Endometrial model, method for producing endometrial model, and endometrial implantation model

Abstract

An endometrium model including: a gel that includes a stroma system extracellular matrix; and endometrial epithelial cells, wherein at least a portion of the endometrial epithelial cells are exposed at the surface of the gel, and at least a portion of the endometrial epithelial cells that are exposed at the surface of the gel form a lumen structure and a non-lumen structure in a continuous manner. Additionally, a method for producing said endometrium model. Additionally, an endometrial implantation model including said endometrium model and embryo cells.

Description

Technical Field

[0001] The present invention relates to an endometrial model, a method for producing the endometrial model, and an endometrial implantation model.

Background Art

[0002] The endometrium is an epithelial tissue present in the uterus and is a tissue necessary for the establishment of pregnancy. During the reproductive period of human adults, the functional layer of the endometrium repeats a cycle of regeneration, differentiation, and shedding every month. After menstruation, the functional layer of the endometrium proliferates in an estrogen-dependent manner, the mucosa regenerates, and differentiates into a progesterone-dominant secretory phase. On about the 7th day after ovulation, a fertilized egg implants in the luminal epithelium on the cilia, providing a microenvironment essential for placenta formation.

[0003] In recent years, attempts have been made to produce a three-dimensional cell structure similar to the tissue structure in vivo called an organoid and use it as an in vivo tissue model. In the endometrium as well, an example of producing an endometrial organoid using endometrial epithelial cells and the like has been reported (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The endometrial organoids in Non-Patent Document 1 have a simple spherical structure and, as a spatial morphological structure, are different from the structure of the endometrium of a living body. Further, since the apical surface for receiving an embryo is inside the structure, it is not possible to simulate embryo implantation.

[0006] Therefore, an object of the present invention is to provide an endometrial model in which the apical surface of endometrial epithelial cells is exposed, a method for producing the endometrial model, and an endometrial implantation model using the endometrial model.

Means for Solving the Problems

[0007] The present invention includes the following aspects. [1] An endometrial model comprising a gel containing a stromal extracellular matrix and endometrial epithelial cells, at least a part of the endometrial epithelial cells being exposed on the surface of the gel, and at least a part of the endometrial epithelial cells exposed on the surface of the gel continuously forming a luminal structure and a non-luminal structure. [2] The endometrial model according to [1], wherein the non-luminal structure is a flat membranous structure. [3] The endometrial model according to [1] or [2], wherein the stromal extracellular matrix is type I collagen. [4] The endometrial model according to any one of [1] to [3], wherein a part of the endometrial epithelial cells is present inside the gel. [5] A method for producing an endometrial model, comprising: (a) a step of causing endometrial epithelial cells to be present inside a gel containing a stromal extracellular matrix; and (b) a step of culturing the endometrial epithelial cells after the step (a). [6] The method for producing an endometrial model according to [4], wherein the stromal extracellular matrix is type I collagen. [7] An endometrial implantation model comprising the endometrial model according to any one of [1] to [4] and embryonic cells. [8] The endometrial implantation model according to [7], wherein the embryonic cells include at least one cell selected from the group consisting of trophoblast stem cells, cytotrophoblast cells, extravillous trophoblast cells, syncytiotrophoblast cells, pluripotent stem cells, and cells derived therefrom. [9] The endometrial implantation model according to [7] or [8], wherein the germ cells form spheroids or organoids.

Advantages of the Invention

[0008] According to the present invention, there are provided an endometrial model in which the apical surface of endometrial epithelial cells is exposed, a method for producing the endometrial model, and an endometrial implantation model using the endometrial model.

Brief Description of the Drawings

[0009]

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[0010] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and redundant descriptions are omitted. The dimensional ratios in each figure are exaggerated for the purpose of explanation and do not necessarily match the actual dimensional ratios.

[0011] [Definitions] The term "comprise" means that it may include components other than the target component. The term "consist of" means that it does not include components other than the target component. The term "consist essentially of" means that it does not include components other than the target component in a manner that exhibits a special function (such as a manner that completely loses the effects of the invention). In this specification, when described as "comprise", it includes the "consist of" aspect and the "consist essentially of" aspect.

[0012] The "cells" described in this specification can be isolated. "Isolated" means a state separated from the natural state. The "cells" described in this specification can be isolated cells.

[0013] [Endometrial model] The first aspect of the present disclosure is an endometrial model. In one embodiment, the endometrial model includes a gel containing a stromal extracellular matrix and endometrial epithelial cells. In one embodiment, at least a part of the endometrial epithelial cells is exposed on the surface of the gel. In one embodiment, at least a part of the endometrial epithelial cells exposed on the surface of the gel continuously forms a luminal structure and a non-luminal structure.

[0014] FIG. 1 is a schematic diagram showing an endometrial model according to one embodiment. The endometrial model 1 includes a gel 20 containing a stromal extracellular matrix and cell aggregates (10a, 10b) of endometrial epithelial cells. The cell aggregate 10a is a cell aggregate of endometrial epithelial cells exposed on the surface of the gel 20 containing a stromal extracellular matrix. The cell aggregate 10b is a cell aggregate of endometrial epithelial cells present inside the gel 20 containing a stromal extracellular matrix.

[0015] The cell aggregate 10a is exposed on the surface of the gel 20 containing a stromal extracellular matrix and has a structure in which a luminal structure 11 and a non-luminal structure 12 are continuously formed. The "luminal structure" refers to a structure in which endometrial epithelial cells are indented toward the gel 20 containing a stromal extracellular matrix. The "non-luminal structure" refers to a structure that is not a luminal structure. Examples of the non-luminal structure include, for example, a flat membranous structure. The structure in which "the luminal structure and the non-luminal structure are continuously formed" refers to a structure that includes at least one each of the luminal structure and the non-luminal structure and in which the luminal structure and the non-luminal structure are connected.

[0016] The number of luminal structures 11 possessed by the cell aggregate 10a is not particularly limited. Depending on the size of the cell aggregate 10a, the cell aggregate 10a may have a plurality of luminal structures 11. When the cell aggregate 10a has a plurality of luminal structures 11, the two luminal structures 11 are connected by a non-luminal structure 12.

[0017] The endometrial epithelial cells that constitute the luminal structure 11 may express progesterone receptor (PGR). The luminal structure 11 may have a function similar to that of mature endometrial glandular epithelium.

[0018] The gel 20 containing the stromal extracellular matrix contains the stromal extracellular matrix. The "stromal extracellular matrix" is an extracellular matrix mainly present in the stroma. Examples of the stromal extracellular matrix include type I collagen, proteoglycans (such as versican, decorin, etc.), fibronectin, etc. Among them, type I collagen is preferable as the stromal extracellular matrix. The stromal extracellular matrix contained in the gel 20 containing the stromal extracellular matrix may be one type or two or more types.

