JP2020130055A - Endometrium-like tissue and method for producing the same - Google Patents

Abstract

To provide an endometrium-like tissue in which changes occurring in a uterine tissue associated with fertilized egg transplant can be observed in vitro, and a method for producing the tissue.SOLUTION: An endometrium-like tissue includes a support body including cell adhesive hydrogel, a first cell layer which is formed on the support body and includes endometrium stromal cell, a second cell layer which is formed on the first cell layer and includes endometrium epithelial cell. A method for producing endometrium-like tissue includes: (1) a process of forming a first cell layer including endometrium stromal cells and a second cell layer including endometrium epithelial cells on a support body including cell adhesive hydrogel, in which the second cell layer is formed on the first cell layer; and (2) a process of culturing a structure obtained in the process (1).SELECTED DRAWING: Figure 8

Description

The present invention relates to endometrial-like tissue and a method for producing the same.

In recent years, due to the effects of late marriage due to the advancement of women into society, the number of elderly pregnancies that are prone to complications of various obstetric diseases is increasing. In addition, the number of patients diagnosed with infertility is increasing, which has become a social problem.

The causes of infertility are divided into female factors and male factors, and female factors are further divided into ovulation factors, fallopian tube factors, uterine factors, cervical factors, immunological factors and the like. Various assisted reproductive technology (ART) and other treatments have been developed as treatments for these infertility, but they are not yet sufficient treatments.

Infertility due to uterine factors is said to be caused by changes in the uterine cavity and endometrium. Regenerative medicine technology aimed at the regeneration of uterine tissue is being developed as a treatment method for diseases associated with adhesion and degeneration of the uterine cavity. For example, it has been reported that when endometrial epithelial cell sheets and endometrial stromal cell sheets are prepared, laminated and transplanted into the uterus of rats, the endometrium is regenerated and pregnancy is established ( Patent Document 1).

In assisted reproductive technology, the ovulation induction method and embryo culture method have been improved year by year, and the embryo transfer rate has improved, but there are problems such as implantation of a good fertilized egg into the uterus with a certain probability. Still remains. Therefore, in order to improve the implantation rate of fertilized eggs, it is required to clarify the causal relationship between the endometrium and the embryo.

Previous in vitro studies conducted to clarify the causal relationship between the endometrium and the embryo are studies using thin structures such as single-layer cultured endometrial cells (Non-Patent Document 1). , An in vitro model capable of observing the infiltration process of fertilized eggs has not yet been developed.

JP-A-2016-204300

Weimar ,. H. , Et al. , In-vitro model systems for the study of human embryo-endometrium interactions. Reprod Biomed Online, 2013.27 (5): p. 461-76.

An object of the present invention is to provide an endometrial-like tissue and a method for producing the same, which enables observation of changes in the uterine tissue associated with fertilized egg transplantation in vitro.

The present inventors have conducted research and development by examining from various angles. as a result,
A support containing cell-adhesive hydrogel, a first cell layer containing endometrial stromal cells formed on the support, and an endometrium formed on the first cell layer. Surprisingly, it was found that the endometrial tissue containing the second cell layer containing epithelial cells can observe changes after fertilized egg transplantation in vitro, leading to the development of the present invention.

That is, the present invention includes the following aspects.

[1] A support containing a cell-adhesive hydrogel and
A first cell layer containing endometrial stromal cells formed on the support,
Endometrial-like tissue formed on the first cell layer and comprising a second cell layer containing endometrial epithelial cells.
[2] The endometrial-like tissue according to [1], wherein the cell-adhesive hydrogel contains collagen.
[3] The endometrial-like tissue according to [1] or [2], wherein the cell-adhesive hydrogel contains fibrin.
[4] The endometrial-like tissue according to any one of [1] to [3], wherein two or more layers of the first cell layer are formed.
[5] The endometrial-like tissue according to any one of [1] to [4], wherein the endometrial stromal cells are primary endometrial stromal cells.
[6] The endometrial-like tissue according to any one of [1] to [5], wherein the endometrial epithelial cells are primary endometrial epithelial cells.
[7] The endometrial-like tissue according to any one of [1] to [6], wherein a fertilized egg is applied on the second cell layer.

[8] A method for producing endometrial-like tissue, which includes the following:
(1) A step of forming a first cell layer containing endometrial stromal cells and a second cell layer containing endometrial epithelial cells on a support containing cell-adhesive hydrogel. Here, the second cell layer is formed on the first cell layer; and (2) the step of culturing the structure obtained in the step (1).
Including methods.
[9] The method according to [8], wherein the cell adhesion hydrogel contains collagen.
[10] The method according to [8] or [9], wherein the cell adhesion hydrogel contains fibrin.
[11] The method according to any one of [8] to [10], wherein two or more layers of the first cell layer are formed.
[12] The step (1)
The method according to any one of [8] to [11], which is a step of placing the laminate having the second cell layer formed on the first cell layer on the support.
[13] The method according to any one of [8] to [12], wherein the first cell layer and the second cell layer are obtained by using a stimulus-responsive culture substrate.
[14] The method according to [13], wherein the stimulus-responsive culture substrate is a temperature-responsive culture substrate.
[15] The method according to any one of [8] to [14], wherein the endometrial stromal cells are primary endometrial stromal cells.
[16] The method according to any one of [8] to [15], wherein the endometrial epithelial cells are primary endometrial epithelial cells.
[17] (3) The method according to any one of [8] to [16], wherein the fertilized egg is applied on the second cell layer.

