. The bladder organoid having the two cell layers is also referred to as " immature bladder organoid ". For example, immature bladder organoid comprises the first cell layer and the second cell layer that is located outward relative to the first cell layer, and does not comprise the cell layer that comprises cells that express KRT5, a basal cell marker.

[0053] Bladder organoids, for example, comprise a first cell layer comprising cells that do not substantially express P63 but express UPK1B and / or UPK2; a second cell layer comprising cells that co-express P63 and UPK1B and / or UPK2, and located outward relative to the first cell layer; and a third cell layer comprising cells that co-express P63 and KRT5, and located outward relative to the second cell layer. Bladder organoids, for example, comprise a first cell layer comprising cells that express UPK1B and / or UPK2; the second cell layer that is located outward relative to the first cell layer; and the third cell layer that is located outward relative to the second cell layer. Bladder organoids having these three cell layers are also referred to as "mature bladder organoids".

[0054] For example, matured bladder organoid comprises the first cell layer having a lumen and facing the lumen; the second cell layer located in the outer direction relative to the first cell layer; and the third cell layer located in the outer direction relative to the second cell layer.For example, matured bladder organoid comprises the first cell layer having a lumen and facing the lumen; the second cell layer located in the outer direction relative to the first cell layer; and the third cell layer located in the outer direction relative to the second cell layer, and the first cell layer and the second cell layer are directly in contact with each other, and the second cell layer and the third cell layer are directly in contact with each other, or the second cell layer is interposed between the first cell layer and the third cell layer.

[0055] The matured bladder organoids, for example, comprise a first cell layer, which is located along its outermost periphery, and comprises cells that co-express P63 and KRT5; a second cell layer, which is located inward relative to the first cell layer, and comprises cells that co-express P63 and UPK1B and / or UPK2; and a third cell layer, which is located inward relative to the second cell layer, and comprises cells that do not substantially express P63 but express UPK1B and / or UPK2. The matured bladder organoids, for example, comprise the first cell layer, which is located along its outermost periphery; the second cell layer, which is located inward relative to the first cell layer; and the third cell layer, which is located inward relative to the second cell layer, and the third cell layer has opposing lumens in the direction from the first cell layer to the third cell layer. In this specification, the cell layers of the matured bladder organoids are also referred to as follows: the first cell layer is also referred to as a basal cell layer, the second cell layer is also referred to as an intermediate cell layer, and the third cell layer is also referred to as a superficial cell layer.

[0056] In the context of organoids, "not substantially expressing" a certain marker means that the expression level of the certain marker is so low that it cannot be characterized by the marker.An organoid that "not substantially expresses" a certain marker may be an organoid in which the expression level of the particular marker in the organoid is reduced to less than 70%, less than 80%, less than 90%, or less than 95% compared to the expression level of the certain marker in a control organoid.

[0057] The term "lumen" refers to the space inside a tubular or sac-like cell structure. The lumen may be, for example, filled with fluid. The lumen of a matured bladder organoid is the space inside a sac-like cell structure. The lumen of a matured bladder organoid is, for example, the cell-free space inside a sac-like cell structure.

[0058] The term "pluripotent stem cells" refers to stem cells that can be cultured in vitro and have the ability to differentiate into tissues derived from three germ layers (ectoderm, mesoderm, and endoderm), i.e., pluripotency. Pluripotent stem cells can be established, for example, from fertilized eggs, cloned embryos, germ stem cells, or stem cells in tissues. Pluripotent stem cells are embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) derived from somatic cells, embryonic tumor cells (EC cells), or embryonic germ stem cells (EG cells). Pluripotent stem cells are preferably ES cells or iPS cells.

[0059] "Definitive endoderm cells" can be induced to differentiate by culturing pluripotent stem cells using induction medium A containing activin A and a GSK3β inhibitor.

[0060] The term "mesenchymal cells" refers to cells derived from the mesenchyme of a multicellular animal during its fetal stage. Mesenchymal cells have the ability to differentiate into, for example, supportive tissue, connective tissue, bone cells, chondrocytes, and adipocytes. An example of a mesenchymal cell is a bladder mesenchymal cell. Bladder mesenchymal cells can be prepared, for example, from a non-human mammal according to a known method or a method described in the Examples herein. Mesenchymal cells prepared from a non-human mammal include, for example, embryonic fibroblasts. An example of an embryonic fibroblast is a mouse embryonic fibroblast (MEF). Mesenchymal cells can be prepared, for example, from ES cells or iPS cells by inducing differentiation according to a known method. Mesenchymal cells can be obtained, for example, commercially.

[0061] The term "mesenchymal stem cells" refers to cells that have the ability to self-renew and differentiate into cells that constitute non-epithelial mesenchyme, such as connective tissue, bone cells, chondrocytes, and adipocytes. Mesenchymal stem cells can be prepared, for example, from ES cells or iPS cells by inducing differentiation according to known methods. Mesenchymal stem cells can be obtained, for example, commercially. Mesenchymal stem cells can be recovered, for example, from living organisms according to known methods. Mesenchymal stem cells are known to exist in living organisms, for example, in dental pulp or bone marrow fluid.

[0062] The term "P63" refers to a protein that is a homolog of the tumor suppressor gene P53 and has functions such as differentiation, proliferation, and maintenance in epithelia. P63 can be used as a bladder epithelial cell marker or a ventral hindgut / Cloaca marker. P63 expressed in cells or organoids can be detected or measured, for example, by immunostaining using an anti-P63 antibody (e.g., anti-P63 antibody rabbit monoclonal (EPR5701)).

[0063] The term "ΔNP63" refers to a p63 isoform lacking the N-terminal transactivation domain (TN). ΔNP63 can be used as a bladder epithelial cell marker or a ventral hindgut marker. ΔNP63 expressed in cells or organoids can be detected or measured, for example, by immunostaining using an anti-ΔNP63 antibody (e.g., Rabbit Anti-ΔNP63 (Cell signaling, #67825)).

[0064] The term "uroplakin (UPK)" refers to a membrane glycoprotein that is involved in the formation of lining cells of transitional epithelium that forms the urinary tract epithelium and has the effect of enhancing the permeability and barrier function of lining cells. Uroplakins include uroplakins Ia, Ib, II, and III. UPK is, for example, UPK1B or UPK2. "UPK1B" can be used, for example, as a bladder epithelium marker or epithelial tissue marker. UPK1B can be detected or measured, for example, by immunostaining using an anti-UPK1B antibody (Mouse Anti-UPK1B monoclonal antibody (clone 1E1) (Sigma-Aldrich, #WH0007348M2). UPK2 is a membrane glycoprotein with a molecular weight of approximately 15 kDa. UPK2 can be used, for example, as a bladder epithelium marker. UPK2 can be detected or measured, for example, by immunostaining using an anti-UPK2 antibody (for example, Mouse Anti-Uroplakin II (BIOCARE MEDICAL, #ACR3051C)).

[0065] The term "keratin 5 (KRT5)" refers to a protein encoded by the KRT5 gene that dimerizes with keratin 14 to form intermediate filaments that constitute the cytoskeleton of basal epithelial cells. KRT5 can be used as a bladder epithelial marker. KRT5 can be detected or measured, for example, by immunostaining using an anti-KRT5 antibody (e.g., Chicken Anti-Keratin 5 (BioLegend, #905903)).

[0066] The term "cytokeratin 20 (CK20)" refers to a type I acidic low-molecular-weight cytokeratin. CK20 is known to be less acidic than other type I keratins and to be expressed in limited tissues. CK20 is present in urothelial cells, gastric and intestinal epithelial cells, and Merkel cells in the skin. CK20 can be used as a surface cell marker. CK20 can be detected or measured, for example, by immunostaining using an anti-CK20 antibody (Dako, M7019).

[0067] The term "ZO1" refers to a membrane phosphoprotein expressed in the tight junctions of epithelial cells and endothelial cells. ZO1 can be used as a tight junction marker. Expression of ZO1 in the cell membrane connecting cells suggests the formation of tight junctions between the cells. Expression of ZO1 in the cell membrane connecting the surface cells in the cell layer of the surface cells of the sheet-shaped cell culture according to the present disclosure suggests that the sheet-shaped cell culture may have a barrier function. ZO1 can be detected or measured, for example, by immunostaining using an anti-ZO1 antibody (Goat Anti-ZO1 (ThermoFisher, #PA5-19090)).

[0068] The term "CDX2" refers to a homeobox protein encoded by the CDX2 gene. CDX2 can be used as a midgut / hindgut marker. The term "SOX2," also known as SRY (sex determining region Y)-box 2, refers to a transcription factor essential for maintaining the self-renewal of undifferentiated ES cells. SOX2 can be used as a dorsal hindgut marker or a lung / stomach lineage marker.

[0069] The term "T," also known as TBXT (T-box transcription factor T), refers to a transcription factor that binds to a DNA sequence called a palindromic T site via an N-terminal region called a T box, and affects the transcription of genes necessary for mesoderm formation and differentiation. T can be used as a dorsal hindgut marker. The term "ISL1" refers to a transcription factor having a LIM homeodomain that acts on the expression regulatory region of the insulin gene. ISL1 can be used as a ventral hindgut marker. The term "SATB2" refers to a DNA-binding protein that binds to AT-rich sequences. SATB2 can be used as a colon or rectum marker or a ventral hindgut marker.

[0070] The term "FOXA2" is an abbreviation for Forkhead box protein A2 and refers to a transcription factor that plays an important role in development. FOXA2 can be used as a marker for early intestinal epithelium or developing bladder epithelial cells. The term "ECAD," also known as E-cadherin, refers to a transmembrane glycoprotein present on the cell surface that acts on cell adhesion. ECAD can be used as an epithelial tissue marker.

[0071] The term "HOXA13" refers to a homeobox protein encoded by the HOXA13 gene in humans. HOXA13 expression in cells or organoids can be detected or measured, for example, by quantitative PCR. The term "Phospho-Smad1 / 5 / 8" or "Smad1 / 5 / 8" refers to a transcription factor that plays an important role in the intracellular TGF-β signaling pathway. Phospho-Smad1 / 5 / 8 can be used as a marker for the ventral hindgut and Cloaca region.

[0072] The term "activin A" refers to a factor belonging to the TGFβ superfamily and a protein that promotes FSH (follicle-stimulating hormone) secretion from the anterior pituitary gland. Activin A exhibits various functions in regulating cell differentiation, proliferation, apoptosis, and carcinogenesis. Induction medium A may contain activin A at, for example, 10 to 500 ng / mL, 30 to 250 ng / mL, or 50 to 150 ng / mL.

[0073] The term "GSK3β inhibitor" refers to a compound that inhibits the action of serine-threonine protein kinase 3β, which is involved in various signaling pathways, including the WNT / β-catenin pathway. The GSK3β inhibitor may be, for example, CHIR99021, SB216763, CHIR-98014, Staurosporine, K252A, WNT (preferably WNT3A), or TWS119, or a combination thereof. The GSK3β inhibitor is preferably CHIR99021 or WNT (preferably WNT3A). Induction Medium A may contain the GSK3β inhibitor at a concentration that exhibits the same level of GSK3β inhibitory effect as that exhibited by CHIR99021 at 0.1 to 5 μM, 0.3 to 2.5 μM, or 0.5 to 1.5 μM.

[0074] The term "pharmaceutical composition" includes the sheet-shaped cell culture or matured bladder organoids disclosed herein. The pharmaceutical composition can be used, for example, to treat bladder damage or bladder disease in mammals. The term "pharmaceutically acceptable carrier" refers to any component other than the sheet-shaped cell culture or matured bladder organoids disclosed herein, which is highly safe and has low allergic reactivity in mammals. Pharmaceutically acceptable carriers include, for example, aqueous or non-aqueous solvents, solutions (e.g., saline, basal medium, or cell suspension preservative), cryoprotectants (e.g., glycerol), water-soluble polymers (e.g., dextran), or buffers (e.g., phosphate buffer) suitable for pharmaceutical administration. Pharmaceutical compositions can be appropriately prepared according to known methods. In one example, the pharmaceutical composition disclosed herein can be prepared by combining the sheet-shaped cell culture or matured bladder organoids with a pharmaceutically acceptable carrier (e.g., basal medium).