[0019] In addition to the stromal extracellular matrix, the gel 20 containing the stromal extracellular matrix may contain other extracellular matrices. Examples of other extracellular matrices include basement membrane extracellular matrices such as laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.; cartilage extracellular matrices such as hyaluronic acid, type II collagen, link protein, etc. Also, commercially available products such as Matrigel (registered trademark) may be used. When the gel 20 containing the stromal extracellular matrix contains other extracellular matrices, the other extracellular matrices may be one type or two or more types.

[0020] The gel 20 containing the stromal extracellular matrix preferably contains a stromal extracellular matrix gel of 50% by volume or more, 55% by volume or more, 60% by volume or more, 65% by volume or more, 70% by volume or more, 75% by volume or more, or 80% by volume or more based on the total gel volume. The gel 20 containing the stromal extracellular matrix may be 100% by volume of the stromal extracellular matrix gel.

[0021] (Maintenance medium for endometrial model) The endometrial model 1 can be maintained in a medium. The medium is not particularly limited as long as it can maintain the endometrial model 1. Examples of the medium include a medium in which growth factors, ROCK inhibitor, GSK3β inhibitor, p38 MAPK inhibitor, maturation inducer, etc. are added to a basal medium generally used for culturing animal cells.

[0022] ≪Basal Medium≫ Examples of the basal medium include Doulbecco’s modified Eagle’s Medium (DMEM) medium, DMEM / F12 medium, Advanced DMEM / F12 medium, IMDM medium, Medium199 medium, Eagle’s Minimum Essential Medium (EMEM) medium, αMEM medium, Ham’s F12 medium, RPMI1640 medium, Fischer’s medium, and mixed media thereof. A preferred basal medium is, for example, DMEM / F12.

[0023] The basal medium may contain serum (such as fetal bovine serum (FBS)) and / or a serum substitute as needed. Examples of the serum substitute include albumin, transferrin, sodium selenite, ITS-X (Invitrogen), Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3’-thioglycerol, etc. The basal medium may contain components such as lipids, amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth factors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. as needed. These can be used in appropriate combinations.

[0024] As the basal medium, for example, a medium obtained by adding bovine serum albumin (BSA), ITS-X, L-ascorbic acid, and antibiotics (such as penicillin and streptomycin) to the above-described basal medium (for example, DMEM / F12) can be mentioned. As a specific example of the basal medium, the TS basal medium used in the examples described later can be mentioned.

[0025] ≪Growth Factor≫ The growth factor is not particularly limited, and examples thereof include epidermal growth factor (EGF), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP).

[0026] EGF binds to the EGF receptor (EGFR) present on the cell surface to induce EGF signal transduction and acts as a mitogenic factor. The organism from which EGF is derived is not particularly limited, but human EGF is preferred. Human FGF may be a recombinant produced by non-human cells. Commercially available EGF can be used. The concentration of EGF in the medium is not particularly limited, but can be, for example, 5 to 200 ng / mL. The concentration of EGF in the medium is preferably 10 to 150 ng / mL, more preferably 10 to 100 ng / mL, still more preferably 10 to 80 ng / mL, and particularly preferably 10 to 60 ng / mL.

[0027] FGF has a high affinity for heparan sulfate proteoglycan, forms a complex with the FGF receptor (FGFR) on the cell surface together with heparan sulfate proteoglycan, induces FGF signal transduction, and acts as a mitogen. The organism from which FGF is derived is not particularly limited, but human FGF is preferred. Human FGF may be a recombinant produced by non-human cells. As FGF, FGF2 (also referred to as bFGF) is preferred. Commercially available FGF can be used. The concentration of FGF (e.g., FGF2) in the medium is not particularly limited, but can be, for example, 5 - 200 ng / mL. The concentration of FGF (e.g., FGF2) in the medium is preferably 10 - 150 ng / mL, more preferably 20 - 100 ng / mL, still more preferably 30 - 80 ng / mL, and particularly preferably 40 - 60 ng / mL.

[0028] When the medium contains FGF, the medium may contain heparin. Heparin has the effect of promoting the activity of FGF. Heparin is preferably in the form of a salt. Examples of the salt of heparin include salts with alkali metals such as lithium, sodium, and potassium; salts with alkaline earth metals such as calcium, barium, and magnesium; salts with metals such as aluminum, zinc, copper, and iron; ammonium salts; salts with organic bases; and salts with amino acids, etc. Commercially available heparin can be used. The concentration of heparin in the medium is not particularly limited, but can be, for example, 0.001 - 10 μg / mL. The concentration of heparin in the medium is preferably 0.005 - 5 μg / mL, more preferably 0.01 - 3 μg / mL, still more preferably 0.05 - 1 μg / mL, and particularly preferably 0.07 - 0.5 μg / mL.

[0029] BMP is a protein belonging to the transforming growth factor β (TGFβ) superfamily and controls induction of cell death, cell differentiation, etc. The organism from which BMP is derived is not particularly limited, but human BMP is preferred. Human BMP may be a recombinant produced by non-human cells. As BMP, BMP4 is preferred. A commercially available product can be used as BMP. The concentration of BMP (for example, BMP4) in the medium is not particularly limited, but can be, for example, 5 to 200 ng / mL. The concentration of BMP (for example, BMP4) in the medium is preferably 10 to 150 ng / mL, more preferably 20 to 100 ng / mL, still more preferably 30 to 80 ng / mL, and particularly preferably 40 to 60 ng / mL.

[0030] ≪ROCK inhibitor≫ A ROCK (Rho associated coiled-coil containing protein kinase) inhibitor is a substance that inhibits the function of Rho binding kinase. Examples of the ROCK inhibitor include trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 1-(5-Isoquinolinylsulfonyl)homopiperazine, or salts thereof. Further, examples include low molecular weight inhibitors such as Fasudil / HA1077, H-1152, and Wf-536, and derivatives thereof. The ROCK inhibitor may be an antisense nucleic acid, siRNA, dominant negative mutant, or expression vector thereof against ROCK. Commercially available products of trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide or a salt thereof include Y27632 ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl·H2O). The ROCK inhibitor may be used alone or in combination of two or more. It is preferable to use Y27632 as the ROCK inhibitor. The concentration of the ROCK inhibitor in the medium is not particularly limited, but for example, it can be 0.1 to 50 μM, preferably 1 to 20 μM, more preferably 1 to 15 μM, and even more preferably 3 to 12 μM.