[18] Endometrial-like tissue obtained by the method according to any one of [8] to [17].

[19] A method for screening a factor that promotes implantation of a fertilized egg using the endometrial-like tissue according to any one of [1] to [7] and [18].

The endometrial-like tissue of the present invention allows in vitro observation of changes that occur in the uterine tissue after fertilized egg transplantation.

FIG. 1 is a schematic view showing a procedure for producing and using the endometrial-like tissue of the present invention in one embodiment. The procedure of (a) preparing the endometrial tissue, (b) preparing the fertilized egg to be used for the endometrial tissue, and (c) the procedure of applying the fertilized egg to the endometrial tissue are shown. FIG. 2 shows an endometrial-like cell sheet (top layer: endometrial epithelial cell sheet, intermediate layer and bottom layer: endometrial stromal cell sheet). FIG. 3 is a photograph showing the results of culturing an endometrial-like cell sheet adhered to a cell culture insert as a comparative example. Average pore size: (a) 1 μm, (b) 3.0 μm, (c) 8.0 μm. FIG. 4 shows the results of culturing an endometrial-like cell sheet adhered to a collagen gel. (A) Endometrial-like cell sheet immediately after transplantation into collagen gel. (B) Endometrial-like cell sheet 3 days after transplantation into collagen gel. (C) HE-stained image of the tissue section of (b). Scale bar = 200 μm. FIG. 5 shows the results of culturing an endometrial-like cell sheet adhered to a fibrin gel / collagen gel. (A) Endometrial-like cell sheet immediately after transplantation into fibrin gel / collagen gel. (B) Endometrial-like cell sheet 3 days after transplantation into fibrin gel / collagen gel. FIG. 6 shows the results of culturing an endometrial-like cell sheet adhered to a fibrin gel / collagen gel. (A) HE-stained image of endometrial-like cell sheet 3 days after transplantation into fibrin gel / collagen gel. Arrow: Epithelial layer, Arrowhead: Interstitium layer. Scale bar = 200 μm. (B) A graph showing the results of measuring the thickness of an endometrial-like cell sheet 3 days after transplantation into a fibrin gel / collagen gel at 1 mm intervals. FIG. 7 shows the results of culturing an endometrial-like cell sheet adhered to a fibrin gel / collagen gel. (A) Fluorescence microscope image of endometrial-like cell sheet immunostained with DAPI (blue), anti-CK18 antibody (green), and anti-vimentin antibody (red). Scale bar = 100 μm. (B) Fluorescence microscope image of endometrial-like cell sheet immunostained with DAPI (blue), anti-estrogen receptor antibody (green), and anti-progesterone receptor antibody (red). Scale bar = 200 μm. FIG. 8 shows the results of culturing a fertilized egg applied to endometrial-like tissue. (A, b) Confocal laser scanning microscope images of endometrial-like tissue immunostained with an anti-GATA3 antibody and an anti-actin antibody. (A) sectional view, (b) three-dimensional view.

Hereinafter, the present invention will be described through embodiments of the invention, but the following embodiments do not limit the invention in the claims, and all combinations of features described in the embodiments are included. It is not always essential for the means of solving the invention. In addition, it is explained in the following "endometrial tissue", "method for producing endometrial tissue" or "method for screening factors that promote implantation of fertilized eggs using an endometrial tissue model". The items are either "endometrial tissue", "method for producing endometrial tissue", or "method for screening factors that promote implantation of fertilized eggs using an endometrial tissue model". Can also be applied in.

In the present specification, terms such as "first" and "second" are used to distinguish one element from another, and for example, the first element is expressed as the second element. Similarly, the second element may be expressed as the first element, which does not deviate from the scope of the present invention.

<< Endometrial tissue >>
The present invention includes a support containing a cell-adhesive hydrogel and
A first cell layer containing endometrial stromal cells formed on the support,
Provided is an endometrial-like tissue formed on the first cell layer and containing a second cell layer containing endometrial epithelial cells. The present invention also provides endometrial-like tissue obtained by the method for producing endometrial-like tissue described later.

As used herein, the term "endometrium-like tissue" refers to a structure that is reconstructed in vitro and mimics the structure of the endometrium of a living body, but includes components that the endometrium of a living body does not have. It may be a thing.

As used herein, the term "cell adhesion hydrogel" is selected from the group consisting of proteins constituting extracellular matrix, chitosan gel, collagen gel, gelatin, peptide gel, laminin gel and fibrin gel, and mixtures thereof. It means what is done. Of these, chitosan gel, collagen gel, gelatin gel, peptide gel and laminin gel are promoted to gel by changing the temperature, pH and / or salt concentration, for example. Fibrin gel is gelled by the action of the monomer fibrinogen with the enzyme thrombin. The cell adhesion hydrogel used in the present invention is preferably a collagen gel and / or a fibrin gel, and more preferably a mixture of a collagen gel and a fibrin gel. In the present specification, the collagen contained in the collagen gel is, for example, type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type. XV, type XVII, or type XVIII collagen or atelocollagen, or a combination thereof. The collagen contained in the collagen gel is, for example, derived from mammals (for example, primates (including humans and non-human primates)), rodents (mouse, rat, hamster, guinea pig, etc.), rabbits, dogs, cows, etc. It may be a horse, a pig, a cat, a goat, a sheep, etc.), or it may be obtained by genetic engineering based on a mammalian collagen gene.

