Intestinal organoid and method for producing same

Abstract

The purpose of the present invention is to provide an intestinal organoid having a lumen.　The present invention provides a method for producing an intestinal organoid, the method comprising: a step for culturing intestinal spheroids on a low-adhesion plate that has a groove-like structure and inducing a string-like intestinal precursor tissue; and a step for conducting rotational suspension culture of the induced string-like intestinal precursor tissue.

Description

Intestinal organoids and their production method CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an application that benefits from the priority of Japanese Patent Application No. 2024-123331 (filing date: July 30, 2024), which is incorporated herein by reference in its entirety.

[0002] The present invention relates to intestinal organoids and a method for producing the same.

[0003] In cases where extensive intestinal resection is necessary for intractable inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, or for congenital childhood diseases, quality of life can be significantly impaired by a condition known as "short bowel syndrome," a condition characterized by a lack of intestinal function. However, small intestine transplantation, a fundamental treatment, has not yet become widespread due to a shortage of donors and post-transplant rejection. In recent years, attempts to generate organs ex vivo from pluripotent stem cells, such as iPS cells, have been promising alternatives to organ transplantation. Methods for generating human intestinal organoids from iPS cells using organoid culture, a three-dimensional culture method for stem cells, have been reported (Non-Patent Documents 1-3). However, the induction method is complex, the size of the induced spheroids is not uniform, and the need for three-dimensional culture makes it difficult to generate large organoids. Furthermore, while it is possible to generate spherical, sac-like structures such as spheroids and organoids, the generation of luminal organs with a single lumen, the most distinctive feature of the gastrointestinal tract, has not yet been achieved.

[0004] Non-Patent Document 7 discloses the introduction of a rotating bioreactor platform to maximize the culture efficiency of large human intestinal organoids (HIOs), but does not teach or suggest anything about the creation of tubular intestinal organoids with a lumen.

[0005] Nature 2011Nat. Protoc. 2011Nat. Med. 2014Cell Report Methods 2022Drug Metab Dispos. 2018Stem Cell Reports 2022STAR Protoc. 2023 Sep 15;4(3):102374.

[0006] The creation of transplantable functional intestinal tissue is one of the most important challenges in gastrointestinal regenerative medicine. Recent advances in intestinal regenerative medicine have included recellularization approaches using decellularized tissue and replacement of colonic epithelium with small intestinal epithelium. However, the need for a scaffolding extracellular matrix is ​​problematic. On the other hand, human intestinal organoids (HIOs) derived from pluripotent stem cells can be used to generate intestinal tissue composed of both epithelial and mesenchymal cells. Because they can reproduce the full-thickness structure of intestinal tissue, they are expected to be useful in intestinal regenerative medicine. Previously, HIOs were induced by collecting midgut and hindgut spheroids (hereafter referred to as "traditional spheroids" or "t-spheroids") that float in the medium during differentiation of pluripotent stem cells into the midgut and hindgut. These spheroids then float in the medium and undergo 3D culture. However, due to the recovery of accidentally released spheroids, the size and yield of t-spheroids are unstable. Furthermore, the spatial constraints of 3D culture limit the size of t-spheroids that can be cultured within the gel. To overcome these issues, the present inventors discovered that midgut and hindgut spheroids can be efficiently generated by dissociating midgut and hindgut cells into single cells and seeding them on low-adhesion spheroid formation plates (suspension spheroids or "s-spheroids"). Furthermore, they discovered that suspension culture can efficiently induce differentiation into HIOs. Furthermore, they discovered that larger HIOs can be cultured using a rotating bioreactor (Non-Patent Document 4). However, current intestinal organoids are spherical and do not yet reproduce the physiological tubular structure of intestinal tissue. Furthermore, differences exist between pluripotent stem cell strains in the development of mesenchymal cells, and the morphological instability of HIOs poses a challenge for clinical application.

[0007] In cases where extensive intestinal resection is necessary, such as in ulcerative colitis, it is known that quality of life is reduced due to a condition called "short bowel syndrome," which is a fundamental treatment for this condition. Small intestine transplantation, which is a fundamental treatment for this condition, has not become common due to a lack of donors and rejection after transplantation. Meanwhile, conventional methods for generating human intestinal organoids are complicated, the size of the induced spheroids is not uniform, and the need for 3D culture in a gel (or "extracellular matrix") makes it difficult to generate large organoids. Furthermore, the generation of a luminal organ with a single lumen, the most distinctive feature of the digestive tract, has not yet been achieved.

[0008] In this study, we successfully created composite suspension intestinal spheroids (assembled suspension spheroids; as-Spheroids) by individually inducing midgut and hindgut cells and visceral mesoderm through endoderm from pluripotent stem cells and fusing them in a suspension state. By maturing these in suspension culture, we successfully induced HIOs accompanied by stable development of mesenchymal tissue. Furthermore, we found that the high fusion properties of as-Spheroids allowed the induction of string-like intestinal precursor tissues (a-HITs), which could be induced into intestinal tissue by suspension culture in a rotating bioreactor. Furthermore, we found that mature intestinal tissue could be constructed by transplantation into mice.

[0009] Specifically, we improved existing methods for inducing human intestinal organoids from iPS cells (Non-Patent Document 1, Non-Patent Document 3). We found that when the induced cells were dispersed into a single cell and seeded on a special low-adhesion spheroid formation plate, they autonomously fused while in a floating state to form spherical intestinal spheroids. Unlike previously reported spheroids, which are irregular and released during the induction process from iPS cells, these spheroids were uniformly spherical and exhibited homogeneous cellular properties. Furthermore, by varying the plate shape and the number of cells seeded, we were able to generate spheroids of any size. While existing intestinal spheroids require three-dimensional culture in a gel, we found that the spheroids induced by this method could be grown in a floating state, eliminating spatial constraints and allowing for the induction and maturation of larger spheroids (Non-Patent Document 4). This floating induction method has been reported by other research groups (Non-Patent Documents 5-6) and is an approach that has attracted attention. A problem with this method of inducing human intestinal organoids is that although mesenchymal cells co-develop, differences in the degree of differentiation lead to instability in the culture. Therefore, the present inventors developed a method in which iPS cells, which were separately induced to differentiate into epithelial cells and mesenchymal cells, were fused to form intestinal spheroids in a suspension state. Furthermore, by utilizing the self-fusing ability of these fused spheroids and seeding them on a specific grooved low-adhesion spheroid formation plate, we successfully fused the spheroids to form string-like structures. Using a rotating bioreactor, we confirmed that these string-like spheroids could mature into luminal spheroids with a single central lumen while remaining in a suspension state. Thus, we demonstrated that floating intestinal organoids induced and cultured in a suspension state could mature and form luminal human intestinal tissue when transplanted into the mesentery of immunodeficient mice.Methods for inducing intestinal cells from pluripotent stem cells to create spherical spheroids and organoids have been reported, but no method has yet been reported for creating human intestinal tissue with the luminal structure characteristic of intestinal tissue outside the body. This method is not only expected to become a fundamental technology for organ creation in regenerative medicine, but also to be useful as a research platform capable of reproducing intestinal tissue in vitro.

[0010] Pluripotent stem cell-derived intestinal organoids are expected to be useful in intestinal regenerative medicine because they can generate intestinal tissue containing both epithelial and mesenchymal cells. However, due to differences between pluripotent stem cell strains, mesenchymal cell development varies widely, resulting in unstable organoid maturation and morphology. This poses challenges to their clinical application. In this study, the inventors successfully generated spheroids from mid- and hindgut cells and visceral mesoderm induced to differentiate individually from pluripotent stem cells, and successfully stabilized mesenchymal cell development in the intestinal organoids. Furthermore, by utilizing the high fusion ability of these spheroids, they generated string-like intestinal precursor structures (assembled human intestinal tubules; a-HITs). In rotary suspension culture, a-HITs developed into a layered structure of epithelial and surrounding mesenchymal tissue. Transplantation into the mesentery of hyperimmunodeficient mice resulted in the formation of mature intestinal tissue several centimeters long. This method for generating luminal intestinal tissue from human pluripotent stem cells holds promise for clinical application in regenerative medicine.

[0011] Specifically, the present inventors discovered that by using a low-adhesion spheroid formation plate, cells autonomously fuse while remaining suspended, leading to the induction of spherical spheroids. According to the present invention, uniform midgut and hindgut spheroids can be produced with high efficiency. According to the present invention, the size of spheroids can be controlled and enlarged. According to the present invention, suspension culture in a medium containing a low concentration of extracellular matrix is ​​possible.

[0012] Furthermore, the inventors have succeeded in fusing different types of cells by mixing hindgut cells and mesenchymal cells in a suspended state, taking advantage of their ability to autonomously fuse while in a suspended state, while adjusting the mixing ratio, etc. This is the first time that multiple cell types, namely hindgut cells and mesenchymal cells, have been successfully fused. The present invention utilizes the high cell aggregation ability to enhance the mesodermal component.

[0013] Furthermore, the present inventors have succeeded in fusing string-like spheroids by using a low-adhesion spheroid formation plate with a grooved structure, due to its high self-sustaining fusion ability, and have further found that by using a rotating bioreactor, the spheroids can be matured into tubular spheroids with a single central lumen while remaining in a floating state.The present invention utilizes the high cell aggregation ability to create an intestinal structure.

[0014] The present invention has the features of eliminating spatial constraints, high aggregation ability, self-organization, and the ability to induce differentiation of a variety of cells.

[0015] That is, the present invention provides the following.

