JP7501051B2 - Cell structure containing small intestinal epithelial cell layer, its use, and its manufacturing method - Google Patents

Description

The present disclosure relates to a cellular structure comprising a small intestinal epithelial cell layer.
The present disclosure also relates to a method for evaluating the effect of a test substance on the small intestine using the cell structure.
The present disclosure also relates to a kit for evaluating the effect of a test substance on the small intestine, comprising the cell structure.
The present disclosure also relates to a method for producing said cell structure.
The present disclosure also relates to an activator of peristalsis of the small intestine or cellular structures.
The present disclosure also relates to an agent for inhibiting the proliferation of small intestinal epithelial cells.
The present disclosure also relates to an agent for promoting the maturation of small intestinal epithelial cells.

Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), can be induced to differentiate into target cells, and are expected to be applied in the field of regenerative medicine. Research is being conducted on methods to culture pluripotent stem cells and induce their differentiation to obtain structures called organoids that resemble human tissue.

In recent years, techniques have been developed to induce spontaneous cell structures through cell culture. In particular, there is a demand for cell structures that mimic in vitro intestinal peristalsis, an important function in the body that moves food and other substances.
For example, Patent Document 1 describes a method for mimicking peristaltic movement by physically causing intestinal epithelial cells attached onto a membrane to contract.

Therefore, attempts have been made to induce target tissues from embryonic stem cells. For example, Patent Document 2 describes that an intestinal tract-like cell structure obtained by adhesion culture of embryoid bodies obtained from mouse iPS cells exhibits peristaltic movement.

Patent Document 3 also describes a method for constructing an intestinal tract-like cell mass with an intramural nervous system, comprising the steps of (a) recovering undifferentiated embryonic stem cells, and inducing embryoid bodies by hanging drop culture of the recovered undifferentiated embryonic stem cells in a medium containing BDNF, and (b) attaching the embryoid bodies induced in step (a) onto a culture dish and further culturing them. Patent Document 3 also describes that the intestinal tract-like cell mass contains nerve cells and Cajal cells, and exhibits peristalsis-like movement. Patent Document 3 also discloses that this tissue is used to perform a toxicity test using tetrodotoxin.

Furthermore, Patent Document 4 describes a method for obtaining mature tissue with a vascular network by transplanting human intestinal organoids and neural crest cells into an immunodeficient animal. It also describes that this tissue has a peristaltic contraction function. There is also a description of drug responses using contraction movements.

Furthermore, Patent Documents 5 and 6 and Non-Patent Document 1 describe a method for obtaining a cell structure containing small intestinal epithelial cells, characterized in that the villus faces outward, useful components can be absorbed from the outside, and all three germ layers are contained, by culturing human ES cells or iPS cells on a cell culture substrate on which a pattern of cell culture regions is formed. Non-Patent Document 1 also describes that the obtained cell structure has peristaltic motility.

Patent No. 6122388 WO2010/143747 JP 2006-239169 A JP 2017-532964 A Patent No. 6151097 JP 2019-000014 A Patent No. 5923492 Patent No. 5881801 WO2013/061608 JP 2004-524516 A Patent No. 5447913

Uchida et al., JCI Insight Vol. 2 e86492 2017 Tanaka, Medical History Vol. 270, pp. 1070-1075, 2019 Takayama et al., Journal of Japanese Society of Gastroenterology, Vol. 98, pp. 922-934, 2001 Fujita et al. Japanese Pharmacological Journal Vol. 123, pp. 170-178, 2004 Salli et al., Experimental Neurology 188:351-364, 2004 Tokuyama et al., Experimental biology and medicine, vol. 235, pp. 649-657, 2010 Lee et al., Stem Cell Reports, Vol. 6, pp. 257-272, 2016 Mustata et al., Cell Reports, vol. 5, pp. 421-432, 2013 Harper et al., Proceeding of the National Academy of the Sciences USA, Vol. 108, pp. 10585-10590, 2011 Cirillo et al., Developmental Biology 168:395-405, 1995 Zachos et al., The Journal of Biological Chemistry, Vol. 291, pp. 3759-3766, 2016 Anderson et al., Gastroenterology 106:414-422, 1994 Matsuki et al., Plos One Vol. 8 e63053 2013 Current Methods in Cellular Neurobiology Volume 4, Jeffery Barker, John Wiley & Sons, Inc., 1983 Spence et al., Nature 470, 105-109, 2011 Hayashi et al. Aichi Prefectural Government Bulletin Vol. 61, pp. 31-38, 2010 Hasegawa et al. Aichi Prefectural Government Bulletin Vol. 64, pp. 23-31, 2014 Tatsuno et al. Yamaguchi Prefectural Environmental Health Center Bulletin No. 50, pp. 47-49, 2008 Chiken et al. Journal of Food Hygiene, Vol. 26, pp. 471-476, 1985 Kitawaki, J. Clinical Gynecology and Obstetrics, Vol. 71, pp. 45-52, 2017

Cell structures that mimic the peristaltic movement of the small intestine are expected to be used in the development of laxatives and laxatives, as well as in evaluating drug toxicity by assessing peristaltic movement, and there is a demand for the establishment of mass production technology.

However, for example, the method described in Patent Document 1 for physically causing intestinal epithelial cells to contract cannot be said to mimic the peristaltic movement of a living organism.

The tissue that causes peristalsis, as described in Patent Document 3, is a cell mass, and the arrangement and shape of the cells are non-uniform, which is an issue. Patent Document 3 also suggests that the rate of tissue that causes peristalsis is low.

In the method described in Patent Document 4, the operation for inducing tissue is difficult to carry out industrially, and a simpler method for inducing tissue that exhibits peristaltic movement was required. Furthermore, in addition to the laborious task of transplanting the tissue into an immunized animal, the method contains foreign animal components and is problematic from the standpoint of animal welfare. Furthermore, the epithelial cells are inward-facing, which poses a problem when used to evaluate absorption.

As described above, Patent Documents 5 and 6 and Non-Patent Document 1 describe methods for producing a cell structure containing small intestinal epithelial cells, which is characterized in that the villus faces outward, useful components can be absorbed from the outside, and cells derived from all three germ layers are contained. However, the methods described in these documents have a problem in that the yield of cell structures having peristaltic motility is low at about 5%.
Therefore, this specification discloses a technology for providing a cell structure having peristaltic motility similar to that of the small intestine, and a method for producing the same.

The present disclosure includes the following inventions.
(1) a small intestinal epithelial cell layer, and
tissues including Cajal cells, nerve cells and muscle cells,
The small intestinal epithelial cell layer contains α-fetoprotein-negative small intestinal epithelial cells.
Cellular structures.
(2) The cell structure is in a sac shape,
The small intestinal epithelial cell layer is present on the outer periphery of the cell structure with the villus layer facing outward.
A cell structure described in (1).
(3) The cell structure according to (1) or (2), wherein the proportion of α-fetoprotein-positive small intestinal epithelial cells among the small intestinal epithelial cells contained in the small intestinal epithelial cell layer is 8% or less.
(4) The cell structure described in any one of (1) to (3), wherein the muscle cells include desmin-positive and smooth muscle actin-positive smooth muscle cells.

(5) A cell structure described in any one of (1) to (4), which has the ability to spontaneously undergo peristaltic movement in the presence of one or more compounds selected from a histamine compound and a serotonin compound.
(6) The cell structure according to any one of (1) to (5), which is maintained in a medium containing a serum replacement component, as well as transferrin, insulin, progesterone, putrescine and selenite.
(7) The cell structure according to any one of (1) to (6), which is induced to differentiate from a pluripotent stem cell.
(8) The cell structure described in (7), wherein the pluripotent stem cells are derived from a human.

(9) A method for evaluating an effect of a test substance on the small intestine, comprising the steps of:
A step 11 of observing the cell structure according to any one of (1) to (8) in the presence of a test substance; and a step 12 of evaluating the effect of the test substance on the small intestine based on the observation results.
The method includes:
(10) The method according to (9), wherein step 11 is carried out in the presence of one or more compounds selected from a histamine compound and a serotonin compound.
(11) After the step 11, the method further comprises a step 13 of washing the cell structure with a liquid medium not containing the test substance,
After step 13, using the cell structure again in step 11;
The method according to (10), comprising:

(12) Small intestinal epithelial cell layer, and
tissues including Cajal cells, nerve cells and muscle cells,
The small intestinal epithelial cell layer contains α-fetoprotein-negative small intestinal epithelial cells.
A method for producing a cell structure, comprising the steps of:
21. Seeding stem cells onto a cell culture substrate having a pattern of cell adhesion sites on its surface;
Step 22: culturing the stem cells to induce differentiation into immature cell structures including a small intestinal epithelial cell layer;
and step 23 of culturing the immature cell structure in suspension in a medium containing transferrin, insulin, progesterone, putrescine and selenite to induce differentiation into the cell structure.
(13) The method according to (12), wherein the culture medium used in step 23 further contains a serum replacement component.

(14) A kit for evaluating the effect of a test substance on the small intestine, comprising a cell structure described in any one of (1) to (8).

(15) transferrin,
Insulin,
Progesterone and/or progestin,
An agent for activating peristalsis in the small intestine or cell structures, comprising putrescine and selenite as active ingredients.
(16) transferrin,
Insulin,
Progesterone and/or progestin,
An inhibitor of small intestinal epithelial cell proliferation, comprising putrescine and selenite as active ingredients.
(17) The inhibitor of small intestinal epithelial cell division according to (16), wherein the small intestinal epithelial cells are contained in a small intestine or a cell structure.

(18) transferrin,
Insulin,
Progesterone and/or progestin,
A maturation promoter for small intestinal epithelial cells, comprising putrescine and selenite as active ingredients.
(19) The agent for promoting maturation of small intestinal epithelial cells according to (18), wherein the small intestinal epithelial cells are contained in a small intestine or a cell structure.

The cellular structure according to one or more embodiments of the present disclosure has peristaltic motility similar to small intestinal tissue.
The cell structures according to one or more embodiments of the present disclosure also have characteristics similar to mature intestinal tissue.

According to the method according to one or more embodiments of the present disclosure, the cell structure can be used to evaluate the effect of a test substance, such as a drug, on the small intestine without relying on animal testing.

According to the method for producing a cell structure according to one or more embodiments of the present disclosure, a cell structure having peristaltic motility similar to that of small intestinal tissue can be produced in a simple and efficient manner.

The agent for activating peristalsis of the small intestine or cellular structure according to one or more embodiments of the present disclosure is capable of activating peristalsis of the small intestine or cellular structure in vivo or in vitro.
The agent for inhibiting the proliferation of small intestinal epithelial cells according to one or more embodiments of the present disclosure can inhibit the proliferation of small intestinal epithelial cells in vivo or in vitro.
The agent for promoting the maturation of small intestinal epithelial cells according to one or more embodiments of the present disclosure can promote the maturation of small intestinal epithelial cells in vivo or in vitro.

Fig. 1 is a schematic diagram of an embodiment of a cell culture substrate having a plurality of annular cell adhesion portions, in which the cell adhesion portions are the exposed surface of a support substrate, used in Experiment 1. Fig. 1(A) is a plan view of the cell culture substrate, and Fig. 1(B) is a schematic cross-sectional view taken along line A-A in Fig. 1(A). FIG. 2 illustrates the media used in Example A, Example B, and Comparative Examples AC. FIG. 3 shows the ratio of cell structures having peristaltic motility in Example A and Comparative Example A. 4 shows photographs of the cultures at each time point of the pattern culture of Comparative Example B and Comparative Example C. The scale bar indicates 500 μm. 5 shows the results of immunostaining of the cellular structures capable of peristaltic motility obtained in Example A. The results show the detection of small intestinal epithelial cells (CDX2 positive) with outward-facing villi (Villin positive), Cajal cells (c-kit positive), nerve cells (βIII tublin positive), and muscle cells (SMA positive). The scale bar indicates 40 μm. 6 shows the results of immunostaining of the cell structures having peristaltic motility among the cell structures obtained in Comparative Example A, and the lower part shows the results of immunostaining of the cell structures not having peristaltic motility among the cell structures obtained in Comparative Example A. Detection results of Cajal cells (c-kit positive) and muscle cells (SMA positive). The scale bar indicates 40 μm. 7 shows the results of immunostaining for E-cadherin and AFP for the cell structure capable of peristaltic motility obtained in Example A, the cell structure capable of peristaltic motility obtained in Example B, and the cell structure capable of peristaltic motility obtained in Comparative Example A. The scale bar indicates 40 μm. 8 shows the results of immunostaining for Ki67 (a dividing cell marker) and CDX2 (a small intestinal epithelial cell marker) for the cell structure having peristaltic motility obtained in Example A and the cell structure not having peristaltic motility obtained in Comparative Example A. The scale bar indicates 40 μm. 9 shows the results of immunostaining for PCNA (a dividing cell marker) and E-cadherin (a small intestinal epithelial cell marker) for the cell structure capable of peristaltic motility obtained in Example A, the cell structure capable of peristaltic motility obtained in Example B, and the cell structure capable of peristaltic motility obtained in Comparative Example A. The scale bar indicates 40 μm. 10 shows images of cells released into the medium during suspension culture of the pouch-like cell structures of Example A and Comparative Example A. The scale bar indicates 500 μm. 11 shows the results of immunostaining in Example C of Experiment 4. The scale bar indicates 200 μm. 12 shows the results of immunostaining for Desmin and SMA of a sample of a cell structure having peristaltic motility ability obtained in Example A in Experiment 7. The scale bar indicates 100 μm. 12 shows the results of immunostaining of Vimentin and SMA of a sample of a cell structure having peristaltic motility obtained in Example A in Experiment 7. The scale bar indicates 100 μm. 14 shows the results of immunostaining for Desmin and SMA of a sample of a cell structure not having peristaltic motility ability obtained in Comparative Example A in Experiment 7. The scale bar indicates 100 μm. 15 shows the results of immunostaining of Vimentin and SMA for the sample of the cell structure not having peristaltic motility obtained in Comparative Example A in Experiment 7. The scale bar indicates 100 μm.