As used herein, the term "support" refers to a structure used for adhering and fixing "cell layers (first cell layer and second cell layer)" described later. The support used in the present invention contains a cell-adhesive hydrogel, which promotes adhesion to the cell layer. The support used in the present invention may be, for example, a culture substrate (for example, a culture vessel) coated with a cell-adhesive hydrogel or a cell culture insert, or may be a cell-adhesive hydrogel itself. .. When the cell adhesion hydrogel is used as a support, for example, a support in which a cell adhesion hydrogel solution before curing is poured into a mold (frame) and cured into a desired shape may be used. For example, the support may be formed by the method described in International Publication No. 2012/036225. In addition, the support may be provided with a flow path through which the medium flows, as described in International Publication No. 2012/036225.

In one embodiment, a "mixture of collagen gel and fibrin gel" that can be used as a support containing a cell-adhesive hydrogel contains the cell layer so that the cell layer does not peel off even after the cell layer is adhered and cultured. You just have to adjust the amount of things. The ratio of collagen to fibrinogen contained in the collagen / fibrin solution before curing may be, for example, about 10: about 1 to about 1: about 10, and about 5: about 1 to about 1: about 5. It may be about 1: about 3. As used herein, the term "about" means a numerical value included in the range of ± 10% of the numerical value.

As used herein, the term "uterine tissue" refers to the female reproductive organs of mammals and the organs that raise the fetus in the body during pregnancy. Uterine tissue in mammals, especially humans, forms a box-shaped cavity. Fertilized eggs are generated and the fetus is raised in the uterine body. The body of the uterus is composed of the endometrium, muscularis, and serosa.

The endometrium can be classified into an endometrial epithelial layer and an endometrial stromal layer from the viewpoint of tissue structure. The endometrial epithelial layer consists of simple columnar cells, contains secretory cells and ciliated cells, and is the cell layer closest to the uterine cavity. The endometrial stromal layer is a structure with many endometrial glands. The entrance of the endometrium line is connected to the epithelium facing the uterine cavity, and the innermost part extends to the deep part of the endometrium. Mucus is secreted from the endometrium glands to cover the surface of the endometrium and support the development of fertilized eggs and embryos.

From a functional point of view, the endometrium has a basal layer in the deep part of the endometrium that does not change in the sexual cycle, and a function that changes the thickness and structure in the sexual cycle and has endometrial glands and helicine arteries. It can be divided into two layers.

The human endometrium depends on the female hormones estrogen and progesterone, and its thickness changes according to the sexual cycle. During the period from the end of menstruation to the ovulation period, estrogen levels increase and the endometrium, especially the functional layer, proliferates (proliferation phase). After the ovulation period, estrogen levels temporarily decrease, then estrogen and progesterone levels increase again, and the endometrium thickens (secretory period). At this time, the uterine gland of the endometrium develops, secretion is promoted, and edema of the endometrial stroma occurs. In this way, an environment suitable for implantation of fertilized eggs is created. After that, the menstrual period occurs on average every 28 days, and the functional layer peels off. At this time, both estrogen and progesterone levels decrease. Thickening and detachment of the uterine mucosa due to the sexual cycle plays an important role in normal pregnancy in women.

As used herein, the term "endometrial stromal cell" refers to a cell that constitutes a supporting tissue of endometrial epithelial cells, and refers to a cell contained in the endometrial stromal layer. The endometrial stromal cells used in the present invention may be primary endometrial stromal cells isolated from endometrial tissue, or established endometrial stromal cells, or ES cells. , IPS cells, Muse cells and other pluripotent stem cells may be endometrial stromal cells. Preferably, the endometrial stromal cells used in the present invention are primary endometrial stromal cells.

In one embodiment, the "first cell layer containing endometrial stromal cells" according to the present invention may contain cells other than endometrial stromal cells, and the type of the cells is not particularly limited. .. In addition, the endometrial stromal cells may be a single type of cell or may contain a plurality of types of cells. In one embodiment, the proportion of endometrial stromal cells contained in the first cell layer according to the present invention is 60% or more, preferably 70% or more, still more preferably 80% or more (for example, 80%, 85%). , 90%, 95%, 96%, 97%, 98%, 99%, 100%).

In the present specification, the "endometrial epithelial cell" is an epithelial cell contained in the uterine mucosa (endometrium) and refers to a cell contained in the endometrial epithelial layer. The endometrial epithelial cells used in the present invention may be primary endometrial epithelial cells isolated from endometrial tissue, or established endometrial epithelial cells, ES cells, iPS cells. , Endometrial epithelial cells derived from pluripotent stem cells such as Muse cells. Preferably, the endometrial epithelial cells used in the present invention are primary endometrial epithelial cells.

In one embodiment, the "second cell layer containing endometrial epithelial cells" according to the present invention may contain cells other than endometrial epithelial cells, and the type of the cells is not particularly limited. In addition, the endometrial epithelial cells may be a single type of cell or may contain a plurality of types of cells. In one embodiment, the proportion of endometrial epithelial cells contained in the second cell layer according to the present invention is 60% or more, preferably 70% or more, still more preferably 80% or more (for example, 80%, 85%, etc.). It can be 90%, 95%, 96%, 97%, 98%, 99%, 100%).