[0016] [Aspect 1] Intestinal organoid having a lumen. [Aspect 2] Intestinal organoid according to Aspect 1, having a single lumen. [Aspect 3] Intestinal organoid according to Aspect 1, having a central lumen. [Aspect 4] Intestinal organoid according to Aspect 1, having a single central lumen. [Aspect 5] Intestinal organoid according to Aspect 1, for constructing intestinal tissue. [Aspect 6] Intestinal organoid according to Aspect 1, for constructing intestinal tissue having a lumen. [Aspect 7] Intestinal organoid according to Aspect 1, for transplantation into the mesentery. [Aspect 8] Intestinal organoid according to Aspect 1, for constructing intestinal tissue in vivo. [Aspect 9] Intestinal organoid according to Aspect 1, for constructing intestinal tissue having a lumen in vivo. [Aspect 10] Intestinal organoid according to Aspect 1, for transplantation into the mesentery in vivo. [Aspect 11] Intestinal organoid according to Aspect 1, derived from pluripotent stem cells. [Aspect 12] A method for producing intestinal organoids, comprising the steps of culturing in suspension on a low-adhesion plate a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, to form intestinal spheroids by fusing the middle and hindgut cells with the visceral mesoderm, and then culturing the formed intestinal spheroids in suspension. [Aspect 13] A method for producing intestinal organoids according to Aspect 12, in which the induction of differentiation from pluripotent stem cells into middle and hindgut cells and the induction of differentiation from pluripotent stem cells into visceral mesoderm are carried out separately. [Aspect 14] A method for producing intestinal organoids according to Aspect 12, in which the induction of differentiation from pluripotent stem cells into middle and hindgut cells passes through endoderm. [Aspect 15] A method for producing intestinal organoids according to Aspect 12, wherein a substance selected from the group consisting of a factor belonging to the TGFβ superfamily, a GSK-3β inhibitor, and a fibroblast growth factor is used in inducing differentiation of pluripotent stem cells into middle and hindgut cells. [Aspect 16] A method for producing intestinal organoids according to Aspect 15, wherein the factor belonging to the TGFβ superfamily is Activin A, the GSK-3β inhibitor is CHIR99021, and the fibroblast growth factor is FGF4.[Aspect 17] A method for producing intestinal organoids according to Aspect 12, wherein the differentiation of pluripotent stem cells into visceral mesoderm is induced via endoderm. [Aspect 18] A method for producing intestinal organoids according to Aspect 12, wherein the differentiation of pluripotent stem cells into visceral mesoderm is induced using a substance selected from the group consisting of a factor belonging to the TGFβ superfamily; a GSK-3β inhibitor; a fibroblast growth factor; a PI3Kα / γ / δ inhibitor; a PORCN inhibitor; a selective inhibitor of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7); a metabolite of vitamin A; and an agonist of Hedgehog signaling. [Aspect 19] The method for producing intestinal organoids according to Aspect 18, wherein the factor belonging to the TGFβ superfamily is Activin A or BMP4, the GSK-3β inhibitor is CHIR99021, the fibroblast growth factor is basic FGF, the PI3Kα / γ / δ inhibitor is PIK90, the PORCN inhibitor is Wnt-C59, the selective inhibitor of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7) is A83-01, the vitamin A metabolite is retinoic acid, and the Hedgehog signal agonist is palmorphamin. [Aspect 20] The method for producing intestinal organoids according to Aspect 12, wherein the mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, contains the middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and the visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, in a cell number ratio of 10:1 to 1:10. [Aspect 21] The mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, is 1 x 10. 4 pieces ~ 1 x 10 8A method for producing intestinal organoids according to Aspect 12, comprising cells. [Aspect 22] The method for producing intestinal organoids according to Aspect 12, wherein a mixture of middle and hindgut cells that have been differentiated from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been differentiated from pluripotent stem cells and dispersed into single cells, is cultured in suspension on a low-adhesion plate, wherein the culture in suspension is performed at 25° C. to 45° C. [Aspect 23] The method for producing intestinal organoids according to Aspect 12, wherein a mixture of middle and hindgut cells that have been differentiated from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been differentiated from pluripotent stem cells and dispersed into single cells, is cultured in suspension on a low-adhesion plate, wherein the culture in suspension is performed in the presence of 1% to 10% CO2. [Aspect 24] A method for producing intestinal organoids according to Aspect 12, wherein a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, is cultured in suspension on a low-adhesion plate for 5 to 72 hours. [Aspect 25] A method for producing intestinal organoids according to Aspect 12, wherein a substance selected from the group consisting of fibroblast growth factor, GSK-3β inhibitor, ROCK inhibitor, and protein kinase C (PKC) activator is used in culturing in suspension on a low-adhesion plate a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells. [Aspect 26] The method for producing intestinal organoids according to aspect 25, wherein the fibroblast growth factor is FGF4, the GSK-3β inhibitor is CHIR99021, the ROCK inhibitor is Y27632, and the activator of protein kinase C (PKC) is PMA.[Aspect 27] A method for producing intestinal organoids according to Aspect 12, wherein a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, is cultured in suspension on a low-adhesion plate using a substance selected from the group consisting of an extracellular matrix, an inhibitor of the bone morphogenetic protein (BMP) signaling pathway, an activator of the Wnt signaling pathway, and an activator of protein kinase C (PKC). [Aspect 28] A method for producing intestinal organoids according to Aspect 27, wherein the extracellular matrix is ​​Matrigel, the inhibitor of the BMP / bone morphogenetic protein (BMP) signaling pathway is Noggin, the activator of the Wnt signaling pathway is R-spondin 1, and the activator of protein kinase C (PKC) is PMA. [Aspect 29] A method for producing intestinal organoids according to Aspect 12, wherein the low-adhesion plate is a plate for spheroid formation. [Aspect 30] A method for producing intestinal organoids according to Aspect 12, wherein the low-adhesion plate is a plate comprising holes on the culture surface. [Aspect 31] A method for producing intestinal organoids according to Aspect 30, wherein the holes have a diameter of 100 to 1,000 μm and a depth of 50 to 500 μm. [Aspect 32] A method for producing intestinal organoids according to Aspect 12, wherein the low-adhesion plate comprises a low-adhesion protein coat on the surface. [Aspect 33] A method for producing intestinal organoids according to Aspect 32, wherein the low-adhesion protein coat is a hydrogel. [Aspect 34] A method for producing intestinal organoids according to Aspect 32, wherein the low-adhesion protein coat is covalently bound to the surface of the low-adhesion plate. [Aspect 35] A method for producing intestinal organoids according to Aspect 33, wherein the hydrogel is hydrophilic, non-ionic, or neutrally charged. [Aspect 36] The method for producing intestinal organoids according to Aspect 12, wherein the culture surface of the low-adhesion plate does not have a flat portion. [Aspect 37] The method for producing intestinal organoids according to Aspect 12, wherein the produced intestinal spheroids are spherical.[Aspect 38] A method for producing intestinal organoids according to Aspect 12, wherein dissociation into single cells is performed using a cell-dissociating enzyme. [Aspect 39] A method for producing intestinal organoids according to Aspect 38, wherein the cell-dissociating enzyme is protease, collagenase, or DNase. [Aspect 40] A method for producing intestinal organoids, comprising the steps of culturing intestinal spheroids on a low-adhesion plate having a grooved structure to induce cord-like intestinal precursor tissue, and subjecting the induced cord-like intestinal precursor tissue to rotary suspension culture. [Aspect 41] A method for producing intestinal organoids according to Aspect 40, wherein the rotary suspension culture is performed using a rotary bioreactor. [Aspect 42] A method for producing intestinal organoids according to Aspect 40, wherein the intestinal organoids have a single lumen in the center. [Aspect 43] A method for producing intestinal organoids according to Aspect 40, wherein the width of the grooved structure in the low-adhesion plate is 100 μm to 10,000 μm. [Aspect 44] A method for producing intestinal organoids according to Aspect 40, wherein the low-adhesion plate has a grooved structure and comprises a protein low-adhesion coat on the surface. [Aspect 45] A method for producing intestinal organoids according to Aspect 44, wherein the protein low-adhesion coat is a hydrogel. [Aspect 46] A method for producing intestinal organoids according to Aspect 44, wherein the protein low-adhesion coat is covalently bound to the surface of the low-adhesion plate. [Aspect 47] A method for producing intestinal organoids according to Aspect 45, wherein the hydrogel is hydrophilic, non-ionic, or neutrally charged. [Aspect 48] A method for producing intestinal organoids according to Aspect 40, wherein 5,000 to 100,000 intestinal spheroids are cultured at the start of culturing the intestinal spheroids on the low-adhesion plate having a grooved structure. [Embodiment 49] The method for producing intestinal organoids according to embodiment 40, wherein intestinal spheroids are cultured on a low-adhesion plate having a grooved structure, a substance selected from the group consisting of a fibroblast growth factor, a GSK-3β inhibitor, a ROCK inhibitor, and a protein kinase C (PKC) activator is used.[Aspect 50] A method for producing intestinal organoids according to Aspect 49, wherein the fibroblast growth factor is FGF4, the GSK-3β inhibitor is CHIR99021, the ROCK inhibitor is Y27632, and the protein kinase C (PKC) activator is PMA. [Aspect 51] A method for producing intestinal organoids according to Aspect 40, wherein the intestinal spheroids are cultured on a low-adhesion plate having a grooved structure, and the culture is carried out at 25°C to 45°C. [Aspect 52] A method for producing intestinal organoids according to Aspect 40, wherein the intestinal spheroids are cultured on a low-adhesion plate having a grooved structure, and the culture is carried out in the presence of 1% to 10% CO2. [Aspect 53] A method for producing intestinal organoids according to Aspect 40, wherein the intestinal spheroids are cultured on a low-adhesion plate having a grooved structure, and the culture is carried out for 5 to 72 hours. [Aspect 54] A method for producing intestinal organoids according to Aspect 40, wherein the step of subjecting the induced cord-like intestinal precursor tissue to rotary suspension culture uses a substance selected from the group consisting of an extracellular matrix, a growth factor, a serum transport protein, a secreted signaling protein, a water-soluble vitamin, and a hormone having a molecular structure similar to insulin. [Aspect 55] A method for producing intestinal organoids according to Aspect 54, wherein the extracellular matrix is ​​Matrigel, the serum transport protein is Afamin, the secreted signaling protein is a substance associated with tumorigenesis, regulation of cell fate, or patterning during embryogenesis, the water-soluble vitamin is a B vitamin, and the hormone having a molecular structure similar to insulin is IGF-1.[Aspect 56] A method for producing intestinal organoids according to Aspect 54, wherein the growth factor is a substance selected from the group consisting of growth factors that stimulate the proliferation of epidermal cells or epithelial cells, activators of the Wnt signaling pathway, inhibitors of the bone morphogenetic protein (BMP / bone morphogenetic proteins) signaling pathway, activators of protein kinase C (PKC), fibroblast growth factors, and selective inhibitors of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7). [Aspect 57] A method for producing intestinal organoids according to Aspect 56, wherein the growth factor that stimulates the proliferation of epidermal cells or epithelial cells is EGF, the activator of the Wnt signaling pathway is R-spondin 1, the inhibitor of the bone morphogenetic protein (BMP / bone morphogenetic proteins) signaling pathway is Noggin, the activator of protein kinase C (PKC) is PMA, the fibroblast growth factor is basic FGF, and the selective inhibitor of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7) is A8301. [Aspect 58] A method for producing intestinal organoids according to Aspect 40, comprising a step of semi-gelling the medium prior to the step of rotary suspension culture of the induced string-like intestinal precursor tissue. [Aspect 59] A method for producing intestinal organoids according to Aspect 40, wherein in the step of subjecting the induced cord-shaped intestinal precursor tissue to rotary suspension culture, the rotary suspension culture is carried out at 25° C. to 45° C. [Aspect 60] A method for producing intestinal organoids according to Aspect 40, wherein in the step of subjecting the induced cord-shaped intestinal precursor tissue to rotary suspension culture, the rotary suspension culture is carried out in the presence of 1% to 10% CO2. [Aspect 61] A method for producing intestinal organoids according to Aspect 40, wherein in the step of subjecting the induced cord-shaped intestinal precursor tissue to rotary suspension culture, the rotary suspension culture is carried out for 5 hours to 10,000 hours.[Aspect 62] A method for producing intestinal organoids according to Aspect 40, wherein, in the step of rotary suspension culturing the induced cord-shaped intestinal progenitor tissue, the rotation is carried out at a rotation speed of 1 rpm to 100 rpm. [Aspect 63] A method for producing intestinal organoids according to Aspect 40, wherein, in the step of rotary suspension culturing the induced cord-shaped intestinal progenitor tissue, the rotation speed is increased. [Aspect 64] A method for producing intestinal organoids according to Aspect 40, wherein, in the step of rotary suspension culturing the induced cord-shaped intestinal progenitor tissue, the medium is replaced once every 2 to 10 days. [Aspect 65] A method for producing intestinal organoids according to Aspect 40, comprising, prior to the step of rotary suspension culturing the induced cord-shaped intestinal progenitor tissue, culturing the induced cord-shaped intestinal progenitor tissue on a low-adhesion plate. [Aspect 66] A method for producing intestinal organoids according to Aspect 65, wherein the low-adhesion plate is a plate for forming spheroids. [Aspect 67] A method for producing intestinal organoids according to aspect 65, wherein the low-adhesion plate is a plate comprising holes on the culture surface. [Aspect 68] A method for producing intestinal organoids according to aspect 67, wherein the holes have a diameter of 100 to 1,000 μm and a depth of 50 to 500 μm. [Aspect 69] A method for producing intestinal organoids according to aspect 65, wherein the low-adhesion plate comprises a low-adhesion protein coat on the surface. [Aspect 70] A method for producing intestinal organoids according to aspect 69, wherein the low-adhesion protein coat is a hydrogel. [Aspect 71] A method for producing intestinal organoids according to aspect 69, wherein the low-adhesion protein coat is covalently bound to the surface of the low-adhesion plate. [Aspect 72] A method for producing intestinal organoids according to aspect 70, wherein the hydrogel is hydrophilic, non-ionic, or neutrally charged. [Aspect 73] A method for producing intestinal organoids according to aspect 65, wherein the low-adhesion plate does not have a flat portion on the culture surface. [Aspect 74] The method for producing intestinal organoids according to Aspect 65, wherein the culture on the low-adhesion plate is performed at 25° C. to 45° C. [Aspect 75] The method for producing intestinal organoids according to Aspect 65, wherein the culture on the low-adhesion plate is performed in the presence of 1% to 10% CO2.[Aspect 76] A method for producing intestinal organoids according to Aspect 65, wherein the culturing on the low-adhesion plate is carried out for 5 to 100 hours. [Aspect 77] A method for producing intestinal spheroids for producing intestinal organoids, comprising the step of culturing in suspension on a low-adhesion plate a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, to produce intestinal spheroids by fusing the middle and hindgut cells with the visceral mesoderm. [Aspect 78] A method for producing string-shaped intestinal progenitor tissue for producing intestinal organoids, comprising the step of culturing intestinal spheroids on a low-adhesion plate having a grooved structure to induce string-shaped intestinal progenitor tissue. [Aspect 79] A method for producing intestinal organoids, comprising the step of culturing intestinal spheroids on a low-adhesion plate having a grooved structure, to induce string-shaped intestinal progenitor tissue, and subjecting the induced string-shaped intestinal progenitor tissue to rotary suspension culture. [Aspect 80] A method for producing intestinal organoids, comprising the steps of: culturing a mixture of middle and hindgut cells, which have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm, which have been induced to differentiate from pluripotent stem cells and dispersed into single cells, in a suspension state on a low-adhesion plate to fuse the middle and hindgut cells with the visceral mesoderm to produce intestinal spheroids; culturing the produced intestinal spheroids on a low-adhesion plate with a grooved structure to induce string-like intestinal precursor tissue; and subjecting the induced string-like intestinal precursor tissue to rotary suspension culture. [Aspect 81] An intestinal organoid having a lumen, the organoid having intestinal tissue containing epithelial cells and mesenchymal cells. [Aspect 82] An intestinal organoid having a lumen, the organoid having intestinal tissue accompanied by epithelium and surrounding mesenchymal tissue. [Aspect 83] An intestinal organoid having a lumen, the organoid having a longitudinal tube length of 2.5 cm or more. [Embodiment 84] An intestinal organoid having a lumen, wherein the outer diameter of the lumen is 1.0 mm or more. [Embodiment 85] An intestinal organoid having a lumen, wherein the inner diameter of the lumen is 600 μm or more.[Embodiment 86] An intestinal organoid having a lumen, which is produced using the production method according to any one of embodiments 12 to 80.

[0017] This is the first successful creation of intestinal organoids with the luminal structure that is the most distinctive feature of the intestinal tract. This is expected to be useful as a fundamental technology for organ creation in regenerative medicine and as a research platform capable of reproducing intestinal tissue in vitro.

[0018] As a fundamental technology for organ creation in regenerative medicine, it can be applied to artificial organs for transplantation. Specifically, it has the potential to be used as a transplant treatment for ulcerative colitis, Crohn's disease, intractable inflammatory bowel disease, and short bowel syndrome resulting from intestinal resection due to congenital childhood disease.