1. Terminology
The terms "stem cells" and "cell culture substrate" used in this specification will now be explained.

<1.1. Stem Cells
The stem cells used in one or more embodiments of the present disclosure may be stem cells capable of differentiating into small intestinal epithelial cells, Cajal cells, nerve cells, and muscle cells, but are preferably endodermal cells (small intestinal epithelial cells etc.), stem cells capable of differentiating into ectodermal cells and mesodermal cells, and more preferably pluripotent stem cells. Pluripotent stem cells are particularly preferably embryonic stem cells (ES cells) or artificially generated stem cells. Pluripotent stem cells (iPS cells) are preferred.

The embryonic stem cells (ES cells) used in one or more embodiments of the present disclosure are preferably mammalian ES cells, and for example, ES cells derived from rodents such as mice or primates such as humans can be used. Particularly preferably, ES cells derived from mice or humans are used. ES cells refer to stem cell lines made from inner cell masses belonging to a part of an embryo at the blastocyst stage, which is an early stage of animal development, and can be proliferated almost indefinitely outside the body while maintaining the pluripotency to theoretically differentiate into all tissues. As ES cells, for example, cells in which a reporter gene has been introduced near the Pdx1 gene can be used to facilitate confirmation of the degree of differentiation. For example, 129/Sv-derived ES cell lines in which the LacZ gene has been incorporated into the Pdx1 locus or ES cell lines SK7 having a GFP reporter transgene under the control of the Pdx1 promoter can be used. Alternatively, the PH3 ES cell line having an mRFP1 reporter transgene under the control of an Hnf3β endoderm-specific enhancer fragment and a GFP reporter transgene under the control of a Pdx1 promoter can be used. In addition, SEES1, SEES2, SEES3, SEES4, SEES5, SEES6, or SEES7, which are ES cell lines established at the Department of Reproductive and Cellular Medicine Research at the National Center for Child Health and Development and disclosed in Akutsu H, et al. Regen Ther. 2015;1:18-29, or cell lines obtained by introducing additional genes into these ES cell lines can also be used.

The induced pluripotent stem cells (iPS cells) used in one or more embodiments of the present disclosure are cells having pluripotency obtained by reprogramming somatic cells. Several groups have succeeded in producing induced pluripotent stem cells, including the group of Professor Shinya Yamanaka at Kyoto University, the group of Rudolf Jaenisch at Massachusetts Institute of Technology, the group of James Thomson at the University of Wisconsin, and the group of Konrad Hochedlinger at Harvard University. For example, International Publication WO2007/069666 describes somatic cell nuclear reprogramming factors that include gene products of Oct family genes, Klf family genes, and Myc family genes, as well as somatic cell nuclear reprogramming factors that include gene products of Oct family genes, Klf family genes, Sox family genes, and Myc family genes, and further describes a method for producing induced pluripotent stem cells by nuclear reprogramming of somatic cells, which includes a step of contacting somatic cells with the nuclear reprogramming factors.

The type of somatic cells used to generate iPS cells is not particularly limited, and any somatic cells can be used. In other words, somatic cells in this disclosure include all cells other than germ cells that constitute the cells of a living organism, and may be differentiated somatic cells or undifferentiated stem cells. The origin of the somatic cells is not particularly limited and may be any of mammals, birds, fish, reptiles, and amphibians, but is preferably mammals (e.g., rodents such as mice, or primates such as humans), and is particularly preferably mice or humans. In addition, when human somatic cells are used, fetal, neonatal, or adult somatic cells may be used. Specific examples of somatic cells include fibroblasts (e.g., skin fibroblasts), epithelial cells (e.g., gastric epithelial cells, hepatic epithelial cells, alveolar epithelial cells), endothelial cells (e.g., blood vessels, lymphatic vessels), nerve cells (e.g., neurons, glial cells), pancreatic cells, blood cells, bone marrow cells, muscle cells (e.g., skeletal muscle cells, smooth muscle cells, cardiac myocytes), hepatic parenchymal cells, non-hepatic parenchymal cells, adipocytes, osteoblasts, cells that constitute periodontal tissue (e.g., periodontal ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), and cells that constitute the kidney, eye, and ear.

An iPS cell is a stem cell that has the ability to self-replicate over a long period of time under specific culture conditions (e.g., conditions for culturing ES cells) and has the ability to differentiate into ectoderm, mesoderm, and endoderm under specific differentiation-inducing conditions. In addition, the iPS cell in this disclosure may be a stem cell that has the ability to form a teratoma when transplanted into a test animal such as a mouse.

To produce iPS cells from somatic cells, first, at least one type of reprogramming gene is introduced into the somatic cells. The reprogramming gene is a gene that encodes a reprogramming factor that has the effect of reprogramming somatic cells to iPS cells. Specific examples of combinations of reprogramming genes include, but are not limited to, the following combinations.
(i) Oct gene, Klf gene, Sox gene, Myc gene (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40 largeT gene (iv) Oct gene, Klf gene, Sox gene

<1.2. Cell culture substrate>
The cell culture substrate used in the present disclosure comprises a pattern of cell adhesion sites on its surface.

The shape of the cell adhesion portion is not particularly limited, but may be, for example, a cell adhesion portion that encapsulates a non-cell adhesion portion, extends continuously or intermittently along the periphery of the non-cell adhesion portion, and surrounds the non-cell adhesion portion.

Other examples of the shape of the cell adhesion portion include polygons such as squares that do not include cell non-adhesive portions, circles, ellipses, etc., with circles being preferred.
In the following description, the area including the cell adhesion portion where the cell structure is cultured by adhesion is referred to as the "cell culture portion."

The cell culture substrate includes one or more cell culture sections, preferably two or more cell culture sections, on the surface of the cell culture substrate. The shapes and dimensions of the two or more cell culture sections included in one cell culture substrate may be the same or different.
The cell adhesion regions are typically arranged in islands among the non-cell adhesion regions.

When the cell culture section includes a non-cell-adhesive section, the non-cell-adhesive section included in the cell-adhesive section may be referred to as a "second non-cell-adhesive section" or a "central section" to distinguish it from a non-cell-adhesive section (a first non-cell-adhesive section, described below) that exists in a section other than the cell culture section.

In the following description, the portion of the cell culture substrate used in the present disclosure other than the cell adhesive portion and the cell non-adhesive portion may be referred to as the "support substrate." In other words, the cell culture substrate used in one or more embodiments of the present disclosure can be said to include a support substrate having a surface including a pattern of one or more cell adhesive portions.

<1.2.1. Support substrate, cell non-adhesive portion, and cell adhesive portion of cell culture substrate>
First, the characteristics of the support substrate and the characteristics other than the shapes of the cell non-adhesive portion and the cell adhesive portion will be described below.

The support substrate used for the cell culture substrate is not particularly limited as long as it is made of a material capable of forming a cell non-adhesive portion and a cell adhesive portion on its surface. Specific examples of the support substrate include inorganic materials such as glass, metal, ceramic, and silicon, and organic materials such as elastomers and plastics (e.g., polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, and polyvinyl chloride resin). In particular, it is preferable to use a glass substrate as the support substrate. The shape of the support substrate is not limited, and examples of the support substrate include flat shapes such as flat plates, flat membranes, films, and porous membranes, and three-dimensional shapes such as cylinders, stamps, multi-well plates, and microchannels.

In this disclosure, "cell adhesiveness" refers to the strength of cell adhesion, i.e., the ease with which cells adhere. A "cell adhesive portion" refers to an area on a surface with good cell adhesiveness, and a "cell non-adhesive portion" refers to an area on a surface with poor cell adhesiveness. Therefore, when cells are seeded on a surface on which cell adhesive portions and non-cell adhesive portions are arranged in a specific pattern, the cells adhere to the cell adhesive portions but do not adhere to the non-cell adhesive portions, resulting in the cells being arranged in a pattern on the surface of the cell culture substrate.

The "cell adhesion portion" is defined as a portion that adheres to cells to be actually cultured, preferably stem cells, when seeded on the cell culture substrate, and the "cell non-adhesive portion" is defined as a portion that does not adhere to cells to be actually cultured, preferably stem cells, when seeded. When cells are seeded on the cell culture substrate, the surface of the cell culture substrate may be coated with a protein or the like to enhance cell adhesion. Specific examples of "stem cells" are as described in this specification. The cell non-adhesive portion may be covered by cells that have adhered to and proliferated on the cell adhesion portion.

As an index for judging whether a cell adhesion portion is a cell adhesion portion or a cell non-adhesion portion, the cell adhesion spreading rate when cells are actually cultured can be used. The surface of the cell adhesion portion having cell adhesiveness is preferably a surface with a cell adhesion spreading rate of 60% or more, and more preferably a surface with a cell adhesion spreading rate of 80% or more. When the cell adhesion spreading rate is high, cells can be cultured efficiently. The cell adhesion spreading rate in the present disclosure is defined as the ratio of cells that are adherent and spread at the time when cells to be cultured at a seeding density of 4000 cells/ ^cm2 or more and less than 30000 cells/ ^cm2 are seeded on the measurement target surface, stored in an incubator at 37°C and a _CO2 concentration of 5%, and cultured for 14.5 hours ({(number of adherent cells)/(number of seeded cells)}×100(%)).

In the above measurement, cells are seeded on the surface to be measured by suspending them in DMEM medium containing 10% FBS, and then slowly shaking the surface to be measured on which the cells have been seeded so that the cells are distributed as uniformly as possible. Furthermore, the cell adhesion spreading rate is measured after replacing the medium immediately before the measurement to remove unattached cells. In measuring the cell adhesion spreading rate, the measurement points are those excluding points where the cell density is likely to be specific (for example, the center of a specified area where the cell density is likely to be high, and the periphery of a specified area where the cell density is likely to be low).

The cell adhesion portion may be a region where a cell adhesion layer is formed on the surface of the support substrate, or, if the surface of the support substrate is cell adhesive (for example, the surface of a glass substrate), may be a region where the surface of the support substrate is exposed, but is preferably a region where the cell adhesive surface of the support substrate is exposed. The cell non-adhesive portion may be a region where a cell non-adhesive layer is formed on the surface of the support substrate. The cell adhesion portion and the cell non-adhesive portion can be formed by various materials and methods. Preferably, the cell non-adhesive portion is a portion where the surface of the support substrate is covered with a cell non-adhesive layer such as a layer containing a hydrophilic organic compound such as a hydrophilic polymer. The average thickness of the cell non-adhesive layer constituting the cell non-adhesive portion is preferably 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, more preferably 1 nm to 10 μm, and most preferably 1.5 nm to 1 μm. If the average thickness is 0.8 nm or more, it is preferable because the adsorption of proteins and the adhesion of cells are less affected by the region of the support substrate that is not covered by the cell non-adhesive layer. In addition, if the average thickness is 500 μm or less, coating is relatively easy. In particular, when the cell non-adhesive layer is formed from a polyethylene glycol layer, the thickness can be, for example, 5 nm to 10 nm. Specific examples of hydrophilic organic compounds are described below.

As a method for producing a cell culture substrate containing polyethylene glycol (PEG) as a hydrophilic polymer as a cell non-adhesive layer, the method described in Patent Document 6 can be used.
Particularly preferred embodiments of the method for forming the cell adhesive portion and the cell non-adhesive portion include the following two embodiments.

In the first embodiment, a cell-nonadhesive layer is formed on the surface of the support substrate, and then a predetermined treatment is applied to a portion of the cell-nonadhesive layer to develop cell adhesion and form a cell-adhesive portion. Specifically, a cell-nonadhesive hydrophilic film containing a hydrophilic organic compound such as a hydrophilic polymer is formed on the surface of the support substrate as a cell-nonadhesive layer, and then a portion of the hydrophilic film that is the cell-nonadhesive layer is selectively subjected to an oxidation treatment and/or decomposition treatment to modify the portion into a cell-adhesive portion having cell adhesion. In this embodiment, a cell-nonadhesive hydrophilic film is formed, and then an oxidation treatment and/or decomposition treatment is applied to the portion where cell adhesion is desired, thereby converting the portion into a portion having cell adhesion and forming the cell-adhesive portion. In the cell culture substrate formed according to the first embodiment, the non-cell-adhesive portion is a portion of the surface of the support substrate covered with a layer containing a hydrophilic organic compound such as a hydrophilic polymer, and the cell-adhesive portion is a portion of the surface of the support substrate where the layer containing a hydrophilic organic compound such as a hydrophilic polymer has been removed by oxidation treatment and/or decomposition treatment to expose the surface of the support substrate, or a portion of the layer containing a hydrophilic organic compound such as a hydrophilic polymer that has been subjected to oxidation treatment and/or decomposition treatment and is covered with a layer (= cell-adhesive layer) that has been modified to have cell-adhesive properties.

The second form is a form in which the cell adhesion portion and the cell non-adhesive portion are formed depending on the density of the organic compound on the surface of the support substrate. In the cell culture substrate formed by the second form, the cell adhesion portion is a surface with a low density of hydrophilic organic compounds such as hydrophilic polymers (including cases where no hydrophilic organic compounds are present), and the cell non-adhesive portion is a surface with a high density of hydrophilic organic compounds such as hydrophilic polymers. The second form utilizes the fact that the surface of a support substrate containing a high density of hydrophilic organic compounds such as hydrophilic polymers has cell non-adhesive properties, while the surface of a support substrate with a low density of the compounds has cell adhesive properties. When a first region to which the compound is easily bound and a second region to which the compound is not easily bound are provided on the surface of the support substrate, and a film of the compound is formed on the surface of the substrate, the first region becomes a cell non-adhesive portion and the second region becomes a cell adhesive region. Alternatively, a part of the surface of the support substrate can be selectively masked with a photoresist or the like, a film of the hydrophilic organic compound is formed on the unmasked region to form a cell non-adhesive portion, and then the masking is removed to expose the surface of the support substrate, thereby forming a cell adhesive portion.