The origin of the animal species of the cells used in the present invention is not particularly limited, but for example, humans or rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees or their immunity. Examples include incomplete animals. Further, it may be a cell derived from the same individual from which the fertilized egg to be applied is collected, or a cell derived from an individual different from the individual from which the fertilized egg was collected. In addition, the animal from which the endometrial cell is derived and the animal from which the fertilized egg is derived do not necessarily have to match. In addition, the animal from which the cells constituting the endometrial tissue are derived and the animal from which the fertilized egg is derived do not necessarily have to match.

In the present specification, the "cell layer (first cell layer and second cell layer)" refers to a sheet-like cell group, and is also referred to as a "cell sheet". In one embodiment, the cell layer (first cell layer or second cell layer) contained in the endometrial tissue of the present invention may be a cell layer having a thickness of one cell, but contracts. It is preferable that the cell layer has a thickness exceeding the thickness of one cell (for example, a thickness of 2 to 10 cells). The cell layer that has been detached from the adhered scaffold and contracted (for example, a contracted cell sheet) maintains the cell junctions (for example, tight junctions and gap junctions) formed between adjacent cells, and Due to its high cell density, it exerts more functions than a single-layer cell layer.

As a method for obtaining a cell layer, for example, cells are cultured on a stimulus-responsive culture medium coated with a polymer that changes the molecular structure by stimuli such as temperature, pH, light, and electricity, and the temperature, pH, By changing the surface properties of the stimulus-responsive culture substrate by changing the stimulus conditions such as light and electricity, the cells are separated from the stimulus-responsive culture substrate into a sheet while maintaining the adhered state between the cells. A method of culturing cells on an arbitrary culture medium and physically peeling the sheet-shaped cell group from the end with a tweezers or the like; and seeding the cells on the culture surface together with hydrogel or the like. Examples thereof include, but are not limited to, a method of exfoliating the formed sheet-like cell group.

In the present specification, the "stimulation-responsive polymer" coated on the stimulation-responsive culture substrate is, for example, poly (N-isopropylacrylamide), poly (N-isopropylacrylamide-acrylic acid) copolymer, or poly. (N-isopropylacrylamide-methylmethacrylate) copolymer, poly (N-isopropylacrylamide-sodium acrylate) copolymer, poly (N-isopropylacrylamide-vinylferrocene) copolymer, γ-ray irradiated poly (vinylmethyl) Examples thereof include resins in which biological substances such as ether) (PVME), poly (oxyethylene) and nucleic acids are incorporated into polymers, and gels prepared by cross-linking the above polymers with a cross-linking agent, but the present invention is limited thereto. Not done.

As used herein, the term "temperature-responsive polymer" refers to a polymer that is one of stimulus-responsive polymers and that changes its shape and / or properties in response to temperature. Examples of the temperature-responsive polymer include poly (N-isopropylacrylamide), poly (N-isopropylacrylamide-acrylic acid) copolymer, poly (N-isopropylacrylamide-methylmethacrylate) copolymer, and poly (N-isopropylacrylamide) copolymer. A gel prepared by cross-linking an isopropylacrylamide-sodium acrylate) copolymer, a poly (N-isopropylacrylamide-vinylferrocene) copolymer, a γ-ray irradiated poly (vinyl methyl ether), and the above polymer with a cross-linking agent. Etc., but are not limited to them. Preferably, for example, a cross-linking agent for poly (N-isopropylacrylamide), poly (N-isopropylacrylamide-methylmethacrylate) copolymer, poly (N-isopropylacrylamide-sodium acrylate) copolymer and the above polymer. Examples include, but are not limited to, gels produced by cross-linking with.

In the present specification, the temperature-responsive polymer coated on the culture substrate is, for example, the upper critical solution temperature (UCST: Upper Critical Solution Temperature) or the lower critical solution temperature (LCST: Lower Critical Solution Temperature) with respect to water. Is 0 to 80 ° C., but is not limited thereto. The critical solution temperature is a threshold temperature at which the shape and / or properties of the polymer are changed. In the present invention, preferably, a cell culture substrate coated with poly (N-isopropylacrylamide) (PIPAAm) can be used.

Poly (N-isopropylacrylamide) (PIPAAm) is known as a polymer having a lower limit critical solution temperature (LCST) at 32 ° C. If it is in a free state, it undergoes dehydration at a temperature of 32 ° C. or higher in water to aggregate PIPAAm. And it becomes cloudy. On the contrary, at a temperature lower than 32 ° C., PIPAAm is hydrated and becomes dissolved in water. The temperature-responsive culture substrate used in one embodiment of the present invention is one in which PIPAAm is coated and fixed on a culture surface such as a petri dish or a cell culture insert. Therefore, at a temperature of 32 ° C. or higher, PIPAAm on the culture surface is similarly dehydrated, but the culture surface is hydrophobic because PIPAAm is fixed to the culture surface. On the contrary, at a temperature of less than 32 ° C., PIPAAm on the culture surface is hydrated, but since PIPAAm is coated on the culture surface, the culture surface is hydrophilic. The hydrophobic culture surface is a surface on which cells can adhere and proliferate, while the hydrophilic culture surface is a surface on which cells are difficult to adhere. Therefore, when the temperature-responsive culture substrate is cooled below 32 ° C., the cells detach from the culture surface. If the cells are cultured until they become confluent on the entire culture surface, the sheet-like cell layer (cell sheet) can be recovered by cooling the culture surface to less than 32 ° C. As the temperature-responsive culture substrate coated with PIPAAm, for example, UpCell (registered trademark) manufactured and sold by CellSeed (Japan) can be used.