[0075] The term "non-human mammal" may be, for example, a rodent such as a mouse, rat, guinea pig, hamster, etc.; a non-human primate such as a chimpanzee; an even-toed ungulate such as a cow, goat, sheep, etc.; a perissodactyla such as a horse; or a companion animal such as a rabbit, dog, cat, etc. The non-human mammal may be, for example, a rodent or a non-human primate.

[0076] The term "bladder" refers to a sac-like organ that temporarily stores urine delivered from the kidneys. The bladder may be a normal bladder without any particular damage or disease, or may be an injured bladder or a bladder affected by a bladder disease. The bladder may be, for example, a normal bladder. The bladder may be, for example, an injured bladder or a bladder affected by a bladder disease.

[0077] The term "urine" refers to a liquid containing substances contained in bodily fluids produced by the kidneys and excreted from the bladder. Urine may be, for example, either urine derived from a living organism or artificial urine, or a mixture thereof. Urine derived from a living organism is commercially available, for example, from Lee BioSolutions. Artificial urine is commercially available, for example, from Hayashi Pure Chemical Industries, Ltd. Artificial urine, for example, contains urea at the same level as urine derived from a living organism. Artificial urine, for example, contains at least one component selected from the group consisting of ammonia, creatinine, uric acid, amino acids, sulfuric acid, phosphorus, oxalic acid, sodium, potassium, and calcium, at the same level as urine derived from a living organism.

[0078] The term "surrounding area" of the bladder refers to tissues or areas adjacent to or near the urinary system. The urinary system includes the kidneys, ureters, bladder, and urethra. The area surrounding the bladder can be, for example, the intraperitoneal cavity or the mesentery. The term "bladder injury" or "bladder disease" can refer to, for example, a bladder damaged by trauma, radiation cystitis, a bladder damaged by diabetes or ischemia, a bladder damaged by a drug harmful to bladder tissue, cystitis, or bladder cancer.

[0079] "Treatment" of bladder injury or disease includes maintaining, reducing, or eliminating the symptoms or pathology.

[0080] The term "mammal" refers to, for example, humans and non-human mammals. Non-human mammals may be, for example, rodents such as mice, rats, guinea pigs, hamsters, etc., non-human primates such as chimpanzees, even-toed ungulates such as cows, goats, sheep, perissodactyls such as horses, or companion animals such as rabbits, dogs, cats, etc. In one embodiment, the mammal is a human.

[0081] The term "mammal in need thereof" refers to a mammal having or suspected of having a bladder injury or disease. A mammal having a bladder injury or disease refers to a mammal that has been diagnosed by a medical professional (e.g., a physician) as having a bladder injury or disease according to predetermined diagnostic criteria. A mammal suspected of having a bladder injury or disease may be a mammal suspected of having a bladder injury or disease based on, for example, the mammal's behavioral history (e.g., trauma, radiation therapy, and having received or having received drugs harmful to bladder tissue) or medical history (e.g., having or having suffered from diabetes, ischemia, cystitis, or bladder cancer). The mammal is preferably a human or non-human primate, more preferably a human.

[0082] The term "test substance" may be, for example, a small molecule compound, a protein (e.g., an antibody), DNA, RNA, small interfering RNA, or an antisense oligonucleotide. The test substance may be, for example, a drug for treating a bladder disorder or disease, or bladder cancer, or a candidate substance thereof. The test substance may be, for example, one type, or a mixture of two or more types. The test substance is preferably one type of substance.

[0083] [Sheet-shaped cell culture and method for producing same] One aspect of the present disclosure provides a sheet-shaped cell culture. Another aspect of the present disclosure provides a method for producing the sheet-shaped cell culture. The method includes culturing a progenitor cell population in a second compartment separated from a first compartment by a porous member to form a sheet-shaped cell culture in the second compartment. The sheet-shaped cell culture comprises, in this order, a cell layer of superficial cells derived from the progenitor cell population, a cell layer of intermediate cells derived from the progenitor cell population, and a cell layer of basal cells derived from the progenitor cell population, and the first compartment holds an induction medium (sheet maturation medium C) containing retinoic acid, bone morphogenetic protein, and fibroblast growth factor. Sheet maturation medium C preferably further comprises an extracellular matrix.

[0084] A method for producing a sheet-shaped cell culture according to this embodiment will be described with reference to Figure 1. In Figure 1, the period from Day 0 to Day 20 represents the step of preparing a progenitor cell population capable of differentiating into superficial cells, intermediate cells, and basal cells. The preparation step includes the steps of inducing differentiation of definitive endoderm (DE) cells from human iPS cells (Day 0 to Day 3), forming ventral hindgut / cloacal spheroids (included in the term "ventral hindgut organoids" in this disclosure) from the DE cells (Day 3 to Day 14), forming immature bladder organoids from the ventral hindgut organoids (Day 14 to Day 20), and preparing a substantially isolated cell population from the immature bladder organoids (Day 20). The step of preparing the progenitor cell population is described in further detail in the "Method for Preparing a Progenitor Cell Population" section below.

[0085] In Figure 1, the period from Day 20 to beyond Day 42 indicates the process of producing a sheet-shaped cell culture from the progenitor cell population. The process of producing the sheet-shaped cell culture includes, for example, introducing the prepared progenitor cell population into a culture vessel 100 (Day 20). The culture vessel 100 includes a cylindrical first container 10 and a cylindrical second container 20. The first container 10 includes a first bottom surface and a first side wall that define a first compartment 1. The second container 20 includes a second bottom surface and a second side wall that define a second compartment 2. The second bottom surface is composed of a porous member 3 (e.g., a membrane filter). The second container 20 can be superimposed on the first container 10 by inserting it into the first compartment 1.

[0086] Introduction of the progenitor cell population into the culture vessel 100 includes, for example, dispensing the cell population into the second compartment 2 using a dispenser such as a pipette. A sheet-shaped cell culture is produced by culturing the progenitor cell population in the second compartment 2 (after Day 42). The method for producing the sheet-shaped cell culture shown in FIG. 1 is an example, and the method for producing a sheet-shaped cell culture according to the present disclosure is not limited to the method exemplified in FIG. 1. Below, a method for producing a sheet-shaped cell culture, including the step of culturing the progenitor cell population in the second compartment, is described in detail.

[0087] In a method for producing a sheet-shaped cell culture, when a progenitor cell population is cultured in the second compartment, a sheet maturation medium C containing retinoic acid (RA), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) is present in the first compartment. The presence of an induction medium in the first compartment via a porous member allows the RA, BMP, FGF, and nutritional components in the sheet maturation medium C to be supplied from the first compartment to the second compartment via the porous member. This allows the progenitor cell population in the second compartment to use the RA, BMP, FGF, and nutritional components for differentiation or proliferation.

[0088] The porous member, for example, includes a first main surface and a second main surface opposite the first main surface. The porous member, for example, includes a first main surface facing the first compartment and a second main surface opposite the first main surface and facing the second compartment. When the progenitor cell population is applied to the second compartment using a dispenser such as a pipette, the second main surface of the second compartment functions, for example, as a culture surface. The progenitor cell population applied to the second compartment includes a plurality of substantially isolated cells.

[0089] A "substantially isolated" cell population includes cells that exist individually and without being linked to other cells. A substantially isolated cell population, for example, contains at least 50% of cells that exist individually and without being linked to other cells per total cell population. A substantially isolated cell population does not exclude that all of the progenitor cells of the cell population exist individually and without being linked to other cells. The degree of isolation is calculated by staining the cell nuclei of a portion of a sample of the substantially isolated cell population with a staining reagent such as DAPI, and then detecting 2,500 cells under a microscope or using a cell sorter. In the context of a substantially isolated cell population, a cluster of cells that are linked to other cells is treated as a single cell. A substantially isolated cell population, for example, contains 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of the total cells of the cell population that exist individually and without being linked to other cells. The thickness of the sheet-shaped cell culture produced by the production method of the present disclosure tends to be more uniform when the progenitor cell population applied to the second compartment contains a higher proportion of substantially isolated cells. The substantially isolated progenitor cell population used in the production method can be prepared according to the method described below in "Method for preparing a progenitor cell population."

[0090] Culturing the progenitor cell population in the second compartment includes providing the progenitor cell population to the second compartment using a dispenser such as a pipette or through a channel in fluid communication with the second compartment. Upon application of the progenitor cell population to the second compartment, the substantially isolated progenitor cell population may reach about 30% to about 110% confluency, about 30 to about 100% confluency, about 30% to about 90% confluency, about 30% to about 80% confluency, about 30% to about 70% confluency, about 40% to about 110% confluency, about 30 to about 100% confluency, about 40% to about 90% confluency, or about 40% to about 80% confluency, relative to the main surface (culture surface) of the porous member. confluency, about 40% to about 70% confluency, about 50% to about 110% confluency, about 30 to about 100% confluency, about 50% to about 90% confluency, about 50% to about 80% confluency, about 50% to about 70% confluency, about 60% to about 110% confluency, about 30 to about 100% confluency, about 60% to about 90% confluency, about 60% to about 80% confluency, or preferably about 60% to about 70% confluency. Since the sheet-shaped cell culture formed tends to have a uniform thickness, it is preferable to apply the progenitor cell population to the second compartment at a cell density that results in about 60% to about 80% confluency.

[0091] A portion of the progenitor cells in the progenitor cell population may be floating, and the remaining progenitor cells may be adhered to the main surface (culture surface) of the porous member. The progenitor cell population in the second compartment is preferably adhered to the main surface (culture surface) of the porous member. The progenitor cell population in the second compartment may be cultured until it adheres to the porous member. The medium used to adhere the progenitor cell population in the second compartment to the porous member may be, for example, a basal medium, a ROCK inhibitor (e.g., 10 μM Y-27632), retinoic acid (e.g., 1 nM ATRA), and BMP (e.g., 10 ng ml －１ BMP4) and FGF (e.g., 100 ng ml －１ FGF7 and 10 ng ml －１ FGF10) and a calcium salt (e.g., 1 mM CaCl ２ ) and heparin (e.g., 1 μg ml－１ ) and the sheet maturation medium C. When the progenitor cell population in the second compartment is attached to the porous member, the first compartment and the second compartment contain, for example, the sheet maturation medium C. In another example, when the progenitor cell population in the second compartment is attached to the porous member, the first compartment may contain the sheet maturation medium C, and the second compartment may be in a gas phase.

[0092] Sheet maturation medium C may be applied to the first compartment using, for example, a dispenser such as a pipette, or may be flowed into the first compartment using a channel that is fluidly connected to the first compartment. Sheet maturation medium C can be prepared, for example, by adding the additive (solid or liquid) to a basal medium (liquid). The concentration of the additive to be added to sheet maturation medium C can be appropriately determined by those skilled in the art, taking into consideration the animal species from which the cells used for culture are provided.

[0093] The "basal medium" may be a cell culture medium that can be prepared according to a known protocol or may be a commercially available cell culture medium. The basal medium may be, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle's (BME), a known stem cell culture medium, or a known medium for differentiating stem cells. The basal medium is preferably a medium for differentiating stem cells, such as STEMdiff APEL2 medium (STEMCELL Technologies; hereinafter also referred to as "APEL2 basal medium"). The medium for differentiating stem cells can be prepared, for example, according to Nature Protocols, Vol. 3, No. 5, pp. 768-776, 2008 (the entire disclosure of which is incorporated herein by reference). The basal medium can be prepared, for example, by referring to the composition of "APEL2 basal medium," and the concentration of each component may be increased or decreased as appropriate according to conventional methods. Sheet maturation medium C may further contain a protein-free medium (e.g., 2% PFHM-II), an antibiotic (e.g., penicillin / streptomycin, gentamicin), or an antibiotic-antimycotic mixture (e.g., antibiotic-antimycotic), or a combination thereof.