In addition, the above embodiment is not limited to the above, and a support substrate having a cell-nonadhesive surface (which may be the surface of a cell-nonadhesive layer) may be prepared, and a part of the surface may be patterned and coated with a cell-adhesive protein such as collagen or fibronectin to form a cell-adhesive pattern. Alternatively, a support substrate having a cell-adhesive surface (which may be the surface of a cell-adhesive layer) may be prepared, and a part of the surface may be coated with a cell-nonadhesive resin such as silicone rubber (e.g., Keiju (registered trademark) manufactured by Mitsubishi Chemical), and the remaining part may be made into a cell-adhesive pattern. Alternatively, a support substrate having a conductive layer of a predetermined pattern on its surface may be prepared, and a cell-nonadhesive layer may be laminated on the surface of the support substrate, and the cell-nonadhesive layer covering the conductive layer may be peeled off by applying a voltage to the conductive layer, and the exposed conductive layer may be made into a cell-adhesive portion (see, for example, JP 2012-120443 A and JP 2013-179910 A).

Below, we will explain the above first and second forms in order, in which cell adhesive and non-adhesive portions are formed on the surface of a support substrate to produce a cell culture substrate having a surface including cell adhesive and non-adhesive portions.

First, the first embodiment will be described.
In the first embodiment, a hydrophilic film containing a hydrophilic organic compound, preferably a hydrophilic polymer, is first provided as a cell non-adhesive layer on the surface of a support substrate. The hydrophilic film is a thin film having water-solubility or water-swellability, and is not particularly limited as long as it has cell non-adhesive properties before oxidation and/or decomposition, and the exposed surface of the support substrate after oxidation and/or decomposition, or the surface of the thin film modified by oxidation treatment and/or decomposition treatment, exhibits cell adhesive properties.

When the cell non-adhesive layer is a hydrophilic film formed by a hydrophilic organic compound, it is preferable to provide a bonding layer between the surface of the support substrate and the hydrophilic film as necessary. The bonding layer is preferably a layer containing a material having a functional group (bonding functional group) that can bond with the functional group of the organic compound of the hydrophilic film. Examples of combinations of the bonding functional group of the material of the bonding layer and the functional group of the hydrophilic organic compound include epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde, etc. In each combination, either one may be the functional group on the bonding layer side. In these methods, before coating with the hydrophilic organic compound, a bonding layer is formed on the support substrate from a material having a predetermined functional group. In the cell non-adhesive layer, the water contact angle of the surface of the bonding layer before forming a thin film of the hydrophilic organic compound is typically 45° or more, preferably 47° or more, when a silane coupling agent having an epoxy group at the end is used as the material having the bonding functional group. Such a bonding layer can be obtained by forming a coating of a material having a bonding functional group on the surface of a supporting substrate.

Examples of hydrophilic organic compounds include hydrophilic polymers (including hydrophilic oligomers), water-soluble organic compounds, surface-active substances, and amphiphilic substances, with hydrophilic polymers being particularly preferred.

Specific examples of hydrophilic polymers include polyalkylene glycols, zwitterionic polymers having phospholipid polar groups, polyacrylamides, polyacrylic acids, polymethacrylic acids, polyvinyl alcohols, polysaccharides, and the like. These specific examples of hydrophilic polymers also include those in the form of derivatives. The molecular shape of the hydrophilic polymer can be linear, branched, or a dendrimer, and the like.

Specific examples of polyalkylene glycols include polyethylene glycol, polypropylene glycol, and copolymers of polyethylene glycol and polypropylene glycol, such as Pluronic F108 and Pluronic F127.

Specific examples of amphoteric polymers having a phospholipid polar group include poly(methacryloyloxyethyl phosphorylcholine) (=MPC polymer), copolymers of methacryloyloxyethyl phosphorylcholine and acrylic monomers, and the like.
A specific example of polyacrylamide is poly(N-isopropylacrylamide).
A specific example of polymethacrylic acid is poly(2-hydroxyethyl methacrylate).
Specific examples of polysaccharides include dextran and heparin.

It is desirable that the surface of the support substrate having the non-cell-adhesive layer has high cell-non-adhesive properties when covered with the non-cell-adhesive layer, and that after the oxidation and/or decomposition treatment of the non-cell-adhesive layer, the exposed surface of the support substrate, or the surface of the layer formed by modifying the non-cell-adhesive layer through the oxidation and/or decomposition treatment, exhibits cell adhesive properties.

As the hydrophilic polymer, polyethylene glycol (PEG) is particularly preferred. PEG contains at least an ethylene glycol chain (EG chain) consisting of one or more ethylene glycol units ((CH ₂ ) ₂ -O), and may be linear or branched. The ethylene glycol chain is, for example, represented by the following formula:
-(( _CH2 ) ₂ -O) _m-
(m is an integer indicating the degree of polymerization)
The symbol m is preferably an integer of 1 to 13, and more preferably an integer of 1 to 10.

PEG also includes ethylene glycol oligomers. PEG also includes those into which functional groups have been introduced. Examples of functional groups include epoxy groups, carboxyl groups, N-hydroxysuccinimide groups, carbodiimide groups, amino groups, glutaraldehyde groups, and (meth)acryloyl groups. The functional groups are preferably introduced at the termini, optionally via a linker. Examples of PEG into which functional groups have been introduced include PEG (meth)acrylate and PEG di(meth)acrylate.

The cell adhesion portion can be formed by subjecting a non-cell adhesion layer containing a hydrophilic organic compound formed on the surface of the support substrate to an oxidation treatment and/or decomposition treatment to expose the surface of the support substrate having cell adhesion properties, or by modifying the non-cell adhesion layer to convert it into a cell adhesion layer.
In the present disclosure, the term "oxidation" is used in the narrow sense to mean a reaction in which an organic compound reacts with oxygen to increase the oxygen content compared to before the reaction.

In this disclosure, "decomposition" refers to a change in which the bonds of an organic compound are broken to produce two or more organic compounds from one type of organic compound. "Decomposition treatment" typically includes, but is not limited to, decomposition by oxidation treatment and decomposition by ultraviolet light irradiation. When the "decomposition treatment" is decomposition involving oxidation (i.e., oxidative decomposition), the "decomposition treatment" and the "oxidation treatment" refer to the same treatment. In addition, decomposition and removal of the non-adhesive cell layer is also included in the "decomposition treatment".

Decomposition by UV irradiation refers to an organic compound absorbing UV light, going through an excited state, and then decomposing. When UV light is irradiated into a system in which an organic compound exists together with molecular species containing oxygen (oxygen, water, etc.), in addition to the UV light being absorbed by the compound and causing decomposition, the molecular species may become activated and react with the organic compound. The latter reaction can be classified as "oxidation." And the reaction in which an organic compound is decomposed by oxidation caused by activated molecular species can be classified as "decomposition by oxidation" rather than "decomposition by UV irradiation."

As described above, "oxidation treatment" and "decomposition treatment" may overlap as operations, and it is not possible to clearly distinguish between the two. Therefore, in this specification, the term "oxidation treatment and/or decomposition treatment" is used.

Next, the second embodiment will be described. In the cell culture substrate formed by the second embodiment, the cell adhesion portion of the surface of the support substrate is a surface with a low density of hydrophilic organic compounds such as hydrophilic polymers (including cases where no hydrophilic organic compounds are present), and the cell non-adhesive portion is a surface with a high density of hydrophilic organic compounds. In other words, the density of the hydrophilic organic compounds differs between the cell adhesion portion and the cell non-adhesive portion. The higher the density, the more difficult it tends to be for cells to adhere. In the cell adhesion portion, the density of the hydrophilic organic compounds is low enough to allow cells to adhere. Preferred examples of hydrophilic organic compounds and hydrophilic polymers are as described above for the first embodiment.

In the second embodiment, when the cell adhesion portion and the cell non-adhesion portion are formed by a hydrophilic film with controlled density, it is preferable to form a binding layer on the support substrate as necessary to increase adhesion to the support substrate, and then form a hydrophilic film made of a hydrophilic organic compound. The binding layer is preferably a layer containing a material containing a functional group (binding functional group) that can bind to a functional group of the hydrophilic organic compound. Examples of combinations of the functional group of the material of the binding layer and the functional group of the hydrophilic organic compound include epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde, etc. In each combination, either one may be the functional group on the binding layer side. In these methods, a binding layer is formed on the support substrate by a material having a predetermined functional group before coating with a hydrophilic material. The density of the material in the binding layer is an important factor that determines the binding force. The density can be easily evaluated using the contact angle of water on the surface of the binding layer as an index. The water contact angle was measured 30 seconds after pure water was dropped from a microsyringe using a CA-Z manufactured by Kyowa Interface Science Co., Ltd.

The density of the material having the binding functional group in the binding layer of the cell adhesion portion is low. In the case where a silane coupling agent having an epoxy group at the end is used as the material having the binding functional group constituting the binding layer, the water contact angle of the surface of the binding layer in the cell adhesion portion before forming a thin film of a hydrophilic organic compound is typically 10° to 43°, preferably 15° to 40°. Examples of the method of forming such a binding layer include a method of forming a coating (binding layer) of a material having a binding functional group on the surface of a support substrate, and then oxidizing and/or decomposing the surface of the binding layer. Examples of the method of oxidizing and/or decomposing the binding layer surface include a method of irradiating the binding layer surface with ultraviolet light, a method of photocatalysis, and a method of treatment with an oxidizing agent. The entire surface of the binding layer may be oxidized and/or decomposed, or may be partially treated. Partial treatment can be performed by using a mask such as a photomask or a stencil mask, or by using a stamp. In addition, the oxidation treatment and/or decomposition treatment may be performed by a direct drawing method such as a method using a laser such as an ultraviolet laser. The same conditions as those for the method of forming a cell adhesion site by oxidation and/or decomposition of a hydrophilic film can be applied. A thin film of a hydrophilic organic compound can be formed on the binding layer thus formed to form a cell adhesion site.

The density of the material having binding functional groups in the binding layer of the non-cell-adhesive portion is high. In the case of using a silane coupling agent having an epoxy group at the end as the material having binding functional groups, the water contact angle of the surface of the binding layer in the non-cell-adhesive portion before forming a thin film of a hydrophilic organic compound is typically 45° or more, preferably 47° or more. Such a binding layer can be obtained by forming a coating of a material having binding functional groups on the surface of the support substrate. When the surface of the binding layer is partially oxidized and/or decomposed, the remaining portion that is not treated becomes a binding layer having the above water contact angle. A thin film of a hydrophilic organic compound is formed on the binding layer thus formed, thereby forming a non-cell-adhesive layer.

In the second form, a portion of the surface of the support substrate may be selectively masked with a photosensitive photoresist or the like, a film of the hydrophilic organic compound may be formed in the unmasked area to form a non-cell adhesive portion, and then the masking may be removed to expose the surface of the support substrate to form a cell adhesive portion.
Next, the characteristics of the cell adhesive portion and the cell non-adhesive portion formed by the above first or second embodiment, or by other methods, will be further described.

The carbon content of the cell adhesion portion (including the binding layer, if present) is preferably lower than the carbon content of the cell non-adhesion portion (including the binding layer, if present). Specifically, the carbon content of the cell adhesion portion is preferably 20-99% of the carbon content of the cell non-adhesion portion. Being within this range is particularly suitable when the thickness of the hydrophilic organic compound layer contained in the cell adhesion portion and the cell non-adhesion portion (the sum of the thickness of the binding layer and the thickness of the hydrophilic film, if a binding layer is present) is 10 μm or less. "Carbon content (atomic concentration, %)" is defined as follows:

In addition, the percentage of carbon bound to oxygen in the cell adhesion portion (including the binding layer if present) is preferably smaller than the percentage of carbon bound to oxygen in the non-cell adhesion portion (including the binding layer if present). Specifically, the percentage of carbon bound to oxygen in the cell adhesion portion is preferably 35 to 99% of the percentage of carbon bound to oxygen in the non-cell adhesion portion. This range is particularly suitable when the thickness of the hydrophilic film (the sum of the thickness of the binding layer and the thickness of the hydrophilic film if a binding layer is present) is 10 μm or less. The "percentage of carbon bound to oxygen (atomic concentration, %)" is defined as follows:

Methods for evaluating the hydrophilic organic compound layer (including the binding layer, if present) contained in the cell adhesion portion and the cell non-adhesion portion can include contact angle measurement, ellipsometry, atomic force microscope observation, electron microscope observation, Auger electron spectroscopy, X-ray photoelectron spectroscopy, various mass spectrometry, and the like. Of these methods, the most quantitative is X-ray photoelectron spectroscopy (XPS/ESCA). This measurement method determines a relative quantitative value, which is generally calculated as an element concentration (atomic concentration, %). The X-ray photoelectron spectroscopy analysis method in this disclosure will be described in detail below.

The "carbon amount" of the cell adhesion portion and the cell non-adhesion portion is defined as "the carbon amount determined from the analysis value of the C1s peak obtained using an X-ray photoelectron spectroscopy device." Furthermore, in this disclosure, the "proportion of carbon bonded to oxygen" of the cell adhesion portion and the cell non-adhesion portion is defined as "the proportion of carbon bonded to oxygen determined from the analysis value of the C1s peak obtained using an X-ray photoelectron spectroscopy device." Specific measurements can be performed as described in JP 2007-312736 A.