Since the cell layer (cell sheet) obtained by using the stimulus-responsive culture substrate used in one embodiment of the present invention does not use proteolytic enzymes such as dispase and trypsin conventionally used for cell recovery. , The lower surface of the cell layer (the surface on the side that was in contact with the culture surface) maintains abundant adhesive proteins, and also retains the cell-cell desmosome structure, and multiple cell layers are laminated. Suitable for conversion.

The number of cells constituting the first cell layer or the second cell layer contained in the endometrial-like tissue of the present invention cannot be limited because it varies depending on the state, type, animal species, etc. of the cells used, but for example. , 0.3 × 10 ⁴ to 10 × 10 ⁶ pieces / cm ² when forming the first cell layer or the second cell layer, 0.5 × 10 ⁴ to 8 × 10 ⁶ pieces / cm ^It may be 2 or it may be formed by sowing at a density of 0.7 × 10 ^{4 to} 5 × 10 ⁶ pieces / cm ² .

In one embodiment, the first cell layer contained in the endometrial tissue of the present invention may be one layer, two or more layers (for example, two layers, three layers, four layers, five layers or more). May be laminated.

In one embodiment, the second cell layer contained in the endometrial tissue of the present invention may be one layer or more (for example, two layers, three layers, four layers, five layers or more). May be laminated.

The cells or cell layers (first cell layer, second cell layer) used in the present invention may be collected and / or cultured, for example, by referring to the method described in JP-A-2016-204300. ..

In one embodiment, the endometrial-like tissue of the present invention may be one in which a fertilized egg is further applied on the second cell layer. As used herein, the term "fertilized egg" refers to a cell to which male and female gametes (sperm and egg) of a species that reproduce eggs are bound, and the cell division progresses, resulting in 2 cells, 4 cells, 8 cells. It refers to a cell mass (cell group) contained in a cell, a mulberry embryo, and a blastomere, and these cell clusters can also be applied to the present invention. Further, in one embodiment, the fertilized egg in which the zona pellucida covering the fertilized egg is opened or removed (hatched) may be applied to the present invention.

In one embodiment, the fertilized eggs that can be applied to the endometrial tissue of the present invention are 1) in vitro fertilization (collecting eggs) and then crossing with sperm, or fertilized eggs obtained by microinsemination; 2) The fertilized egg collected from any mammal female after mating may be used, and the fertilized egg obtained by 1) or 2) may be a thawed fresh or frozen embryo. Further, the fertilized egg that can be applied is a cell mass (for example, 2 cells, 4 cells, 8 cells, morula, or blastocyst) that has been cultured in vitro by a known method to promote cell division. May be good.

In one embodiment, the endometrial tissue of the present invention may be provided, for example, as a kit in which each of the configurations described above is packaged separately and the endometrial tissue can be prepared at the desired time. Good.

<< Method for producing endometrial-like tissue >>
The present invention
(1) A step of forming a first cell layer containing endometrial stromal cells and a second cell layer containing endometrial epithelial cells on a support containing cell-adhesive hydrogel. Here, the second cell layer is formed on the first cell layer; and (2) the step of culturing the structure obtained in the step (1).
Provided is a method for producing endometrial-like tissue, including.

In one embodiment, step (1) is
(1-1) A first cell layer containing endometrial stromal cells is laminated on a support containing cell adhesion hydrogel, and a second layer containing endometrial epithelial cells is laminated on the first cell layer. It may be a step of laminating cell layers.

In another embodiment, step (1) is
(1-2) The step may be a step of placing the laminate having the second cell layer formed on the first cell layer on a support containing a cell-adhesive hydrogel. By preparing a laminate in which the second cell layer is formed on the first cell layer in advance before placing it on a support containing cell-adhesive hydrogel, adhesion between the cell layers of the laminate is promoted. Therefore, the handling of the laminated body becomes easy. In addition, the number of steps of placing the cell layer on the support can be reduced, and the risk of contamination such as bacteria can be reduced.

The method for laminating the first cell layer and the second cell layer in the present invention is not particularly limited, but for example, cells are seeded on a cell culture substrate, and an extracellular matrix protein is formed therein. After applying a gel containing proteins (eg, laminin, collagen, cadoherin, fibronectin, elastin or vitronectin, etc.) and / or typical cell-adhesive hydrogel materials (eg, gelatin, hyaluronic acid, chitin or chitosan, etc.) Further, a method of seeding cells to obtain a laminate; a method of peeling cultured cells in a sheet form and, if necessary, laminating cell layers using a cultured cell transfer jig or the like. The cultured cell transfer jig used is not limited in terms of material and shape as long as it can capture and move the exfoliated cell layer, but for example, it is in the form of a film, a porous film, a non-woven fabric, or a weave. It may be in the form of cloth, and the material may be polyvinylidene fluoride (PVDF), silicon, polyester, polyvinyl alcohol, urethane, cellulose and its derivatives, chitin, chitosan, collagen, gelatin, fibring loop and the like. Further, as the cultured cell transfer jig to be used, for example, the apparatus described in JP-A-2007-222153 may be used.