[0094] Sheet maturation medium C contains retinoic acid (preferably ATRA or 9-cis-retinoic acid, more preferably ATRA) at concentrations of, for example, 0.1 to 20 nM, 0.1 to 15 nM, 0.1 to 10 nM, 0.1 to 7.5 nM, 0.1 to 5 nM, 0.1 to 4 nM, 0.1 to 3 nM, 0.1 to 2 nM, 0.2 to 20 nM, 0.2 to 15 nM, 0.2 to 10 nM, 0.2 to 7.5 nM, 0.2 to 5 nM, 0.2 to 4 nM, 0.2 to 3 nM, 0.2 to 2 nM, 0.4 to 20 nM, 0.4 to 15 nM, 0.4 to 1 Contains 0 nM, 0.4-7.5 nM, 0.4-5 nM, 0.4-4 nM, 0.4-3 nM, 0.4-2 nM, 0.6-20 nM, 0.6-15 nM, 0.6-10 nM, 0.6-7.5 nM, 0.6-5 nM, 0.6-4 nM, 0.6-3 nM, 0.6-2 nM, 0.6-1.5 nM, 0.8-20 nM, 0.8-15 nM, 0.8-10 nM, 0.8-7.5 nM, 0.8-5 nM, 0.8-4 nM, 0.8-3 nM, 0.8-2 nM, 0.8-1.5 nM, 0.8-1.2 nM, or 1 nM.

[0095] The sheet maturation medium C contains a bone morphogenetic protein (e.g., at least one selected from the group consisting of BMP2, BMP4, and BMP7, preferably BMP4) at concentrations of, for example, 1 to 100 ng / mL, 1 to 75 ng / mL, 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 30 ng / mL, 1 to 20 ng / mL, 1 to 15 ng / mL, 2 to 100 ng / mL, 2 to 75 ng / mL, 2 to 50 ng / mL, 2 to 40 ng / mL, 2 to 30 ng / mL, 2 to 20 ng / mL, 2 to 15 ng / mL, 3 to 100 ng / mL, 3 to 100 ng / mL, 3 to 20 ng / mL, 3 to 15 ng / mL, 3 to 100 ng / mL, 3 to 20 ng / mL, 3 to 20 ng / mL, 3 to 15 ... ~75ng / mL, 3~50ng / mL, 3~40ng / mL, 3~30ng / mL, 3~20ng / mL, 3~15ng / mL, 5~100ng / mL, 5~75ng / mL, 5~50ng / mL, 5~40ng / mL, 5~30ng / mL, 5~20 Contains ng / mL, 5 to 15 ng / mL, 7.5 to 100 ng / mL, 7.5 to 75 ng / mL, 7.5 to 50 ng / mL, 7.5 to 40 ng / mL, 7.5 to 30 ng / mL, 7.5 to 20 ng / mL, 7.5 to 15 ng / mL, or 10 ng / mL.

[0096] The sheet maturation medium C contains a fibroblast growth factor (e.g., at least one selected from the group consisting of FGF4, FGF7, and FGF10; a combination of FGF4 and FGF7, a combination of FGF7 and FGF10, or a combination of FGF4 and FGF10; preferably a combination of FGF7 and FGF10) at a concentration of, for example, 10 to 1000 ng, 10 to 750 ng, 10 to 500 ng, 10 to 400 ng, 10 to 300 ng, 10 to 200 ng, 10 to 150 ng, 25 to 1000 ng, 25 to 750 ng, 25 to 500 ng, 25 to 400 ng, 25 to 300 ng, 25 to 200 ng, 25 to 150 ng, 40 ~1000ng, 40-750ng, 40-500ng, 40-400ng, 40-300ng, 40-200ng, 40-150ng, 60-1 000ng, 60-750ng, 60-500ng, 60-400ng, 60-300ng, 60-200ng, 60-150ng, 75-1000 Sheet maturation medium C contains the above-mentioned concentrations of FGF, for example, FGF7:FGF10 = 30:1, 25:1, 20:1, 15:1, 10:1, 7.5:1, 5:1, 2.5:1, preferably 10:1.

[0097] The sheet maturation medium C further contains, for example, a calcium salt. The sheet maturation medium C contains calcium salt at concentrations of, for example, 0.1 to 10 mM, 0.1 to 7.5 mM, 0.1 to 5 mM, 0.1 to 4 mM, 0.1 to 3 mM, 0.1 to 2 mM, 0.1 to 1.5 mM, 0.2 to 10 mM, 0.2 to 7.5 mM, 0.2 to 5 mM, 0.2 to 4 mM, 0.2 to 3 mM, 0.2 to 2 mM, 0.2 to 1.5 mM, 0.4 to 10 mM, 0.4 to 7.5 mM, 0.4 to 5 mM, The calcium salt may be, for example, an anhydrate or a hydrate, or a mixture thereof. The calcium salt may be, for example, calcium chloride (CaCl ２ ), calcium carbonate (CaCO ３ ), and calcium bicarbonate (Ca(HCO ３ ) ２ ), or a combination thereof. The calcium salt is preferably calcium chloride.

[0098] Sheet maturation medium C may further contain, for example, an extracellular matrix. When the adhesion of the progenitor cell population to the porous member is insufficient to prevent the cell culture from detaching from the porous member during the culture period for forming the sheet-shaped cell culture, it is preferable that sheet maturation medium C contain an extracellular matrix. The extracellular matrix (e.g., Matrigel) is present in an amount of, for example, 10% v / v or less, 7% v / v or less, 5% v / v or less, 4% v / v or less, 3% v / v or less, 2.5% v / v, and preferably 2% v / v per volume of the induction medium. Sheet maturation medium C, which is used until the progenitor cell population forms a thin layer of cell culture, may contain extracellular matrix in an amount of 0.1 to 10% v / v, 0.1 to 7% v / v, 0.1 to 5% v / v, 0.1 to 4% v / v, 0.1 to 3% v / v, 0.1 to 2.5% v / v, 0.1 to 2% v / v, 1 to 10% v / v, 1 to 7% v / v, 1 to 5% v / v, 0.1 to 4% v / v, 0.1 to 3% v / v, 1 to 2.5% v / v, 0.1 to 2% v / v, 1.5 to 10% v / v, 1.5 to 7% v / v, 1.5 to 5% v / v, 1.5 to 4% v / v, 1.5 to 3% v / v, 1.5 to 2.5% v / v, or 1.5 to 2% v / v. When the extracellular matrix contains a biological product (e.g., a basement membrane preparation from mouse sarcoma cells), the concentration of the extracellular matrix in sheet maturation medium C is preferably low from the viewpoint of preventing or reducing contamination of the sheet-shaped cell culture to be produced.

[0099] Culturing the progenitor cell population in the second compartment includes, for example, culturing a population containing progenitor cells floating or adhered to the surface of the porous member in the presence of sheet maturation medium C in the first compartment. The culturing is carried out, for example, until the progenitor cell population forms a thin layer cell culture. A "thin layer cell culture" refers to a membrane-like cell culture containing a cell monolayer region. A thin layer cell culture may, for example, contain a cell monolayer region and a cell multilayer region. A thin layer cell culture may, for example, contain only a cell monolayer region. The culturing is carried out for, for example, 3 to 12 days, 4 to 12 days, 5 to 12 days, 3 to 11 days, 4 to 11 days, 5 to 11 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 3 to 9 days, 4 to 9 days, 5 to 9 days, 3 to 8 days, 4 to 8 days, 5 to 8 days, 3 to 7 days, 4 to 7 days, 5 to 7 days, or 6 days. The sheet maturation medium C is changed, for example, every day, every two days, or every three days.

[0100] The culturing is carried out, for example, until the area of ​​the cell layer in the formed thin layer cell culture is 30% or more of the total area of ​​the cell culture, for example, 30% to 100%, 30% to 90%, 30% to 80%, 30% to 70%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 60% to 100%, 60% to 90%, 60% to 80%, or 60% to 70%.

[0101] The sheet maturation medium C used until a thin layer of cell culture of progenitor cells is formed may further contain, for example, a ROCK inhibitor in addition to a basal medium, retinoic acid, bone morphogenetic protein, and fibroblast growth factor. The sheet maturation medium C may further contain, for example, an extracellular matrix (e.g., Matrigel™). The sheet maturation medium C may further contain, for example, a calcium salt (e.g., CaCl ２ ) may further comprise.

[0102] Sheet maturation medium C, which is used until a thin layer of cell culture of progenitor cells is formed, may contain, for example, a basal medium, an extracellular matrix (e.g., Matrigel™), retinoic acid (e.g., 1 nM ATRA), bone morphogenetic protein (e.g., 10 ng / ml BMP4), fibroblast growth factors (e.g., 100 ng / ml FGF7 and 10 ng / ml FGF10), a ROCK inhibitor (e.g., Y-27632), and a calcium salt (e.g., CaCl ２ The sheet maturation medium C preferably contains a basal medium, an extracellular matrix (e.g., 2% Matrigel™), retinoic acid (e.g., 1 nM ATRA), bone morphogenetic protein (e.g., 10 ng / ml BMP4), fibroblast growth factors (e.g., 100 ng / ml FGF7 and 10 ng / ml FGF10), a ROCK inhibitor (e.g., 10 μM Y-27632), and 1 mM calcium salt (e.g., CaCl ２ ) is included.

[0103] Sheet maturation medium C, which is used until a thin layer of cell culture of progenitor cells is formed, contains a ROCK inhibitor (e.g., Y-27632) at, for example, 1 to 100 μM, 1 to 75 μM, 1 to 50 μM, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, 1 to 15 μM, 2.5 to 100 μM, 2.5 to 75 μM, 2.5 to 50 μM, 2.5 to 40 μM, 2.5 to 30 μM, 2.5 to 20 μM, 2. The sheet maturation medium C may contain a ROCK inhibitor other than Y-27632 at a concentration that exhibits an effect similar to that of Y-27632.

[0104] After a thin layer of cell culture of the progenitor cell population is formed in the second compartment, the thin layer of cell culture may be cultured in the first compartment in the presence of sheet maturation medium C. The culture may be carried out for, for example, 5 to 50 days, 5 to 45 days, 5 to 40 days, 5 to 35 days, 8 to 50 days, 8 to 45 days, 8 to 40 days, 8 to 35 days, 10 to 50 days, 10 to 45 days, 10 to 40 days, 10 to 35 days, 12 to 50 days, 12 to 45 days, 12 to 40 days, 12 to 35 days, 14 to 50 days, 14 to 45 days, 14 to 40 days, or 14 to 35 days. The sheet maturation medium C may be replaced, for example, every day, every two days, or every three days. A multilayered sheet-shaped cell culture (also referred to as a "first sheet-shaped cell culture") may be formed by the culture.

[0105] The sheet maturation medium C used until the sheet-shaped cell culture comprising multilayered cell layers is formed contains the same concentration of additives as the sheet maturation medium C used until the cell culture of a thin layer of progenitor cells is formed, except that it is substantially free of a ROCK inhibitor. The sheet maturation medium C used until the sheet-shaped cell culture comprising multilayered cell layers is formed contains, for example, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000 or less of the ROCK inhibitor concentration in the sheet maturation medium C used until the cell culture of a thin layer of progenitor cells is formed. Preferably, the sheet maturation medium C used until the sheet-shaped cell culture comprising multilayered cell layers is formed does not contain a ROCK inhibitor.