<1.2.2. Preferred examples of the shape of the cell adhesion portion>
As described above, an example of the shape of the cell adhesion part is a polygon such as a square not including a cell non-adhesive part, a circle, an ellipse, etc., and a circle is preferable. In the case of a circle, the diameter can be preferably a diameter that satisfies the above-mentioned area range, and specifically, the diameter of the circle can be 350 μm or more, preferably 800 μm or more, preferably 1000 μm or more, preferably 1200 μm or more, more preferably 1500 μm or more, preferably 6000 μm or less, more preferably 4000 μm or less, even more preferably 3000 μm or less, and even more preferably 2000 μm or less. In this example, the cell culture part consists of only the cell adhesion part.

Another example of the shape of the cell adhesive portion can be a cell adhesive portion that contains the non-cell adhesive portion, extends continuously or intermittently along the periphery of the non-cell adhesive portion, and surrounds the non-cell adhesive portion. In this example, the cell culture portion is composed of the non-cell adhesive portion and the cell adhesive portion that surrounds it. The characteristics of the cell culture substrate having the cell culture portion according to this example will be described below with reference to FIG. 1.
A cell culture substrate 1 , which is an example of the present disclosure, has a surface S including a cell culture portion 20 .

The cell culture section 20 includes a cell non-adhesive section (central section) 21 and a cell adhesive section 22 that extends continuously or intermittently along the periphery P of the cell non-adhesive section 21 (FIG. 1 shows an example in which the cell adhesive section extends continuously) and surrounds the cell non-adhesive section 21. This embodiment is an example in which one or more cell culture sections 20 are included on the surface S of the cell culture substrate 1, and each of the one or more cell culture sections 20 has the above-mentioned characteristics.

In the example shown in FIG. 1, one or more cell culture sections 20 are scattered in an island shape in the cell non-adhesive section 10. In this example, the cell non-adhesive section 10 may be referred to as the "first cell non-adhesive section," and the cell non-adhesive section 21 of the cell adhesive section 20 may be referred to as the "second cell non-adhesive section." In the following description, the "cell non-adhesive section 21" may be referred to as the "central section 21" or the "central section 21 which is a cell non-adhesive section."

The portion of the cell culture substrate 1 on which the first cell non-adhesive portion 10 and the cell adhesive portion 20 are arranged is referred to as the "support substrate 30."

In the example shown in FIG. 1, the first cell non-adhesive portion 10 and the central portion 21, which is the second cell non-adhesive portion, are the surfaces of the first cell non-adhesive layer 10A and the second cell non-adhesive layer 21A laminated on the surface of the support substrate 30.

In the example shown in FIG. 1, the cell adhesion portion 22 is the exposed surface of the support substrate 30. Although not shown, the cell adhesion portion 22 may be the surface of a cell adhesion layer laminated on the surface of the support substrate 30.

For ease of explanation, FIG. 1(B) emphasizes the thickness of the cell non-adhesive layer 10A and the cell non-adhesive layer 21A, as well as the step between the cell adhesive portion 22 and the cell non-adhesive layer 10A or the cell non-adhesive layer 21A. However, the thickness and step are sufficiently small compared to the dimensions of the cells and cell structures to be cultured, so that the surface S including one or more cell culture portions 20 can support cells as a substantially flat surface.

In FIG. 1(B), an example is shown in which the support substrate 30 is in direct contact with the first non-cell adhesive layer 10A and the second non-cell adhesive layer 21A, but as described above, a bonding layer may be interposed between them.

Specific examples and manufacturing methods of the support substrate 30, the first cell non-adhesive portion 10, the first cell non-adhesive layer 10A, the second cell non-adhesive portion 21, the second cell non-adhesive layer 21A, and the cell adhesive portion 22 are as described above.

When stem cells are cultured on a cell culture substrate 1 having such a structure, the stem cells adhere to and grow in the cell adhesion portion 22 surrounding the cell non-adhesive portion (central portion) 21, forming an aggregation portion in which the cells are densely aggregated, and in the formed cell aggregation portion, the cells can differentiate into small intestinal epithelial cells that express trophectoderm cell markers, and a bag-like cell structure (tissue) is easily formed. The obtained cell structure contains small intestinal epithelial cells and functions as an intestinal organoid. When differentiation of stem cells is induced using a cell culture substrate having the above structure, the cell structure containing small intestinal epithelial cells can be released and collected from the cell culture substrate in a relatively short time.

The shape and dimensions of the cell culture section 20 are not particularly limited, but a preferred embodiment has the following features:

Let L be the straight line that passes through the midpoint C of the two most distant points A1, A2 that face each other across the central portion 21 on the inner circumference Q of the cell adhesion portion 22. Let X be the distance between the two intersection points A3, A4 of this line L and the inner circumference Q of the cell adhesion portion 22. Let W be the width of the cell adhesion portion 22 in the direction along the line L that passes through the midpoint C.

The distance X is preferably more than 80 μm and not more than 880 μm, more preferably 180 μm or more and not more than 880 μm, particularly preferably 180 μm or more and not more than 600 μm, particularly preferably 180 μm or more and not more than 500 μm. If the distance X is too small, the central portion 21 is immediately covered by the cells during growth culture, and it is difficult to obtain a specific bag-like structure on the outer periphery of the cell structure. On the other hand, if the distance X is too large, it takes a long time for the cells to grow and completely cover the central portion 21, and the production efficiency of the cell structure decreases. When the distance X is within the above range, a bag-like cell structure can be cultured with high yield in a relatively short time. The distance X refers to the diameter of the circle when the shape of the central portion 21 is a circle as shown in FIG. 1, and when the circle is a perfect circle, the distance X is the same regardless of the straight line L. Although not shown, when the central portion 21 is rectangular, the distance X is maximum when the straight line L is in the diagonal direction and minimum when the straight line L is in the short direction. In this disclosure, preferably, the distance X is within the above range over the entire circumference (i.e., for all straight lines L).

The width W is preferably more than 30 μm and not more than 400 μm, more preferably 40 μm or more and not more than 400 μm, and particularly preferably 60 μm or more and not more than 300 μm. If the width W is too small, there is a problem that the cells are easily peeled off during culture. In addition, in order to induce a bag-shaped cell structure, it is desirable for a plurality of cells to adhere to the cell adhesion portion 22 in the width direction to form an aggregate portion, and therefore a larger width W is preferable, and therefore the width W is preferably 40 μm or more, and more preferably 60 μm or more, as described above. On the other hand, if the width W is too large, the density of the cells adhered to the cell adhesion portion 22 is likely to be biased, making it difficult to form a uniform cell aggregate portion in the width direction, and making it difficult to obtain a cell structure with a uniform structure. When the width W is within the above range, the cell structure can be cultured with a high yield in a relatively short time. In the present disclosure, the width W is preferably within the above range over the entire circumference (i.e., for all straight lines L).

The ratio X/W is preferably 0.5 or more, more preferably 1.0 or more, more preferably 1.3 or more, and preferably 20.0 or less, more preferably 15.0 or less, more preferably 10.0 or less. When the ratio X/W is within the above range, the cell structure can be cultured with high yield in a relatively short time. In the present disclosure, the ratio X/W is preferably within the above range over the entire circumference (i.e., for all straight lines L).

In FIG. 1, the central portion 21 is circular, and the cell adhesion portion 22 is annular in shape, concentrically surrounding the circular central portion 21, and is highly symmetrical, which is particularly preferable for obtaining a uniform cell structure. However, the present invention is not limited to this example, and the central portion 21 may be rectangular (square or oblong), and the cell adhesion portion 22 may have an inner and outer rectangular shape. Although not shown, the central portion may be elliptical, and the cell adhesion portion may have an elliptical annular shape extending along the central portion. In the above example, the inner and outer shapes of the cell adhesion portion are similar, but the present invention is not limited to this example, and for example, the inner shape of the cell adhesion portion (i.e., the outer shape of the central portion) may be polygonal, such as a rectangle, and the outer shape of the cell culture portion may be circular or elliptical, or conversely, the inner shape of the cell adhesion portion (i.e., the outer shape of the central portion) may be circular or elliptical, and the outer shape of the cell culture portion may be polygonal, such as a rectangle. The central portion 21 may also be semicircular.

In the example shown in FIG. 1, the cell adhesion portion 22 extends continuously along the periphery P of the central portion 21, which is the second cell non-adhesive portion, and surrounds the central portion 21 over the entire circumference. However, the cell adhesion portion may extend intermittently. Even in such a structure, the cells adhered to the cell adhesion portion 22 can form a tissue that connects the cut portions of the cell adhesion portion 22 through proliferation. In an embodiment in which the cell adhesion portion 22 extends intermittently along the periphery P of the central portion 21, each interrupted portion has a length of preferably less than half, more preferably less than a quarter, more preferably less than a sixth, and more preferably less than an eighth of the entire circumference of the periphery P of the central portion 21, and when multiple interrupted portions are included, the total length of the interrupted portions is preferably less than half, more preferably less than a quarter, more preferably less than a sixth, and more preferably less than an eighth of the entire circumference of the periphery P of the central portion 21.

In the cell culture substrate used in the present disclosure, the cell adhesive portion extends so as to surround the non-cell adhesive central portion, so that when cells adhere and proliferate thereon, the cells become dense, which facilitates differentiation into cells having the properties of trophectoderm cells, and the proliferated cells are easily layered. As a result, a bag-shaped cell structure can be efficiently obtained in which cells having the properties of trophectoderm cells are distributed on the periphery.

When multiple cell culture sections 20 are present, as in the cell culture substrate 1, they are isolated from each other, and are preferably spaced apart by at least 0.20 mm, and more preferably at least 0.30 mm. By isolating each cell culture section 20 by at least a certain distance, the cells in each cell culture section 20 are cultured uniformly at a certain interval without forming intercellular bonds with the cells in the adjacent cell culture sections 20, allowing the construction of a highly reproducible experimental system.

For example, Non-Patent Document 2 describes how stem cells are adherently cultured on a cell culture substrate that includes a pattern (ring pattern) of cell adhesion parts as shown in Figure 1 to obtain a bag-shaped cell structure.

2. Method for producing cell structure
Disclosed herein are
Small intestinal epithelial cell layer, and
tissues including Cajal cells, nerve cells and muscle cells,
The small intestinal epithelial cell layer contains α-fetoprotein-negative small intestinal epithelial cells.
The method for producing a cell structure includes the steps of:
21. Seeding stem cells onto a cell culture substrate having a pattern of cell adhesion sites on its surface;
Step 22: culturing the stem cells to induce differentiation into immature cell structures including a small intestinal epithelial cell layer;
The method is characterized in that it includes a step 23 of floating-culture the immature cell structure in a medium containing transferrin, insulin, progesterone, putrescine and selenite to induce differentiation into the cell structure.

The method according to the present disclosure is preferably capable of producing a cell structure that is characterized by (1) having peristaltic motility, (2) containing mature small intestinal epithelial cells that are negative for the expression of alpha-fetoprotein (AFP), an indicator of immaturity, and (3) having developed muscle tissue.

(1) Peristaltic Motility Peristaltic movement in the living body occurs when pacemaker cells, Cajal cells, receive signals from nerve cells or hormones and then stimulate muscle cells to cause contraction, as described in Non-Patent Documents 3 and 4. Therefore, in order to cause peristaltic movement, it is necessary to form connections between nerve cells and muscle cells and connections between Cajal cells and muscle cells.

The presence of c-kit and/or S100 positive Cajal cells is described in the above-mentioned Non-Patent Document 1. The same document also describes the difference in the development of nerve cells as a difference between tissues in which peristalsis occurs and those in which peristalsis does not occur. However, up to now, there has been no known method for inducing a cellular structure that contains Cajal cells, nerve cells, and muscle cells and has peristaltic motility.

As shown in the Examples of this specification, muscle cells are observed even in tissues that do not have peristaltic motility. However, in tissues that do not have peristaltic motility, the number of Cajal cells is small, and it is thought that the peristaltic rhythm cannot be controlled via nerve cells.
It is believed that in order to obtain a cellular structure capable of peristalsis, it is necessary to develop nerve cells and Cajal cells.

In the method according to the present disclosure, in step 22, adhesion culture (pattern culture) is performed on a cell culture substrate, and a pouch-shaped immature cell structure containing a small intestinal epithelial cell layer is induced. Then, in step 23, the immature cell structure is suspension cultured in a medium containing transferrin, insulin, progesterone, putrescine, and selenite. The factors in the medium change the properties, growth, and proliferation of the cells, promoting the development of tissues containing Cajal cells, nerve cells, and muscle cells that enable peristalsis.

Transferrin, insulin, progesterone, putrescine and selenite used in the suspension culture in step 23 are known as factors of "N-2 supplement."

Patent Document 7 describes that N-2 supplements promote the growth of nerve cells, particularly in an adherent culture system. This document describes an example in which small intestinal cells obtained from fetal rats are cultured in a medium containing N-2 supplements to obtain enteric nerves, which is thought to promote their development and differentiation.
Furthermore, Non-Patent Documents 5 and 6 report cases in which serotonergic neurons were induced to differentiate from mouse ES cells using a medium containing N-2 supplement.

However, there is no suggestion in the prior art that the development of tissues including Cajal cells, nerve cells, and muscle cells that enable peristalsis is promoted by culturing immature cell structures including a small intestinal epithelial cell layer in a suspension culture medium containing transferrin, insulin, progesterone, putrescine, and selenite.

(2) Reduction in the expression of alpha-fetoprotein (AFP), an indicator of juvenility The cell structure produced by the method according to the present disclosure is further characterized in that it contains small intestinal epithelial cells that are negative for AFP, an indicator of juvenility, in the small intestinal epithelial cell layer.

In step 23 of the method according to the present disclosure, the immature cell structure containing the small intestinal epithelial cell layer obtained in step 22 is cultured in suspension in a medium containing transferrin, insulin, progesterone, putrescine and selenite, thereby promoting the development of small intestinal epithelial cells.