[0019] As a research platform capable of reproducing intestinal tissue in vitro, it can be applied to artificial organs for transplantation. It can meet needs such as reducing the number of experimental animals and eliminating differences in drug efficacy due to differences in animal species. In other words, there is a need for an organ for intestinal disease experiments, and it can be used as a test material (intestinal model organ). It can also be used as an in vitro safety evaluation system and test material without using animals.

[0020] Figure 1 shows the construction of as-spheroids by fusion of mid-hindgut cells and visceral mesoderm, followed by their induction into a-HIOs through suspension culture. (A) Schematic diagram of as-spheroid construction and culture. (B) Construction of as-spheroids by fusion of GFP-labeled mid-hindgut cells and tdTomato-labeled visceral mesoderm. (C) Immunostaining of as-spheroids on Day 0: CDX2 (mid-hindgut marker) and FOXF1 (visceral mesoderm marker). (D) Immunostaining of as-spheroids on Day 11: CDX2 (mid-hindgut marker) and FOXF1 (visceral mesoderm marker). (E) s-spheroids and as-spheroids on Day 15. Successful stromal development is observed following visceral mesoderm fusion. (F) Fluorescent image of an a-HIO (day 28). Successful development of tdTomato-labeled stromal cells is observed around the GFP-labeled epithelium. (G) Immunostaining of a-HIO (day 28). Vimentin-positive mesenchymal tissue surrounds the E-cadherin-positive epithelial layer. Figure 2 shows the maturation of A-HIO in vivo. (A) HE staining of the engrafted tissue. The epithelial layer with crypt structures and the underlying stratified stromal layer are visible. (B-E) Immunostaining of the engrafted tissue. Muc2-positive goblet cells (B), lysozyme-positive Paneth cells (C), Ki67-positive proliferative cells (D), and villin-positive absorptive epithelial cells (E) are present among the E-cadherin-positive epithelial cells, confirming the presence of various differentiated cells. (F) In situ hybridization of LGR5. LGR5-positive intestinal epithelial stem cells are localized at the crypt base. Figure 3 shows the construction of an intestinal tract structure (a-HIT) utilizing the high fusion-proneness of As-Spheroids. (A) Schematic diagram of the creation and culture of a-HIT. (B) Fluorescence image of a-HIT on day 0. Developed from GFP-labeled mid-hindgut cells and tdTomato-labeled visceral mesoderm. (C) Immunostaining of day 0 a-HIT: CDX2 (mid-hindgut marker) and FOXF1 (visceral mesoderm marker). Abundant FOXF1-positive cells are observed. (D) Fluorescent image of day 28 a-HIT cultured in assembloid medium. GFP-labeled epithelial cells are surrounded by tdTomato-labeled stromal cells.Midgut and hindgut cells were generated from GFP-labeled pluripotent stem cells, and visceral mesoderm was generated from tdTomato-labeled pluripotent stem cells. (E) Stereomicroscope image of a-HIT on day 14 cultured in assembloid medium. A lumen is visible in the center. Figure 4 shows that a-HIT reconstruct mature intestinal tissue in vivo. (A) Transplantation of a-HIT into the mesentery (left) and engraftment and maturation (right). (B) HE staining of the engrafted tissue. An epithelial layer with crypt structures and a stratified stromal layer are visible. A lumen is visible in the center. (C) E-cadherin and SMA immunostaining of the engrafted tissue. E-cad-positive epithelium and SMA-positive smooth muscle layer are visible. (DJ) Immune cells in the engrafted tissue. Expression of CDX2, a small intestinal epithelial-specific marker, is confirmed (D). Various differentiated epithelial cells can be seen, including Ki67-positive proliferative cells (E), MUC2-positive goblet cells (F), chromogranin A-positive neuroendocrine cells (G), villin1- and sucrase isomaltase-positive absorptive epithelial cells (H1), and lysozyme-positive Paneth cells (J). (K) In situ hybridization of LGR5 (a marker specific to intestinal stem cells). LGR5-positive intestinal epithelial stem cells are localized at the crypt base. Figure 5 shows the characterization of as-spheroids (related to Figure 1). (A) Immunostaining of FOXF1 in visceral mesoderm. Highly efficient induction of pluripotent stem cells into visceral mesoderm is confirmed. (B) Enlargement of as-spheroids. Produced from GFP-labeled middle hindgut cells and tdTomato-labeled visceral mesoderm. (C-D) PCR comparison of day 0 s-spheroids and as-spheroids. Fusion of visceral mesoderm (as-Spheroid) shows higher expression of FOXF1 compared to non-fusion (s-Spheroid). Figure 6 shows the analysis of the properties of suspension-cultured as-Spheroid (related to Figure 2). (A) Comparison of day 15 s-Spheroid and as-Spheroid by quantitative PCR. (B) Photographs of suspension-cultured s-Spheroid and as-Spheroid. S-Spheroid shows large strain-to-strain differences in mesenchymal tissue development. As-Spheroid shows stable stromal development.(C) Suspension culture in medium containing (left) and not containing (right) 10% Matrigel. The addition of Matrigel is necessary for self-organization. Figure 7 shows the creation of intestinal tissue (Human Intestinal Tubules) using a specially designed cell culture plate. (A) Specially designed cell culture plate (OrgSP plate). Approximately 30 rows of 1000-um-wide grooves are excavated on the bottom of the ultra-low attachment plate. (B) Creation of HIT using s-Spheroids. (C) Immunostaining of CDX2 and FOXF1 in s-Spheroid-derived HIT. Compared to as-Spheroid-derived HIT (Figure 3C), the amount of visceral mesoderm components (FOXF1) is significantly lower. (D) HIT derived from t-Spheroids. They have a heterogeneous shape. Figure 8 shows the characterization of a-HITs cultured in suspension under ENRh conditions (related to Figure 3). (A) Fluorescence image of da17 a-HITs cultured under ENRh conditions. (B) Immunostaining of day 11 a-HIOs cultured in ENR. E-cadherin-positive epithelium is separated into vimentin-positive stromal cells. Figure 9 shows the culture conditions for a-HIOs that promote the proliferation of epithelial components (related to Figure 3). (A) Photograph of s-spheroids cultured in ENR and assembloid medium. (B) Immunostaining of s-spheroids cultured in ENR and assembloid medium. Many Ki67-positive epithelial cells are observed in the assembloid medium, suggesting epithelial proliferation. (C) Ki67 positivity in s-spheroids cultured in ENR and assembloid medium. The positivity rate is higher in the assembloid medium. (D) Photograph of day 11 as-spheroids cultured in assembloid medium. Mesenchymal tissue is observed surrounding the epithelium. (EG) Immunostaining of day 11 as-spheroids cultured in assembloid medium. Vimentin-positive stromal cells are present around E-cadherin-positive epithelial cells (E), the intestinal lineage of the epithelium (CDX2) and the visceral mesoderm lineage of the stroma (FOXF1) are maintained (F), and the epithelium exhibits a high proliferation rate, Ki67 (G).Figure 10 shows the characterization of a-HIT cultured in assembloid medium (related to Figure 3). (AB) Immunostaining of day 11 a-HIT cultured in assembloid medium. Vimentin-positive stromal cells are present around E-cadherin-positive epithelial cells (A). The small intestinal lineage of the epithelium (CDX2) and the visceral mesoderm lineage of the interstitium (FOXF1) are maintained (B). (CD) Immunostaining of day 26 a-HIT cultured in assembloid medium. The visceral mesoderm is differentiated from tdTomato-labeled pluripotent stem cells. Vimentin-positive stromal cells are tdTomato-positive, confirming their origin from the fused visceral mesoderm (C). Some SMA-positive cells are also observed, demonstrating differentiation into myofibroblasts (D). (E) Immunostaining of day 26 a-HIT cultured in assembloid medium. A lumen is observed in the center. Figure 11 shows the maturation of a-HIT cultured in ENR medium by mesentery transplantation (related to Figure 4). No lumen is observed in the center.

[0021] The present invention provides intestinal organoids having a lumen. Organoids are three-dimensional organs created in vitro, for example, in a test tube.

[0022] The intestinal organoid of the present invention preferably has a single lumen.The intestinal organoid of the present invention preferably has a lumen in the center.The intestinal organoid of the present invention preferably has a single lumen in the center.

[0023] The intestinal organoids having a lumen of the present invention are preferably used to construct intestinal tissue. The intestinal organoids having a lumen of the present invention are preferably used to construct intestinal tissue with a lumen. The intestinal organoids having a lumen of the present invention are preferably used to transplant into the mesentery. The intestinal organoids having a lumen of the present invention are preferably used to construct intestinal tissue ex vivo or in vivo. The intestinal organoids having a lumen of the present invention are preferably used to construct intestinal tissue with a lumen ex vivo or in vivo. The intestinal organoids having a lumen of the present invention are preferably used to transplant into the mesentery ex vivo or in vivo.

[0024] The intestinal organoids having a lumen in the present invention are preferably derived from pluripotent stem cells, more preferably from induced pluripotent stem cells.

[0025] The intestinal organoid of the present invention preferably has the organoid of intestinal tissue that comprises epithelial cell and mesenchymal cell.The intestinal organoid of the present invention preferably has the organoid of intestinal tissue that comprises epithelial cell and mesenchymal tissue and that is associated with epithelium and its surrounding mesenchymal tissue.

[0026] The present invention provides a method for producing intestinal organoids, comprising the steps of culturing in suspension on a low-adhesion plate a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have also been induced to differentiate from pluripotent stem cells and dispersed into single cells, thereby fusing the middle and hindgut cells with the visceral mesoderm to produce intestinal spheroids, and of culturing the produced intestinal spheroids in suspension.

[0027] Preferably, the induction of differentiation from pluripotent stem cells into middle / hindgut cells and the induction of differentiation from pluripotent stem cells into visceral mesoderm are carried out separately.

[0028] The induction of differentiation from pluripotent stem cells into middle and hindgut cells preferably occurs via the endoderm.

[0029] Inducing differentiation of pluripotent stem cells into middle and hindgut cells, one, two, or three substances selected from the group consisting of a factor belonging to the TGFβ superfamily, a GSK-3β inhibitor, and a fibroblast growth factor are preferably used. Preferably, the factor belonging to the TGFβ superfamily is Activin A, the GSK-3β inhibitor is CHIR99021, and the fibroblast growth factor is FGF4.

[0030] The induction of differentiation of pluripotent stem cells into visceral mesoderm preferably passes through endoderm.

[0031] In inducing differentiation of pluripotent stem cells into visceral mesoderm, preferably, one, two, three, four, five, six, seven, or eight substances selected from the group consisting of factors belonging to the TGFβ superfamily; GSK-3β inhibitors; fibroblast growth factors; PI3Kα / γ / δ inhibitors; PORCN inhibitors; selective inhibitors of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7); vitamin A metabolites; and Hedgehog signaling agonists are used. Selective inhibitors of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7) include, for example, selective inhibitors of one, two, or three substances selected from the group consisting of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), and type I nodal receptor-like kinase (ALK7). Preferably, the factor belonging to the TGFβ superfamily is Activin A or BMP4, the GSK-3β inhibitor is CHIR99021, the fibroblast growth factor is basic FGF, the PI3Kα / γ / δ inhibitor is PIK90, the PORCN inhibitor is Wnt-C59, the selective inhibitor of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7) is A83-01, the metabolite of vitamin A is retinoic acid, and the Hedgehog signal agonist is palmorfamine.