In one embodiment, in the method of the present invention, the first cell layer and the second cell layer may be cell layers obtained by using a stimulus-responsive culture substrate. This results in a cell layer that is rich in adhesive proteins, thereby creating an endometrial-like tissue that is tightly adhered between the cell layers and between the cell layers and the support. In one embodiment, the stimulus-responsive culture substrate may be, for example, a temperature-responsive culture substrate.

In one embodiment, by the step of culturing the construct obtained in the step (1), the first cell layer, the second cell layer and the support containing the cell adhesion hydrogel are firmly adhered to each other, and the fertilized egg is transplanted. It is possible to provide endometrial-like tissue in which changes that occur later in the uterine tissue can be observed in vitro.

The method of the present invention in one embodiment may further include (3) applying a fertilized egg onto the second cell layer.

<< Method for screening factors that promote implantation of fertilized eggs using an endometrial tissue model >>
The endometrial-like tissue of the present invention, or the endometrial-like tissue obtained by the method of the present invention, can be used as a method for evaluating factors that promote implantation of fertilized eggs. In one embodiment, the method of the invention is, for example, not limited to:
(1) Step of applying candidate factor to endometrial tissue;
(2) A step of applying and culturing a fertilized egg on the second cell layer of the endometrial tissue;
(3) A step of evaluating the implantation of the fertilized egg,
May include.

In one embodiment, the candidate factor applied in step (1) may be applied to endometrial tissue (eg, added to the medium) in any step of the method for producing endometrial tissue described above. It may be adapted (eg, added to the medium).

In one embodiment, the method for evaluating implantation of the fertilized egg in step (3) is a known staining method (for example, but not limited to, hematoxylin-eosin (H & E) staining, fertilized egg and endometrial tissue. Implantation of fertilized eggs by staining endometrial-like tissue with immunohistochemical staining or immunofluorescent staining with staining antibodies (eg, anti-GATA3 antibody and anti-actin antibody) and then using any microscope. The floor degree can be evaluated. Thereby, the effect of the applied candidate factor can be evaluated. In determining the effect of any candidate factor, for example, molecular biological techniques such as changes in gene expression in endometrial tissue or fertilized eggs and analysis of produced proteins are used for evaluation. You can also do it.

In one embodiment, the candidate factor is, for example, a low molecular weight compound, peptide, nucleic acid, protein, cell, tissue extract or cell culture of a mammal (eg, mouse, rat, pig, bovine, sheep, monkey, human, etc.). It may be a supernatant, a plant-derived compound or extract (for example, a crude drug extract, a crude drug-derived compound), a microorganism-derived compound or extract, or a culture product.

In one embodiment, endometrial-like tissue may be used, for example, which comprises cells from an individual having impaired implantation of a fertilized egg.

Hereinafter, the present invention will be described in more detail based on examples, but these are not intended to limit the present invention in any way. The experimental protocol using rats in this example was approved by the Ethics Committee on Animal Experiments at Tokyo Women's Medical University, and was published by the National Institutes of Health (NIH) in "Experimental Animal Management". And guidelines for use ”(revised in 1996).

<Method of producing endometrial-like cell sheet>
A 3-week-old SD rat (systematic name: Slc: SD, Japan SLC Co., Ltd.) was purchased and killed by CO ₂ . The SD rat was laparotomized, the ligaments connecting the ovaries and the pelvis on both sides of the uterus were dissected, the uterine broad membrane was expanded, and then the cervix was resected to remove the uterus. The removed uterus was then separated into the myometrium and endometrium under a microscope.

A 0.25% trypsin / 0.1% EDTA solution was added to the separated endometrial tissue and treated at 37 ° C. with shaking at 150 rpm for 20 minutes. Then, pipetting was performed 70 to 100 times with a 5 mL pipettor and about 200 times with a 1000 μL pipetman. Equal amounts of medium with 10% FBS were added and filtered through 100 μm and 40 μm cell strainers. All the obtained cells were seeded in a 100 mm dish and allowed to stand for 2 hours (hereinafter referred to as "cell selection step").

Two hours later, the supernatant was collected and washed three times with PBS. PBS during washing was also collected. The supernatant and the washed PBS were combined to obtain an "endometrial epithelial cell suspension" containing mainly endometrial epithelial cells. After washing, the cells adhering to the dish were reacted with 0.25% trypsin / 0.1% EDTA for 5 minutes at 37 ° C to exfoliate, and "endometrial stromal cell suspension" mainly containing endometrial stromal cells. A turbid liquid was obtained.

Endometrial epithelial cells were seeded in cell culture inserts immobilized with a temperature-responsive polymer at a density of 5.0 × 10 ⁶ / dish. Endometrial stromal cells were seeded in a 35 mm temperature responsive culture dish (UpCell® CellSeed) at a density of 2.5 × 10 ⁶ / dish.

Endometrial epithelial cells are a medium having the following composition:
・ DMEM / F12 (1: 1)
・ Insulin 10 μg / mL
・ Transferrin 10 μg / mL
・ Selenite 0.038 μM
・ Hydrocortisone 100 μg / mL
・ Retinoic acid 2.5nM
・ L-ascorbic acid 100 μM
・ EGF 10 ng / mL
Was cultured using.

Endometrial stromal cells have the following composition:
・ DMEM / F12 (1: 1)
・ 10% FBS
・ 1% Penicillin
・ 1% Streptomycin
・ 1% Hepes
Was cultured using.