In the adherent culture on the cell culture substrate in step 22 of the method according to the present disclosure, the stem cells aggregate and differentiate into immature small intestinal epithelial cells, which form a pouch-like structure (immature cell structure). It has been found that the small intestinal epithelial cell layer of this immature cell structure also has properties as a trophectoderm.

Non-Patent Document 7 describes how immature small intestinal epithelial cells may have properties of both CDX2-positive endodermal cells and trophectoderm cells.

In fact, Non-Patent Document 8 describes the preparation of pouch-like cell structures by culturing intestinal stem cells obtained from mouse fetuses or newborns, and shows that trophectoderm markers Trop2 and Connexin43 remain in epithelial cells at early juvenile stages.

In addition, it has been described in Non-Patent Documents 9 and 10 that alpha-fetoprotein (AFP), which is mainly expressed in the liver, is also expressed in the intestines of mouse fetuses and newborns. It has also been confirmed that AFP can be used as an indicator of immature intestinal cells.

In step 23 of the method disclosed herein, a cell structure of AFP-negative small intestinal epithelial cells is obtained, which suggests that the cells have developed and matured into small intestinal tissue in step 23.

Non-Patent Document 11 also suggests that organoids derived from embryonic stem cells generally have juvenility even when they are produced from cells of adult origin. Previously, no means were known for promoting the maturation of organoids.

The method disclosed herein is based on the unexpected finding that the development of small intestinal epithelial cells is promoted by culturing a cell structure containing a small intestinal epithelial cell layer in a suspension culture medium containing transferrin, insulin, progesterone, putrescine, and selenite.

Putrescine, one of the five factors added to the medium, is a type of polyamine that is contained in breast milk and is known to contribute to the growth of infants. Patent Document 8 also describes that polyamines contribute to maintaining intestinal health.

It has also been suggested in Non-Patent Document 12 that transferrin, another of the five factors, is involved in the transport of iron into enterocytes via endocytosis.

However, the combination of transferrin, insulin, progesterone, putrescine and selenite promotes the maturation of small intestinal epithelial cells in cellular structures, which was previously unknown and unexpected.

As shown in the examples of this specification, even if the cell structure is obtained by suspension culture in a medium containing the five factors in step 23 and then further suspension cultured in a medium not containing the five factors, the expression of AFP remains reduced and spontaneous peristaltic motility is maintained.

(3) Muscle Tissue Development A further characteristic of the cell structure produced by the method according to the present disclosure is that it has mature and developed muscle tissue.
Developed muscle tissue preferably contains desmin-positive and smooth muscle actin (SMA)-positive smooth muscle cells.

According to the description in Supplemental Figure 14 of Non-Patent Document 15, in the interstitial cells found in organoids containing intestinal epithelial cells, desmin-positive and smooth muscle actin-positive cells are smooth muscle-like cells, and vimentin-positive and SMA-positive cells have the properties of intestinal myofibroblasts.

Peristaltic movement is performed by smooth muscle, so cell structures containing desmin-positive and smooth muscle actin-positive smooth muscle cells have peristaltic movement ability. In contrast, intestinal myofibroblasts exist just below the intestinal epithelial basement membrane and are thought to be involved in molecular transport between epithelial cells and basement membrane cells, and therefore are thought to contribute little to movement.

In the examples of this specification, when the immature cell structure from step 22 was subjected to suspension culture in a medium to which transferrin, insulin, progesterone, putrescine and selenite were not added, the majority of the smooth muscle actin-positive cells in the cell structure were vimentin-positive cells, whereas when the immature cell structure from step 22 was subjected to suspension culture in a medium to which N-2 supplement containing transferrin, insulin, progesterone, putrescine and selenite was added, desmin-positive and smooth muscle actin-positive smooth muscle cells were confirmed in the cell structure.

Thus, according to the method of the present disclosure, the development of muscle tissue in the cell structure is promoted by carrying out the suspension culture of the cell structure in step 23 in a medium containing transferrin, insulin, progesterone, putrescine and selenite. The five factors are thought to contribute to the maturation of not only nerves but also muscle tissue.

(4) Inhibition of small intestinal epithelial cell division When the cell structure produced by the method according to the present disclosure is in a medium containing transferrin, insulin, progesterone, putrescine, and selenite, the division of small intestinal epithelial cells may be inhibited.

In the method according to the present disclosure, in step 23, the immature cell structure containing the small intestinal epithelial cell layer obtained in step 22 is cultured in suspension in a medium containing transferrin, insulin, progesterone, putrescine and selenite, which is believed to inhibit the division of small intestinal epithelial cells.

For example, Non-Patent Document 13 has reported that epithelial cell division is suppressed by the addition of acetate and butyrate produced by lactic acid bacteria, but it was not previously known that transferrin, insulin, progesterone, putrescine, and selenite have a similar effect.

In addition, it has been described in Non-Patent Document 14 that N-2 supplements, which contain transferrin, insulin, progesterone, putrescine, and selenite, do not promote the growth of cells other than nerve cells, but the effect of N-2 supplements on the division of small intestinal epithelial cells was unknown.
That is, the unexpected effect of step 23 of the method according to the present disclosure is that it inhibits the division of small intestinal epithelial cells.

It is preferable to continue to maintain the cell structure obtained through step 23 in a medium containing transferrin, insulin, progesterone, putrescine and selenite even after completion of step 23, from the viewpoint of suppressing an increase in the proportion of dividing cells.
Below, preferred embodiments of each step of the method for producing a cell structure according to the present disclosure are described.

<2.1. Step 21>
Step 21 of the method for producing a cell structure according to the present disclosure is seeding stem cells onto a cell culture substrate having a pattern of cell adhesion sites on its surface.
Here, the preferred embodiments of the cell culture substrate and the stem cells are as described above.

The cell culture substrate is preferably precoated with a precoating agent for the purpose of promoting adhesion of stem cells to the cell adhesion sites. The precoating agent is a component that is applied to the cell culture substrate in advance to promote adhesion of cells to the cell adhesion sites. Precoating agents include extracellular matrices (collagen, fibronectin, proteoglycan, laminin, vitronectin), gelatin, lysine, peptides, gel-like matrices containing these, serum, etc. By carrying out the precoating treatment, adhesion of stem cells with low adhesiveness to the cell adhesion sites can be promoted, and cell adhesion culture and differentiation induction can be effectively carried out.

Stem cells can be cultured under conditions that maintain the undifferentiated state before seeding. The medium used for the culture is not particularly limited as long as it does not induce differentiation of stem cells. For example, the medium may include a medium containing a leukemia inhibitory factor that is known to have the property of maintaining the undifferentiated state of mouse embryonic stem cells and mouse induced pluripotent stem cells, and a medium containing basic FGF that is known to have the property of maintaining the undifferentiated state of human iPS cells.

Step 21 and step 22 described later may be performed in a medium capable of proliferating stem cells and inducing differentiation, and the medium is not particularly limited. As the basal medium used in step 21 and step 22 described later, Knockout DMEM (KDMEM) medium, DMEM medium, EMEM medium, MEM medium, DMEM-F12 medium, BME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. may be used. Various growth factors, serum or serum substitutes, antibiotics, amino acids, etc. may be added to the medium. For example, 0.1-2% pyruvic acid, 0.1-2% non-essential amino acids, 0.1-2% penicillin/streptomycin, 0.1-1% glutamine, 0.1-2% β-mercaptoethanol, and 1 mM-20 mM ROCK inhibitor (e.g., Y27632) may be added. Specific examples of the medium used in step 21 and step 22 described below include XF32 medium described in the Examples, and commercially available media such as StemFit (Ajinomoto Co., Ltd.), StemFlex (Life Technologies, Inc.), and ReproFF (ReproCell Co., Ltd.). The medium may be a serum-containing medium or a serum-free medium containing a serum replacement component, but is preferably a serum-free medium containing a serum replacement component.

The seeding density of stem cells on the cell culture substrate is not particularly limited and may be in accordance with conventional methods. In the present disclosure, stem cells are preferably seeded on the cell culture substrate at a density of 3×10 ⁴ cells/cm ² or more, more preferably at a density of 3×10 ⁴ to 5×10 ⁷ cells/cm ² , and even more preferably at a density of 3×10 ⁴ to 2.5×10 ⁷ cells/cm ² .

<2.2. Step 22>
In step 22 of the method for producing a cell structure according to the present disclosure, the stem cells seeded in step 21 are cultured on the cell culture substrate to induce differentiation into an immature cell structure including a small intestinal epithelial cell layer. This is the process of making the material
The medium that can be used in step 22 is as described above.
The culture temperature in step 22 is usually 37° C. It is preferable to culture in an atmosphere with a _CO2 concentration of about 5% using a _CO2 cell culture device or the like.

The culture period in step 22 varies depending on the initial cell seeding density and the shape and size of the cell adhesion portion, but is preferably about 2 to 4 weeks. When stem cells are cultured and induced to differentiate on a cell culture substrate having the structure described in this specification, immature cell structures including the differentiated small intestinal epithelial cell layer naturally float and detach, and can be collected 2 to 4 weeks after seeding. In addition, the detachment of the immature cell structures from the cell culture substrate may be promoted using various methods such as mild enzyme treatment (e.g., Accutase or TrypLE) that does not destroy the immature cell structures, EDTA treatment, spraying with a liquid such as culture medium, and physical detachment with a scraper.

The immature cell structure containing the small intestinal epithelial cell layer obtained in step 22 is typically a pouch-shaped cell structure in which the small intestinal epithelial cell layer is present on the outer periphery of the cell structure with the villus layer facing outward.

The immature cell structure containing the small intestinal epithelial cell layer obtained in step 22 can be a pouch-shaped cell structure with a long axis length of 0.5 mm to 5 mm when detached from the cell culture substrate.

<2.3. Step 23>
Step 23 of the method for producing a cell structure according to the present disclosure comprises: soaking the immature cell structure containing the small intestinal epithelial cell layer obtained in step 22 in a medium containing transferrin, insulin, progesterone, putrescine and selenite. and culturing the small intestinal epithelial cell layer in suspension in a culture medium to produce a cellular structure comprising a tissue containing a small intestinal epithelial cell layer and Cajal cells, nerve cells and muscle cells, the small intestinal epithelial cell layer comprising α-fetoprotein-negative small intestinal epithelial cells. This is the process of inducing differentiation.

In step 23, the immature cell structure containing the small intestinal epithelial cell layer obtained in step 22 is cultured while suspended in a liquid medium. In order to maintain the suspended state, it is preferable to culture the immature cell structure together with the liquid medium in a Petri dish or a culture vessel having a surface that has been polymer-coated to reduce cell adhesion.

The suspension culture in step 23 can be carried out in a state where multiple cell structures are accommodated in one container. There is no particular limit to the number of cell structures accommodated in one container, but the more the number of cell structures accommodated in one container, the higher the production efficiency, and the fewer the number of cell structures, the less likely the maturation of the cell structures is to be inhibited and the higher the yield of cell structures having peristaltic motility. Therefore, as a guideline, about 5 to 10 cells are preferable.
When suspension culture is performed in a 3.5 cm Petri dish, it is preferable to carry out suspension culture by placing about 2 to 3 mL of medium and 5 to 10 immature cell structures therein.

The medium used for the suspension culture in step 23 is not particularly limited, except that the five predetermined factors are added, and can be selected from the same range as the medium used in steps 21 and 22 above. For example, as the basal medium, Knockout DMEM (KDMEM) medium, DMEM medium, EMEM medium, MEM medium, DMEM-F12 medium, BME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. can be used. Various growth factors, serum or serum replacement components, antibiotics, amino acids, etc. may be added to the medium. A specific example of the medium used in step 23 is the XF32 medium described in the Examples. The medium used in step 23 may be a serum-containing medium or a serum-free medium containing a serum replacement component, but is preferably a serum-free medium containing a serum replacement component. In the examples, it has been found that suspension culture in a serum-free medium containing serum alternatives containing the five specified factors is more efficient at inducing cell structures with peristaltic motility than suspension culture in a serum-containing medium containing the five specified factors. In addition, since the cell structures may adhere to the culture vessel due to the influence of adhesive proteins contained in the serum-containing medium, it is preferable to use a serum-free medium. The concentration of the serum alternative in the serum-free medium is preferably 1% or more.

The medium used for the suspension culture in step 23 is characterized by containing transferrin, insulin, progesterone, putrescine, and selenite. These five factors are known as "N-2 supplement." An example of the composition of N-2 supplement before dilution is 1 mM transferrin, 0.086 mM insulin, 0.002 mM progesterone, 10 mM putrescine, and 0.003 mM selenite, and step 23 can be performed in a medium containing this concentration of N-2 supplement diluted, for example, to 1/100. An example of the selenite is sodium selenite.

The medium used for the suspension culture in step 23 may contain transferrin, insulin, progesterone, putrescine, and selenite, and the final concentrations are not particularly limited. For example, the concentration of transferrin in the medium can be 1 μM to 100 μM. For example, the concentration of insulin in the medium can be 0.086 μM to 8.6 μM. For example, the concentration of progesterone in the medium can be 0.002 μM to 0.2 μM. For example, the concentration of putrescine in the medium can be 10 μM to 1 mM. For example, the concentration of selenite in the medium can be 0.003 μM to 0.3 μM.
The proteins, transferrin and insulin, are not particularly limited in the organism of origin, but may be human transferrin and human insulin. Human transferrin and human insulin can be biosynthesized from microorganisms such as Escherichia coli or yeast that have human genes incorporated therein. Progesterone, putrescine, and selenite can be obtained by chemical synthesis. In recent years, research has been conducted to obtain insulin by chemical synthesis.

Transferrin, insulin, progesterone, putrescine and selenite may also be added to the medium as B-27 (registered trademark) supplement (manufactured by Invitrogen), which contains all of these ingredients. Note that the exact composition of B-27 (registered trademark) supplement has not been disclosed, but examples of the ingredients contained therein are described, for example, in Patent Document 9. Furthermore, a medium containing both N-2 supplement and B-27 (registered trademark) supplement may also be used, and an example of such a medium is N2B27 medium.