[0032] Preferably, the mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells is a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells in a ratio of 10:1 or more, 9:1 or more, 8:1 or more, 7:1 or more, 6:1 or more, 5:1 or more, 4:1 or more, 3:1 or more, 2:1 or more, 1:1 or more, 1:2 or more, 1:3 or more, 1:4 or more, 1:5 or more, 1:6 or more, 1:7 or more, 1:8 or more, 1:9 or more, 1:10 or more, 1:11 or more, 1:12 or more, 1:13 or more, 1:14 or more, 1:15 or more, 1:16 or more, 1:17 or more, 1:18 or more, 1:19 or more, 1:19 or more, 1:20 or more, 1:21 or more, 1:22 or more, 1:23 or more, 1:24 or more, 1:25 or more, 1:26 or more, 1:27 or more, 1:28 or more, 1:2 ...9 or more, 10 or more, 10:2 or more, 9:2 or more, 8:2 or more, 7:2 or more, 6:2 or more, 5:2 or more, 4:2 or more, 3:2 or more, 2:2 or more, 2:2 or more, 2:2 or more, 2:2 or more, 2:3 or more, 2:4 or more, 2:5 or more, 2:6 or more, 2:7 or more, 2:8 or more, 2:9 or more, 2:10 or more, 10:3 or more, 9:3 or more, 8:3 or more, 7:3 or more, 6:3 or more, 5:3 or more, 4:3 or more, 3:3 or more, 2:3 or more, 3:3 or more, 3:2 or more, 3:3 or more, 3:4 or more, 3:5 or more, 3:6 or more, 3:7 or more, 3:8 or more, 3:9 or more, 3:10 or more, 10:4 or more, 9:4 or more, 8:4 or more, 7:4 or more, 6:4 or more, 5:4 or more, 4:4 or more, 3:4 or more, 2:4 or more, 4:4 or more, 4:2 or more, 4:3 or more, 4:4 or more, 4:5 or more, 4:6 or more, 4:7 or more, 4:8 or more, 4:9 or more, 4:10 or more, 10:5 or more, 9:5 or more, 8:5 or more, 7:5 or more, 6:5 or more, 5:5 or more, 4:5 or more, 3:5 or more, 2:5 or more, 5:5 or more, 5:2 or more, 5:3 or more, 5:4 or more, 5:5 or more, 5:6 or more, 5:7 or more, 5:8 or more, 5:9 or more, 5:10 or more, 10:6 or more, 9:6 or more, 8:6 or more, 7:6 or more, 6:6 or more, 5:6 or more, 4:6 or more, 3:6 or more, 2:6 or more, 6:6 or more, 6:2 or more, 6:3 or more, 6:4 or more, 6:5 or more, 6:6 or more, 6:7 or more, 6:8 or more, 6:9 or more, 6:10 or more, 10:7 or more, 9:7 or more, 8:7 or more, 7:7 or more, 6:7 or more, 5:7 or more, 4:7 or more, 3:7 or more, 2:7 or more, 7:7 or more, 7:2 or more, 7:3 or more, 7:4 or more, 7:5 or more, 7:6 or more, 7:7 or more, 7:8 or more, 7:9 or more, 7:10 or more, 10:8 or more, 9:8 or more, 8:8 or more, 7:8 or more, 6:8 or more, 5:8 or more, 4:8 or more, 3:8 or more,2:8 or more, 8:8 or more, 8:2 or more, 8:3 or more, 8:4 or more, 8:5 or more, 8:6 or more, 8:7 or more, 8:8 or more, 8:9 or more, 8:10 or more, 10:9 or more, 9:9 or more, 8:9 or more, 7:9 or more, 6:9 or more, 5:9 or more, 4:9 or more, 3:9 or more, 2:9 or more, 9:9 or more, 9:2 or more, 9:3 or more, 9:4 or more, 9:5 or more, 9:6 or more, 9:7 or more, 9:8 or more, 9:9 or more, 9:10 or more, 10:10 or more, 9:10 or more, 8:10 or more, 7:10 or more, 6:10 or more, 5:10 or more, 4:10 or more, 3:10 or more, 2:10 or more, 10 Contains a cell number ratio of 10:10 or more, 10:2 or more, 10:3 or more, 10:4 or more, 10:5 or more, 10:6 or more, 10:7 or more, 10:8 or more, 10:9 or more, or 10:10 or more, and 10:1 or less, 9:1 or less, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less, 3:1 or less, 2:1 or less, 1:1 or less, 1:2 or less, 1:3 or less, 1:4 or less, 1:5 or less, 1:6 or less, 1:7 or less, 1:8 or less, 1:9 or less, 1:10 or less, 10:2 or less, 9:2 or less, 8:2 or less, 7:2 or less, 6:2 or less, 5:2 or less, 4:2 or less, 3:2 or less, 2: 2 or less, 2:2 or less, 2:2 or less, 2:3 or less, 2:4 or less, 2:5 or less, 2:6 or less, 2:7 or less, 2:8 or less, 2:9 or less, 2:10 or less, 10:3 or less, 9:3 or less, 8:3 or less, 7:3 or less, 6:3 or less, 5:3 or less, 4:3 or less, 3:3 or less, 2:3 or less, 3:3 or less Bottom, 3:2 or less, 3:3 or less, 3:4 or less, 3:5 or less, 3:6 or less, 3:7 or less, 3:8 or less, 3:9 or less, 3:10 or less, 10:4 or less, 9:4 or less, 8:4 or less, 7:4 or less, 6:4 or less, 5:4 or less, 4:4 or less, 3:4 or less, 2:4 or less, 4:4 or less, 4:2 or less, 4 : 3 or less, 4:4 or less, 4:5 or less, 4:6 or less, 4:7 or less, 4:8 or less, 4:9 or less, 4:10 or less, 10:5 or less, 9:5 or less, 8:5 or less, 7:5 or less, 6:5 or less, 5:5 or less, 4:5 or less, 3:5 or less, 2:5 or less, 5:5 or less, 5:2 or less, 5:3 or less, 5:4 Below, 5:5 or less, 5:6 or less, 5:7 or less, 5:8 or less, 5:9 or less, 5:10 or less, 10:6 or less, 9:6 or less, 8:6 or less, 7:6 or less, 6:6 or less, 5:6 or less, 4:6 or less, 3:6 or less, 2:6 or less, 6:6 or less, 6:2 or less, 6:3 or less, 6:4 or less, 6:5 or less,6:6 or less, 6:7 or less, 6:8 or less, 6:9 or less, 6:10 or less, 10:7 or less, 9:7 or less, 8:7 or less, 7:7 or less, 6:7 or less, 5:7 or less, 4:7 or less, 3:7 or less, 2:7 or less, 7:7 or less, 7:2 or less, 7:3 or less, 7:4 or less, 7:5 or less, 7:6 or less, 7:7 or less, 7:8 Below, 7:9 or less, 7:10 or less, 10:8 or less, 9:8 or less, 8:8 or less, 7:8 or less, 6:8 or less, 5:8 or less, 4:8 or less, 3:8 or less, 2:8 or less, 8:8 or less, 8:2 or less, 8:3 or less, 8:4 or less, 8:5 or less, 8:6 or less, 8:7 or less, 8:8 or less, 8:9 or less, 8:10 or less, Contains a cell number ratio of 10:9 or less, 9:9 or less, 8:9 or less, 7:9 or less, 6:9 or less, 5:9 or less, 4:9 or less, 3:9 or less, 2:9 or less, 9:9 or less, 9:2 or less, 9:3 or less, 9:4 or less, 9:5 or less, 9:6 or less, 9:7 or less, 9:8 or less, 9:9 or less, 9:10 or less, 10:10 or less, 9:10 or less, 8:10 or less, 7:10 or less, 6:10 or less, 5:10 or less, 4:10 or less, 3:10 or less, 2:10 or less, 10:10 or less, 10:2 or less, 10:3 or less, 10:4 or less, 10:5 or less, 10:6 or less, 10:7 or less, 10:8 or less, 10:9 or less, or 10:10 or less.

[0033] A mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells is prepared, for example, at a concentration of 1 x 10 4 pcs or more, 1 x 10 5 pcs or more, 2 x 10 5 pcs or more, 3 x 10 5 pcs or more, 4 x 10 5 5 x 10 pieces or more 5 pcs or more, 6 x 10 5 7 x 10 pieces or more 5 pcs or more, 8 x 10 5 pcs or more, 9 x 10 5 pcs or more, 1 x 10 6 pcs or more, 2 x 10 6 pcs or more, 3 x 10 6 pcs or more, 4 x 10 6 5 x 10 pieces or more 6 pcs or more, 6 x 10 6More than 7 x 10 6 More than one, 8 x 10 6 More than 9 x 10 6 More than one, 1 x 10 7 More than one, 2 x 10 7 More than one, 3 x 10 7 More than one, 4 x 10 7 More than one, 5 x 10 7 More than one, 6 x 10 7 More than 7 x 10 7 More than one, 8 x 10 7 More than 9 x 10 7 More than one, and also 1 x 10 8 More than 1 cell, 1 x 10 8 9 x 10 7 Below one, also 8 x 10 7 Less than 1, 7 x 10 7 Less than 6 x 10 7 Less than 5 x 10 7 Less than 1, 4 x 10 7 Less than 1, 3 x 10 7 Less than one, 2 x 10 7 Less than one, 1 x 10 7 Less than 1, 9 x 10 6 Less than 8 x 10 6 Less than 1, 7 x 10 6 Less than 6 x 10 6 Less than 5 x 10 6 Less than 1, 4 x 10 6 Less than 1, 3 x 10 6 Less than one, 2 x 10 6 Less than one, 1 x 10 6 Less than 1, 9 x 10 5 Less than 8 x 10 5 Less than 1, 7 x 10 5 Less than 6 x 10 5 Less than 5 x 10 5 Less than 1, 4 x 10 5 Less than 1, 3 x 10 5 Less than one, 2 x 10 5 Less than one, 1 x 10 5 Below one, also 1 x 10 4 Contains less than one cell.

[0034] In culturing a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, in a suspension state on a low-adhesion plate, the suspension culture may be performed at temperatures, for example, 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, 36°C or higher, 37°C or higher, 38°C or higher, 39°C or higher, 40°C or higher, 41°C or higher, 42°C or higher. or above, 43°C or above, 44°C or above, 45°C or above, 46°C or above, 47°C or above, 48°C or above, 49°C or above, or 50°C or above, and 50°C or below, 49°C or below, 48°C or below, 47°C or below, 46°C or below, 45°C or below, 44°C or below, 43°C or below, 42°C or below, 41°C or below, 40°C or below, 39°C or below, 38°C or below, 37°C or below, 36°C or below, 35°C or below, 34°C or below, 33°C or below, 32°C or below, 31°C or below, 30°C or below, 29°C or below, 28°C or below, 27°C or below, 26°C or below, or 25°C or below.

[0035] When a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells is cultured in suspension on a low-adhesion plate, the culture in suspension is carried out in the presence of, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more of CO2, or in the presence of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of CO2.

[0036] When a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells is cultured in a suspension state on a low-adhesion plate, the culture in a suspension state may be for, for example, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours or more, 24 hours or more, 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 hours or more, 34 hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more, 46 hours or more, 47 hours or more, 48 hours or more, 49 hours or more, 50 hours or more, 51 hours or more, 52 hours or more, 53 hours or more, 54 hours or more, 55 hours or more, 56 hours or more, 57 hours or more, 58 hours or more, 59 hours or more, 60 hours or more, 61 hours or more, 62 hours or more, 63 hours or more, 64 hours or more, 65 hours or more, 66 hours or more, 67 hours or more, 68 hours or more, 69 hours or more, 70 hours or more, 71 hours or more, or 72 hours or more, 71 hours or less, 70 hours or less, 69 hours or less, 68 hours or less, 67 hours or less, 66 hours or less, 65 hours or less, 64 hours or less, 63 hours or less, 62 hours or less, 61 hours or less, 60 hours or less, 59 hours or less, 58 hours or less, 57 hours or less, 56 hours or less, 55 hours or less, 54 hours or less, 53 hours or less, 52 hours or less, 51 hours or less, 50 hours less than 49 hours, less than 48 hours, less than 47 hours, less than 46 hours, less than 45 hours, less than 44 hours, less than 43 hours, less than 42 hours, less than 41 hours, less than 40 hours, less than 39 hours, less than 38 hours, less than 37 hours, less than 36 hours, less than 35 hours, less than 34 hours, less than 33 hours, 3 2 hours or less, 31 hours or less, 30 hours or less, 29 hours or less, 28 hours or less, 27 hours or less, 26 hours or less, 25 hours or less, 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less,It is performed for 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, or 5 hours or less.

[0037] A mixture of middle and hindgut cells, which have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm, which have been induced to differentiate from pluripotent stem cells and dispersed into single cells, is cultured in suspension on a low-adhesion plate, preferably using one, two, three, or four substances selected from the group consisting of a fibroblast growth factor, a GSK-3β inhibitor, a ROCK inhibitor, and a protein kinase C (PKC) activator. Preferably, the fibroblast growth factor is FGF4, the GSK-3β inhibitor is CHIR99021, the ROCK inhibitor is Y27632, and the protein kinase C (PKC) activator is PMA.

[0038] A mixture of middle and hindgut cells, which have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm, which have been induced to differentiate from pluripotent stem cells and dispersed into single cells, is cultured in suspension on a low-adhesion plate, preferably using one, two, three, or four substances selected from the group consisting of an extracellular matrix, an inhibitor of the bone morphogenetic protein (BMP) signaling pathway, an activator of the Wnt signaling pathway, and an activator of protein kinase C (PKC). Preferably, the extracellular matrix is ​​Matrigel, the inhibitor of the bone morphogenetic protein (BMP) signaling pathway is Noggin, the activator of the Wnt signaling pathway is R-spondin 1, and the activator of protein kinase C (PKC) is PMA.

[0039] The low-adhesion plate is preferably a plate for spheroid formation. The low-adhesion plate is preferably a plate having holes on the culture surface. The diameter of the holes is, for example, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, 600 μm or more, 700 μm or more, 800 μm or more, 900 μm or more, or 1,000 μm or more, and 1,000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, or 100 μm or less. The depth of the holes may be, for example, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 130 μm or more, 140 μm or more, 150 μm or more, 160 μm or more, 170 μm or more, 180 μm or more, 190 μm or more, 200 μm or more, 210 μm or more, 220 μm or more, 230 μm or more, 240 μm or more, 250 μm or more, 260 μm or more, 270 μm 280μm or more, 290μm or more, 300μm or more, 310μm or more, 320μm or more, 330μm or more, 340μm or more, 350μm or more, 360μm or more, 370μm or more, 380μm or more, 39 0μm or more, 400μm or more, 410μm or more, 420μm or more, 430μm or more, 440μm or more, 450μm or more, 460μm or more, 470μm or more, 480μm or more, 490μm or more, or 500μm or more and 500μm or less, 490μm or less, 480μm or less, 470μm or less, 460μm or less, 450μm or less, 440μm or less, 430μm or less, 420μm or less, 410μm or less, 400μm or less, 390μm or less, 380μm or less, 370μm or less, 360μm or less, 350μm or less, 340μm or less, 330μm or less, 320μm or less, 310μm or less, 300μm or less, 290μm or less, 280μm or less, 270 μm or less, 260 μm or less, 250 μm or less, 240 μm or less, 230 μm or less, 220 μm or less, 210 μm or less, 200 μm or less, 190 μm or less, 180 μm or less, 170 μm or less, 160 μm or less, 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, or 50 μm or less.The low-adhesion plate preferably includes a low-adhesion protein coat on its surface. The low-adhesion protein coat is preferably a hydrogel. The low-adhesion protein coat is preferably covalently bonded to the surface of the low-adhesion plate. The hydrogel is preferably hydrophilic, non-ionic, or neutrally charged. Preferably, the low-adhesion plate does not have any flat areas on the culture surface.