[0106] The second compartment is, for example, a gas phase or a liquid phase. In one example, until the progenitor cell population reaches confluence in the second compartment, the second compartment preferably contains the same medium as the gas phase or the medium present in the first compartment. After the progenitor cell population reaches confluence in the second compartment, the second compartment preferably contains the gas phase or a medium different from the medium maintained in the first compartment. When the medium maintained in the first compartment is sheet maturation medium C, "a medium different from the medium maintained in the first compartment" means a medium that is substantially free of additives according to the present disclosure, such as retinoic acid, bone morphogenetic protein, and fibroblast growth factor. In this context, a medium "substantially free" of an additive contains the additive (e.g., at least one additive selected from the group consisting of retinoic acid, BMP, and FGF) at a concentration of, for example, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, or 1 / 1000 of the concentration of the additive in sheet maturation medium C maintained in the first compartment. When the medium maintained in the first compartment is sheet maturation medium C, the medium different from the medium maintained in the first compartment preferably does not contain additives according to the present disclosure, such as retinoic acid, bone morphogenetic protein, and fibroblast growth factor. Culturing a thin layer of cell culture in a second compartment using a medium substantially free of additives according to the present disclosure includes culturing the progenitor cell population in a second compartment "substantially free of an induction medium containing retinoic acid, bone morphogenetic protein, and fibroblast growth factor." The second compartment is preferably in the gas phase.

[0107] After the progenitor cell population reaches confluence in the second compartment, culturing the thin layer of cell culture until a sheet-shaped cell culture is formed in the second compartment may include, for example, a culture period in the gas phase and a culture period in the liquid phase (medium substantially free of the additives disclosed herein).

[0108] In one embodiment, the production method includes applying a substantially isolated progenitor cell population to a second compartment separated from the first compartment via a porous member. Specifically, the production method includes applying the substantially isolated progenitor cell population to the second compartment separated from the first compartment via a porous member, and culturing the progenitor cell population in the second compartment separated from the first compartment via the porous member in the presence of sheet maturation medium C in the first compartment to form a sheet-shaped cell culture in the second compartment (preferably, culturing the progenitor cell population in the second compartment in the presence of sheet maturation medium C further containing a ROCK inhibitor in the first compartment to form a thin cell culture in the second compartment, and then further culturing the progenitor cell population in the presence of sheet maturation medium C substantially free of a ROCK inhibitor in the first compartment to form a sheet-shaped cell culture in the second compartment).

[0109] To obtain a further matured sheet-shaped cell culture, the production method may include culturing the first sheet-shaped cell culture in the second compartment in the presence of urine. The sheet-shaped cell culture further matured by culturing in the presence of urine is also referred to as a "second sheet-shaped cell culture." Culturing in the presence of urine in the second compartment includes, for example, applying urine to the second compartment. The urine contacts the second main surface of the sheet-shaped cell culture, which is opposite the first main surface that contacts the surface of the porous member. Culturing the sheet-shaped cell culture in the presence of urine is carried out for, for example, 3 to 20 days, 3 to 18 days, 3 to 15 days, 3 to 13 days, 3 to 10 days, 5 to 20 days, 5 to 18 days, 5 to 15 days, 5 to 13 days, 5 to 10 days, 7 to 20 days, 7 to 18 days, 7 to 15 days, 7 to 13 days, 7 to 10 days, or 9 days. The urine is replaced, for example, every day, every two days, or every three days.

[0110] In the second sheet-shaped cell culture, the expression level of either or both of CK20 and ZO1, preferably both, in the superficial cells is at least two-fold higher than that of the first sheet-shaped cell culture formed by culturing for the same number of days in the absence of urine, for example, 2-fold to 10-fold, 2-fold to 8-fold, 2-fold to 7-fold, 2-fold to 6-fold, 2-fold to 5-fold, 3-fold to 10-fold, 3-fold to 8-fold, 3-fold to 7-fold, 3-fold to 6-fold, or 3-fold to 5-fold.

[0111] The sheet-shaped cell culture formed according to the production method of the present disclosure comprises, in this order, a cell layer of basal cells derived from a progenitor cell population, a cell layer of intermediate cells derived from a progenitor cell population, and a cell layer of superficial cells derived from a progenitor cell population. The sheet-shaped cell culture comprises, for example, a cell layer of the basal cells, a cell layer of the intermediate cells, and a cell layer of the superficial cells, with the cell layer of the intermediate cells interposed between the cell layer of the basal cells and the cell layer of the superficial cells. The sheet-shaped cell culture comprises a cell layer of the basal cells, a cell layer of the intermediate cells on the cell layer of the basal cells, and a cell layer of the superficial cells on the cell layer of the intermediate cells.

[0112] The sheet-shaped cell culture according to the present disclosure is not limited to a sheet-shaped cell culture produced by the method for producing a sheet-shaped cell culture according to the present disclosure. The sheet-shaped cell culture also includes a sheet-shaped cell culture having the characteristics according to the present disclosure, produced by a modified or alternative method of the above-described production method. The sheet-shaped cell culture may, for example, comprise a cell layer of superficial cells, a cell layer of intermediate cells, and a cell layer of basal cells, in this order, wherein the superficial cells do not substantially express P63 but express uroplakin (UPK), the intermediate cells co-express P63 and UPK, and the basal cells co-express P63 and keratin 5 (KRT5). The sheet-shaped cell culture may, for example, comprise a cell layer of basal cells, a cell layer of intermediate cells, and a cell layer of superficial cells, wherein the cell layer of intermediate cells is interposed between the cell layer of basal cells and the cell layer of superficial cells. The sheet-shaped cell culture comprises a cell layer of the basal cells, a cell layer of the intermediate cells on the cell layer of the basal cells, and a cell layer of the superficial layer cells on the cell layer of the intermediate cells.

[0113] Basal cells co-express P63 and keratin 5. Intermediate cells co-express P63 and uroplakin. Surface layer cells, for example, do not substantially express P63 but express uroplakin. Surface layer cells, for example, express uroplakin and either or both of CK20 and ZO1. Surface layer cells, for example, do not substantially express P63 but express uroplakin and either or both of CK20 and ZO1. Surface layer cells preferably express uroplakin and both CK20 and ZO1. Surface layer cells preferably do not substantially express P63 but express uroplakin and both CK20 and ZO1.

[0114] The sheet-shaped cell culture according to the present disclosure and the sheet-shaped cell culture formed according to the production method according to the present disclosure have, for example, the following properties. The sheet-shaped cell culture comprises, for example, at least 7 layers of cells, e.g., 7 to 20 layers, 7 to 19 layers, 7 to 18 layers, 7 to 17 layers, 7 to 16 layers, 7 to 15 layers, 8 to 20 layers, 8 to 19 layers, 8 to 18 layers, 8 to 17 layers, 8 to 16 layers, 8 to 15 layers, 9 to 20 layers, 9 to 19 layers, 9 to 18 layers, 9 to 17 layers, 9 to 16 layers, 9 to 15 layers, 10 to 20 layers, 10 to 19 layers, 10 to 18 layers, 10 to 17 layers, 10 to 16 layers, or 10 to 15 layers.

[0115] The number of cell layers in a sheet-shaped cell culture is analyzed and measured at the center of a circumscribing circle of the cell culture. When a torn or distorted surface is present in the sheet-shaped cell culture, the number of cell layers in the sheet-shaped cell culture is analyzed and measured at the center of a circumscribing circle of a substantially flat area having a substantially uniform thickness in the sheet-shaped cell culture.

[0116] The thickness of the sheet-shaped cell culture in the direction in which the first main surface and the second main surface face each other is, for example, 50 μm or more, 50 μm or more to 300 μm or less, 50 μm or more to 250 μm or less, 50 μm or more to 200 μm or less, 50 μm or more to 190 μm or less, 50 μm or more to 180 μm or less, 50 μm or more to 170 μm or less, 60 μm or more to 200 μm or less, 60 μm or more to 190 μm or less, 60 μm or more to 180 μm or less, 60 μm or more to 170 μm or less, 70 μm or more to 200 μm or less, 70 μm or more to 190 μm or less, 70 μm or more and 180 μm or less, 70 μm or more and 170 μm or less, 80 μm or more and 200 μm or less, 80 μm or more and 190 μm or less, 80 μm or more and 180 μm or less, 80 μm or more and 170 μm or less, 90 μm or more and 200 μm or less, 90 μm or more and 190 μm or less, 90 μm or more and 180 μm or less, 90 μm or more and 170 μm or less, 100 μm or more and 200 μm or less, 100 μm or more and 190 μm or less, 100 μm or more and 180 μm or less, or 100 μm or more and 170 μm or less.

[0117] The sheet-shaped cell culture has, for example, elasticity. The elasticity of the sheet-shaped cell culture can be measured by stretching the width of the sheet in one direction. The elasticity of the sheet-shaped cell culture can be measured, for example, by pinching both ends of a straight line passing through the center of the circumscribed circle of the sheet-shaped cell culture with tweezers, fixing one end, and pulling the other end in the opposite direction from the fixed end. The sheet-shaped cell culture can be stretched to 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 times the size (distance from the fixed end to the other end) of the sheet-shaped cell culture in a static state. The elasticity of the sheet-shaped cell culture can be measured, for example, within a range in which the sheet-shaped cell culture does not tear when stretched in one direction and then returned to its original size.

[0118] The sheet-shaped cell culture has, for example, resistance to urine. The urine resistance of the sheet-shaped cell culture can be analyzed or measured by placing urine in the second compartment containing the sheet-shaped cell culture and culturing the sheet-shaped cell culture in the presence of urine. The culturing in the presence of urine is, for example, for 9 days.

[0119] The sheet-shaped cell culture according to the present disclosure and the sheet-shaped cell culture formed according to the production method according to the present disclosure can be used, for example, as a main component of a pharmaceutical composition for treating bladder damage or bladder disease. The sheet-shaped cell culture can be used, for example, as a biomaterial called an epithelial sheet with barrier function.

[0120] [Method for preparing progenitor cell population] One aspect of the present disclosure provides a method for preparing progenitor cell population.The method comprises subjecting ventral hindgut organoid or bladder organoid to cell dissociation treatment.Preferably, the method uses the ventral hindgut organoid of the present disclosure, immature bladder organoid or mature bladder organoid, more preferably, immature bladder organoid.

[0121] The progenitor cell population of the present disclosure can be used in the method for producing a sheet-shaped cell culture of the present disclosure. The progenitor cell population includes progenitor cells that can differentiate into superficial cells, intermediate cells, and basal cells. The ventral hindgut organoids or bladder organoids can be prepared from pluripotent stem cells, for example, according to the method disclosed in WO 2022 / 025269 (the entire disclosure of which is incorporated herein by reference).

[0122] For example, subjecting ventral hindgut organoids or bladder organoids to cell dissociation treatment includes incubating them with a cell dissociation enzyme such as trypsin or TrypLE™ (e.g., TrypLE™ Express or TrypLE™ Select). By subjecting the organoids to cell dissociation treatment, a substantially isolated population of progenitor cells can be obtained. The proportion of progenitor cells that exist individually and not linked to other progenitor cells per total cell of the progenitor cell population can be adjusted by the time the organoids are treated with a cell detachment agent, the type and concentration of the cell detachment agent, and the extent of the treatment of the organoids treated with the cell detachment agent by pipetting or the like. A substantially isolated cell population can be prepared, for example, by subjecting the ventral hindgut organoids or bladder organoids to cell dissociation treatment, and then passing them through a cell strainer to remove impurities and cell clumps. The progenitor cell population prepared by the method according to this embodiment includes progenitor cells that can differentiate into basal cells, intermediate cells, or surface cells.

[0123] Ventral hindgut organoids can be produced by, for example, culturing pluripotent stem cells using induction medium A to induce their differentiation into definitive endoderm cells; culturing the definitive endoderm cells using induction medium B, and then culturing them using hindgut induction medium B in the presence of an extracellular matrix (preferably in an extracellular matrix gel) to induce their differentiation into hindgut organoids; and culturing the hindgut organoids using ventralization induction medium B in the presence of an extracellular matrix (preferably in an extracellular matrix gel) to induce their differentiation into ventral hindgut organoids.

[0124] Ventral hindgut organoids (e.g., ventral hindgut spheroids or cloacal spheroids) can be produced, for example, by culturing pluripotent stem cells using induction medium A to induce differentiation into definitive endoderm cells; culturing the definitive endoderm cells using induction medium B, and then culturing them using ventral hindgut induction medium B in the presence of an extracellular matrix (preferably in an extracellular matrix gel) to induce differentiation into ventral hindgut / cloacal spheroids.