The time for suspension culture in step 23 is not particularly limited, but in order to increase the yield of cell structures having peristaltic motility, 2 weeks or more is preferable, 3 weeks or more is more preferable, and 4 weeks or more is particularly preferable.
The frequency and amount of medium replacement during the suspension culture is not particularly limited. For example, half of the medium can be replaced once a week.

The size of the cell structure obtained through step 23 is not particularly limited, and even a small cell structure has peristaltic motility. The cell structure obtained through step 23 can be, for example, a bag-shaped cell structure with a major axis length of 0.5 mm to 5 mm.
The cell structure obtained through step 23 is capable of peristaltic movement in whole or in part.

3. Characteristics of cell structures
A preferred embodiment of the cell structure disclosed herein comprises a small intestinal epithelial cell layer and a tissue comprising Cajal cells, nerve cells and muscle cells, characterized in that the small intestinal epithelial cell layer comprises α-fetoprotein-negative small intestinal epithelial cells.

The cell structure according to the present disclosure can spontaneously move in a peristaltic manner due to the functions of Cajal cells, nerve cells, and muscle cells. Therefore, the cell structure according to the present disclosure can be used to evaluate the effect of test substances such as drugs and poisons on the small intestine. The tissue containing Cajal cells, nerve cells, and muscle cells is typically located on the opposite side of the small intestinal epithelial cell layer to the villus layer.

The cell type in the cell structure is preferably identified by immunostaining or in situ hybridization using a protein or nucleic acid (marker) that is specifically present in the target cell type as an indicator. Alternatively, the cell type may be identified by genetic analysis such as PCR, microarrays, and RNA-seq, or protein analysis using an antibody that specifically binds to a specific protein, such as Western blotting.

Markers for small intestinal epithelial cells include CDX2, Villin, E-cadherin, and HNF4. In particular, Villin is a marker for the villus layer, and the presence of the villus layer strongly suggests that cells expressing Villin have the properties of intestinal cells. Cells expressing the above markers can be considered to be small intestinal epithelial cells.

Cajal cells (also called interstitial cells of Cajal) function as pacemaker cells. Markers of Cajal cells include c-kit, CD34, CD117, S-100β, etc. Cells expressing the above markers can be considered as Cajal cells.

Examples of markers for nerve cells include βIII tubulin (Tuj-1), MAP-2, MAP2ab, Neurofilament, HuC/D, Doublecortin, and PSA-NCAM. Examples of markers for the cell nucleus of nerve cells include NeuN and PAX6. Furthermore, synaptic markers such as Synapsin can also be used as markers for nerve cells. Cells expressing the above markers can be considered as nerve cells.

Markers for muscle cells include smooth muscle actin (SMA), desmin myosin D, vimentin, and the like. Cells expressing the above markers can be considered muscle cells. Particularly preferably, cells positive for both SMA and desmin can be considered smooth muscle cells involved in peristalsis. Smooth muscle cells that are desmin-positive and smooth muscle actin-positive are known to be involved in peristalsis. That is, in a preferred embodiment of the cell structure according to the present disclosure, the muscle cells include smooth muscle cells that are desmin-positive and smooth muscle actin-positive.

The cell structure according to the present disclosure is characterized in that the small intestinal epithelial cell layer contains small intestinal epithelial cells that are negative for alpha-fetoprotein (AFP), an indicator of immaturity. Conventional small intestinal organoids, such as those described in Patent Document 6, have the characteristics of immature cells. The present inventors have discovered that by culturing a cell structure in suspension in a medium containing transferrin, insulin, progesterone, putrescine, and selenite, it is possible to promote the maturation of small intestinal epithelial cells and obtain a cell structure containing mature small intestinal epithelial cells that are negative for AFP.

Besides AFP, cytokeratin 7 may be an indicator of the immaturity of small intestinal epithelial cells. Examples of markers that are indicators of the maturity of small intestinal epithelial cells include dipeptidyl peptidase 4 (DPPIV), sucrose isomaltase (SIM), alkaline phosphatase (ALPI), lactase (LCT), and gastric inhibitory peptide (GIP), as described in Patent Document 4. The maturity of small intestinal epithelial cells can also be determined by observing the development of crypts.

In one or more aspects, the cell structure according to the present disclosure includes AFP-negative small intestinal epithelial cells in the small intestinal epithelial cell layer means that the small intestinal epithelial cell layer contains cells that are positive for a marker for small intestinal epithelial cells (e.g., CDX2, Villin, E-cadherin, etc.) and AFP-negative cells. In another one or more aspects, the cell structure according to the present disclosure includes AFP-negative small intestinal epithelial cells in the small intestinal epithelial cell layer means that the expression of AFP is reduced in the tissue of the small intestinal epithelial cell layer compared to other reference tissues. In addition, the cell structure according to the present disclosure can be evaluated as including AFP-negative small intestinal epithelial cells in the small intestinal epithelial cell layer when only small intestinal epithelial cells are separated from the cells contained in the cell structure according to the present disclosure by flow cytometry or the like, and when AFP-negative cells or cells with reduced AFP expression are present among the separated small intestinal epithelial cells, it can be evaluated that the cell structure according to the present disclosure includes AFP-negative small intestinal epithelial cells in the small intestinal epithelial cell layer. In a preferred embodiment of the cell structure according to the present disclosure, the proportion of AFP-positive small intestinal epithelial cells among the small intestinal epithelial cells contained in the small intestinal epithelial cell layer (based on the number of cells) is, for example, 70% or less (i.e., the proportion of AFP-negative small intestinal epithelial cells is 30% or more), preferably 50% or less (i.e., the proportion of AFP-negative small intestinal epithelial cells is 50% or more), more preferably 30% or less (i.e., the proportion of AFP-negative small intestinal epithelial cells is 70% or more), and particularly preferably 8% or less (i.e., the proportion of AFP-negative small intestinal epithelial cells is 92% or more). A method for calculating this proportion includes a method of measuring the number of positive cells that co-express AFP among the marker-positive cells of small intestinal epithelial cells using immune cells. The proportion can also be defined by comparing the expression level of AFP in the small intestinal epithelial cell layer of the cell structure according to the present disclosure with the expression level of AFP in another reference tissue, and by the relative ratio of the former to the latter. Here, the gene expression level of AFP may be the expression level based on genetic analysis of AFP, or may be the expression level based on the protein amount of AFP. The amount of protein can be quantified, for example, based on band intensity in Western blotting.

The cell structure according to the present disclosure preferably has the ability to spontaneously move in a peristaltic manner in the presence of one or more compounds selected from histamine compounds and serotonin compounds.

The cell structure according to the present disclosure preferably has a pouch-like structure, and is characterized in that the small intestinal epithelial cell layer is present on the outer periphery of the cell structure with the villus layer facing outward. A cell structure having this configuration can absorb and incorporate surrounding substances into the inside of the cell structure. In this embodiment, the tissue containing Cajal cells, nerve cells, and muscle cells is preferably adjacent to the small intestinal epithelial cell layer and is located inside the pouch-like structure. The villus layer can be identified using the presence of Villin as an indicator.

In a preferred embodiment of the cell structure according to the present disclosure, the division of small intestinal epithelial cells contained in the small intestinal epithelial cell layer is inhibited, and specifically, the proportion of dividing cells (based on cell number) among the small intestinal epithelial cells contained in the small intestinal epithelial cell layer is 1% or less. Markers for dividing cells include Ki67, PCNA, CyclinD, etc., and cells expressing the markers can be identified as dividing cells. It is also possible to identify cells as dividing cells by incorporating thymidine analogs such as BrdU and IdU and detecting the substance by immunostaining or by using a radioisotope such as tritium.

The cell structure according to the present disclosure is preferably maintained in a medium containing a serum replacement component, as well as transferrin, insulin, progesterone, putrescine, and selenite. Examples of such a medium include those disclosed as the medium for suspension culture in step 23 above. The cell structure according to the present disclosure maintained in a medium containing a serum replacement component, as well as transferrin, insulin, progesterone, putrescine, and selenite is preferred because it stably maintains the above-mentioned characteristics, particularly the suppressed division ability of small intestinal epithelial cells.

The cell structure disclosed herein is preferably differentiated from pluripotent stem cells, more preferably from human-derived pluripotent stem cells, and most preferably from human-derived iPS cells or ES cells.

The cell structure according to the present disclosure is particularly preferably produced by the above-described method including steps 21, 22 and 23.

4. Method for evaluating the effect of a test substance on the small intestine using a cell structure, and a kit therefor
The disclosure herein also provides
A method for evaluating an effect of a test substance on the small intestine, comprising the steps of:
Step 11 of observing the cell structure according to the present disclosure in the presence of a test substance; and Step 12 of evaluating the effect of the test substance on the small intestine based on the observation results.
The present invention relates to a method comprising the steps of:
The test substance here includes poisons, drugs, and the like.

In the past, to evaluate the effects of test substances such as poisons and drugs on the small intestine, the test substances were often administered to animals such as mice, but this method has a high degree of variability, is costly and time-consuming, and there was a demand for alternative methods from the perspective of animal welfare.

Alternative methods to animal testing include, for example, the immunochromatography method described in Patent Document 10, and the determination of viability or death of cultured cells and identification by LC/MS described in Patent Document 11 and Non-Patent Documents 16 to 18.

In addition to the time required for preparation, these alternative methods are performed by comparison with known compounds, so there is a risk that, for example, changes in the compound may go undetected and be overlooked. This is also true for single-cell culture evaluation systems, where sensitivity is unknown.

The evaluation method according to the present disclosure makes it possible to directly evaluate the effect of a test substance on the small intestine. In addition, since the cell structure according to the present disclosure contains small intestinal epithelial cells, nerve cells, Cajal cells, and muscle cells, it is possible to evaluate the effect of a test substance on these cells at once.

In addition, if the pouch structure of the cell structure disclosed herein is destroyed or the expression level of a functional protein is reduced in the presence of the test substance, it can be determined that the test substance is toxic to small intestinal epithelial cells.

More preferably, step 11 in the evaluation method according to the present disclosure is a step of observing the peristaltic movement of the cell structure according to the present disclosure in the presence of a test substance.

As evidence that changes in peristaltic movement can be a subject of evaluation, Non-Patent Document 19 describes a change in peristaltic movement when a poison derived from paralytic shellfish poison is administered to the digestive tract of a guinea pig. The toxicity or efficacy of a test substance can be evaluated by detecting the change in peristaltic movement when a test substance is administered to a cell structure according to the present disclosure, which exhibits spontaneous peristaltic movement.

When observing peristaltic movement, it is preferable to administer the test substance while promoting the peristaltic movement of the cell structure according to the present disclosure by adding one or more compounds selected from histamine compounds and serotonin compounds. The compound is preferably in the form of a salt so that it can be easily dissolved in water.

When observing peristalsis, the test substance may be administered while promoting the peristaltic movement of the cell structure according to the present disclosure by electrical stimulation. Promotion of peristalsis by electrical stimulation is described, for example, in Patent Document 4.

Peristaltic movement can be observed by capturing a video of the cell structure according to the present disclosure in a culture medium or buffer solution containing the test substance, and evaluating changes in the shape of the cell structure according to the present disclosure from the video. Changes can be easily captured by observing the captured video at an increased playback speed rather than at a constant speed. There are no limitations to the imaging device or imaging method. Furthermore, in order to quantify changes, the cell structure according to the present disclosure may be imaged in a state where it is fixed to a substrate rather than in a floating state.

When observing peristaltic movement in step 11, the cell structure according to the present disclosure is cultured in suspension in a liquid medium, and in a state in which it can easily contract and swell, a test substance is added to the liquid medium, and changes in peristaltic movement are observed. The concentration of the test substance added and the time for which the test substance is allowed to act can be set appropriately depending on the substance.

If in step 11 the peristaltic movement of the cell structure according to the present disclosure stops or attenuates in the presence of the test substance, in step 12 it can be determined that the test substance contains a substance that is toxic to the small intestine.

The evaluation method according to the present disclosure preferably further includes, after step 11, step 13 of washing the cell structure according to the present disclosure with a liquid medium not containing the test substance, and includes using the cell structure again in step 11 after step 13. Unlike cultured cells which cannot be reused, the cell structure according to the present disclosure can be reused repeatedly, and is therefore preferable in terms of reducing costs.

In addition, in step 11, if the peristaltic movement of the cell structure of the present disclosure stops or attenuates in the presence of the test substance, in order to confirm that this stop or attenuation of peristaltic movement is not due to cell death of the cell structure, the cell structure can be washed with a liquid medium such as a liquid culture medium or buffer that does not contain the test substance, and transferred to a culture medium that does not contain a toxic substance, thereby confirming that peristaltic movement occurs again.

Toxins that may cause diarrhea or the like are known to have the effect of enhancing peristaltic movement of the small intestine. Specific examples include toxins derived from Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Salmonella, Clostridium perfringens, and Bacillus cereus, which are generally classified as enterotoxins. A sample that may contain a poison having such an effect is used as a test substance, and in step 11, changes in the peristaltic movement of the cell structure according to the present disclosure are observed. If peristaltic movement is promoted, in step 12, the sample can be evaluated as containing a poison having the above effect. Evaluation of the presence or absence of enterotoxins has traditionally been performed using test animals such as monkeys. The evaluation method according to the present disclosure is useful as an alternative to animal testing.

Other examples of test substances include substances that affect cells or tissues contained in the cell structure of the present disclosure, such as sodium channel blockers of nerve cells, such as tetrodotoxin; various paralytic shellfish toxins, such as palytoxin and okadaic acid; etc.