Four days after seeding, the culture substrate in which the endometrial epithelial cells and endometrial stromal cells were cultured was incubated at 20 ° C. for 1 hour, respectively, to obtain cell sheets (hereinafter, endometrial epithelial cell suspension). The cell sheet obtained from the fluid is referred to as "epithelial cell sheet", and the cell sheet obtained from the endometrial stromal cell suspension is referred to as "stromal cell sheet"). The exfoliated cell sheet was suspended in medium or PBS.

Two layers of the stromal cell sheet prepared above were laminated, and one layer of the epithelial cell sheet was laminated on the layer to obtain a total of three layers of laminated cell sheets (hereinafter referred to as "endometrium-like cell sheets") (hereinafter referred to as "endometrial-like cell sheets"). 1a and 2).

<Comparative example 1>
It was verified whether the cell culture insert, which is a network structure, can be used as a scaffold for adhering the endometrial-like cell sheet prepared by the above procedure.

Using transparencies, endometrial cell sheets of cell culture inserts with pore sizes of 0.4 μm, 1.0 μm, 3.0 μm or 8.0 μm (FALCON, # 353493, # 353102, # 353092, # 353093) It was moved up and allowed to stand for 1.5 hours.

The endometrial-like cell sheet engrafted in the cell culture insert was immersed in DMEM / F12 medium (Nacalai Tesque, # 05177-15) containing 1% Penicillin and Streptomycin, and 3 at 37 ° C. and 5% CO ₂ . Incubated for days. Endometrial cell sheets cultured on cell culture inserts were completely exfoliated and contracted after 3 days on pore sizes 1.0 μm, 3.0 μm and 8.0 μm cell culture inserts (Fig.). 3). Similarly, a cell culture insert having a pore size of 0.4 μm (FALCON, # 353493) completely peeled off and contracted after 3 days (not shown).

<Example 1>
It was verified whether the collagen gel can be used as a scaffold for adhering the endometrial-like cell sheet prepared by the above procedure.

Collagen gel is as follows:
-2.4 mL of 5 mg / mL collagen acidic solution I-AC (KOKEN, # IAC-50)
-0.3 mL 10-fold concentrated medium (Gibco)
-0.03 mL of 1000 mM NaHCO ₃ (Wako Pure Drug, # 191-01305)
0.03 mL of 1000 mM Hepes (Sigma-aldrich, # H3375-500G)
-It was prepared by mixing 0.24 mL of distilled water, pouring it into an acrylic frame described in Sakaguchi et al. (International Publication No. 2012/036225), and curing it.

The endometrial-like cell sheet was placed on an OHP film, transferred onto the prepared collagen gel, and allowed to stand for 1.5 hours.

The endometrial-like cell sheet engrafted in the collagen gel was immersed in DMEM / F12 medium (Nacalai Tesque, # 05177-15) containing 1% Penicillin and Streptomycin, and 3 at 37 ° C. and 5% CO ₂ . Incubated for days. As a result, the cell sheet engrafted on the collagen gel, but part of it was exfoliated (Fig. 4b). As a result of performing HE staining and observing the cell sheet adhered on the collagen gel, a gap was observed between the cell sheet and the gel (Fig. 4c).

<Example 2>
Next, it was verified whether the scaffold prepared by mixing collagen gel and fibrin gel could be used as a scaffold for adhering the endometrial-like cell sheet prepared by the above procedure.

Fibrin is a protein involved in blood coagulation and has cell adhesion. Thrombin reacts with fibrinogen to change into fibrin, and fibrin reacts in a chain reaction to form a fibrin membrane, filling the gaps in tissues. Since the fibrin membrane is decomposed by an enzyme called plasmin that exists in the living body, aprotinin that stops the action of this enzyme is added. When used as a scaffold for adhering cell sheets, fibrinogen, thrombin and aprotinin are mixed to prepare fibrin. Collagen provides the basis for supporting fibrinogen, thrombin, and aprotinin, making it possible to create gels with microchannels.

Fibrin gel / collagen gel is as follows:
1.5 mL 25 mg / mL fibrinogen (Sigma-aldrich, # F8630-5G)
-1.5 mL of 2.5 U / mL thrombin (Sigma-aldrich, # T4648-1KU)
0.15 mL 75000 KIU / mL aprotinin (Wako Junyakusha, # 016-11836)
-2.4 mL of 5 mg / mL collagen acidic solution I-AC (KOKEN, # IAC-50)
-0.3 mL 10-fold concentrated medium (Gibco)
-0.03 mL of 1000 mM NaHCO ₃ (Wako Pure Drug, # 191-01305)
0.03 mL of 1000 mM Hepes (Sigma-aldrich, # H3375-500G)
-0.24 mL of distilled water was mixed, poured into an acrylic frame described by Sakaguchi et al. (International Publication No. 2012/036225), and cured to prepare a product.

The endometrial-like cell sheet was placed on an OHP film, transferred onto a fibrin gel / collagen gel, and allowed to stand for 1.5 hours.

Endometrial cell sheets engrafted in fibrin gel / collagen gel are immersed in DMEM / F12 medium (Nacalai Tesque, # 05177-15) containing 1% Pencillin and Streptomycin, and brought to 37 ° C. and 5% CO ₂ . Was cultured for 3 days. As a result, the endometrial-like cell sheet cultured on the fibrin gel / collagen gel did not peel off even after 3 days, and an endometrial-like cell sheet having the same size as immediately after transplantation was observed (Fig. 5). As a result of performing HE staining and observing the endometrial-like cell sheet adhered on the fibrin gel / collagen gel, there was no gap between the endometrial-like cell sheet and the gel, and there were two layers, an epithelial layer and an stromal layer. The structure was confirmed (Fig. 6a). This structure was similar to the normal endometrial structure.