[0040] Preferably, the intestinal spheroids produced are spherical.

[0041] Dispersion into single cells is preferably carried out using a cell dissociation enzyme, which is preferably a protease, collagenase, or DNase.

[0042] The present invention provides a method for producing intestinal organoids, comprising the steps of culturing intestinal spheroids on a low-adhesion plate having a grooved structure to induce string-like intestinal precursor tissues, and subjecting the induced string-like intestinal precursor tissues to rotary suspension culture.

[0043] Rotary suspension culture is preferably carried out using a rotary bioreactor.

[0044] Intestinal organoids preferably have a lumen.Intestinal organoids preferably have a single lumen.Intestinal organoids preferably have a lumen in the center.Intestinal organoids preferably have a single lumen in the center.

[0045] In the low-adhesion plate having a grooved structure, the width of the grooved structure is preferably 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, 600 μm or more, 700 μm or more, 800 μm or more, 900 μm or more, 1,000 μm or more, 2,000 μm or more, 3,000 μm or more, 4,000 μm or more, 5,000 μm or more, 6,000 μm or more, 7,000 μm or more, 8,000 μm or more, 9,000 μm or more. or more, or 10,000 μm or more, and 10,000 μm or less, 9,000 μm or less, 8,000 μm or less, 7,000 μm or less, 6,000 μm or less, 5,000 μm or less, 4,000 μm or less, 3,000 μm or less, 2,000 μm or less, 1,000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, or 100 μm or less.

[0046] In the low-adhesion plate having a groove-like structure, the low-adhesion plate preferably includes a low-adhesion protein coating on the surface. The low-adhesion protein coating is preferably a hydrogel. The low-adhesion protein coating is preferably covalently bonded to the surface of the low-adhesion plate. The hydrogel is preferably hydrophilic, nonionic, or neutrally charged.

[0047] At the start of culturing intestinal spheroids on a low-adhesion plate having a grooved structure, the number of cells is preferably 5,000 or more, 6,000 or more, 7,000 or more, 8,000 or more, 9,000 or more, 10,000 or more, 20,000 or more, 30,000 or more, 40,000 or more, 50,000 or more, 60,000 or more, 70,000 or more, 80,000 or more, 90,000 or more, or More than 100,000 intestinal spheroids are cultured, and 100,000 or less, 90,000 or less, 80,000 or less, 70,000 or less, 60,000 or less, 50,000 or less, 40,000 or less, 30,000 or less, 20,000 or less, 10,000 or less, 9,000 or less, 8,000 or less, 7,000 or less, 6,000 or less, or 5,000 or less intestinal spheroids are cultured.

[0048] In culturing intestinal spheroids on a low-adhesion plate having a grooved structure, one, two, three, or four substances selected from the group consisting of a fibroblast growth factor, a GSK-3β inhibitor, a ROCK inhibitor, and a protein kinase C (PKC) activator are preferably used. Preferably, the fibroblast growth factor is FGF4, the GSK-3β inhibitor is CHIR99021, the ROCK inhibitor is Y27632, and the protein kinase C (PKC) activator is PMA.

[0049] In culturing intestinal spheroids on a low-adhesion plate having a grooved structure, the culture may be performed at temperatures such as 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, 36°C or higher, 37°C or higher, 38°C or higher, 39°C or higher, 40°C or higher, 41°C or higher, 42°C or higher, 43°C or higher, 44°C or higher, 45°C or higher, 46°C or higher, 47°C or higher, 48°C or higher, 49°C or higher, 50°C or higher, 51°C or higher, 52°C or higher, 53°C or higher, 54°C or higher, 55°C or higher, 56°C or higher, 57°C or higher, 58°C or higher, 59°C or higher, 60°C or higher, 61°C or higher, 62°C or higher, 63°C or higher, 64°C or higher, 65°C or higher, 66°C or higher, 67°C or higher, 68°C or higher, 69°C or higher, 70°C or higher, 71°C or higher, 72°C or higher, 73°C or higher, 74°C or higher, 75°C or higher, 76°C or higher, 77°C or higher, 78°C or higher, 79°C or higher, 79°C or higher, 80°C or higher, 81°C or higher, 82°C or higher, 83°C It is performed at temperatures above 8°C, above 49°C, or above 50°C, and at temperatures below 50°C, below 49°C, below 48°C, below 47°C, below 46°C, below 45°C, below 44°C, below 43°C, below 42°C, below 41°C, below 40°C, below 39°C, below 38°C, below 37°C, below 36°C, below 35°C, below 34°C, below 33°C, below 32°C, below 31°C, below 30°C, below 29°C, below 28°C, below 27°C, below 26°C, or below 25°C.

[0050] When culturing intestinal spheroids on a low-adhesion plate having a grooved structure, the culture is carried out in the presence of, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more CO2, or in the presence of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less CO2.

[0051] When culturing intestinal spheroids on a low-adhesion plate having a grooved structure, the culture may be continued for, for example, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours or more, 24 hours or more, 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 hours or more, 34 hours or more, or hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more, 46 hours or more, 47 hours or more, 48 hours or more, 49 hours or more, 50 hours or more, 51 hours or more, 52 hours or more, 53 hours or more, 54 hours or more, 55 hours or more, 56 hours or more, 57 hours or more, 58 hours or more, 59 hours or more, 60 hours or more, 61 hours or more, 62 hours or more, 63 hours or more, 64 hours or more, 65 hours or more, 66 hours or more, 67 hours or more, 68 hours or more, 69 hours or more, 70 hours or more, 71 hours or more, or 72 hours or more, 72 hours or less, 71 hours or less, 70 hours or less, 69 hours or less, 68 hours or less, 67 hours or less, 66 hours or less, 65 hours or less, 64 hours or less, 63 hours or less, 62 hours or less, 61 hours or less, 60 hours or less, 59 hours or less, 58 hours or less, 57 hours or less, 56 hours or less, 55 hours or less, 54 hours or less, 53 hours or less, 52 hours or less, 51 hours or less, 50 hours or less, 49 hours or less, 48 ​​hours or less, 47 hours or less, 46 hours or less, 45 hours or less, 44 hours or less, 43 hours or less, 42 hours or less, 1 hour or less, 40 hours or less, 39 hours or less, 38 hours or less, 37 hours or less, 36 hours or less, 35 hours or less, 34 hours or less, 33 hours or less, 32 hours or less, 31 hours or less, 30 hours or less, 29 hours or less, 28 hours or less, 27 hours or less, 26 hours or less, 25 hours or less, 24 hours or less Lower, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less,Or it will be done for 5 hours or less.

[0052] In the step of subjecting the induced cord-like intestinal precursor tissue to rotary suspension culture, preferably one, two, three, four, five, or six substances selected from the group consisting of extracellular matrix, growth factor, serum transport protein, secreted signaling protein, water-soluble vitamin, and hormone with a molecular structure similar to insulin are used. Preferably, the extracellular matrix is ​​Matrigel, the serum transport protein is Afamin, the secreted signaling protein is a substance associated with tumorigenesis, cell fate regulation, or embryonic patterning such as Wnt3a, the water-soluble vitamin is a B vitamin such as nicotinamide, and the hormone with a molecular structure similar to insulin is IGF-1. Preferably, the growth factors are one, two, three, four, five, or six substances selected from the group consisting of growth factors that stimulate the proliferation of epidermal or epithelial cells, activators of the Wnt signaling pathway, inhibitors of the bone morphogenetic protein (BMP) signaling pathway, activators of protein kinase C (PKC), fibroblast growth factors, and selective inhibitors of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7). Selective inhibitors of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7) include, for example, selective inhibitors of one, two, or three substances selected from the group consisting of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), and type I nodal receptor-like kinase (ALK7).Preferably, the growth factor that stimulates the proliferation of epidermal or epithelial cells is EGF, the activator of the Wnt signaling pathway is R-spondin 1, the inhibitor of the bone morphogenetic protein (BMP) signaling pathway is Noggin, the activator of protein kinase C (PKC) is PMA, the fibroblast growth factor is basic FGF, and the selective inhibitor of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7) is A8301.

[0053] The production method of the present invention preferably includes a step of semi-gelling the medium prior to the step of subjecting the induced cord-like intestinal precursor tissue to rotary suspension culture. The rotary suspension culture may be, for example, three-dimensional culture performed within a gel or extracellular matrix.

[0054] In the step of subjecting the induced cord-like intestinal precursor tissue to rotary suspension culture, the rotary suspension culture may be performed at a temperature of, for example, 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, 36°C or higher, 37°C or higher, 38°C or higher, 39°C or higher, 40°C or higher, 41°C or higher, 42°C or higher, 43°C or higher, 44°C or higher, 45°C or higher, 46°C or higher, 47°C or higher, 48°C or higher. or above, 49°C or above, or 50°C or above, and 50°C or below, 49°C or below, 48°C or below, 47°C or below, 46°C or below, 45°C or below, 44°C or below, 43°C or below, 42°C or below, 41°C or below, 40°C or below, 39°C or below, 38°C or below, 37°C or below, 36°C or below, 35°C or below, 34°C or below, 33°C or below, 32°C or below, 31°C or below, 30°C or below, 29°C or below, 28°C or below, 27°C or below, 26°C or below, or 25°C or below.

[0055] In the step of subjecting the induced string-like intestinal precursor tissue to rotary suspension culture, the rotary suspension culture is carried out in the presence of, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more CO2, or in the presence of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less CO2.

[0056] In the step of subjecting the induced cord-like intestinal precursor tissue to rotary suspension culture, the rotary suspension culture may be performed for, for example, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 20 hours or more, 30 hours or more, 40 hours or more, 50 hours or more, 60 hours or more, 70 hours or more, 80 hours or more, 90 hours or more, 100 hours or more, 200 hours or more, 300 hours or more, 400 hours or more, 500 hours or more, 600 hours or more, 700 hours or more, 800 hours or more, 900 hours or more, 1000 hours or more, 2000 hours or more, 3000 hours or more, 4000 hours or more, 5000 hours or more, 6000 hours or more, 7000 hours or more, 8000 hours or more, 900 hours or more, The test is carried out for 0 hours or more, or 10,000 hours or more, or for 10,000 hours or less, 9,000 hours or less, 8,000 hours or less, 7,000 hours or less, 6,000 hours or less, 5,000 hours or less, 4,000 hours or less, 3,000 hours or less, 2,000 hours or less, 1,000 hours or less, 900 hours or less, 800 hours or less, 700 hours or less, 600 hours or less, 500 hours or less, 400 hours or less, 300 hours or less, 200 hours or less, 100 hours or less, 90 hours or less, 80 hours or less, 70 hours or less, 60 hours or less, 50 hours or less, 40 hours or less, 30 hours or less, 20 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, or 5 hours or less.

[0057] In the step of rotary suspension culture of the induced cord-like intestinal precursor tissue, rotation is preferably carried out at a rotation speed of 1 rpm or more, 2 rpm or more, 3 rpm or more, 4 rpm or more, 5 rpm or more, 6 rpm or more, 7 rpm or more, 8 rpm or more, 9 rpm or more, 10 rpm or more, 20 rpm or more, 30 rpm or more, 40 rpm or more, 50 rpm or more, 60 rpm or more, 70 rpm or more, 80 rpm or more, 90 rpm or more, or 100 rpm or more, or at a rotation speed of 100 rpm or less, 90 rpm or less, 80 rpm or less, 70 rpm or less, 60 rpm or less, 50 rpm or less, 40 rpm or less, 30 rpm or less, 20 rpm or less, 10 rpm or less, 9 rpm or less, 8 rpm or less, 7 rpm or less, 6 rpm or less, 5 rpm or less, 4 rpm or less, 3 rpm or less, 2 rpm or less, or 1 rpm or less.

[0058] In the step of subjecting the induced cord-like intestinal precursor tissue to rotary suspension culture, the rotation speed is preferably increased.

[0059] In the step of rotary suspension culture of the induced string-like intestinal precursor tissue, the medium is preferably changed at least once every 2 days, at least once every 3 days, at least once every 4 days, at least once every 5 days, at least once every 6 days, at least once every 7 days, at least once every 8 days, at least once every 9 days, or at least once every 10 days, and not more than once every 10 days, not more than once every 9 days, not more than once every 8 days, not more than once every 7 days, not more than once every 6 days, not more than once every 5 days, not more than once every 4 days, not more than once every 3 days, or not more than once every 2 days.

[0060] The manufacturing method of the present invention preferably includes a step of culturing the induced cord-like intestinal precursor tissue on a low-adhesion plate prior to a step of rotary suspension culture of the induced cord-like intestinal precursor tissue.