[0031] ≪GSK3β inhibitor≫ GSK (Glycogen Synthase Kinase) 3β inhibitors are substances that inhibit the functions of GSK3β, such as kinase activity (e.g., phosphorylation ability against β-catenin). Examples of GSK3β inhibitors include 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile, Kenpaullone, 1-Azakenpaullone, CHIR98014, AR-A014418, CT99021, CT20026, SB216763, AR-A014418, lithium, SB415286, TDZD-8, BIO, BIO-acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopentadienylruthenium complex, TDZD-8 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT, alpha-4-dibromoacetophenone, AR-AO 144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione; TWS119 pyrrolopyrimidine compound, L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form; 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, SB216763, and small molecule inhibitors such as SB415286. GSK3β inhibitors may also be antisense nucleic acids, siRNAs, dominant negative mutants, and their expression vectors against GSK3β. Commercially available products of 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile include CHIR99021 and the like. GSK3β inhibitors may be used alone or in combination of two or more. It is preferable to use CHIR99021 as the GSK3β inhibitor. The concentration of the GSK3β inhibitor in the medium is not particularly limited, and for example, it can be 0.1 to 20 μM, preferably 0.2 to 10 μM, more preferably 0.5 to 5 μM, and even more preferably 0.5 to 3 μM.

[0032] ≪p38 MAPK inhibitor≫ A p38 MAPK inhibitor is a substance that inhibits the function of p38 MAPK (P38 mitogen-activated protein kinase). Examples of p38 MAPK inhibitors include SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole), SB203580 (4-[4-(4-fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-1H-imidazol-5-yl]pyridine), VX702 (6-(N-carbamoyl-2,6-difluoroanilino)-2-(2,4-difluorophenyl)pyridine-3-carboxamide), VX745 (5-(2,6-dichlorophenyl)-2-[2,4-difluorophenyl)thio]-6H-pyrimido[1,6-b]pyridazin-6-one), PD169316 (4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole), RO4402257 (6-(2,4-difluorophenoxy)-2-{[3-hydroxy-1-(2-hydroxyethyl)propyl]amino}-8-methylpyrido[2,3-D]pyrimidin-7(8h)-one), BIRB796 (1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea), etc. The p38 MAPK inhibitor may be an antisense nucleic acid, siRNA, dominant negative mutant, and their expression vectors, etc. against p38 MAPK. The p38 MAPK inhibitor may be used alone or in combination of two or more. It is preferable to use SB202190 as the p38 MAPK inhibitor. The concentration of the p38 MAPK inhibitor in the medium is not particularly limited, but can be, for example, 0.1 to 20 μM, preferably 0.2 to 10 μM, more preferably 0.5 to 5 μM, and even more preferably 0.5 to 3 μM.

[0033] <<Maturation-inducing factor>> The maturation-inducing factor is a factor that induces the maturation of endometrial cells. The maturation-inducing factor is not particularly limited as long as it can induce the maturation of endometrial cells. Examples of the maturation-inducing factor include estrogen receptor ligands, progesterone receptor ligands, cyclic AMP (cAMP) or its derivatives, prolactin, placental lactogen, chorionic gonadotropin, and the like.

[0034] An estrogen receptor ligand is a substance that binds to an estrogen receptor. Examples of estrogen receptor ligands include estrone, estradiol, estriol, and the like. Examples of estradiol include β-estradiol, 17β-estradiol, and the like. The estrogen receptor ligand may be used alone or in combination of two or more. It is preferable to use estradiol as the estrogen receptor ligand. The concentration of the estrogen receptor ligand in the medium is not particularly limited, but can be, for example, 0.1 to 50 μM, preferably 0.5 to 30 μM, more preferably 1 to 20 μM, and even more preferably 5 to 15 μM.

[0035] A progesterone receptor ligand is a substance that binds to a progesterone receptor. Examples of progesterone receptor ligands include progesterone, medroxyprogesterone acetate, and the like. The progesterone receptor ligand may be used alone or in combination of two or more. It is preferable to use medroxyprogesterone acetate as the progesterone receptor ligand. The concentration of progesterone or its derivative in the medium is not particularly limited, but can be, for example, 0.1 to 20 μM, preferably 0.2 to 10 μM, more preferably 0.5 to 5 μM, and even more preferably 0.5 to 3 μM.

[0036] Examples of cAMP or its derivative include 8-bromo-adenosine-3'-5'-cyclic monophosphate (8-Br-cAMP), dibutyryl-cAMP, 8-CPT-2-Me-cAMP sodium salt (8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate sodium salt), etc. cAMP or its derivative may be used alone or in combination of two or more. It is preferable to use 8-Br-cAMP as cAMP or its derivative. The concentration of cAMP or its derivative in the medium is not particularly limited, but can be, for example, 0.1 to 200 μM, preferably 1 to 150 μM, more preferably 10 to 100 μM, and even more preferably 20 to 80 μM.

[0037] Prolactin is a hormone secreted from prolactin-secreting cells in the anterior pituitary gland. The organism from which prolactin is derived is not particularly limited, but human prolactin is preferred. Human prolactin may be a recombinant produced by non-human cells. Commercially available prolactin can be used. The concentration of prolactin in the medium is not particularly limited, but can be, for example, 5 to 200 ng / mL. The concentration of prolactin in the medium is preferably 10 to 150 ng / mL, more preferably 10 to 100 ng / mL, even more preferably 10 to 80 ng / mL, and particularly preferably 10 to 60 ng / mL.

[0038] Placental lactogen is a peptide hormone secreted from the placenta. The organism from which placental lactogen is derived is not particularly limited, but human placental lactogen is preferred. Human placental lactogen may be a recombinant produced by non-human cells. Commercially available placental lactogen can be used. The concentration of placental lactogen in the medium is not particularly limited, but can be, for example, 5 - 200 ng / mL. The concentration of placental lactogen in the medium is preferably 10 - 150 ng / mL, more preferably 10 - 100 ng / mL, even more preferably 10 - 80 ng / mL, and particularly preferably 10 - 60 ng / mL.

[0039] Chorionic gonadotropin is a peptide hormone secreted from the trophoblast syncytium. The organism from which chorionic gonadotropin is derived is not particularly limited, but human chorionic gonadotropin is preferred. Human chorionic gonadotropin may be a recombinant produced by non-human cells. Commercially available chorionic gonadotropin can be used. The concentration of chorionic gonadotropin in the medium is not particularly limited, but can be, for example, 0.01 - 100 μg / mL. The concentration of chorionic gonadotropin in the medium is preferably 0.05 - 50 μg / mL, more preferably 0.1 - 20 μg / mL, even more preferably 0.2 - 10 μg / mL, and particularly preferably 0.5 - 5 μg / mL.