The test substance may also be a drug candidate expected to have a beneficial effect on the small intestine, such as a drug candidate for treating a peristalsis-related disease such as irritable bowel syndrome, or a drug candidate for promoting peristalsis, such as a laxative. The test substance may also be a substance that stimulates or inhibits intestinal peristalsis, or a substance that has an effect on peristalsis when co-cultured with intestinal bacteria.

According to the evaluation method of the present disclosure, the test substance is directly contacted with the cell structure, which is a small intestine-like tissue, and therefore the detection time can be shortened. Conventional evaluation methods using cultured cells require several hours after administration of the test product in addition to preparation for cell proliferation, but the evaluation method of the present disclosure allows evaluation in a relatively short time.
The disclosure of the present specification also relates to a kit for evaluating the effect of a test substance on the small intestine, comprising the cell structure according to the present disclosure.

In addition to the cell structure, the kit according to the present disclosure preferably further includes one or more selected from a container for containing the cell structure, a culture medium for culturing the cell structure, and a liquid medium for washing the cell structure. There are no particular limitations on the container. The culture medium can be selected from the same range as the culture medium used for the suspension culture in step 23, or the culture medium used in step 21 or step 22. The liquid medium may be the same as the culture medium, or may be an appropriate buffer solution.

The kit according to the present disclosure may further comprise a substance for promoting peristalsis, for example, one or more compounds selected from histamine-based compounds and serotonin-based compounds.
Kits according to the present disclosure may further include an agent for inhibiting peristalsis, for example, a drug such as norepinephrine.
The kit according to the present disclosure may further include a container or fixture for fixing the cell structure to facilitate observation.
The kit according to the present disclosure may further include an imaging device and analysis software for measuring changes in size of the cell structure in the image.

5. Activators of peristaltic movement of small intestine or cell structure
The disclosure of the present specification also relates to an activator of peristalsis of the small intestine or cellular structures, comprising transferrin, insulin, progesterone and/or progestin, putrescine and selenite as active ingredients.

A first embodiment of the activator according to the present disclosure can be used as an activator for small intestinal peristalsis in a human or non-human animal body. The activator according to this embodiment can be used to promote peristalsis in a human or non-human animal body. The activator according to this embodiment can be in the form of a pharmaceutical composition or a food composition.

The second embodiment of the activator according to the present disclosure can be used as an activator for the peristaltic movement of a cellular structure existing outside the body, preferably a small intestinal organoid having peristaltic motility. It can be used for the purpose of inducing a cellular structure having peristaltic motility.

The first embodiment of the activator according to the present disclosure is preferably a composition containing transferrin, insulin, progesterone and/or progestin, putrescine, and selenite as active ingredients, in a form that can be administered to humans or non-human animals. The route of administration can be oral or parenteral, and preferably oral, injection, or infusion. Formulations for oral administration can be capsules or tablets. The first embodiment of the activator according to the present disclosure can contain, in addition to the active ingredients, one or more components that are acceptable as a medicine or food.

A second embodiment of the activator according to the present disclosure is preferably a composition containing transferrin, insulin, progesterone and/or progestin, putrescine, and selenite as active ingredients, in a form applicable to cell structures existing outside a living body. For example, the second embodiment of the activator according to the present disclosure may be a medium composition containing the active ingredients and medium components.
Progestins are a group of substances that have the effect of artificially synthesizing progesterone analogues, which are easily metabolized in the liver after intestinal absorption. Progestins are also called progestagens. Specific types of progestins include those described in Non-Patent Document 20. Examples of progestins include 17α-hydroxyprogesterone, medroxyprogesterone acetate, norethisterone, levonorgestrel, dienogest, dydrogesterone, and drospirenone.

In the first or second embodiment of the activator according to the present disclosure, the types and concentrations of transferrin, insulin, progesterone and/or progestin, putrescine, and selenite can be selected from the same ranges as the types and concentrations of each component in the medium used for the suspension culture in step 23. Progestin can be used at the same concentration as progesterone.

<6. Inhibitors of Small Intestinal Epithelial Cell Division>
The disclosure of the present specification also relates to an agent for inhibiting the proliferation of small intestinal epithelial cells, which comprises transferrin, insulin, progesterone and/or progestin, putrescine and selenite as active ingredients.

The small intestinal epithelial cells may be small intestinal epithelial cells isolated ex vivo, small intestinal epithelial cells present in a living body or contained in a small intestine isolated ex vivo, or small intestinal epithelial cells contained in a cell structure present ex vivo, preferably a small intestinal organoid having peristaltic motility.

A first embodiment of the division inhibitor according to the present disclosure can be used as an inhibitor of division of small intestinal epithelial cells contained in the small intestine in the living body of a human or non-human animal. The division inhibitor according to this embodiment can be used to inhibit the division of small intestinal epithelial cells in the living body. The division inhibitor according to this embodiment may be in the form of a pharmaceutical composition or a food composition.

The second embodiment of the mitotic inhibitor according to the present disclosure can be used as a mitotic inhibitor for isolated small intestinal epithelial cells, small intestinal epithelial cells contained in a small intestine isolated ex vivo, or small intestinal epithelial cells contained in a cell structure present ex vivo, preferably small intestinal epithelial cells contained in a small intestinal organoid having peristaltic motility.

The first embodiment of the mitotic inhibitor according to the present disclosure is preferably a composition containing transferrin, insulin, progesterone and/or progestin, putrescine, and selenite as active ingredients, in a form that can be administered to humans or non-human animals. The route of administration can be oral or parenteral, and preferably oral, injection, or infusion. Formulations for oral administration can be capsules or tablets. The first embodiment of the mitotic inhibitor according to the present disclosure can contain, in addition to the active ingredients, one or more components that are acceptable as a medicine or food.

A second embodiment of the mitotic inhibitor according to the present disclosure is preferably a composition containing transferrin, insulin, progesterone and/or progestin, putrescine, and selenite as active ingredients, and is applicable to isolated small intestinal epithelial cells, small intestinal epithelial cells contained in a small intestine isolated ex vivo, or small intestinal epithelial cells contained in a cell structure present ex vivo. For example, the second embodiment of the mitotic inhibitor according to the present disclosure can be a medium composition containing the active ingredients and medium components.

In the first or second embodiment of the mitotic inhibitor of the present disclosure, the types and concentrations of transferrin, insulin, progesterone and/or progestin, putrescine, and selenite can be selected from the same ranges as the types and concentrations of each component in the medium used for the suspension culture in step 23. Progestin can be used at the same concentration as progesterone.

<7. Small intestinal epithelial cell maturation promoter>
The disclosure of the present specification also relates to an agent for promoting the maturation of small intestinal epithelial cells, comprising transferrin, insulin, progesterone and/or progestin, putrescine and selenite as active ingredients.

The small intestinal epithelial cells may be small intestinal epithelial cells isolated ex vivo, small intestinal epithelial cells present in a living body or contained in a small intestine isolated ex vivo, or small intestinal epithelial cells contained in a cell structure present ex vivo, preferably a small intestinal organoid having peristaltic motility.

A first embodiment of the maturation promoter according to the present disclosure can be used as a maturation promoter for small intestinal epithelial cells contained in the small intestine of a human or non-human animal body. The maturation promoter according to this embodiment can be used to promote the maturation of small intestinal epithelial cells in a body. The maturation promoter according to this embodiment may be in the form of a pharmaceutical composition or a food composition.

The second embodiment of the maturation promoter according to the present disclosure can be used as a maturation promoter for isolated small intestinal epithelial cells, small intestinal epithelial cells contained in a small intestine isolated ex vivo, or small intestinal epithelial cells contained in a cell structure present ex vivo, preferably small intestinal epithelial cells contained in a small intestinal organoid having peristaltic motility.

The first embodiment of the maturation accelerator according to the present disclosure is preferably a composition containing transferrin, insulin, progesterone and/or progestin, putrescine, and selenite as active ingredients, in a form that can be administered to humans or non-human animals. The route of administration can be oral or parenteral, and preferably oral, injection, or infusion. Formulations for oral administration can be capsules or tablets. The first embodiment of the maturation accelerator according to the present disclosure can contain, in addition to the active ingredients, one or more components that are acceptable as medicines or foods.

A second embodiment of the maturation promoter according to the present disclosure is preferably a composition containing transferrin, insulin, progesterone and/or progestin, putrescine, and selenite as active ingredients, and is in a form applicable to isolated small intestinal epithelial cells, small intestinal epithelial cells contained in a small intestine isolated ex vivo, or small intestinal epithelial cells contained in a cell structure present ex vivo. For example, the second embodiment of the maturation promoter according to the present disclosure can be a medium composition containing the active ingredients and medium components.

In the first or second embodiment of the maturation promoter according to the present disclosure, the types and concentrations of transferrin, insulin, progesterone and/or progestin, putrescine, and selenite can be selected from the same ranges as the types and concentrations of each component in the medium used for suspension culture in step 23. Progestin can be used at the same concentration as progesterone.
Hereinafter, the present disclosure will be described with reference to specific experimental results, but the scope of the present disclosure is not limited to the scope of the experimental results.

<Experiment 1>
(Preparation of cell culture substrate)
As a cell culture substrate, a cell culture substrate was prepared that includes a cell adhesion portion (see FIG. 1) formed on a glass substrate and consisting of a circular pattern with an inner diameter of 600 μm and a width of 100 μm, which is a region formed by oxidative decomposition of a layer of polyethylene glycol 400, and a cell non-adhesive portion, which is a region in which the surface of the glass substrate is coated with polyethylene glycol 400 on the inside and outside of the circular pattern of the cell adhesion portion. The cell culture substrate includes a plurality of cell adhesion portions consisting of the circular patterns formed at intervals of 300 to 500 μm (see FIG. 1). In the following description, the cell adhesion portion consisting of a circular pattern is referred to as an "annular cell adhesion portion."
The cell culture substrate was prepared according to the procedure described in Japanese Patent No. 5070565. The outline of the procedure is described below.

(First stage reaction)
39.0 g of toluene, 0.48 g of epoxy silane TSL8350 (GE Toshiba Silicone), and 0.97 g of triethylamine were mixed and stirred at room temperature for 10 minutes. A 10 cm square glass substrate that had been UV-cleaned was immersed in this silane solution with the cleaned surface facing upward. After leaving it at room temperature for 16 hours, the substrate was washed with ethanol and water and dried with a nitrogen blower. As a result, a thin film containing epoxy groups was formed on the surface of the glass substrate.

(Second stage reaction)
25 μl of concentrated sulfuric acid was added drop by drop to 50 g of polyethylene glycol (PEG400) with an average molecular weight of 400 while stirring. After stirring for several minutes, the entire amount was transferred to a glass dish. The above substrate was immersed in the mixture and reacted at 80° C. for 20 minutes. After the reaction, the substrate was thoroughly washed with water and dried with nitrogen blowing. This resulted in the formation of a uniform hydrophilic thin film on the glass surface.

(Oxidation Treatment)
A photomask was prepared by coating the entire surface with a titanium oxide photocatalyst. The photomask had a plurality of openings formed at intervals of 300 to 500 μm in shape corresponding to the above-mentioned annular cell adhesion portion, and had an opening of about 1.5 cm in width around the periphery. The illuminance of the exposure machine was measured in advance at a wavelength of 350 nm to set the exposure time as a guide. The photocatalyst layer of this photomask was brought into contact with the hydrophilic thin film on the surface of the substrate, and the substrate was placed in the exposure machine so that light was irradiated from the photomask side. The substrate was exposed to light for 50 seconds with a mercury lamp with an illuminance of 20 mW/cm ² at a wavelength of 350 nm, and the hydrophilic thin film on the surface of the substrate was partially oxidized and decomposed. The substrate was cut into a size of 25 mm x 15 mm and used as a cell adhesion substrate. Before use in cell culture, the cell culture substrate was subjected to EOG sterilization for 22 hours.

The cell culture substrate was placed on the bottom of a 3.5 cm Petri dish (Corning), and coated with vitronectin (Life Technologies) diluted 1/100 with phosphate buffered saline (PBS) at room temperature for 30 minutes or more, and then washed three times with PBS before use.
The cell culture substrate thus obtained has a cross-sectional structure as shown in FIG. 1(B).

(culture)
The National Center for Child Health and Development (National Research and Development Agency) has established Edom iPS cells, a human iPS cell line, by transiently expressing the four Yamanaka factors in cells obtained from menstrual blood using a Sendai virus vector (PLOS Genet. 2011 May; 7(5): e1002085. Published online 2011 May 26. doi: 10.1371/journal.pgen.1002085 PMCID: PMC3102737). Edom iPS cells were pre-grown in StemFit medium (Ajinomoto) in a 10 cm cell culture dish (Corning) coated with vitronectin (Vitronectin, Life Technologies) diluted 1/100 in PBS in the same manner as above. The grown cells were detached from the dish by treatment with EDTA solution (Life Technologies) diluted to 0.5 mM in PBS.

The Edom iPS cells, which had been detached and collected, were seeded on the cell culture substrate at 5×10 ⁶ cells and cultured. The following XF32 medium was used as the medium. By culturing in this XF32 medium, the cells proliferated on the annular cell adhesion parts and formed a bag-shaped cell structure, which was naturally detached and collected 3 to 4 weeks after the start of culture. The culture process from seeding cells on the cell culture substrate and culturing the cells until the bag-shaped cell structure was detached is called "pattern culture". In the pattern culture, the medium was changed every 3 to 4 days. The bag-shaped cell structures obtained by pattern culture were collected, and approximately 6 to 10 cells were placed in a 3.5 cm Petri dish.