[0061] The low-adhesion plate is preferably a plate for spheroid formation. The low-adhesion plate is preferably a plate having holes on the culture surface. The diameter of the holes is, for example, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, 600 μm or more, 700 μm or more, 800 μm or more, 900 μm or more, or 1,000 μm or more, and 1,000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, or 100 μm or less. The depth of the holes may be, for example, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 130 μm or more, 140 μm or more, 150 μm or more, 160 μm or more, 170 μm or more, 180 μm or more, 190 μm or more, 200 μm or more, 210 μm or more, 220 μm or more, 230 μm or more, 240 μm or more, 250 μm or more, 260 μm or more, 270 μm 280μm or more, 290μm or more, 300μm or more, 310μm or more, 320μm or more, 330μm or more, 340μm or more, 350μm or more, 360μm or more, 370μm or more, 380μm or more, 39 0μm or more, 400μm or more, 410μm or more, 420μm or more, 430μm or more, 440μm or more, 450μm or more, 460μm or more, 470μm or more, 480μm or more, 490μm or more, or 500μm or more and 500μm or less, 490μm or less, 480μm or less, 470μm or less, 460μm or less, 450μm or less, 440μm or less, 430μm or less, 420μm or less, 410μm or less, 400μm or less, 390μm or less, 380μm or less, 370μm or less, 360μm or less, 350μm or less, 340μm or less, 330μm or less, 320μm or less, 310μm or less, 300μm or less, 290μm or less, 280μm or less, 270 μm or less, 260 μm or less, 250 μm or less, 240 μm or less, 230 μm or less, 220 μm or less, 210 μm or less, 200 μm or less, 190 μm or less, 180 μm or less, 170 μm or less, 160 μm or less, 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, or 50 μm or less.The low-adhesion plate preferably includes a low-adhesion protein coat on its surface. The low-adhesion protein coat is preferably a hydrogel. The low-adhesion protein coat is preferably covalently bonded to the surface of the low-adhesion plate. The hydrogel is preferably hydrophilic, non-ionic, or neutrally charged. Preferably, the low-adhesion plate does not have any flat areas on the culture surface.

[0062] Culturing on low-adhesion plates can be performed at temperatures such as 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, 36°C or higher, 37°C or higher, 38°C or higher, 39°C or higher, 40°C or higher, 41°C or higher, 42°C or higher, 43°C or higher, 44°C or higher, 45°C or higher, 46°C or higher, 47°C or higher, 48°C or higher, 49°C or higher, or is performed at 50°C or higher, 50°C or lower, 49°C or lower, 48°C or lower, 47°C or lower, 46°C or lower, 45°C or lower, 44°C or lower, 43°C or lower, 42°C or lower, 41°C or lower, 40°C or lower, 39°C or lower, 38°C or lower, 37°C or lower, 36°C or lower, 35°C or lower, 34°C or lower, 33°C or lower, 32°C or lower, 31°C or lower, 30°C or lower, 29°C or lower, 28°C or lower, 27°C or lower, 26°C or lower, or 25°C or lower.

[0063] Culturing on low-adhesion plates is carried out, for example, in the presence of 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more CO2, or in the presence of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less CO2.

[0064] Culturing on low-adhesion plates can be carried out for, for example, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 20 hours or more, 30 hours or more, 40 hours or more, 50 hours or more, 60 hours or more, 70 hours or more, 80 hours or more, 90 hours or more, 100 hours or more, 200 hours or more, 300 hours or more, 400 hours or more, or 500 hours or more, or 500 hours or less, 400 hours or less, 300 hours or less, 200 hours or less, 100 hours or less, 90 hours or less, 80 hours or less, 70 hours or less, 60 hours or less, 50 hours or less, 40 hours or less, 30 hours or less, 20 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, or 5 hours or less.

[0065] The present invention provides a method for producing intestinal spheroids for producing intestinal organoids, comprising the steps of culturing a mixture of middle and hindgut cells, which have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm, which have been induced to differentiate from pluripotent stem cells and dispersed into single cells, in a floating state on a low-adhesion plate, and fusing the middle and hindgut cells with the visceral mesoderm to produce intestinal spheroids.

[0066] The present invention provides a method for producing string-like intestinal progenitor tissue for producing intestinal organoids, comprising the step of culturing intestinal spheroids on a low-adhesion plate having a grooved structure to induce string-like intestinal progenitor tissue.

[0067] The present invention provides a method for producing intestinal organoids, which comprises a step of culturing intestinal spheroids on a low-adhesion plate with a grooved structure to induce string-like intestinal precursor tissue, and then subjecting the tissue to rotary suspension culture.

[0068] The present invention provides intestinal organoids having a lumen, which are produced using any of the production methods described herein.

[0069] The present invention provides organoids with a lumen, and the organoids have a longitudinal length of the lumen of 0.5 cm or more, 1.0 cm or more, 1.5 cm or more, 2.0 cm or more, 2.5 cm or more, 3.0 cm or more, 3.5 cm or more, or 4.0 cm or more.The upper limit of the longitudinal length of the lumen can be, for example, 10.0 cm, but is not limited thereto, and can be, for example, 6.0 cm or less, 5.0 cm or less, or 4.0 cm or less.The length of the lumen-containing intestinal organoid can be, for example, 1.5 cm to 3.5 cm, for example, about 2.5 cm.

[0070] The present invention provides an intestinal organoid with a lumen, wherein the outer diameter of the tube is 0.5 mm or more, 0.6 mm or more, 0.7 mm or more, 0.8 mm or more, 0.9 mm or more, 1.0 mm or more, 1.1 mm or more, 1.2 mm or more, 1.3 mm or more, 1.4 mm or more, 1.5 mm or more, or 2.0 mm or more.The upper limit of the outer diameter of the tube can be, for example, 5.0 mm, but is not limited thereto, and can be, for example, 4.0 mm or less, 3.0 mm or less, or 2.0 mm or less.The outer diameter of the intestinal organoid with a lumen can be, for example, 0.5 mm to 1.5 mm, for example, about 1.0 mm.

[0071] The present invention provides organoids of the intestinal tract with lumen, wherein the internal diameter of the tract is 300 μ m or more, 400 μ m or more, 500 μ m or more, 600 μ m or more, 700 μ m or more, 800 μ m or more, 900 μ m or more, 1000 μ m or more, 1100 μ m or more, 1200 μ m or more, 1300 μ m or more, or 1400 μ m or more.The upper limit of the external diameter of the tract can be, for example, 2000 μ m, but is not limited thereto, and can be, for example, 1800 μ m or less, 1600 μ m or less, or 1500 μ m or less.The internal diameter of the intestinal tract with lumen can be, for example, 500 μ m to 800 μ m, for example, about 600 μ m.

[0072] The present invention provides a method for constructing intestinal tissue, which is characterized by transplanting intestinal organoids having a lumen into the mesentery.

[0073] The present invention provides a method for treating the intestinal tract, which comprises transplanting intestinal organoids having a lumen into the mesentery.

[0074] The present invention provides use of intestinal organoids having a lumen for producing a pharmaceutical composition for transplanting into the mesentery to construct intestinal tissue.

[0075] The present invention provides use of intestinal organoids having a lumen for producing a pharmaceutical composition for transplanting into the mesentery to treat the intestinal tract.

[0076] The concentration of each component in the medium used in the production method of the present invention may be, for example, 1 ng / ml or more, 2 ng / ml or more, 3 ng / ml or more, 4 ng / ml or more, 5 ng / ml or more, 6 ng / ml or more, 7 ng / ml or more, 8 ng / ml or more, 9 ng / ml or more, 10 ng / ml or more, 20 ng / ml or more, 30 ng / ml or more, 40 ng / ml or more, 50 ng / ml or more, 60 ng / ml or more, 70 ng / ml or more, 80 ng / ml or more, 90 ng / ml or more, 100 ng / ml or more, 200 ng / ml or more, 300 ng / ml or more, 400 ng / ml or more, 500 ng / ml or more, 600 ng / ml or more, 700 ng / ml or more, 800 ng / ml or more, 900 ng / ml or more, 1000 ng / ml or more, 2000 ng / ml or more, ng / ml or more, 3000 ng / ml or more, 4000 ng / ml or more, 5000 ng / ml or more, 6000 ng / ml or more, 7000 ng / ml or more, 8000 ng / ml or more, or 9000 ng / ml or more and 9000 ng / ml or less, 8000 ng / ml or less, 7000 ng / ml or less, 6000 ng / ml or less, 5000 ng / ml or less, 4000 ng / ml or less, 3000 ng / ml or less, 2000 ng / ml or less, 1000 ng / ml or less, 900 ng / ml or less, 800 ng / ml or less, 700 ng / ml or less, 600 ng / ml or less, 500 ng / ml or less, 400 ng / ml or less, 300 ng / ml or less, 200 ng / ml or less, 100 ng / ml or less, 90 ng / ml or less, 80 ng / ml or less, 70 ng / ml or less, 60 ng / ml or less, 50 ng / ml or less, 40 ng / ml or less, 30 ng / ml or less, 20 ng / ml or less, 10 ng / ml or less, 9 ng / ml or less, 8 ng / ml or less, 7 ng / ml or less, 6 ng / ml or less, 5 ng / ml or less, 4 ng / ml or less, 3 ng / ml or less, 2 ng / ml or less, or 1 ng / ml or less.The concentration of each component in the medium used in the production method of the present invention may be, for example, 1 nM or more, 2 nM or more, 3 nM or more, 4 nM or more, 5 nM or more, 6 nM or more, 7 nM or more, 8 nM or more, 9 nM or more, 10 nM or more, 20 nM or more, 30 nM or more, 40 nM or more, 50 nM or more, 60 nM or more, 70 nM or more, 80 nM or more, 90 nM or more, 100 nM or more, 200 nM or more, 300 nM or more, 400 nM or more, 500 nM or more, 600 nM or more, 700 nM or more, 800 nM or more, 900 nM or more, 1000 nM or more, 2000 nM or more, 3000 nM or more, 4000 nM or more, 5000 nM or more, 6000 nM or more, 7000 nM or more, 8000 nM or more, 9000 nM or more, nM or more, 10000 nM or more, 20000 nM or more, 30000 nM or more, 40000 nM or more, 50000 nM or more, 60000 nM or more, 70000 nM or more, 80000 nM or more, or 90000 nM or more and 90000 nM or less, 80000 nM or less, 70000 nM or less, 60000 nM or less, 50000 nM or less, 40000 nM or less, 30000 nM or less, 20000 nM or less, 10000 nM or less, 9000 nM or less, 8000 nM or less, 7000 nM or less, 6000 nM or less, 5000 nM or less, 4000 nM or less, 3000 nM or less, 2000 nM or less, 1000 nM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less.

[0077] <Experimental Methods> Animals: 8-10 week old female NSG mice were used in all experiments. They were kept in the animal facility at Tokyo Medical and Dental University, and experiments were conducted with the approval of the university's Animal Experiment Ethics Committee (A2023-202A).

[0078] iPS cell lines and culture: The HiPS-RIKEN-2F, Tkd3-4, and PB001 strains were used. The HiPS-RIKEN-2F strain was transfected with GFP protein using electroporation. The PB001 strain was lentivirally labeled with tdTomato. iPS cells were maintained and cultured using the Cellartis DEF-CS culture system (Takara Bio).

[0079] Induction of differentiation of iPS cells into mid- and hindgut cells: iPS cells were differentiated into mid- and hindgut cells according to a previously reported method (Day 0: Activin A 100ng / ml, CHIR99021 3μM; Day 1-Day 2: Activin A 100ng / ml; Day 3-Day 6: FGF4 100ng / ml, CHIR99021 3μM). On Day 7, single cells were isolated using TrypLE Express and used to form spheroids.

[0080] Induction of iPS cell differentiation into visceral mesoderm: iPS cells were differentiated into visceral mesoderm as previously reported (Day 0: Activin A (30 ng / ml), CHIR99021 (6 μM), basic FGF (20 ng / ml), BMP4 (40 ng / ml, R&D), and PIK90 (100 nM); Day 1: Wnt-C59 (1 μM, BMP4 (30 ng / ml), and A83-01 (1 μM); Day 2-3: basic FGF (20 ng / ml), Wnt-C59 (1 μM), BMP4 (30 ng / ml), A83-01 (1 μM), and retinoic acid (2 μM); Day 4-5: retinoic acid (2 μM) and purmorphamine (PMA) (2 μM)). The basal medium was AdDMEM / F12, GlutaMAX (1x), HEPES (15 mM), N-2 supplement (1x), B-27 supplement without vitamin A (1x), and Penicillin-Streptomycin Mixed Solution (1x). On day 6, single cells were isolated using Accumax and used to form spheroids.

[0081] Creation of S-Spheroids and As-Spheroids: To create As-Spheroids (fusion of visceral mesoderm), single-celled mid- and hindgut cells and visceral mesoderm were mixed at a 3:1 ratio, and the cell suspension was transferred to a 15 mL low-attachment tube. For S-Spheroid creation, only mid- and hindgut cells were used. The cells were suspended at 300 G for 3 minutes, the supernatant was discarded, and the cells were suspended in medium (composition described below) and plated at 1.6 x 10 per well in an EZSPHERE 6-well plate. 6Cells were seeded and cultured at 37°C with 5% CO2 for 24 hours to form spheroids. The culture medium was HIO basal medium (AdDMEM / F12, GlutaMAX (1x), HEPES (15 mM), N-2 supplement (1x), B-27 (1x), and Penicillin-Streptomycin Mixed Solution (1x)) supplemented with FGF4 (100 ng / ml), CHIR99021 (3 μM), Y27632 (10 μM), and PMA (2 μM). For the formation of large spheroids, 1 x 10 cells were seeded per well of an EZ-BindShut 96-well plate. 5 cells were seeded.