[0040] Examples of the maintenance medium for the endometrial model include, for example, a medium obtained by adding growth factors (such as EGF), ROCK inhibitors (such as Y27632), GSK3β inhibitors (such as CHIR99021), and p38 MAPK inhibitors (such as SB202190) to a basal medium for animal cells (such as TS basal medium, etc.). Specific examples of such a medium include, for example, the ECSY medium used in the examples described later.

[0041] In addition, as a maintenance medium for the endometrial model, for example, a medium obtained by adding growth factors (such as EGF), ROCK inhibitors (such as Y27632), GSK3β inhibitors (such as CHIR99021), p38 MAPK inhibitors (such as SB202190), maturation-inducing factors (such as estradiol, medroxyprogesterone acetate, 8-Br-cAMP, etc.) to a basal medium for animal cells (such as TS basal medium, etc.) can be mentioned. Specific examples of such a medium include, for example, the ECSY + EPC medium used in the examples described later.

[0042] In addition, when inducing further maturation of endometrial epithelial cells in the endometrial model, further maturation-inducing factors (such as prolactin, placental lactogen, chorionic gonadotropin, etc.) can be added to the above-mentioned maintenance medium. For example, a medium obtained by adding growth factors (such as EGF), ROCK inhibitors (such as Y27632), GSK3β inhibitors (such as CHIR99021), p38 MAPK inhibitors (such as SB202190), maturation-inducing factors (such as estradiol, medroxyprogesterone acetate, 8-Br-cAMP, prolactin, placental lactogen, chorionic gonadotropin, etc.) to a basal medium for animal cells (such as TS basal medium, etc.) can be mentioned. Specific examples of such a medium include, for example, a medium (such as ECSY + EPC + PLG medium) obtained by adding human prolactin (such as 20 ng / mL), human placental lactogen (such as 20 ng / mL), and human chorionic gonadotropin (such as 1 μg / mL) to the ECSY + EPC medium used in the examples described later.

[0043] The endometrial model of this aspect has a structure in which endometrial epithelial cells are exposed on the surface of the gel. Furthermore, it has a luminal structure similar to that of endometrial glandular epithelium. These structural features are similar to those of the endometrium in vivo. Therefore, the endometrial model of this aspect can be applied to the screening and evaluation of drugs for endometrial diseases (such as endometriosis, endometrial cancer, etc.). In addition, since the apical surface of the endometrial epithelial cells is exposed, it is possible to simulate implantation. Therefore, it can be applied to the elucidation of the implantation mechanism or implantation failure, the screening of drugs for treating implantation failure, and the evaluation of drugs for treating implantation failure.

[0044] [Method for producing an endometrial model] The second aspect of the present disclosure is a method for producing an endometrial model. The method according to this aspect includes: (a) a step of causing endometrial epithelial cells to be present inside a gel containing a stromal extracellular matrix; and (b) a step of culturing the endometrial epithelial cells.

[0045] [Step (a)] In step (a), endometrial epithelial cells are caused to be present inside a gel containing a stromal extracellular matrix.

[0046] [Endometrial epithelial cells] The endometrial epithelial cells may be isolated from endometrial epithelial tissue or a cultured strain of endometrial epithelial cells may be used. As a method for isolating from endometrial tissue, a known method can be used. As the endometrial epithelial tissue, decidua may be used. The endometrial epithelial cells can be isolated, for example, by appropriately using mechanical and / or enzymatic treatment on endometrial epithelial tissue to separate the cells. Examples of mechanical treatment include cutting with a scalpel. Examples of enzymatic treatment include treatment with a protease having extracellular matrix degrading activity (such as dispase, collagenase, etc.). After mechanical and / or enzymatic treatment of the endometrial epithelial tissue, the endometrial epithelial cells can be recovered using a strainer, filter, or the like. The recovered endometrial epithelial cells may be appropriately washed with a medium or the like.

[0047] The isolated endometrial epithelial cells may be prepared by appropriately adding ice-cold Matrigel or the like and culturing. The culture temperature in Matrigel may be, for example, 30 to 40 °C (for example, 37 °C). The culture time is not particularly limited, and examples include 5 minutes or more, 10 minutes or more, or 15 minutes or more. The upper limit of the culture time is not particularly limited, and examples include 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, or 20 minutes or less. The prepared endometrial epithelial cells can be maintained with ECSY medium or the like.

[0048] (Gel containing stromal extracellular matrix) As the gel containing stromal extracellular matrix, the same one as described above can be used. The gel containing stromal extracellular matrix preferably contains type I collagen as the stromal extracellular matrix.

[0049] (Seeding method of endometrial epithelial cells) The method of allowing endometrial epithelial cells to be present inside the gel containing stromal extracellular matrix is not particularly limited. For example, the cells may be recovered from the culture solution of endometrial epithelial cells by centrifugation or the like and mixed with the gel containing stromal extracellular matrix. Alternatively, the endometrial epithelial cells may be suspended in an appropriate medium to prepare a cell suspension, and the cell suspension may be injected into the gel containing stromal extracellular matrix.

[0050] The number of endometrial epithelial cells seeded in the gel containing stromal extracellular matrix is not particularly limited. Examples of the number of seeded cells include 1 or more, 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, etc. The upper limit of the number of seeded cells is not particularly limited. For example, 10 20 or less, 10 15less than or equal to 10 14 less than or equal to 10 13 less than or equal to, or 10 12 less than or equal to, etc. can be mentioned. The lower limit value and the upper limit value can be combined as appropriate.

[0051] (Step (b)) In step (b), after step (a), endometrial epithelial cells are cultured.

[0052] Culturing is started with endometrial epithelial cells present inside a gel containing stromal extracellular matrix. The culturing may be performed by adding an appropriate medium to the gel containing stromal extracellular matrix containing endometrial epithelial cells. The culture vessel is not particularly limited, and examples include untreated well plates, untreated dishes, etc.

[0053] As the medium, those mentioned above, etc. can be used. During culturing, it is preferable to perform medium replacement as appropriate. The frequency of medium replacement is not particularly limited, and examples include 2 to 3 times a week.

[0054] The medium may be appropriately added with a maturation-inducing factor in order to promote the maturation of endometrial epithelial cells. For example, in the initial stage of culturing, culturing may be performed in a maturation-inducing factor-free medium. Subsequently, it may be replaced with a maturation-inducing factor-containing medium and further cultured.