[0125] Ventral hindgut organoids express the ventral hindgut marker P63, HOXA13 and CK8 / KRT8, and do not substantially express the dorsal hindgut markers SOX2 and / or CDX2, or even if they express SOX2 and / or CDX2, the expression levels are lower than those of SOX2 and / or CDX2 in hindgut organoids. Ventral hindgut organoids, for example, have a lumen; express at least one (e.g., one, two, three, or four or more) "ventral hindgut marker" selected from the group consisting of P63, ΔN63, GATA3, ISL1, and SATB2; express at least one (e.g., one, two, three, four, or five or more) marker selected from the group consisting of Phospho-Smad1 / 5 / 8, HOXA13, FOXA2, CK8 / KRT8, and ECAD; and do not substantially express at least one (e.g., one, two, or three, preferably three) marker selected from the group consisting of SOX2, T, and CDX2.

[0126] Immature bladder organoids can be induced to differentiate by culturing (preferably suspension culture) ventral hindgut organoids (e.g., ventral hindgut spheroids or cloacal spheroids) using immature organoid induction medium C. Differentiation induction of immature bladder organoids is preferably carried out in the presence of an extracellular matrix. Differentiation induction of immature bladder organoids includes culturing for, for example, 5 to 30 days, 5 to 25 days, 5 to 20 days, 5 to 15 days, 5 to 10 days, or 5 to 7 days.

[0127] When immature bladder organoids are present in an extracellular matrix gel, the immature bladder organoids are removed from the extracellular matrix gel. Immature bladder organoids in the extracellular matrix gel can be removed, for example, using Gentle Cell Dissociation Reagent. Immature bladder organoids in the extracellular matrix gel can be removed, for example, using tweezers and a scalpel under a microscope. The extracellular matrix gel preferably has a similar elasticity to that of a gel formed with a Matrigel solution having a protein concentration of 1-8 mg / mL, 1.5-6 mg / mL, 2.5 mg / mL, or 5 mg / mL. In the present disclosure, the term "similar elasticity" refers to an elasticity within ±30%, ±20%, or ±10% of the target elasticity. The extracellular matrix gel is preferably formed with Matrigel at a protein concentration of 1-8 mg / mL, 1.5-6 mg / mL, or 1.5-3.5 mg / mL or 4-6 mg / mL.

[0128] Immature bladder organoids can be prepared, for example, by culturing pluripotent stem cells using induction medium A to induce differentiation into definitive endoderm cells; culturing the definitive endoderm cells using induction medium B, and then culturing them using ventral hindgut induction medium B in the presence of an extracellular matrix to induce differentiation into ventral hindgut organoids; and culturing the ventral hindgut organoids using immature organoid induction medium C.

[0129] Induction medium A may be, for example, STEMdiff APEL2 medium (STEMCELL Technologies) supplemented with 100 ng / mL Activin A, 1 to 1.25 μM CHIR99021, 2% PFHM-II, and antibiotic-antimycotic. Ventral hindgut induction medium B used to induce differentiation of ventral hindgut / cloacal spheroids from definitive endoderm cells may be, for example, a medium obtained by adding 200 ng / mL FGF4, 10 ng / mL BMP4, 4 μM CHIR99021, 1 μg / mL Heparin, 2% PFHM-II, and antibiotic-antimycotic to STEMdiff APEL2 medium.

[0130] Ventralization induction medium B used to induce differentiation of ventral hindgut organoids from hindgut spheroids / organoids may be, for example, a medium in which 30 ng / mL BMP4, 200 ng / mL FGF4, 8 μM CHIR99021, 1 μg / mL Heparin, 2% PFHM-II, and antibiotic-antimycotic are added to STEMdiff APEL2 medium.

[0131] Immature organoid induction medium C is, for example, an APEL2 basal medium (hereinafter also referred to as "APEL2 / 2% PFHMII / AA") supplemented with 2% Protein Free Hybridoma Medium II (PFHMII) (ThermoFisher Scientific) and Antibiotic-Antimycotic (Life Technologies), and contains 0 to 10 ng / mL BMP4 (e.g., substantially no BMP, 2.5 ng / mL, 5 ng / mL, or 10 ng / mL BMP4), 1 μM all-trans retinoic acid, 10 ng / mL FGF10, 1 μg / mL heparin, 2% The medium may be PFHM-II, a medium supplemented with antibiotic-antimycotic.

[0132] For example, immature bladder organoids comprise a first cell layer comprising cells that do not substantially express P63 but express UPK1B and / or UPK2; and a second cell layer that is located outward relative to the first cell layer and comprises cells that co-express P63 and UPK1B and / or UPK2. For example, immature bladder organoids comprise the first cell layer and the second cell layer that is located outward relative to the first cell layer, and do not comprise a cell layer that comprises cells that express KRT5, a basal cell marker.

[0133] Matured bladder organoids can be induced to differentiate from ventral hindgut organoids (e.g., ventral hindgut / cloacal spheroids) according to the method described below in "Method for producing matured bladder organoids."

[0134] When preparing the progenitor cell population used to produce the sheet-shaped cell culture of the present disclosure, organoids prepared by suspension culture are preferred from the viewpoint of ease of recovery.

[0135] The substantially isolated progenitor cell population prepared according to the method of the present disclosure includes progenitor cells that can differentiate into superficial cells, intermediate cells, and basal cells. The progenitor cell population can be used in the culture method described below to prepare the sheet-shaped cell culture of the present disclosure.

[0136] [Method for maturing bladder organoids and method for producing matured bladder organoids] One aspect of the present disclosure provides a method for maturing bladder organoids. The method comprises floating culture of immature bladder organoids in an induction medium C (organoid maturation medium C) containing retinoic acid, BMP, and FGF to mature the bladder organoids. The organoid maturation medium C further comprises, for example, a calcium salt. The organoid maturation medium C is, for example, substantially free of extracellular matrix. The organoid maturation medium C comprises, for example, less than 0.1%, less than 0.05%, or less than 0.01% extracellular matrix.

[0137] The organoid maturation medium C is preferably the same as the sheet maturation medium C, and more preferably the same as the sheet maturation medium C used to induce differentiation of a sheet-shaped cell culture containing a multilayered cell layer. The FGF is preferably FGF7 or FGF10. Organoid maturation medium C may contain retinoic acid (e.g., atRA at 0.1-20 nM, 0.1-15 nM, 0.1-10 nM, 0.1-7.5 nM, 0.1-5 nM, 0.1-4 nM, 0.1-3 nM, 0.1-2 nM, 0.2-2 nM, 0.4-2 nM, 0.6-1.5 nM, 0.8-1.2 nM, or 1 nM), fibroblast growth factor (e.g., FGF7:FGF10 at a ratio of 30:1, 25:1, 20:1, 15:1, 10:1, or 10-1000 ng, 10-750 ng, 10-500 ng, 10-400 ng, 10-300 ng), or 10-200 ng of FGF7:FGF10. ng, 10-200 ng, 10-150 ng, 25-150 ng, 40-150 ng, 60-150 ng, 75-150 ng, 90-150 ng, or 110 ng of a combination of FGF7 and FGF10), and a bone morphogenetic protein (e.g., 1-100 ng / mL, 1-75 ng / mL, 1-50 ng / mL, 1-40 ng / mL, 1-30 ng / mL, 1-20 ng / mL, 1-15 ng / mL, 2-15 ng / mL, 3-15 ng / mL, 5-15 ng / mL, 7.5-15 ng / mL, or 10 ng / mL BMP4). Organoid maturation medium C preferably contains a calcium salt (e.g., 0.1 to 10 mM, 0.1 to 7.5 mM, 0.1 to 5 mM, 0.1 to 4 mM, 0.1 to 3 mM, 0.1 to 2 mM, 0.1 to 1.5 mM, 0.2 to 1.5 mM, 0.4 to 1.5 mM, 0.5 to 1.5 mM, 0.7 to 1.5 mM, or 1 mM calcium chloride). Organoid maturation medium C may, for example, contain APEL2 / 2% PFHMII / AA, 1 nM atRA, 10 ng / mL BMP4, 100 ng / mL FGF7, 10 ng / mL FGF10, and 1 mM CaCl ２ , 1 μg ml －１ Contains heparin.

[0138] In one embodiment of this aspect, the method for maturing bladder organoids may further comprise culturing ventral hindgut organoids using immature organoid induction medium C, thereby inducing differentiation of immature bladder organoids.The method for maturing bladder organoids comprises, for example, culturing ventral hindgut organoids using immature organoid induction medium C, thereby inducing differentiation of immature bladder organoids; and using organoid maturation medium C, culturing the immature bladder organoids in suspension, thereby maturing bladder organoids.

[0139] The components of immature organoid induction medium C are the same as those of organoid maturation medium C, except that bone morphogenetic protein is an optional component. The differences between the composition of immature organoid induction medium C and that of organoid maturation medium C are described below.

[0140] Immature organoid induction medium C contains retinoic acid (preferably all-trans retinoic acid), for example, 0.1 to 10 μM, 0.1 to 7.5 μM, 0.1 to 5 μM, 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1.5 μM, 0.2 to 10 μM, 0.2 to 7.5 μM, 0.2 to 5 μM, 0.2 to 4 μM, 0.2 to 3 μM, 0.2 to 2 μM, 0.2 to 1.5 μM, 0.3 to 10 μM, 0.3-7.5μM, 0.3-5μM, 0.3-4μM, 0.3-3μM, 0.3-2μM, 0.3-1.5μM, 0.4-10μM, 0.4-7.5μM, 0.4-5μM, 0.4-4μM, 0.4-3μM, 0.4-2μM, 0.4-1.5μM, 0.5-10μM, 0.5-7.5μM, 0.5-5μM, 0.5 ~4μM, 0.5-3μM, 0.5-2μM, 0.5-1.5μM, 0.6-10μM, 0.6-7.5μM, 0.6-5μM, 0.6-4μM, 0.6-3 μM, 0.6-2 μM, 0.6-1.5 μM, 0.7-10 μM, 0.7-7.5 μM, 0.7-5 μM, 0.7-4 μM, 0.7-3 μM, 0.7-2 μM , 0.7 to 1.5 μM, 0.8 to 10 μM, 0.8 to 7.5 μM, 0.8 to 5 μM, 0.8 to 4 μM, 0.8 to 3 μM, 0.8 to 2 μM, 0.8 to 1.5 μM, 0.9 to 10 μM, 0.9 to 7.5 μM, 0.9 to 5 μM, 0.9 to 4 μM, 0.9 to 3 μM, 0.9 to 2 μM, 0.9 to 1.5 μM, or 1 μM.

[0141] Immature organoid induction medium C contains fibroblast growth factor (e.g., FGF10) in the following concentrations: 1 to 100 ng, 1 to 75 ng, 1 to 50 ng, 1 to 40 ng, 1 to 30 ng, 1 to 20 ng, 1 to 15 ng, 2.5 to 100 ng, 2.5 to 75 ng, 2.5 to 50 ng, 2.5 to 40 ng, 2.5 to 30 ng, 2.5 to 20 ng, 2.5 to 15 ng, 4 to 100 ng, 4 to 75 ng, 4 to 50 ng, 4 to 40 ng, 4 to 30 ng , 4 to 20 ng, 4 to 15 ng, 6 to 100 ng, 6 to 75 ng, 6 to 50 ng, 6 to 40 ng, 6 to 30 ng, 6 to 20 ng, 6 to 15 ng, 7.5 to 100 ng, 7.5 to 75 ng, 7.5 to 50 ng, 7.5 to 40 ng, 7.5 to 30 ng, 7.5 to 20 ng, 7.5 to 15 ng, 9 to 100 ng, 9 to 75 ng, 9 to 50 ng, 9 to 40 ng, 9 to 30 ng, 9 to 20 ng, 9 to 15 ng, or 10 ng.