[0082] Generation of S-spheroids and as-spheroids: Spheroids were harvested from EZSPHERE plates and cultured on ultra-low attachment plates. The HIO basal medium was supplemented with 10% Matrigel, EGF (100 ng / ml), Noggin (100 ng / ml; for the first 3 days only), R-spondin1 (500 ng / ml), and PMA (2 μM). Large spheroids generated using EZ-bindshut were cultured in a rotating bioreactor (CellPet 3D-iPS). The medium was changed every 3–4 days.

[0083] Creation of special plates: In collaboration with AGC Technoglass, we created an ultra-low adhesion 6-well plate (Org-SP plate) with parallel grooves 1000 μm wide drilled on the bottom.

[0084] Assembled Human Intestinal Tubule (a-HIT) generation: Spheroids were harvested from EZSPHERE plates into 50 mL low-attachment plates, centrifuged at 100 g for 3 minutes, and the supernatant was discarded. Approximately 32,400 spheroids (approximately 32,400 cells) were used for one well of an OrgSP plate (6-well format). The culture medium was based on HIO basal medium (AdDMEM / F12, GlutaMAX (1x), HEPES (15 mM), N-2 supplement (1x), B-27 (1x), and penicillin-streptomycin mixed solution (1x)), supplemented with FGF4 (100 ng / ml), CHIR99021 (3 μM), Y27632 (10 μM), and PMA (2 μM). 12 mL of medium was used per well of the OrgSP plate. The cells were cultured at 37°C with 5% CO2 for 24 hours to promote spheroid fusion.

[0085] a-HIT culture: a-HIT were cultured in HIO basal medium supplemented with Matrigel (20% for the first 3 days, then 10% thereafter) and growth factors. The growth factor combinations were as follows: (1) ENR: EGF (100 ng / ml), Noggin (100 ng / ml for the first 3 days), R-Spondin1 (500 ng / ml), and 2 μM PMA. (2) The assembloid medium: Afamin / Wnt3a conditioned medium (10% (vol / vol), MBL Lifescience), EGF (100 ng / ml), R-Spondin1 (500 ng / ml), nicotinamide (10 mM, Sigma), IGF-1 (50 ng / ml, BioLegend), basic FGF (25 ng / ml), A8301 (500 nM), and PMA (2 μM). Prior to a-HIT collection, medium was added to an ultra-low attachment 6-well plate and incubated at 37°C for 20 minutes to allow the medium to semi-gel. a-HIT were collected from the OrgSP plate using a wide-bore 1000 μl pipette and placed in the aforementioned 6-well plate for 3 days of culture at 37°C with 5% CO2. The a-HIT were then transferred to a culture syringe and cultured in a rotating bioreactor (CellPet 3D-iPS). The rotation speed was started at 10 rpm and gradually increased as the a-HIT grew. The medium was changed every 3–4 days.

[0086] Mesenteric transplantation of a-HIO and a-HIT: Mesenteric transplantation was performed according to a previously reported method. Prior to mesenteric transplantation, a-HIO was cultured for 4 weeks, and a-HIT for 2 weeks. After anesthesia with 2% isoflurane, a 2-cm midline incision was made in the abdominal wall and peritoneum, and the distal ileum was pulled and exposed to the outside of the body using a cotton swab. A-HIO or a-HIT was placed alongside the distal ileum and fixed with Beriplast. After transplantation, the intestine was returned to the abdominal cavity, and the peritoneum and abdominal wall were sutured. Mice were sacrificed after 8 weeks, and the grafted tissue was analyzed.

[0087] <Methods> (1) Construction of as-Spheroids, Establishment of Culture Method, and Characterization: Human iPS cells were individually induced to differentiate into midgut and hindgut cells and visceral mesoderm via endoderm. The induced midgut and hindgut cells were dissociated into single cells and seeded on ultra-low attachment spheroid formation plates (EZ-sphere AGC Technoglass) with microwells to promote spheroid formation in a suspended state. The resulting spheroids were named assembled suspension-spheroids (as-Spheroids). The as-Spheroids were cultured in suspension using an ultra-low attachment culture plate and a rotating bioreactor (CellPet 3D-iPS, JTEC Corporation) in a medium containing Matrigel and growth factors, and then induced to differentiate into intestinal organoids (assembled HIOs; a-HIOs). The a-HIOs were transplanted into the mesentery of severely immunodeficient mice to evaluate their maturation in vivo. The characteristics and histology of As-Spheroids, a-HIOs, and transplanted tissues were analyzed by quantitative PCR, immunostaining, in situ hybridization, etc.

[0088] (2) Construction of assembled human intestinal tubules: An ultra-low-attachment spheroid formation plate (OrgSP plate, AGC Technoglass) with a 1000 μm-wide groove at the bottom was specially designed and fabricated for this invention. As-spheroids were seeded onto the OrgSP plate and cultured in suspension for 24 hours to promote fusion of spheroids within the grooves. The string-like structures formed by fusion were named assembled human intestinal tubules (a-HITs). a-HITs were cultured in a rotary suspension culture in a rotating bioreactor, and their structure was analyzed by immunostaining. Culture conditions for a-HITs were compared and examined (EGF, Noggin, R-spondin1; assemble medium: Afamin-Wnt3A conditioned medium, EGF, R-spondin1, Nicotinamide, IGF1, FGF2, A8301) to identify medium conditions that promote tube formation. a-HIT was transplanted into the mesentery of hyperimmunodeficient mice to evaluate their maturation in vivo. The a-HIT and the transplanted tissues were characterized and histologically analyzed by quantitative PCR, immunohistochemistry, in situ hybridization, etc.

[0089] Results: Single-cell midgut and hindgut cells and visceral mesoderm (Fig. 5A) were seeded onto spheroid formation plates, where they rapidly aggregated and formed spheroids within the microwells (Fig. 1A-B, Fig. 5B). The as-spheroids (as-spheroids) were confirmed by immunostaining and quantitative PCR to express the gut-specific marker CDX2 and the visceral mesoderm marker FOXF1 (Fig. 1C, Fig. 5C-D). Following suspension culture, the as-spheroids self-organized and differentiated into intestinal organoids (assembled suspension HIOs; as-HIOs), which were layered structures with intestinal epithelial cells surrounded by abundant mesenchymal tissue (Fig. 1D-G, Fig. 6A). Fusion of the visceral mesoderm resulted in the development of mesenchymal cells, regardless of the strain (Fig. 6B). The addition of a certain concentration of extracellular matrix (e.g., Matrigel) to the culture medium was required for proper self-organization (Fig. 6C). When as-HIOs were transplanted onto the mesentery of severely immunodeficient mice, we confirmed that the intestinal tissue structure could be reproduced, including mature intestinal epithelium (presence of various differentiated cells and intestinal epithelial stem cells) and mesenchymal tissue (including the submucosa and smooth muscle layer) (Figure 2).

[0090] We investigated whether the high aggregation and self-organization abilities of as-Spheroids could be applied to the construction of intestinal structures. We designed and fabricated an ultra-low-adhesion spheroid formation plate with 1000 μm-wide grooves (Fig. 7A). As-Spheroids were seeded on the plate, they aggregated within the grooves and, due to their high fusion ability, formed string-like intestinal precursor structures (assembled human intestinal tubules; a-HITs) several centimeters long within 24 hours of seeding (Fig. 3A and B). While s-Spheroids also formed string-like intestinal precursor structures (Fig. 7B), the use of as-Spheroids ensured stable construction of visceral mesodermal components (Fig. 3C and Fig. 7C). We also attempted to construct intestinal precursor structures by seeding t-spheroids in the same manner, but the difficulty in obtaining the necessary yield and the low fusion ability compared to as-Spheroids made it difficult to form similar string-like structures (Fig. 7D). These results demonstrate that as-Spheroid is the most suitable material for creating stable, string-like intestinal precursor structures.

[0091] The constructed a-HITs were cultured in a rotary bioreactor in a rotating suspension culture system, where they self-organized into intestinal tissues containing epithelial and surrounding mesenchymal tissues (Figure 8A). Stable tube formation was difficult in the ENR (EGF, Noggin, R-spondin1) medium commonly used for HIO culture (Figure 8B). After optimizing the culture conditions, we found that culturing as-spheroids in a medium containing Afamin-Wnt CM, EGF, R-spondin1, nicotinamide, IGF-1, FGF-2, and A8301 (assembloid medium) promoted the proliferation of epithelial components while maintaining the intestinal epithelial lineage (Figure 9). Culture in assembloid medium promoted the proliferation of epithelial components and central lumen formation while ensuring the development of mesenchymal tissues (Figures 3D-E, Figure 10). After growth and maturation in rotary suspension culture, a-HIT were transplanted onto the mesentery of hyperimmunodeficient mice, where they survived and formed large, mature intestinal tissues measuring several centimeters (Fig. 4). a-HIT cultured in ENR medium showed no visible lumen even after maturation in vivo due to multicystic formation (Fig. 11), whereas a-HIT cultured in assembloid medium showed continuous lumen formation even after maturation.

[0092] <Discussion> In this study, we demonstrated that spheroids (as-Spheroids) can be formed using mid- and hindgut cells and visceral mesoderm individually induced to differentiate from pluripotent stem cells, and that intestinal organoids (a-HIOs) with stable mesenchymal tissue can be induced by suspension culture. Using as-Spheroids, which have a high fusion potential, we demonstrated that it is possible to design and construct intestinal tissues several centimeters in size, and further demonstrate that mature intestinal tissue can be constructed in vivo.

[0093] Previously, mesenchymal tissue development in existing HIOs relied on mesodermal cells generated spontaneously during midgut and hindgut differentiation, and was strongly influenced by differences between pluripotent stem cell strains and individual organoids. Recently, methods for forming fused spheroids (assembloids) using multiple progenitor cells derived from pluripotent stem cells have been reported in various fields. The inventors suspected that this assembloid technology could also be applied to the field of intestinal organoids. Midgut and hindgut cells and visceral mesoderm individually induced from pluripotent stem cells exhibited high fusion potential using a suspension fusion method developed by the inventors on low-adhesion spheroid formation plates, and uniform spheroids were efficiently constructed (as-spheroids). Furthermore, the formed as-spheroids self-organized similarly to conventional spheroids and differentiated into intestinal organoids (a-HIOs) with stably developed mesenchymal tissue. This finding indicates that the fused mesoderm self-organizes in a manner similar to endogenously developed mesenchymal components. This method not only overcomes the problem of differences in mesenchymal cell development between strains, but also suggests that various cell populations may be fused in a similar manner.

[0094] Previous research reports have primarily used this high fusion ability to increase the complexity of organoids (increase in cell population) and to verify cell-cell interactions. In this invention, the inventors have demonstrated that this high fusion ability can be used to create large structures aimed at organ construction. Specifically, they have successfully created an intestinal structure by using as-Spheroids, which have high fusion ability, as the building blocks of the structure.

[0095] While several attempts to construct large tissue structures have been reported in recent years, most of these require cell placement using specialized machines such as bioprinters. The present inventors have achieved large-scale tissue construction using only spheroid formation plates, leveraging the cell's inherent aggregation, fusion, and self-organization abilities, which is considered a more versatile method. Furthermore, this method not only forms large tissues but also promotes organ formation by relying on the cells' inherent self-organization ability, making it useful as a platform for exploring the process of organogenesis.

[0096] In this study, we demonstrated that a-HIT can be efficiently cultured in suspension culture using a rotating bioreactor. Furthermore, we demonstrated that mature intestinal tissue measuring several centimeters can be constructed after transplantation into the mesentery, proposing proof-of-concept that luminal intestinal construction is possible from pluripotent stem cells. Our intestinal construction method, which utilizes high aggregation ability and suspension culture, is also expected to be applicable to other endodermal organs.

Claims

1. Intestinal organoids with a lumen.

2. The intestinal organoid of claim 1, having a single lumen.

3. The intestinal organoid of claim 1, having a lumen in the center.

4. The intestinal organoid of claim 1, having a single lumen in the center.

5. The intestinal organoid of claim 1 for constructing intestinal tissue.

6. The intestinal organoid of claim 1 for constructing intestinal tissue having a lumen.

7. The intestinal organoid of claim 1 for transplantation into the mesentery.

8. The intestinal organoid of claim 1 for constructing intestinal tissue in a living body.

9. The intestinal organoid of claim 1 for constructing intestinal tissue with a lumen in a living body.

10. The intestinal organoid of claim 1 for transplantation into the mesentery in a living body.

11. The intestinal organoid of claim 1, which is derived from pluripotent stem cells.

12. A method for producing intestinal organoids, comprising the steps of culturing in suspension on a low-adhesion plate a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, thereby fusing the middle and hindgut cells with the visceral mesoderm to create intestinal spheroids, and culturing the created intestinal spheroids in suspension.

13. A method for producing intestinal organoids as described in claim 12, in which the induction of differentiation from pluripotent stem cells into middle and hindgut cells and the induction of differentiation from pluripotent stem cells into visceral mesoderm are carried out separately.

14. A method for producing intestinal organoids as described in claim 12, wherein the differentiation of pluripotent stem cells into middle and hindgut cells is induced via the endoderm.

15. A method for producing intestinal organoids as described in claim 12, in which a substance selected from the group consisting of factors belonging to the TGFβ superfamily, GSK-3β inhibitors, and fibroblast growth factors is used in inducing differentiation of pluripotent stem cells into middle and hindgut cells.

16. A method for producing intestinal organoids as described in claim 15, wherein the factor belonging to the TGFβ superfamily is Activin A, the GSK-3β inhibitor is CHIR99021, and the fibroblast growth factor is FGF4.