[0055] Examples of the culturing period in a maturation factor-free medium (for example, ECSY medium) include 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more, etc. The upper limit of the culturing period in a maturation factor-free medium (for example, ECSY medium) is not particularly limited, and examples include 30 days or less, 25 days or less, 20 days or less, 15 days or less, 12 days or less, or 10 days or less, etc. The lower limit value and the upper limit value can be combined as appropriate.

[0056] The culture period in a medium containing a maturation factor (for example, ECSY + EPC medium) can be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more, etc. The upper limit of the culture period in a medium containing a maturation factor (for example, ECSY medium) is not particularly limited, but can be, for example, 30 days or less, 25 days or less, 20 days or less, 15 days or less, 12 days or less, 10 days or less, etc. The lower limit value and the upper limit value can be combined as appropriate.

[0057] When further promoting the maturation of endometrial epithelial cells, as a maturation-inducing factor, a medium containing 3 or more, 4 or more, 5 or more maturation-inducing additive factors selected from the group consisting of an estrogen receptor ligand, a progesterone receptor ligand, cAMP or its derivative, prolactin, placental lactogen, and chorionic gonadotropin may be used. For example, a medium containing all of an estrogen receptor ligand, a progesterone receptor ligand, cAMP or its derivative, prolactin, placental lactogen, and chorionic gonadotropin (for example, ECSY + EPC + PLG medium) may be used.

[0058] During the culture, the endometrial epithelial cells inside the gel containing the stromal extracellular matrix move within the gel, and some cells reach the gel surface. The cells that reach the gel surface form a structure in which the luminal structure and the non-luminal structure are continuous on the gel surface. The culture in a medium containing a maturation-inducing factor can continue until the endometrial epithelial cells form the said structure.

[0059] The culture conditions can be the conditions generally used for culturing animal cells. For example, the culture temperature can be 32 - 40 °C (preferably 35 - 38 °C, typically 37 °C), and the CO2 concentration can be 2 - 5% (preferably 5%).

[0060] By the method of this aspect, the endometrial model according to the first aspect can be prepared.

[0061] [Endometrial Implantation Model] The third aspect of the present disclosure is an endometrial implantation model. The endometrial implantation model according to this aspect includes the endometrial model according to the first aspect and germ cells.

[0062] FIG. 2 is a schematic diagram showing an endometrial implantation model according to an embodiment. The endometrial implantation model 2 includes an endometrial model 1 and germ cells 30. The germ cells 30 may or may not be implanted on the top surface of the endometrial model 1.

[0063] (Germ cells) The "germ cells" refer to cells that can simulate implantation into the endometrium. The fertilized egg moves in the fallopian tube and reaches the uterus. The fertilized egg divides while moving through the fallopian tube and becomes a blastocyst through the 2-cell stage embryo, 4-cell stage embryo, 8-cell stage embryo, and morula. When the blastocyst that has reached the uterus becomes an expanded blastocyst, the zona pellucida becomes thinner, and when the zona pellucida ruptures, it becomes a hatched blastocyst. The hatched blastocyst adheres in contact with the endometrium. The blastocyst is composed of an inner cell mass, a blastocyst cavity, and a trophoblast, and the trophoblast cells invade the endometrium to establish implantation. Germ cells are typically cells that make up an embryo and can be any of a fertilized egg, 2-cell stage embryo, 4-cell stage embryo, 8-cell stage embryo, morula, and blastocyst. The germ cells may be cells isolated from an embryo as described above, or cells derived from the isolated cells. The germ cells may be pluripotent stem cells or cells derived from pluripotent stem cells. The germ cells may be cells isolated from the placenta or cells derived from the cells.

[0064] Examples of germ cells include trophoblast stem cells, cytotrophoblast cells, extravillous trophoblast cells, syncytiotrophoblast cells, pluripotent stem cells, and cells derived therefrom.

[0065] Trophoblast stem cells (TS cells) may be derived from blastocysts, may be derived from cytotrophoblast cells (CT cells), or may be derived from pluripotent stem cells. Induction of TS cells from blastocysts can be performed by known methods. For example, mechanical and / or enzymatic treatment is appropriately used on placental tissue to isolate cells. Then, after culturing the cells in a medium for inducing TS cells, TS cells can be established using the expression of TS cell markers (such as positive for GATA2, GATA3, TFAP2, ELF5, ZNF750, and negative for CDX2, etc.) as an indicator. As a method for inducing TS cells from CT cells, for example, the method described in Patent No. 6400832 can be used. As a method for inducing TS cells from pluripotent stem cells, for example, the methods described in International Publication No. 2020 / 250438, etc. can be used.

[0066] For CT cells, for example, those isolated from the placenta can be used. Isolation of CT cells from the placenta can be performed, for example, by appropriately using mechanical and / or enzymatic treatment on placental tissue to isolate cells, and using the expression of CT cell markers (such as positive for CD49f, E-cadherin, etc.) as an indicator to isolate CT cells (Patent No. 6400832 Gazette, Haider, S., et al., Stem Cell Reports 11, 537 - 551 (2018)).

[0067] Syncytiotrophoblast cells (ST cells) may be isolated from the placenta or may be derived from TS cells. Isolation of ST cells from the placenta can be performed, for example, by appropriately using mechanical and / or enzymatic treatment on placental tissue to isolate cells, and using the expression of ST cell markers (such as positive for Syndecan 1 (SDC1), human chorionic gonadotropin (hCG), etc.) as an indicator to isolate ST cells. Examples of methods for inducing ST cells from TS cells include the methods described in International Publication No. 2020 / 250438 and the like.

[0068] The extravillous trophoblast cell (EVT cell) may be isolated from the placenta or may be induced from TS cells. Isolation of EVT cells from the placenta can be achieved, for example, by appropriately using mechanical and / or enzymatic treatment on placental tissue to separate the cells and isolating EVT cells using the expression of EVT cell markers (such as HLA-G positive) as an indicator. Examples of methods for inducing EVT cells from TS cells include the methods described in International Publication No. 2020 / 250438 and the like.

[0069] Pluripotent stem cells are cells with pluripotency and may be embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), or cells with pluripotency induced from these stem cells. The method for producing pluripotent stem cells is not particularly limited, and known methods can be used. Cell lines of ES cells and iPS cells are also available from cell banks such as the RIKEN BioResource Center (BRC). Pluripotent stem cells are preferably ES cells or iPS cells, and more preferably ES cells.

[0070] Embryonic cells may form spheroids or organoids. Formation of spheroids or organoids from embryonic cells can be carried out by known methods. For example, by using a medium as described above (for example, a medium obtained by adding growth factors, ROCK inhibitors, GSKβ inhibitors, p38 MAPK inhibitors to a basal medium) to perform suspension culture of embryonic cells, spheroids or organoids of embryonic cells can be produced.