[0142] When bone morphogenetic protein (e.g., BMP4) is included, immature organoid induction medium C contains bone morphogenetic protein at concentrations of, for example, 0.1 to 10 ng / mL, 0.1 to 7.5 ng / mL, 0.1 to 5 ng / mL, 0.1 to 2.5 ng / mL, 0.1 to 1 ng / mL, 0.2 to 10 ng / mL, 0.2 to 7.5 ng / mL, 0.2 to 5 ng / mL, 0.2 to 2.5 ng / mL, 0.2 to 1 ng / mL, 0.3 to 10 ng / mL, 0.3 to 7.5 ng / mL, ng / mL, 0.3-5 ng / mL, 0.3-2.5 ng / mL, 0.3-1 ng / mL, 0.5-10 ng / mL, 0.5-7.5 ng / mL, 0.5-5 ng / mL, 0.5-2.5 ng / mL, 0.5-1 ng / mL, 0.75-10 ng / mL, 0.75-7.5 ng / mL, 0.75-5 ng / mL, 0.75-2.5 ng / mL, 0.75-1 ng / mL, 2.5 ng / mL, 5 ng / mL, or 10 ng / mL. Immature organoid induction medium C does not contain or is substantially free of BMP, for example.

[0143] The culture may be carried out for, for example, 20 days or more, 30 days or more, 40 days or more, 50 days or more, 60 days or more, 20 to 100 days, 20 to 90 days, 20 to 80 days, 20 to 70 days, 20 to 60 days, 20 to 50 days, 30 to 100 days, 30 to 90 days, 30 to 80 days, 30 to 70 days, 30 to 60 days, 30 to 50 days, The induction medium C is changed for 0 days, 40 to 100 days, 40 to 90 days, 40 to 80 days, 40 to 70 days, 40 to 60 days, 40 to 50 days, 50 to 100 days, 50 to 90 days, 50 to 80 days, 50 to 70 days, 50 to 60 days, 60 to 100 days, 60 to 90 days, 60 to 80 days, or 60 to 70 days. The induction medium C is changed, for example, every day, every two days, or every three days.

[0144] Suspension culture for maturation of bladder organoids involves rotary suspension culture using organoid maturation medium C. Rotary suspension culture can be performed, for example, using a whole embryo culture system or a 3D rotary suspension culture device, CellPet 3D-iPS (registered trademark) (J-Tech Corporation). The rotation speed in rotary suspension culture is appropriately set by those skilled in the art so that cell clusters do not fall due to gravity and touch the culture vessel. The rotation speed may be, for example, 1 to 50 rpm, 1 to 30 rpm, 1 to 15 rpm, 1 to 10 rpm, 2 to 50 rpm, 2 to 30 rpm, 2 to 15 rpm, 2 to 10 rpm, 3 to 50 rpm, 3 to 30 rpm, 3 to 15 rpm, or 3 to 10 rpm. Rotary suspension culture is performed, for example, under known cell culture conditions.

[0145] One aspect of the present disclosure provides a method for producing matured bladder organoids.The method comprises, for example, using organoid maturation medium C to culture immature bladder organoids in suspension, and inducing differentiation into mature bladder organoids.The method comprises, for example, using immature organoid induction medium C to culture ventral hindgut organoids (for example, ventral hindgut spheroids or cloacal spheroids) in immature organoid induction medium C to induce differentiation into immature bladder organoids; and using organoid maturation medium C to culture immature bladder organoids in suspension, and inducing differentiation into mature bladder organoids.In the method, the immature bladder organoids are preferably present in an extracellular matrix gel.

[0146] Organoids embedded in extracellular matrix gel can be obtained, for example, by suspending one or more organoids in an extracellular matrix solution and gelling the resulting suspension. Organoids in the extracellular matrix gel can be removed, for example, using Gentle Cell Dissociation Reagent (4°C, 30 minutes, shaking at 30 rpm) or using tweezers under a microscope. The removed organoids can be re-embedded in extracellular matrix gel. The removed organoids may be washed with medium, for example, before being embedded in extracellular matrix gel. When multiple organoids are embedded in extracellular matrix gel, it is preferable to set the density so that adjacent organoids do not come into contact with each other. For example, when using Matrigel, gelation of the suspension containing organoids can be achieved by dropping approximately 50 μl of the suspension onto parafilm and incubating the droplet at 37°C for 75 minutes.

[0147] The extracellular matrix gel may be, for example, a gel (e.g., Matrigel) having the same degree of elasticity as a gel formed from a Matrigel solution having a protein concentration of 3 to 13 mg / mL, 3 to 12 mg / mL, 3 to 11 mg / mL, 4 to 13 mg / mL, 4 to 12 mg / mL, 4 to 11 mg / mL, 5 to 13 mg / mL, 5 to 12 mg / mL, 5 to 11 mg / mL, 6 to 13 mg / mL, 6 to 12 mg / mL, 6 to 11 mg / mL, 7 to 13 mg / mL, 7 to 12 mg / mL, 7 to 11 mg / mL, 8 to 13 mg / mL, 8 to 12 mg / mL, or 8 to 11 mg / mL, preferably 7 to 12 μg / mL.

[0148] The organoid maturation medium C for the method for producing matured bladder organoids is, for example, the same as the organoid maturation medium C for the method for maturing bladder organoids.

[0149] The matured bladder organoids produced by the production method according to this embodiment comprise a cell layer surrounding a lumen, the cell layer comprising, in this order, a cell layer of surface cells that do not substantially express P63 but express UPK, a cell layer of intermediate cells that co-express P63 and UPK, and a cell layer of basal cells that co-express P63 and KRT5, the surface cells facing the lumen, and the surface cells further express CK20 and ZO1. The matured bladder organoids produced by the production method according to this embodiment comprise a cell layer surrounding a lumen, the cell layer comprising, in this order, a cell layer of surface cells that express UPK and further express CK20 and ZO1, a cell layer of intermediate cells that co-express P63 and UPK, and a cell layer of basal cells that co-express P63 and KRT5, the surface cells facing the lumen. The surface cells do not substantially express P63. The matured bladder organoid may have a major axis of, for example, 80 μm or more, 100 μm or more, 120 μm or more, 150 μm or more, or 200 μm or more. The matured bladder organoid does not contain mesenchymal stem cells or cells derived from mesenchymal cells.

[0150] The suspension culture of the ventral hindgut organoids is characterized by the suspension culture being carried out under conditions in which the ventral hindgut organoids and mesenchymal stem cells or mesenchymal cells are not substantially in contact with each other. In the context of suspension culture of organoids, "not substantially in contact" includes, for example, less than 100, less than 30, less than 10, less than 3, or 1 mesenchymal stem cell or mesenchymal cell per ventral hindgut organoid during suspension culture. "Not substantially in contact" does not exclude the absence of contact between the ventral hindgut organoids and mesenchymal stem cells or mesenchymal cells during suspension culture. "Not substantially in contact" includes, for example, the absence of mesenchymal stem cells or mesenchymal cells in the culture system in which the ventral hindgut organoids are suspension cultured.

[0151] The matured bladder organoid produced by the method for producing the matured bladder organoid of this embodiment can be used as the material for preparing a substantially isolated progenitor cell population.The matured bladder organoid can be used, for example, to evaluate the drug responsiveness to test substance.The matured bladder organoid can be used, for example, as the active ingredient of the pharmaceutical composition of the present disclosure.

[0152] [Culturing Method] One aspect of the present disclosure provides a culturing method comprising: applying a progenitor cell population capable of differentiating into surface cells, intermediate cells, and basal cells to a second compartment separated from a first compartment via a porous member; and culturing the progenitor cell population in the second compartment, wherein the first compartment holds a culture medium containing retinoic acid, bone morphogenetic protein, and fibroblast growth factor. The culture medium preferably further contains an extracellular matrix. The progenitor cell population is prepared according to the [Method for Preparing a Progenitor Cell Population] disclosed herein. The progenitor cell population is, for example, a progenitor cell population prepared from ventral hindgut organoids or immature bladder organoids, preferably a progenitor cell population prepared from immature bladder organoids.

[0153] The progenitor cell population can be applied to the second compartment using, for example, a dispenser such as a pipette. The progenitor cell population can be applied to the second compartment by, for example, introducing it through a channel that is fluidly connected to the second compartment. The progenitor cell population is cultured, for example, under known cell culture conditions. The method for culturing a progenitor cell population according to this embodiment may further include culturing the progenitor cell population according to the culture method described in the "Method for producing a sheet-shaped cell culture" according to the present disclosure.

[0154] [Pharmaceutical composition or method for producing same] One aspect of the present disclosure provides a pharmaceutical composition comprising the sheet-shaped cell culture or matured bladder organoid of the present disclosure and a pharmaceutically acceptable carrier.

[0155] The pharmaceutical composition is administered to a mammal in need thereof, for example, by surgically transplanting it into a predetermined site of the bladder, or by injecting it into a predetermined site of the bladder using an instrument such as a syringe. From the viewpoint of reducing graft rejection, the mammal to which the pharmaceutical composition is administered and the animal species of the pluripotent stem cells used to produce the sheet-shaped cell culture or mature bladder organoids are preferably the same species, and more preferably the same individual.

[0156] The pharmaceutical composition of the present disclosure can be used in a method for treating bladder damage or disease in a mammal. One embodiment provides a method for treating bladder damage or disease, comprising administering a pharmaceutical composition comprising the sheet-shaped cell culture or matured bladder organoid of the present disclosure to a mammal in need thereof.

[0157] [Method of treating bladder injury or disease] One aspect of the present disclosure provides a method of treating bladder injury or disease in a mammal. The method of treating bladder injury or disease in a mammal comprises introducing a sheet-shaped cell culture, a matured bladder organoid, or a pharmaceutical composition according to the present disclosure into the bladder or a surrounding area of ​​a mammal in need thereof. In one embodiment, the method of treating bladder injury or disease in a mammal comprises introducing a pharmaceutical composition according to the present disclosure into the bladder or a surrounding area of ​​a mammal in need thereof.

[0158] From the viewpoint of reducing transplant rejection, it is preferable that the animal species of the pluripotent stem cells used to produce the sheet-shaped cell culture, matured bladder organoid, or pharmaceutical composition to be introduced into a mammal in need thereof is the same species or the same individual as the mammal in need thereof.

[0159] [Non-human mammal having sheet-shaped cell culture or matured bladder organoid, or method for producing same] One aspect of the present disclosure provides a method for producing a non-human mammal having a sheet-shaped cell culture according to the present disclosure in the bladder or a surrounding area thereof. Another aspect of the present disclosure provides a non-human mammal having a sheet-shaped cell culture according to the present disclosure in the bladder or a surrounding area thereof. One aspect of the present disclosure provides a method for producing a non-human mammal having matured bladder organoids according to the present disclosure in the bladder or a surrounding area thereof. Another aspect of the present disclosure provides a non-human mammal having matured bladder organoids according to the present disclosure in the bladder or a surrounding area thereof.

[0160] A non-human mammal having a sheet-shaped cell culture or matured bladder organoids in its bladder or its surrounding area can be produced by a method comprising introducing the sheet-shaped cell culture or matured bladder organoids according to the present disclosure into the bladder or its surrounding area of ​​a non-human mammal. A non-human mammal having the sheet-shaped cell culture or matured bladder organoids in its bladder or its surrounding area can be produced by a method comprising introducing the sheet-shaped cell culture or matured bladder organoids according to the present disclosure into the bladder or its surrounding area of ​​a non-human mammal, and raising the non-human mammal. Raising the non-human mammal can include, for example, feeding the non-human mammal with a known diet.