17. A method for producing intestinal organoids as described in claim 12, wherein the differentiation of pluripotent stem cells into visceral mesoderm is induced via endoderm.

18. A method for producing intestinal organoids as described in claim 12, wherein the differentiation of pluripotent stem cells into visceral mesoderm is induced using a substance selected from the group consisting of factors belonging to the TGFβ superfamily; GSK-3β inhibitors; fibroblast growth factors; PI3Kα / γ / δ inhibitors; PORCN inhibitors; selective inhibitors of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7); vitamin A metabolites; and Hedgehog signal agonists.

19. The method for producing intestinal organoids described in claim 18, wherein the factor belonging to the TGFβ superfamily is Activin A or BMP4, the GSK-3β inhibitor is CHIR99021, the fibroblast growth factor is basic FGF, the PI3Kα / γ / δ inhibitor is PIK90, the PORCN inhibitor is Wnt-C59, the selective inhibitor of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7) is A83-01, the vitamin A metabolite is retinoic acid, and the Hedgehog signal agonist is palmorfamine.

20. A method for producing intestinal organoids as described in claim 12, wherein the mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells contains middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells in a cell number ratio of 10:1 to 1:

10.

21. A mixture of middle and hindgut cells induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm induced to differentiate from pluripotent stem cells and dispersed into single cells, was cultured at a concentration of 1 x 10 4 pieces ~ 1 x 10 8 The method for producing the intestinal organoid of claim 12, comprising cells.

22. A method for producing intestinal organoids as described in claim 12, wherein a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, is cultured in a floating state on a low-adhesion plate, and the floating culture is performed at 25°C to 45°C.

23. A method for producing intestinal organoids as described in claim 12, wherein a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, is cultured in a floating state on a low-adhesion plate, and the floating culture is performed in the presence of 1% to 10% CO2.

24. A method for producing intestinal organoids as described in claim 12, wherein a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, is cultured in a floating state on a low-adhesion plate, and the culture in a floating state is carried out for 5 to 72 hours.

25. A method for producing intestinal organoids as described in claim 12, in which a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, is cultured in a floating state on a low-adhesion plate, using a substance selected from the group consisting of fibroblast growth factor, GSK-3β inhibitor, ROCK inhibitor, and protein kinase C (PKC) activator.

26. A method for producing intestinal organoids as described in claim 25, wherein the fibroblast growth factor is FGF4, the GSK-3β inhibitor is CHIR99021, the ROCK inhibitor is Y27632, and the protein kinase C (PKC) activator is PMA.

27. A method for producing intestinal organoids as described in claim 12, in which a mixture of middle and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, is cultured in a floating state on a low-adhesion plate, and a substance selected from the group consisting of extracellular matrix, inhibitors of the bone morphogenetic protein (BMP) signaling pathway, activators of the Wnt signaling pathway, and activators of protein kinase C (PKC) is used.

28. A method for producing intestinal organoids as described in claim 27, wherein the extracellular matrix is ​​Matrigel, the inhibitor of the bone morphogenetic protein (BMP) signaling pathway is Noggin, the activator of the Wnt signaling pathway is R-spondin 1, and the activator of protein kinase C (PKC) is PMA.

29. A method for producing intestinal organoids described in claim 12, wherein the low-adhesion plate is a plate for spheroid formation.

30. A method for producing intestinal organoids described in claim 12, wherein the low-adhesion plate is a plate containing holes on the culture surface.

31. A method for producing intestinal organoids according to claim 30, wherein the diameter of the holes is 100 to 1,000 μm and the depth of the holes is 50 to 500 μm.

32. A method for producing intestinal organoids described in claim 12, wherein the low-adhesion plate comprises a low-adhesion protein coating on its surface.

33. A method for producing intestinal organoids described in claim 32, wherein the low-adhesion protein coat is a hydrogel.

34. A method for producing intestinal organoids described in claim 32, wherein the protein low-adhesion coat is covalently bound to the surface of the low-adhesion plate.

35. A method for producing intestinal organoids described in claim 33, wherein the hydrogel is hydrophilic, nonionic, or neutrally charged.

36. A method for producing intestinal organoids as described in claim 12, wherein the low-adhesion plate does not have any flat areas on the culture surface.

37. A method for producing intestinal organoids described in claim 12, wherein the intestinal spheroids produced are spherical.

38. A method for producing intestinal organoids as described in claim 12, wherein dispersing into single cells is performed using a cell dissociation enzyme.

39. A method for producing intestinal organoids described in claim 38, wherein the cell dissociation enzyme is protease, collagenase, or DNase.

40. A method for producing intestinal organoids, comprising the steps of culturing intestinal spheroids on a low-adhesion plate having a grooved structure to induce string-like intestinal precursor tissue, and culturing the induced string-like intestinal precursor tissue in a rotary suspension culture.

41. A method for producing intestinal organoids according to claim 40, wherein the rotary suspension culture is carried out using a rotary bioreactor.

42. A method for producing intestinal organoids according to claim 40, wherein the intestinal organoids have a single lumen in the center.

43. A method for producing intestinal organoids described in claim 40, wherein the width of the grooved structure in the low-adhesion plate is 100 μm to 10,000 μm.

44. A method for producing intestinal organoids as described in claim 40, wherein the low-adhesion plate has a grooved structure and comprises a protein low-adhesion coating on its surface.

45. A method for producing intestinal organoids described in claim 44, wherein the low-adhesion protein coat is a hydrogel.

46. ​​A method for producing intestinal organoids described in claim 44, wherein the protein low-adhesion coat is covalently attached to the surface of the low-adhesion plate.

47. A method for producing intestinal organoids described in claim 45, wherein the hydrogel is hydrophilic, nonionic, or neutrally charged.

48. A method for producing intestinal organoids described in claim 40, wherein 5,000 to 100,000 intestinal spheroids are cultured at the start of culturing the intestinal spheroids on a low-adhesion plate having a grooved structure.

49. A method for producing intestinal organoids described in claim 40, wherein a substance selected from the group consisting of a fibroblast growth factor, a GSK-3β inhibitor, a ROCK inhibitor, and a protein kinase C (PKC) activator is used when culturing intestinal spheroids on a low-adhesion plate having a grooved structure.

50. A method for producing intestinal organoids as described in claim 49, wherein the fibroblast growth factor is FGF4, the GSK-3β inhibitor is CHIR99021, the ROCK inhibitor is Y27632, and the protein kinase C (PKC) activator is PMA.

51. A method for producing intestinal organoids described in claim 40, wherein intestinal spheroids are cultured on a low-adhesion plate having a grooved structure, the culture is carried out at 25°C to 45°C.

52. A method for producing intestinal organoids described in claim 40, wherein intestinal spheroids are cultured on a low-adhesion plate having a grooved structure, the culture is carried out in the presence of 1% to 10% CO2.

53. A method for producing intestinal organoids described in claim 40, wherein intestinal spheroids are cultured on a low-adhesion plate having a grooved structure, the culture is carried out for 5 to 72 hours.

54. A method for producing intestinal organoids as described in claim 40, wherein a substance selected from the group consisting of extracellular matrix, growth factor, serum transport protein, secreted signaling protein, water-soluble vitamin, and hormone having a molecular structure similar to insulin is used in the process of rotary suspension culture of the induced string-like intestinal precursor tissue.

55. A method for producing intestinal organoids as described in claim 54, wherein the extracellular matrix is ​​Matrigel, the serum transport protein is Afamin, the secreted signaling protein is a substance associated with tumorigenesis, regulation of cell fate, or patterning during embryogenesis, the water-soluble vitamin is a B vitamin, and the hormone having a molecular structure similar to insulin is IGF-1.

56. A method for producing intestinal organoids as described in claim 54, wherein the growth factor is a substance selected from the group consisting of growth factors that stimulate the proliferation of epidermal cells or epithelial cells, activators of the Wnt signaling pathway, inhibitors of the bone morphogenetic protein (BMP / bone morphogenetic proteins) signaling pathway, activators of protein kinase C (PKC), fibroblast growth factors, and selective inhibitors of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7).

57. A method for producing intestinal organoids as described in claim 56, wherein the growth factor that stimulates the proliferation of epidermal cells or epithelial cells is EGF, the activator of the Wnt signaling pathway is R-spondin 1, the inhibitor of the bone morphogenetic protein (BMP / bone morphogenetic proteins) signaling pathway is Noggin, the activator of protein kinase C (PKC) is PMA, the fibroblast growth factor is basic FGF, and the selective inhibitor of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), or type I nodal receptor-like kinase (ALK7) is A8301.

58. A method for producing intestinal organoids described in claim 40, comprising a step of semi-gelling the medium prior to the step of rotating and floating-culture of the induced string-like intestinal precursor tissue.

59. A method for producing intestinal organoids described in claim 40, wherein in the step of subjecting the induced string-like intestinal precursor tissue to rotary suspension culture, the rotary suspension culture is carried out at 25°C to 45°C.

60. A method for producing intestinal organoids described in claim 40, wherein in the step of subjecting the induced string-like intestinal precursor tissue to rotary suspension culture, the rotary suspension culture is carried out in the presence of 1% to 10% CO2.

61. A method for producing intestinal organoids described in claim 40, wherein in the step of subjecting the induced string-like intestinal precursor tissue to rotary suspension culture, the rotary suspension culture is carried out for 5 to 10,000 hours.

62. A method for producing intestinal organoids described in claim 40, wherein in the step of rotary suspension culture of the induced string-like intestinal precursor tissue, the rotation is performed at a rotation speed of 1 rpm to 100 rpm.

63. A method for producing intestinal organoids as described in claim 40, wherein the rotation speed is increased during the process of rotating and floating culture of the induced string-like intestinal precursor tissue.

64. A method for producing intestinal organoids described in claim 40, wherein, in the process of rotating and floating culture of the induced cord-like intestinal precursor tissue, the medium is changed once every 2 to 10 days.

65. A method for producing intestinal organoids as described in claim 40, comprising a step of culturing the induced string-like intestinal precursor tissue on a low-adhesion plate prior to a step of rotating and floating-culture the induced string-like intestinal precursor tissue.

66. A method for producing intestinal organoids described in claim 65, wherein the low-adhesion plate is a plate for spheroid formation.

67. A method for producing intestinal organoids described in claim 65, wherein the low-adhesion plate is a plate containing holes on the culture surface.

68. A method for producing intestinal organoids according to claim 67, wherein the diameter of the holes is 100 to 1,000 μm and the depth of the holes is 50 to 500 μm.

69. A method for producing intestinal organoids described in claim 65, wherein the low-adhesion plate comprises a protein low-adhesion coat on the surface.

70. A method for producing intestinal organoids described in claim 69, wherein the low-adhesion protein coat is a hydrogel.

71. A method for producing intestinal organoids described in claim 69, wherein the protein low-adhesion coat is covalently attached to the surface of the low-adhesion plate.

72. A method for producing intestinal organoids described in claim 70, wherein the hydrogel is hydrophilic, nonionic, or neutrally charged.

73. A method for producing intestinal organoids described in claim 65, in which the culture surface of the low-adhesion plate does not have any flat areas.

74. A method for producing intestinal organoids described in claim 65, wherein the culture on a low-adhesion plate is carried out at 25°C to 45°C.

75. A method for producing intestinal organoids described in claim 65, wherein the culture on a low-adhesion plate is carried out in the presence of 1% to 10% CO2.

76. A method for producing intestinal organoids described in claim 65, wherein the culture on a low-adhesion plate is carried out for 5 to 100 hours.

77. A method for producing intestinal spheroids for producing intestinal organoids, comprising the steps of culturing a mixture of mid- and hindgut cells that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm that have been induced to differentiate from pluripotent stem cells and dispersed into single cells, in a floating state on a low-adhesion plate, and fusing the mid- and hindgut cells with the visceral mesoderm to produce intestinal spheroids.

78. A method for producing string-like intestinal precursor tissue for producing intestinal organoids, comprising the step of culturing intestinal spheroids on a low-adhesion plate having a grooved structure to induce string-like intestinal precursor tissue.

79. A method for producing intestinal organoids, comprising a step of culturing intestinal spheroids on a low-adhesion plate with a grooved structure to induce string-like intestinal precursor tissue, and then culturing the tissue in a rotary suspension culture.

80. A method for producing intestinal organoids, comprising the steps of: culturing a mixture of middle and hindgut cells, which have been induced to differentiate from pluripotent stem cells and dispersed into single cells, and visceral mesoderm, which have been induced to differentiate from pluripotent stem cells and dispersed into single cells, in a floating state on a low-adhesion plate to fuse the middle and hindgut cells with the visceral mesoderm to produce intestinal spheroids; culturing the produced intestinal spheroids on a low-adhesion plate with a grooved structure to induce string-like intestinal precursor tissue; and subjecting the induced string-like intestinal precursor tissue to rotary floating culture.

81. An intestinal organoid having a lumen and intestinal tissue containing epithelial cells and mesenchymal cells.

82. An intestinal organoid having a lumen, the organoid having intestinal tissue with epithelium and surrounding mesenchymal tissue.

83. An intestinal organoid having a lumen, the longitudinal length of the lumen being 2.5 cm or more.

84. An intestinal organoid having a lumen, the outer diameter of the lumen being 1.0 mm or more.

85. An intestinal organoid having a lumen, the inner diameter of the lumen being 600 μm or more.

86. An intestinal organoid having a lumen, the organoid being produced using a production method described in any one of claims 12 to 80.

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