[0071] Embryonic cell spheroids or organoids may be cell aggregates derived from TS cells. For example, cell aggregates can be induced by culturing TS cells in suspension in a TS medium (Okae et al Cell Stem Cell 2018). The cell aggregates may contain one or more cells selected from the group consisting of TS cells, CT cells, ST cells, and EVT cells, or may contain one or more cells selected from the group consisting of TS cells, CT cells, and ST cells.

[0072] Embryonic cell spheroids or organoids may be blastocyst-like structures. A blastocyst-like structure is a cell structure having a structure similar to that of a blastocyst derived from pluripotent cells such as pluripotent stem cells. Blastocyst-like structures can be induced by known methods (e.g., Yu L, et al., Nature. 2021 Mar;591(7851):620-626.). For example, pluripotent stem cells are cultured using a 5i / L / A medium, then cultured in an HDM medium, and then cultured in a TDM medium to obtain blastocyst-like structures. As the pluripotent stem cells, the naive type or the primed type may be used, but the naive type is preferably used.

[0073] (Preparation of endometrial implantation model) An endometrial implantation model can be prepared by co-culturing an endometrial model with embryonic cells. The medium used for co-culture is not particularly limited, but in addition to the above-mentioned media, the IVC1 medium, IVC2 medium, etc. used in the examples can be mentioned.

[0074] Examples of the medium for co-culture include a medium (e.g., ECSY+EPC medium) obtained by adding growth factors (such as EGF), ROCK inhibitors (such as Y27632), GSK3β inhibitors (such as CHIR99021), p38 MAPK inhibitors (such as SB202190), maturation-inducing factors (such as estradiol, medroxyprogesterone acetate, 8-Br-cAMP, etc.) to a basal medium for animal cells (such as TS basal medium).

[0075] Alternatively, as the co-culture medium, a medium further supplemented with maturation-inducing factors such as prolactin, placental lactogen, and chorionic gonadotropin may be used. For example, a medium (such as ECSY+EPC+PLG medium) obtained by adding growth factors (such as EGF), ROCK inhibitors (such as Y27632), GSK3β inhibitors (such as CHIR99021), p38 MAPK inhibitors (such as SB202190), and maturation-inducing factors (such as estradiol, medroxyprogesterone acetate, 8-Br-cAMP, prolactin, placental lactogen, chorionic gonadotropin, etc.) to a basal medium for animal cells (such as TS basal medium, etc.) can be used.

[0076] Alternatively, as the co-culture medium, a medium (such as IVC1 medium, IVC2 medium, etc.) obtained by adding serum (such as FBS), serum substitutes (such as ITS-X, KSR, etc.), amino acids (such as L-glutamine, N-acetyl-L-cysteine, etc.), organic acids (such as pyruvic acid, lactic acid, etc.), and maturation-inducing factors (such as estrogen receptor ligands, progesterone receptor ligands, etc.) to a basal medium for animal cells (such as DMEM / F12 medium, Advanced DMEM / F12 medium, etc.) can be used. For example, co-culture may be performed in IVC1 medium for a predetermined period (such as half a day to 3 days, half a day to 2 days, or half a day to 1 day, etc.) from the start of co-culture, and then co-culture may be performed in IVC2 medium.

[0077] The culture conditions can be those generally used for culturing animal cells. For example, the culture temperature can be 32 to 40 °C (preferably 35 to 38 °C, typically 37 °C), and the CO2 concentration can be 2 to 5% (preferably 5%).

[0078] When a spheroid or organoid of embryonic cells is co-cultured with an endometrial model, the spheroid or organoid of embryonic cells can implant on the apical surface of the endometrial model. In the implanted spheroid or organoid, the cells at the implantation site may differentiate into SDC1-positive cells (such as ST cells).

[0079] The endometrial implantation model according to this aspect includes the endometrial model of the first aspect and embryonic cells, so it can simulate implantation into the endometrium. Therefore, the endometrial implantation model according to this aspect can be applied to the elucidation of the mechanism of implantation or implantation failure, the screening of drugs for implantation failure, and the like.

Example

[0080] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0081] <Medium> (TS basal medium) The following components were added to DMEM / F12 medium (FUJIFILM Wako) to prepare TS basal medium. The concentrations shown below are the final concentrations of each component in the TS basal medium. Bovine serum albumin (BSA) (FUJIFILM Wako) 0.15% Penicillin 5,000 units / mL Streptomycin (Thermo Fisher Scientific) 5,000 μg / mL ITS-X (FUJIFILM Wako) 1% KSR (Thermo Fisher Scientific) 1% L-ascorbic acid (FUJIFILM Wako) 0.2 mM

[0082] (ECSY medium) The following additive factors were added to the TS basal medium to prepare the ECSY medium. The concentrations shown below are the final concentrations in the ECSY medium. Human EGF, recombinant (FUJIFILM Wako) 50 ng / mL CHIR99021 (FUJIFILM Wako) 2 μM SB202190 (FUJIFILM Wako) 2 μM Y27632 (FUJIFILM Wako) 10 μM

[0083] (ECSY + EPC medium) The following maturation-inducing additives were added to the ECSY medium to prepare the ECSY + EPC medium. The concentrations shown below are the final concentrations in the ECSY + EPC medium. Estradiol (SIGMA) 10 μM Medroxyprogesterone acetate (MPA) (FUJIFILM Wako) 2 μM 8-Bromo-cAMP (SIGMA) 50 μM

[0084] (ECSY + EPC + PLG medium) The following maturation-inducing additives were added to the ECSY + EPC medium to prepare the ECSY + EPC + PLG medium. The concentrations shown below are the final concentrations in the ECSY + EPC + PLG medium. Human prolactin, recombinant (Peprotech) 20 ng / mL Human placental lactogen (hPL) (R&D systems) 20 ng / mL Human chorionic gonadotropin (hCG) (SIGMA) 1 μg / mL

[0085] (5i / L / A medium) The following components were mixed to prepare the 5i / L / A medium (heunissen, T. W. et al. Cell Stem Cell 15, 471 - 487 (2014)). The concentrations shown below are the final concentrations in the 5i / L / A medium. DMEM / F12 medium (Invitrogen) 250 mL Neurobasal medium (Invitrogen) 250 mL N2 supplement (Invitrogen) 5 mL B27 supplement (Invitrogen) 10 mL 1× GlutaMAX (Gibco) 1× Non-essential amino acids (Gibco) β-Mercaptoethanol (Gibco) 0.1 mM Penicillin-Streptomycin (Gibco) 0.5% Bovine Serum Albumin (BSA, Sigma) 50 mg / mL PD0325901 (Stemgent) 1 μM IM-12 (Enzo) 0.5 μM or 1 μM SB590885 (R&D systems) 0.5 μM WH-4-023 (A Chemtek) 1 μM Recombinant human LIF (Peprotech) 20 ng / mL Activin A (Peprotech) 10 ng / mL