[0161] In one example, by mixing tumor cells or tumor fragments when producing the sheet-shaped cell culture or mature bladder organoids according to the present disclosure, a sheet-shaped cell culture or mature bladder organoid containing the tumor cells or tumor fragments can be produced. By introducing the sheet-shaped cell culture or mature bladder organoids into the bladder or its surrounding area of ​​a non-human mammal, a non-human mammal having bladder epithelial tissue in its bladder or its surrounding area as a bladder epithelial cancer model can be produced. A non-human mammal having bladder epithelial tissue in its bladder or its surrounding area as a bladder epithelial cancer model can be used in a method for evaluating the effectiveness of a candidate therapeutic agent for bladder epithelial cancer, which includes contacting the non-human mammal with a candidate therapeutic agent for bladder epithelial cancer and evaluating the effectiveness of the candidate therapeutic agent.

[0162] [Method for assessing drug responsiveness to a test substance] A method for assessing drug responsiveness to a test substance comprises contacting the test substance with a cell culture or matured bladder organoid described below, and detecting changes in the test substance, the sheet-shaped cell culture, or the matured bladder organoid. The cell culture or matured bladder organoid is a sheet-shaped cell culture according to the present disclosure, a non-human mammal having a sheet-shaped cell culture according to the present disclosure in its bladder or a surrounding area thereof (or a sheet-shaped cell culture according to the present disclosure in the non-human mammal), a matured bladder organoid according to the present disclosure, or a non-human mammal having a matured bladder organoid according to the present disclosure in its bladder or a surrounding area thereof (or a matured bladder organoid according to the present disclosure in the non-human mammal).

[0163] "Contacting" the sheet-shaped cell culture, the sheet-shaped cell culture in a non-human mammal, the matured bladder organoids, or the matured bladder organoids in a non-human mammal with a test substance means placing the sheet-shaped cell culture, the sheet-shaped cell culture in a non-human mammal, the matured bladder organoids, or the matured bladder organoids in a non-human mammal under conditions that allow contact between the test substance and the cell culture. Contacting the sheet-shaped cell culture or matured bladder organoids with the test substance may, for example, involve mixing the test substance with a culture medium containing the sheet-shaped cell culture or matured bladder organoids. Contacting the sheet-shaped cell culture in a non-human mammal or matured bladder organoids with the test substance may, for example, involve administering the test substance orally or parenterally to the non-human mammal.

[0164] The changes in the sheet-shaped cell culture, the matured bladder organoid, or the sheet-shaped cell culture or matured bladder organoid in the non-human mammal include, for example, structural or functional changes in the sheet-shaped cell culture or the matured bladder organoid.The changes in the test substance include, for example, structural or functional changes or concentration changes of the test substance.

[0165] In this specification, ordinal numbers such as first and second are used for the purpose of conveniently distinguishing between substances and spaces having the same or similar structures or properties. The ordinal numbers do not in any way limit the structures or properties.

[0166] The term "comprising" means that the recited elements and / or steps are present, and that other elements and / or steps may be present. The term "consisting of" means that the recited elements and / or steps are present, and that other elements and / or steps are not present. The term "consisting essentially of" means that the recited elements and / or steps are present, and that other elements and / or steps may be present to the extent that they do not adversely affect the technical features of the present disclosure. As used herein, the term "substantially free" does not exclude "completely free."

[0167] The terms and descriptions referred to in this disclosure for a particular aspect or embodiment also apply to other aspects or embodiments as appropriate, unless expressly stated otherwise.

[0168] Specific examples will be described below, but they are intended to illustrate preferred embodiments of the present invention and are not intended to limit the invention described in the appended claims in any way.

[0169] Example 1: Bladder epithelial cell sheets were prepared from human iPS cells. (1) Induction of Definitive Endoderm (DE) Cells: Day 0-3: The day before differentiation induction, human iPS cells were dissociated into single cells using 1x TripLE™ select (Life Technologies). The isolated cell solution was mixed with 10 μM Y-27632 (Tocris Bioscience) and an amount of iMatrix silk-511 solution (0.25 μg cm) depending on the plate surface area. －２ The cells were then resuspended in StemFit AK02N supplemented with 1000 mM MgCl 2 HCl. The cells were then plated in 6-well plates at 45,000 cells cm －２ After overnight culture, the cells were incubated with 1.25 μM CHIR99021 (Tocris Bioscience) and 100 ng ml －１ DE cells were induced by culturing for 3 days in APEL2 / 2% PFHMII / AA in the presence of Activin A (Ajinomoto). The medium was changed on days 2 and 3.

[0170] (2) Induction of ventral hindgut / cloacal spheroids: Day 3-6 To induce ventral hindgut spheroids (also referred to as "cloacal spheroids"), DE cells were cultured in APEL2 / 2% PFHMII / AA containing 4 μM CHIR99021, 200 ng ml －１ FGF4, 10ng ml －１ BMP4 and 1 μg ml －１The cells were cultured in the presence of heparin for 11 days (Days 3-14). During ventral hindgut / cloacal induction, specifically from Day 4 or 5 to Day 6, the culture plate was placed on an orbital shaker (TAITECH) rotating at 100 rpm. On Day 6, floating spheroids spontaneously formed.

[0171] On Day 6-14, the floating spheroids were embedded for 3D culture using 100% Matrigel Growth Factor Reduced (Corning) (50 μl droplet-1). After the Matrigel™ solidified, the spheroids were cultured in ventral hindgut / cloacal induction medium to induce differentiation into ventral hindgut / cloacal spheroids (which are included in the ventral hindgut organoids in this disclosure). The medium was changed daily before embedding the spheroids in Matrigel™ and every two days thereafter. The ventral hindgut / cloacal induction medium contained 10 μM BMP4, 200 ng / mL －１ FGF4, APEL2 / 2% PFHMII / AA supplemented with 4 μM CHIR99021 (1 μg mL －１ Heparin) was used.

[0172] (3) Induction of Immature Bladder Organoids Day 14-20: To induce immature bladder organoids, ventral hindgut / cloacal spheroids were cultured in APEL2 / 2% PFHMII / AA containing 1 μM ATRA (Sigma), 10 ng / ml －１ BMP4, 10ng ml －１ FGF10 (R&D Systems), 1mM CaCl ２ (Promocell) and 1 μg ml －１ The organoids were cultured for 6 days in the presence of heparin. The induced organoids were observed under a microscope. A dual structure of epithelial and luminal structures was observed in the induced organoids, but cells expressing the basal cell marker KRT5 were not observed. These results indicate that the organoids differentiated from human iPS cells on day 20 are immature bladder organoids in the process of bladder development.

[0173] As described below, the immature bladder organoids were further cultured to mature, and the three-layer structure observed in mature bladder epithelium (a layer of P63-positive and KRT5-positive basal cells, a layer of UPK-positive and P63-positive intermediate cells, and a layer of UPK-positive and P63-negative superficial cells) was observed. This result suggests that the immature bladder organoids formed in Example 1 contain progenitor cells capable of differentiating into the three types of cells that make up the three-layer structure.

[0174] (4) Cultivation of Bladder Epithelial Cell Sheets Day 20 and After: 100% Matrigel™, in which immature bladder organoids on day 20 of differentiation induction were embedded, was treated with Gentle Cell Dissociation Reagent (4°C, 30 minutes) to isolate immature bladder organoids, which were then washed twice with ice-cold APEL2 / 2% PFHMII / AA. The immature bladder organoids were dissociated into single cells using TripLE select. The isolated cell population was cultured in sheet maturation medium C (APEL2 / 2% PFHMII / AA, 1 nM ATRA, 10 ng / ml) containing 10 μM Y-27632. －１ BMP4, 100ng ml －１ FGF7, 10ng ml －１ FGF10, 1mM CaCl ２ and 1 μg ml －１ The cells were then suspended in 2% Matrigel-coated 96-well Transwell plates (Transwell insert-integrated 96-well 0.4 μm polyester membrane (Corning, #7369)) at 100,000 cells per well. －１ The cells were plated so that the media was 10 μM Y-27632. The lower surface of the Transwell contained the same medium as the medium in which the cell population was suspended. On day 22 of differentiation induction, the medium on the upper surface of the Transwell was removed and air-liquid interface culture was initiated. The medium on the lower surface of the Transwell was changed to sheet maturation medium C containing 10 μM Y-27632 and 2% Matrigel™ from day 2 of air-liquid interface culture, and from day 6 onwards, it was changed to sheet maturation medium C containing 2% Matrigel™ but without 10 μM Y-27632. The medium was changed every two days.

[0175] [Modification of Example 1] (1) Induction of definitive endoderm (DE) cells: Day 0-3 A differentiation-inducing basal medium was prepared according to Nature Protocols, Vol. 3, No. 5, pp. 768-776, 2008, except that the albumin concentration in the medium was changed to 0.0312%, and human iPS cells were cultured to induce DE cells in substantially the same manner as described in (1) of Example 1.

[0176] (2) Induction of ventral hindgut / cloacal spheroids: Days 3-6 and 6-14 Ventral hindgut spheroids (cloacal spheroids) were induced from the DE cells in substantially the same manner as described in (2) of Example 1, except that the concentration of CHIR99021 added to the medium used to culture the DE cells was changed from 4 μM to 2 μM, and the ventral hindgut / cloacal induction medium was replaced daily at 5 ml / well from day 4 to day 6.

[0177] (3) Induction of immature bladder organoids. Day 14-20: The concentration of BMP4 added to the medium when culturing ventral hindgut / cloacal spheroids was increased to 10 ng / ml. －１ From 0ng ml －１ , 2.5ng ml －１ or 5ng ml －１ Immature bladder organoids were induced from the ventral hindgut / cloacal spheroids in substantially the same manner as described in Example 1 (3), except that the method was changed to: In the induced organoids, a dual structure of epithelial and luminal structures was observed (particularly, expression of FOXA2, an early intestinal epithelial marker or a developing bladder epithelial cell marker, was observed), but cells expressing KRT5, a basal cell marker, were not observed. This result indicates that the induced organoids are immature bladder organoids in the process of bladder development.

[0178] Example 2 The following tests were performed using cultured cell sheets derived from human iPS cells prepared according to the procedures described in Example 1. (1) Brightfield Observation of Bladder Epithelial Cell Sheets The cultured cell sheets on the Transwells were observed under brightfield conditions on day 26 after differentiation induction ( Figure 2A ). Figure 2A shows that the cultured cell sheets contain regions with relatively thin cell layers and regions with relatively thick cell layers. The cultured cell sheets on the Transwells were observed under brightfield conditions on day 35 after differentiation induction. Compared to the cultured cell sheets on day 26 after differentiation induction, the cultured cell sheets on day 35 after differentiation induction had an increased cell layer thickness. The cells on the Transwells were observed under brightfield conditions on day 42 after differentiation induction ( Figure 2B ). Figure 2B shows that the cultured cell sheets on day 42 after differentiation induction contain a relatively thicker cell layer than the cultured cell sheets on day 26 after differentiation induction. The cultured cell sheets removed from the Transwells on day 42 after differentiation induction were observed under brightfield conditions ( Figure 2C ). FIG. 2C shows that a cultured cell sheet of relatively uniform thickness was formed that conformed to the shape of the Transwell.

[0179] (2) Three-layer structure in bladder epithelial cell sheets. Cultured cell sheets on day 57 of differentiation induction were observed under a fluorescent microscope (Figure 3). Figure 3 shows a cross-section of a cultured cell sheet cultured on a Transwell. The right side of the cross-section is the surface that was in contact with the Transwell membrane (referred to as the "lower surface"), and the left side of the cross-section is the surface facing the open end of the Transwell (referred to as the "upper surface"). Figure 3D is a fluorescent image of DAPI, which stains cell nuclei, demonstrating the formation of a layer of DAPI-stained cells. Figure 3A shows the presence of a layer of UPK1B-expressing cells on the upper surface of the cultured cell sheet. Figure 3B shows the presence of a layer of P63-expressing cells extending from the center of the cultured cell sheet to the lower surface. Figure 3C shows the presence of a layer of KRT5-expressing cells extending from the center of the cultured cell sheet to the lower surface. Figure 3E shows the presence of a layer of UPK-positive, P63-negative cells on the upper surface of the cultured cell sheet, suggesting that this layer corresponds to the layer of surface cells of mature bladder epithelium. Figure 3E shows that a layer of P63- and KRT5-positive cells was present on the underside of the cultured cell sheet, suggesting that this layer corresponds to the basal cell layer of mature bladder epithelium. Figure 3E shows that a layer of UPK- and P63-positive cells was present near the center of the cultured cell sheet, suggesting that this layer corresponds to the intermediate cell layer of mature bladder epithelium. These results indicate that on day 57 of differentiation induction, a bladder epithelial cell sheet was formed with cell layers corresponding to the three-layer structure of bladder epithelium.