When the thickness of the endometrial-like cell sheet was measured at 1 mm intervals from the edge, the average thickness was 51.7 μm (FIG. 6b).

Tissue sections of the endometrial cell sheet were immunostained with DAPI (blue), anti-CK18 antibody (green) (Abcam, # ab53118), anti-vimentin antibody (red) (Abcam, # ab20346). It was observed with a fluorescence microscope. As a result, it was confirmed that the uppermost layer was positive for the epithelial marker CK18 (Fig. 7a). In addition, a layer positive for vimentin, which is an interstitial marker, was confirmed immediately below it.

Tissue sections of the endometrial cell sheet were immunostained with DAPI (blue), anti-estrogen receptor antibody (green) (Abcam, # ab3577), and anti-progesterone antibody (red) (Abcam, # ab2764). , Observed with a fluorescence microscope. As a result, as shown in FIG. 7b, an estrogen receptor positive and a progesterone positive layer were confirmed.

From the above results, it was clarified that the scaffold made of fibrin gel / collagen gel can stably maintain the endometrial-like cell sheet in vitro.

<Example 3>
Evaluation of transplantation of fertilized eggs using endometrial-like tissue

The fertilized egg repeats division, passes through two cells, and reaches the blastocyst in about 3 days. In rats, fertilized eggs implant in the state of blastocysts. It was verified whether the endometrial tissue of the present invention can reproduce the implantation of a fertilized egg in the same manner as the living body.

Commercially available frozen fertilized eggs (Arc Resources) were thawed and cultured in a fertilized egg medium (mR1ECM (Arc Resources) for 1 day to form blastocysts. The obtained blastocysts were classified according to grade. It was 4AA and was a good embryo.

The blastocyst hatched from the zona pellucida is placed on a fibrin gel / collagen gel + endometrial cell sheet (hereinafter referred to as "endometrial tissue") obtained by the same method as in Example 2. , Immersed in fertilized egg medium, cultured at 37 ° C. and 5% CO _{2 for} 36 hours.

The endometrial tissue was then immunostained with an anti-GATA3 antibody (red) (Abcam, # ab106625) and an anti-actin antibody (green) (invitrogen, # 1941122) and confocal laser scanning microscope (FV3000; Olympus). Observed at.

As a result, it was observed that the GATA3 positive site (that is, the fertilized egg) was surrounded by the actin positive site (that is, the endometrial tissue). This is thought to mimic the initial infiltration process that occurs when a fertilized egg (blastocyst) implants. It was suggested that this experimental system could be used to elucidate the relationship between fertilized eggs, epithelium, and stromal tissue during the initial infiltration process.

All publications and patent documents cited herein are incorporated herein by reference in their entirety. Although specific embodiments of the present invention have been described herein for purposes of illustration, it will be appreciated by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.

Claims (18)

With a support containing cell adhesion hydrogel,
A first cell layer containing endometrial stromal cells formed on the support,
Endometrial-like tissue formed on the first cell layer and comprising a second cell layer containing endometrial epithelial cells. The endometrial-like tissue according to claim 1, wherein the cell-adhesive hydrogel contains collagen. The endometrial-like tissue according to claim 1 or 2, wherein the cell-adhesive hydrogel contains fibrin. The endometrial-like tissue according to any one of claims 1 to 3, wherein two or more layers of the first cell layer are formed. The endometrial-like tissue according to any one of claims 1 to 4, wherein the endometrial stromal cells are primary endometrial stromal cells. The endometrial-like tissue according to any one of claims 1 to 5, wherein the endometrial epithelial cells are primary endometrial epithelial cells. The endometrial-like tissue according to any one of claims 1 to 6, wherein a fertilized egg is applied on the second cell layer. A method for producing endometrial-like tissue, such as:
(1) A step of forming a first cell layer containing endometrial stromal cells and a second cell layer containing endometrial epithelial cells on a support containing cell-adhesive hydrogel. Here, the second cell layer is formed on the first cell layer; and (2) the step of culturing the structure obtained in the step (1).
Including methods. The method of claim 8, wherein the cell-adhesive hydrogel comprises collagen. The method of claim 8 or 9, wherein the cell adherent hydrogel comprises fibrin. The method according to any one of claims 8 to 10, wherein two or more layers of the first cell layer are formed. The step (1)
The method according to any one of claims 8 to 11, which is a step of placing the laminate having the second cell layer formed on the first cell layer on the support. The method according to any one of claims 8 to 12, wherein the first cell layer and the second cell layer are obtained by using a stimulus-responsive culture substrate. 13. The method of claim 13, wherein the stimulus-responsive culture substrate is a temperature-responsive culture substrate. The method according to any one of claims 8 to 14, wherein the endometrial stromal cells are primary endometrial stromal cells. The method according to any one of claims 8 to 15, wherein the endometrial epithelial cells are primary endometrial epithelial cells. (3) The method according to any one of claims 8 to 16, further comprising a step of applying a fertilized egg on the second cell layer. Endometrial-like tissue obtained by the method according to any one of claims 8 to 17.