[0086] (HDM medium) The following components were added to a 1:1 (v / v) mixed medium of DMEM / F12 medium and Neurobasal medium to prepare the HDM medium. The concentrations shown below are the final concentrations in the HDM medium. 1× N2 supplement 1× B27 supplement 1× GlutaMAX 1× Non-essential amino acids β-Mercaptoethanol 0.1 mM Penicillin-Streptomycin 0.5% bFGF (Peprotech) 20 ng / mL Activin A (Peprotech) 20 ng / mL CHIR99021 3 μM

[0087] (TDM medium) The following components were added to a 1:1 (v / v) mixed medium of DMEM / F12 medium and Neurobasal medium to prepare the TDM medium. The concentrations shown below are the final concentrations in the TDM medium. 0.5× N2 supplement 0.5× B27 supplement ITS-X 0.5% 0.5× GlutaMAX 0.5× Non-essential amino acids β-mercaptoethanol 0.1 mM Knockout Serum Replacement (KSR, Gibco) 0.5% (v / v) FBS 0.1% BSA 50mg / mL Penicillin-Streptomycin 0.5% PD0325901 1μM A83-01 0.5 μM SB590885 0.25μM WH-4-023 0.5μM IM-12 0.25μM CHIR99021 1μM SB431542 0.5μM Recombinant human LIF 10ng / mL EGF 25ng / mL L-ascorbic acid 0.75μg / mL VPA 0.4mM

[0088] (IVC1 medium) The following components were added to Advanced DMEM / F12 medium (Gibco) to prepare IVC1 medium. The concentrations shown below are the final concentrations in the IVC1 medium. FBS 20% (v / v) L-glutamine (Gibco) 2mM 1× ITS-X β-oestradiol (Sigma) 8nM Progesterone (Sigma) 200ng / mL N-acetyl-L-cysteine (Sigma) 25μM Sodium Lactate (Sigma) 0.22% (v / v) Sodium Pyruvate (Sigma) 1mM Y27632 10 μM

[0089] (IVC2 Medium) The following components were added to Advanced DMEM / F12 medium to prepare IVC2 medium. The concentrations shown below are the final concentrations in the IVC2 medium. KSR 30% (v / v) L-glutamine 2 mM 1× ITS-X β-oestradiol 8 nM Progesterone 200 ng / mL N-acetyl-L-cysteine 25 μM Sodium Lactate 0.22% (v / v) Sodium Pyruvate 1 mM Y27632 10 μM

[0090] (TS Medium) The following components were added to TS basal medium to prepare TS medium. The concentrations shown below are the final concentrations of each component in the TS medium. Y27632 (FUJIFILM Wako) 2.5 μM EGF (FUJIFILM Wako) 25 ng / mL VPA (FUJIFILM Wako) 0.8 mM A83-01 (FUJIFILM Wako) 5 μM CHIR99021 (FUJIFILM Wako) 2 μM

[0091] <Isolation of Endometrial Epithelial Cells> Endometrial epithelial cells were isolated according to the following procedure. 1. Under a stereomicroscope, the decidua was manually isolated using forceps. 2. The decidua was washed with washing medium (RPMI1640). 3. The decidua was transferred to a new dish and cut into pieces of 0.5 - 1 mm 3 using a scalpel. 4. Added enzyme (dispase / collagenase), and reacted with shaking at 37°C for 1 - 2 hours. 5. Added medium to neutralize. 6. Passed through a 100μm filter 1 - several times to capture cells on the filter.

[0092] <Preparation of endometrial epithelial cells> Endometrial epithelial cells were prepared according to the following procedure. 1. The endometrial epithelial cells captured on a 100μm filter in 6 of <Isolation of endometrial epithelial cells> were recovered by inverting the 100μm filter and washing with a washing medium (RPM1640). 2. Centrifuged at 500g for 5 minutes. 3. Discarded the supernatant, added 1 mL of Advanced DMEM / F - 12 (Thermo Fisher Scientific), and suspended the cells by pipetting. 4. Transferred the cell suspension to a new 1.5 mL tube and centrifuged at 500g for 5 minutes. 5. Discarded the supernatant, estimated the volume of the pellet, and tapped on ice for 2 - 3 minutes. 6. Added approximately 20 - fold volume of ice - cold Matrigel® (Corning) to the pellet volume, gently pipetted several times, and then returned it to ice. 7. Dropped 20 μL of the cell suspension onto a non - treated 48 - well plate pre - warmed to 37°C. 8. Incubated at 37°C for 15 minutes or more. 9. Added 250 μL of ECSY medium and maintained.

[0093] <Preparation of endometrial model> An endometrial model was prepared according to the following procedure. 1. Removed the medium from the maintenance culture solution of endometrial organoids. 2. Added 0.5 mL of DMEM / F - 12 (cold), and pipetted 20 times to suspend the cells. 3. Centrifuged at 500g for 3 minutes and removed the supernatant. 4. Matrigel (registered trademark) (Corning) and type I collagen (Cellmatrix (registered trademark) Type I-A; Nitta Gelatin) were added (Matrigel / type I collagen = 20 / 80 (volume ratio)). For the control, only Matrigel was added. 5. 20 μL of the cell-containing gel was dropped onto an untreated 48-well plate and incubated at 37°C for 30 minutes. 6. ESCY medium was added and the cells were cultured at 37°C in a 5% CO2 concentration. During the culture, the medium was changed 2 - 3 times a week. 7. On the 7th day of culture, the medium was changed to ECSY + EPC medium.

[0094] <Example 1> An endometrial model was prepared using Matrigel or type I collagen gel by the above method. Figure 3 shows a bright-field phase-contrast microscopic image of the endometrial model. When cultured using Matrigel, the endometrial epithelial cells formed a spherical structure inside the Matrigel. The endometrial epithelial cells remained inside the Matrigel and did not expose on the gel surface. When cultured using type I collagen gel, the endometrial epithelial cells moved to the gel surface without remaining inside the type I collagen gel. The endometrial epithelial cells were exposed on the gel surface and gel shrinkage was observed.