[0180] (3) Elasticity of Bladder Epithelial Cell Sheets A bladder epithelial cell sheet on day 42 of differentiation induction was pinched with tweezers (Figure 4A), stretched approximately 1.6 times in one direction (Figure 4B), and then returned to its original size (Figure 4C). Figure 4B shows that the bladder epithelial cell sheet did not break even when stretched approximately 1.6 times. No breakage was observed even after the bladder epithelial cell sheet was stretched seven times in the same manner. This result suggests that the bladder epithelial cell sheet prepared according to the preparation procedure described in Example 1 has elasticity similar to that of in vivo bladder epithelium.

[0181] (4) Reactivity of bladder epithelial cell sheets to human urine. Bladder epithelial cell sheets containing cells on day 48 of differentiation induction (day 20 + day 28) prepared according to the preparation procedure described in Example 1 ( Figures 5A and 5B ) were added to the top surface of the sheets, and the sheets were cultured for an additional 9 days ( Figure 5D , day 20 + day 37). As a control, bladder epithelial cell sheets containing cells on day 48 of differentiation induction prepared according to the preparation procedure described in Example 1 were cultured for 9 days without the addition of urine ( Figure 5D , day 20 + day 37). Urine was replaced every 2 days.

[0182] The sheet-shaped cell culture shown in Figure 5D (urine addition) showed a morphology similar to that of the sheet-shaped cell culture shown in Figure 5C (no urine addition), suggesting that the bladder epithelial cell sheet prepared according to the preparation procedure described in Example 1 is resistant to urine.

[0183] A sheet-shaped cell culture (57 days after differentiation induction) prepared in a similar manner to the sheet-shaped cell culture shown in Figure 5D (urine addition) was observed under a fluorescent microscope (Figure 6). Figure 6E suggests the presence of a layer of UPK-positive, P63-negative superficial cells on the upper surface of the cultured cell sheet, a layer of P63-positive, KRT5-positive basal cells on the lower surface, and a layer of UPK-positive, P63-positive intermediate cells near the center of the cultured cell sheet. The UPK1B-positive, P63-negative superficial cell layer observed in Figure 6E (57 days after differentiation induction, including 9 days of culture with urine addition) was thicker than the UPK1B-positive, P63-negative superficial cell layer observed in Figure 3E (57 days after differentiation induction, without urine addition).

[0184] The expression level of CK20, a terminal differentiation marker of surface cells, was examined. Compared to the expression level of CK20 in bladder epithelial cell sheets containing cells on day 57 of differentiation induction without urine addition (Figure 7A), the expression level of CK20 in bladder epithelial cell sheets containing cells on day 57 of differentiation induction, including a 9-day culture period after urine addition, was elevated (Figure 7E). Expression of tight junction protein (ZO1) was observed between surface cells (Figures 7A and 7F).

[0185] [Example 3] Maturation of immature bladder organoids without using mouse embryonic bladder mesenchyme Day 20 and later To induce mature bladder organoids, immature bladder organoids in Matrigel drops (day 20 of differentiation induction) were cultured in APEL2 / 2% PFHMII / AA with 1 nM ATRA, 10 ng ml －１ BMP4, 100ng ml －１ FGF7 (R&D Systems), 10ng ml －１ FGF10, 1mM CaCl ２ , 1 μg ml －１ In the presence of heparin, embryos were cultured in a whole embryo culture system (Nakayama, Cat. No. 10-0310) at 37°C and 5% CO ２ , 20% O ２ , gas flow rate 50 ml min －１ ) for 47 days. The medium was changed every 2 days. During the maturation of the immature bladder organoids, the Matrigel in which the bladder organoids were embedded was digested with Gentle Cell Dissociation Reagent (STEMCELL Technologies) for 30 minutes at 4°C to refresh the Matrigel, followed by washing twice with APEL2 basal medium and placing the bladder organoids in fresh Matrigel (50 μl droplets). －１ ) and Matrigel droplets were prepared again.

[0186] [Example 4] The following test was performed using bladder organoids prepared according to the preparation procedure described in Example 3 on the 67th day after differentiation induction. (1) Three-layer structure in mature bladder organoids Bladder organoids on the 67th day after differentiation induction were observed using fluorescence (Figure 8A). Figure 8A shows a bladder organoid containing a lumen, with the lumen comprising, in order from the outside, a layer of UPK-positive and P63-negative surface cells, a layer of UPK-positive and P63-positive intermediate cells, and a layer of P63-positive and KRT5-positive basal cells. Figure 8B shows that the surface cell layer expresses CK20, a terminal differentiation marker for surface cells, and tight junction protein (ZO1) is expressed between the surface cells. These results indicate that by using a combination of appropriate additives (differentiation inducers) in vitro, mature bladder organoids with a three-layer structure of bladder epithelium were formed.

[0187] (2) Elasticity and Barrier Function of Matured Bladder Organoids In vivo, the bladder has elasticity, expanding during urine storage and shrinking after urine is excreted. The bladder also has a barrier function that allows urine storage. We tested whether the matured bladder organoids we created have the same elasticity as the bladder in vivo. A green food dye solution was injected into the lumen of the matured bladder organoids (Figure 9A). Figure 9A shows that the injection of the green dye solution caused the matured bladder organoids to expand. This suggests that the matured bladder organoids have elasticity.

[0188] We tested whether the matured bladder organoids possessed barrier function based on intercellular tight junctions. An FITC-dextran (10 kDa) solution was injected into the lumen of matured bladder organoids, and fluorescence intensity was measured (Figure 9B, Control). Figure 9B shows that fluorescence intensity was detected 3, 5, and 8 hours after FITC injection. This suggests that matured bladder organoids possess barrier function. To reduce intercellular junctions, the calcium chelator EGTA was added to the culture medium 3 hours after FITC injection (Figure 9B, EGTA). Fluorescence intensity was detected 2 hours after EGTA addition (5 hours after FITC addition), but was significantly reduced compared to the control without EGTA. Changes in fluorescence intensity in this test were recorded for 8 hours (Figure 9C). Figure 9C shows that the fluorescence intensity in matured bladder organoids disappeared 3 hours after the addition of EGTA (6 hours after the addition of FITC), whereas the fluorescence intensity was maintained in the control group without EGTA, suggesting that matured bladder organoids have a barrier function based on tight junctions between cells.

Claims

1. A method for producing a sheet-shaped cell culture, comprising culturing a progenitor cell population in a second compartment separated from a first compartment by a porous member to form a sheet-shaped cell culture in the second compartment, wherein the sheet-shaped cell culture comprises, in this order, a cell layer of superficial cells derived from the progenitor cell population, a cell layer of intermediate cells derived from the progenitor cell population, and a cell layer of basal cells derived from the progenitor cell population, and wherein the first compartment holds an induction medium containing retinoic acid, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF).

2. The method of claim 1, wherein the induction medium further comprises an extracellular matrix.

3. The method of claim 1, wherein the progenitor cell population comprises progenitor cells derived from ventral hindgut organoids or bladder organoids.

4. The method of claim 1, wherein the BMP comprises at least one selected from the group consisting of BMP2, BMP4, and BMP7.

5. The manufacturing method according to claim 1, wherein the FGF comprises at least one selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

6. The method of claim 1, wherein culturing the progenitor cell population comprises culturing the progenitor cell population in a second compartment that is substantially free of an induction medium containing retinoic acid, bone morphogenetic protein, and fibroblast growth factor.

7. The method of claim 1, wherein culturing the progenitor cell population comprises culturing the progenitor cell population in the second compartment under a gas phase.

8. A culture method comprising: applying a progenitor cell population capable of differentiating into superficial cells, intermediate cells, and basal cells to a second compartment separated from a first compartment by a porous member; and culturing the progenitor cell population in the second compartment, wherein the first compartment holds a culture medium containing retinoic acid, a bone morphogenetic protein, and a fibroblast growth factor.

9. A sheet-shaped cell culture produced according to the method of any one of claims 1 to 7.

10. A sheet-shaped cell culture comprising a cell layer of superficial cells, a cell layer of intermediate cells, and a cell layer of basal cells in this order, wherein the superficial cells do not substantially express P63 but express uroplakin (UPK), the intermediate cells co-express P63 and UPK, and the basal cells co-express P63 and keratin 5 (KRT5).

11. The sheet-shaped cell culture according to claim 10, wherein the surface layer cells further express either or both of CK20 and ZO1.

12. A method for producing matured bladder organoids, comprising culturing immature bladder organoids in suspension using an induction medium containing retinoic acid, BMP, and FGF.

13. A method for producing mature bladder organoids, comprising culturing ventral hindgut organoids in an induction medium containing retinoic acid and FGF to induce differentiation into immature bladder organoids; and floating-culture the immature bladder organoids in an induction medium containing retinoic acid, BMP, and FGF to induce differentiation into mature bladder organoids.

14. The manufacturing method described in claim 13, wherein the induction medium used to induce the immature bladder organoids further contains BMP.

15. Matured bladder organoids produced according to the production method described in any one of claims 12 to 14.

16. A mature bladder organoid comprising a cell layer surrounding a lumen, the cell layer comprising, in this order, a cell layer of superficial cells that do not substantially express P63 but express UPK, a cell layer of intermediate cells that co-express P63 and UPK, and a cell layer of basal cells that co-express P63 and KRT5, the superficial cells facing the lumen, and the superficial cells further express CK20 and ZO1.

17. A matured bladder organoid comprising a cell layer surrounding a lumen, the cell layer comprising, in this order: a cell layer of superficial cells that express UPK and further express CK20 and ZO1; a cell layer of intermediate cells that co-express P63 and UPK; and a cell layer of basal cells that co-express P63 and KRT5, the superficial cells facing the lumen.

18. A pharmaceutical composition comprising: a sheet-shaped cell culture produced according to the production method of any one of claims 1 to 7, the sheet-shaped cell culture of claim 10 or 11, a matured bladder organoid produced according to the production method of any one of claims 12 to 14, or the matured bladder organoid of claim 16 or 17; and a pharmaceutically acceptable carrier.

19. A method for producing a non-human mammal having a sheet-shaped cell culture or mature bladder organoid in the bladder or a surrounding area thereof, comprising introducing into the bladder or a surrounding area a sheet-shaped cell culture produced according to the production method of any one of claims 1 to 7, a sheet-shaped cell culture according to claim 10 or 11, a matured bladder organoid produced according to the production method of any one of claims 12 to 14, or a matured bladder organoid according to claim 16 or 17.

20. A non-human mammal having, in the bladder or a peripheral region thereof, a sheet-shaped cell culture produced according to the production method of any one of claims 1 to 7, a sheet-shaped cell culture of claim 10 or 11, a matured bladder organoid produced according to the production method of any one of claims 12 to 14, or a matured bladder organoid of claim 16 or 17.

21. A method for evaluating drug responsiveness to a test substance, comprising: contacting a sheet-shaped cell culture produced according to the production method of any one of claims 1 to 7, the sheet-shaped cell culture of claim 10 or 11, matured bladder organoids produced according to the production method of any one of claims 12 to 14, matured bladder organoids of claim 16 or 17, or a non-human mammal having in its bladder or a peripheral site thereof a sheet-shaped cell culture produced according to the production method of any one of claims 1 to 7, the sheet-shaped cell culture of claim 10 or 11, matured bladder organoids produced according to the production method of any one of claims 12 to 14, or matured bladder organoids of claim 16 or 17, with the test substance; and detecting a change in the test substance, the sheet-shaped cell culture, or the mature bladder organoids.

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