[0095] <Example 2> An endometrial model was prepared using Matrigel or type I collagen gel by the above method. Then, sections of the endometrial model were prepared and immunostained with anti-Laminin antibody and Hoechst staining were performed. Figure 4 shows the immunostained image of the endometrial model section. When cultured using Matrigel, the endometrial epithelial cells were surrounded by Laminin and did not expose on the gel surface. When cultured using type I collagen gel, it was confirmed that the endometrial epithelial cells were exposed on the gel surface (arrow).

[0096] <Example 3> An endometrial model was prepared using type I collagen gel in the same manner as above, except that ECSY+EPC+PLG medium was used instead of ECSY+EPC medium. Subsequently, sections of the endometrial model were prepared, and immunostaining with anti-EpCAM antibody or anti-PGR antibody, F-actin staining with phalloidin, and Hoechst staining were performed. EpCAM is an epithelial cell marker and is widely expressed in epithelial cells. F-actin is a structural protein of the cytoskeleton. PGR is a progesterone receptor and serves as an indicator of hormone sensitivity.

[0097] Figure 5 shows immunostaining images of sections of the endometrial model. In the endometrial model, a luminal structure and a non-luminal structure were formed continuously. PGR was expressed in the luminal structure, and it was confirmed that it was hormone-sensitive. The luminal structure had a structure similar to the glandular epithelium of the endometrium.

[0098] <Example 4> The endometrial cells were cultured in a planar manner using type I collagen gel or Matrigel. Figure 6 shows the phase-contrast microscope image (Phase) and the staining image with Rhodamine B (Rhocamine B) of the endometrial cells on the third day of planar culture. When cultured in a planar manner using Matrigel, the endometrial cells grew in a colony-like manner. On the other hand, when cultured in a planar manner using type I collagen gel, it was confirmed that the endometrial cells were dispersed and migrating radially. From this result, it was shown that the migration of endometrial cells was promoted on the surface of type I collagen gel.

[0099] <Example 5> To simulate the implantation of embryonic cells into the endometrial model, a co-culture test was performed between ES cells (transferred from the laboratory of Dr. Umekawa, National Center for Child Health and Development) or TS cells and the endometrial model. As the TS cells, TS cells genetically modified to express EGFP (EGFP-TS cells) were used.

[0100] ES cells were induced into blastoids and co-cultured with an endometrial model (Yu L et al Nature 2021). The induction into blastoids was performed as follows. Naive ES cells were seeded into 256-well microwells made of agarose at a density of 25 - 50 cells / well. After culturing overnight in 5i / L / A medium, the medium was changed to HDM medium. Three days after starting the culture in HDM medium, the medium was changed to TDM medium. Thereafter, the culture was continued in HDM medium while changing the medium once every two days. Around 10 - 12 days after seeding the ES cells, cells that formed blastoids were collected using a mouse pipette and used for co-culture with the endometrial model.

[0101] TS cells were cultured in suspension for 3 days in TS medium (Okae et al Cell Stem Cell 2018) using a Petri dish to form aggregates. These aggregates were used for co-culture with the endometrial model.

[0102] An endometrial model was prepared using type I collagen gel by the above method. Subsequently, ES cells or EGFP-TS cells were co-cultured with the endometrial model to prepare an endometrial implantation model. IVC1 medium and IVC2 medium were used as the co-culture medium. Co-culture was performed using IVC1 medium from day 1 to day 1 after the start of co-culture. On day 2 after the start of co-culture, the medium was changed to IVC2 medium, and co-culture was performed using IVC2 medium. On day 5 after the start of co-culture, sections of the endometrial implantation model were prepared. Immunostaining with anti-OCT4 antibody or anti-SDC1 antibody, F-actin staining with phalloidin, and Hoechst staining were performed on the sections. OCT4 is an undifferentiated marker and is expressed in undifferentiated cells. SDC1 is an ST cell marker.

[0103] Figure 7 shows an immunostained image of a section of an implantation model using ES cells. Figure 8 shows an immunostained image of a section of an implantation model using EGFP-TS cells. From Figures 7 and 8, it was confirmed that ES cells or EGFP-TS cells were attached to the surface of the endometrial model. In the cells at the implantation site in contact with the endometrial model, the expression of SDC1 was confirmed in both ES cells and TS cells. From this, it was considered that the cells at the implantation site had differentiated into ST cells. From these results, it was shown that the endometrial implantation can be simulated using the endometrial model prepared by the above method.

Industrial Applicability

[0104] According to the present invention, there are provided an endometrial model in which the apical surface of endometrial epithelial cells is exposed, a method for producing the endometrial model, and an endometrial implantation model using the endometrial model.

[0105] As described and illustrated above are the preferred embodiments of the present invention, which are to be regarded as illustrative of the present invention and not as restrictive. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit or scope of the present invention. Therefore, the present invention is not to be considered limited by the foregoing description, but only by the scope of the appended claims.

Explanation of Signs

[0106] 1 Endometrial model 2 Endometrial implantation model 10a, 10b Aggregates of endometrial epithelial cells 11 Lumen structure 12 Non-lumen structure 20 Gel containing stromal extracellular matrix 30 Embryonic cells

Claims

1. A gel containing an interstitial extracellular matrix, and endometrial epithelial cells, wherein at least a part of the endometrial epithelial cells is exposed on the surface of the gel, and at least a part of the endometrial epithelial cells exposed on the surface of the gel continuously forms a luminal structure and a non-luminal structure, An endometrial model.

2. The endometrial model according to claim 1, wherein the non-luminal structure is a flat membranous structure.

3. The endometrial model according to claim 1 or 2, wherein the interstitial extracellular matrix is type I collagen.

4. The endometrial model according to any one of claims 1 to 3, wherein a part of the endometrial epithelial cells is present inside the gel.

5. (a) A step of seeding a cell suspension of endometrial epithelial cells into a gel containing an interstitial extracellular matrix, and (b) a step of culturing the endometrial epithelial cells after the step (a), A method for producing an endometrial model, wherein at least a part of the endometrial epithelial cells exposed on the surface of the gel continuously forms a luminal structure and a non-luminal structure.

6. The method for producing an endometrial model according to claim 5, wherein the interstitial extracellular matrix is type I collagen.

7. An endometrial implantation model comprising the endometrial model according to any one of claims 1 to 4, and embryonic cells.

8. The endometrial implantation model according to claim 7, wherein the embryonic cells comprise at least one cell selected from the group consisting of trophoblast stem cells, cytotrophoblast cells, extravillous trophoblast cells, syncytiotrophoblast cells, pluripotent stem cells, and cells derived therefrom.

9. The endometrial implantation model according to claim 7 or 8, wherein the embryonic cells form spheroids or organoids. ​

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