The composition of XF32 medium is as follows, and it was prepared by mixing the following ingredients (the contents concentrations were taken from Non-Patent Document 1).
Knockout DMEM (KDMEM; ThermoFisher Scientific): Approximately 85% by volume of the entire medium
XenoFree-Knockout Serum Replacement (XF-KSR; ThermoFisher Scientific): Approximately 15% by volume of the total medium
Basic Fibroblast Growth Factor (bFGF; ThermoFisher Scientific): 20 ng/mL
Insulin growth factor-I (IGF-I; Nichirei Corporation): 200 ng/mL
Hereglin (FUJIFILM Wako Pure Chemical Industries): 10 ng/mL
Glutamax-I (L-alanyl-L-glutamine; ThermoFisher Scientific): 2 mM
Non Essential Amino Acid Solution (NEAA; ThermoFisher Scientific): 0.1 mM for all non-essential amino acids
Sodium Pylvate (ThermoFisher Scientific): 1 mM
Streptomycin (ThermoFisher Scientific): 50 U/mL
Penicillin (ThermoFisher Scientific) 50 μg/mL

Then, 3 ml of medium containing N-2 supplement (Life Technologies) diluted 1/100 in XF32 medium was added to a 3.5 cm Petri dish containing 6 to 10 bag-shaped cell structures, and the bag-shaped cell structures were cultured in suspension in the medium for 3 weeks to 1 month. The medium was replaced once a week, roughly half the amount. This is Example A. The process of culturing the bag-shaped cell structures in suspension in a Petri dish containing medium is called "suspension culture". Here, the N-2 supplement before dilution contains 1 mM transferrin, 0.086 mM insulin, 0.002 mM progesterone, 10 mM putrescine, and 0.003 mM selenite, and the N-2 supplement-added XF medium used for suspension culture contains each component at 1/100 times the concentration of the above. Regarding the methods of producing these substances, insulin and transferrin are obtained by biosynthesis using E. coli or yeast that have human genes inserted into them, while the low molecular weight compounds progesterone, putrescine, and selenite are obtained by chemical synthesis.

As Comparative Example A, suspension culture was performed in the same manner as in Example A, except that suspension culture was performed for 3 weeks to 1 month in XF32 medium without the addition of N-2 supplement.

The characteristics of the media used in pattern culture and suspension culture in Example A and Comparative Example A are shown in Figure 2.

The presence or absence of peristalsis was examined using the method described below. A solution of serotonin-creatinine sulfate monohydrate (FUJIFILM Wako Pure Chemical Industries, Ltd.) prepared at a concentration of 10 mM in phosphate buffer (PBS) was added to the medium containing the bag-shaped cell structure of Example A or Comparative Example A after suspension culture at a final concentration of 10 μM at a dilution of 1/1000 to stimulate the cells. After that, a video was captured for 2 to 5 minutes using an all-in-one fluorescent microscope BF-X710 (Keyence Corporation), and the presence or absence of spontaneous contraction was confirmed by observing the obtained video. The playback speed was up to 16 times faster to examine the presence or absence of movement. When saving the file, ImageJ (NIH) was used to increase the speed to 16 times, and the avi format was converted to wmv format to reduce the file size.

As a result, spontaneous peristaltic movement was observed in 19 of the 22 cell structures (approximately 86.4%) in Example A. Note that peristaltic movement of the cell structures in Example A was observed even when serotonin-creatinine sulfate monohydrate was not added, but greater movement was observed when serotonin-creatinine sulfate monohydrate was added.

In Example A, spontaneously moving cell structures were not observed one day after the start of suspension culture in XF32 medium supplemented with N-2 supplement. This suggests that the addition of N-2 supplement itself does not have the effect of facilitating peristaltic movement, and that spontaneous movement occurred due to changes in the organization of the cell structures during suspension culture in the presence of N-2 supplement.

Furthermore, when the cell structure after suspension culture in which peristaltic movement was observed in Example A was maintained in XF32 medium without N-2 supplement for another week (Example B), peristaltic movement was observed when it was stimulated with a serotonin substance in the same way. This fact also suggests that N-2 supplement does not induce peristaltic motility by itself, but acts as a growth factor for tissues involved in peristaltic movement. Furthermore, this result suggests that N-2 supplement is not necessary to maintain peristaltic motility after the development of tissues involved in peristaltic movement by N-2 supplement.

In addition, for the cell structure obtained in Example A, it was observed that peristalsis was also promoted by adding histamine dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) to the medium at a final concentration of 2 μM as another stimulating substance, as described in Non-Patent Document 4. This suggests that there is no limit to the type of stimulating substance for promoting peristalsis.

On the other hand, in the cell structures of Comparative Example A after suspension culture, spontaneous peristalsis was observed in only 2 out of 24 (approximately 8.3%). From this, it can be concluded that treating the bag-shaped cell structures in a medium containing N-2 supplement as in Example A promoted the formation of tissues involved in peristalsis. Figure 3 shows the percentage of cell structures capable of peristalsis in Example A and Comparative Example A.

Separately from the above, a cell culture substrate was prepared in which a cell non-adhesive area, which is an area covered with a polyethylene glycol layer, and multiple circular cell adhesive areas with a diameter of 1500 μm were formed by oxidative decomposition of the polyethylene glycol layer, as described in Non-Patent Document 1. Using this cell culture substrate, Edom iPS cells were seeded in the same manner as above, and pattern culture was performed in XF32 medium for one month to obtain a bag-shaped cell structure released from the substrate. The obtained cell structure was subjected to suspension culture for one month in a medium containing N-2 supplement (Life Technologies) diluted 1/100 in XF32 medium, as in Example A above. Thereafter, the presence or absence of peristaltic motility was confirmed by applying a stimulant. As a result, it was observed that peristaltic motility was generated by adding 10 μM serotonin-creatinine sulfate monohydrate and 2 μM histamine dihydrochloride as stimulants. This suggests that the peristaltic motility of the bag-like cell structures obtained by culturing on a cell culture substrate with a cell adhesion pattern on its surface does not depend on the shape of the cell culture area.

The above-mentioned Example B was carried out under the following conditions. For up to 4 weeks after the start of suspension culture, the cell structures were cultured in suspension in a medium containing N-2 supplement (Life Technologies) diluted 1/100 in XF32 medium, at which point it was confirmed that peristalsis occurred. After that, the cell structures were cultured in suspension in XF32 medium without N-2 supplement until one week later, but pattern culture and suspension culture of the bag-shaped cell structures were carried out using the same procedures as in Example A.

<Experiment 2>
In Example A of Experiment 1, a cell structure capable of peristaltic motility was obtained by performing pattern culture in XF32 medium without N-2 supplement, followed by suspension culture in XF32 medium containing N-2 supplement. In this experiment, the following analysis was performed to examine whether a cell structure capable of peristaltic motility could be obtained even when N-2 supplement was added at different times.

In Comparative Example B, pattern culture was performed under the same conditions as in Example A, except that XF32 medium supplemented with 1/100 diluted N-2 supplement was used from the start, and bag-like cell structures that naturally detached and floated were collected 3 to 4 weeks after the start of culture. The medium was replaced every 3 to 4 days, as in Example A.

In Comparative Example C, XF32 medium without N-2 supplement was used for 2 weeks after seeding, and then XF32 medium with 1/100 diluted N-2 supplement was used. Except for this, pattern culture was performed under the same conditions as in Example A, and 3 to 4 weeks after the start of culture, bag-like cell structures that naturally detached and floated were collected. The medium was replaced every 3 to 4 days, as in Example A.
FIG. 4 shows photographs of the cultures at various time points of the pattern culture of Comparative Example B and Comparative Example C.

In Comparative Example B, as shown in the upper part of Figure 4, bag-shaped cell structures were obtained from some patterns. However, when 10 μM serotonin-creatinine monohydrate was added to the eight recovered cell structures, no peristaltic movement occurred.

In Comparative Example C, as shown in the lower part of Figure 4, numerous pouch-shaped cell structures with major axes of approximately 0.5 to 1 mm were obtained after 3 to 4 weeks of culturing. These cell structures were collected and maintained in a Petri dish in XF32 medium alone for 1 to 1 week, after which 2 μM histamine dihydrochloride or 10 μM serotonin-creatinine monohydrate was added as in Example A. However, of the 30 tissues observed, no tissue exhibiting peristaltic movement was observed.

These results show that adding N-2 supplement to the medium used in the pattern culture stage does not result in a cell structure capable of peristaltic motility. In order to obtain a cell structure capable of peristaltic motility, it is considered preferable to add N-2 supplement to the medium used in the suspension culture stage.

<Experiment 3>
The tissue state and differences of the cell structures induced by culture in N-2 supplement-added medium were examined by immunostaining. The method is described below.

In Experiment 1, the cell structure with peristaltic motility obtained in Example A, the cell structure with peristaltic motility obtained in Comparative Example A, and the cell structure without peristaltic motility obtained in Comparative Example A were each embedded in iPGel (Genostaff), fixed overnight or more in 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.), and then embedded in paraffin to prepare 5 μm-thick tissue section slides, which were then subjected to immunostaining.
The immunostaining method was carried out following the method described in Non-Patent Document 1.

The primary antibodies used were as follows:
Rabbit IgG-labeled anti-CDX2 antibody (Abcam, dilution ratio 1/1000)
Mouse IgG1-labeled anti-Villin antibody (Santa Cruz, dilution ratio 1/200)
Mouse IgG1-labeled anti-c-kit antibody (Proteintech, dilution ratio 1/500)
Rabbit IgG-labeled anti-βIII tubulin antibody (Abcam, dilution ratio 1/1000)
Mouse IgG1-labeled anti-αfetoprotein (AFP; ThermoFisher Scientific, dilution ratio 1/500)
Mouse IgG1-labeled anti-Ki67 antibody (DAKO, undiluted)
Mouse IgG2a-labeled anti-Smooth Muscle Actin antibody (SMA; Sigma, dilution ratio 1/500)
Mouse IgG2a-labeled anti-E-cadherin antibody (BD Pharmingen, dilution ratio 1/1000)
Rabbit IgG-labeled anti-PCNA antibody (Santa Cruz, dilution ratio 1/200)

The secondary antibodies used were as follows: All were manufactured by Life Technologies, Inc., at a dilution rate of 1/1000.
Alexa488-labeled anti-rabbit IgG antibody Alexa488-labeled anti-mouse IgG1 antibody Alexa543-labeled anti-mouse IgG antibody Alexa568-labeled anti-mouse IgG2a antibody Alexa543-labeled anti-rabbit IgG antibody

After immunostaining, the cell nuclei were stained with DAPI (Sigma) diluted 1/1000 in PBS for 10 minutes at room temperature. Then, the cells were mounted with Fluorescence Mounting Medium (DAKO) and a cover glass, and observed using a BF-X710 microscope. When counting cells positive for markers expressed in the cell nucleus, such as CDX2, Ki67, and PCNA, a cell was considered to be one positive cell if the marker expressed in the cell nucleus was co-stained with DAPI. For other cells positive for markers expressed in the cell membrane or cytoplasm, a cell was considered to be one positive cell if it was clear that the cells were separated and contained DAPI.

Figure 5 shows the results of immunostaining of the cell structure with peristaltic motility obtained in Example A. In this cell structure, tissue containing CDX2-positive small intestinal epithelial cells with Villin-positive villi facing outward relative to the inside of the pouch was confirmed. This tissue was the same as the tissue of the pouch-shaped cell structure prepared by culturing in XF32 medium without N-2 supplement, as described in Non-Patent Document 1. This confirmed that N-2 supplement does not inhibit the formation of a pouch-shaped cell structure with small intestinal epithelial cells with villi facing outward.

In addition, in the cell structure with peristaltic motility obtained in Example A, as shown in the middle and bottom panels of Figure 5, c-kit-positive Cajal cells, βIII tublin-positive nerve cells, and SMA-positive muscle cells were examined, and it was confirmed that these cells were present in close proximity to the lower layer of the tissue containing the small intestinal epithelial cells.

The upper part of Figure 6 shows the results of immunostaining of the cell structures obtained in Comparative Example A that had peristaltic motility, and the lower part shows the results of immunostaining of the cell structures obtained in Comparative Example A that did not have peristaltic motility. In the cell structures that had peristaltic motility, tissues in which c-kit positive Cajal cells and SMA positive muscle cells were in close proximity were observed. On the other hand, in the cell structures that did not have peristaltic motility, SMA positive muscle cells and βIII tublin positive nerve cells (not shown) were observed, but c-kit positive Cajal cells were not observed. This suggests that the lack of peristaltic motility is due to poor development of pacemaker cells.

Based on these results, it is presumed that culturing cell structures in a medium containing N-2 supplements promotes differentiation into Cajal cells, and that the formation of tissue in which Cajal cells come into contact with muscle cells and nerve cells facilitates peristalsis.

Figure 7 shows the results of immunostaining for E-cadherin and AFP for the cell structure with peristaltic motility obtained in Example A, the cell structure with peristaltic motility obtained in Example B, and the cell structure with peristaltic motility obtained in Comparative Example A. In the cell structure with peristaltic motility obtained in Comparative Example A, AFP expression was observed in almost all small intestinal epithelial cells (E-cadherin positive cells). On the other hand, in Example A, the AFP positivity rate among small intestinal epithelial cells was reduced to 4.7-8%. The positivity rate was also 2.6% in Example B. These results suggest that the expression of AFP tends to decrease by suspension culture of the bag-shaped cell structure in the presence of N-2 supplement, and suggest that the property was maintained.

Figure 8 shows the results of immunostaining for Ki67 and CDX2 for the cell structure with peristaltic motility obtained in Example A and the cell structure without peristaltic motility obtained in Comparative Example A. In Example A, there were no Ki67-positive cells (i.e., dividing cells) among the CDX2-positive cells. In contrast, Ki67-positive CDX2-positive cells were observed in Comparative Example A, and their proportion was 6.4% of all CDX2-positive cells.

Figure 9 shows the results of immunostaining for PCNA (another dividing cell marker) and E-cadherin (small intestinal epithelial cell marker) for the cell structure with peristaltic motility obtained in Example A, the cell structure with peristaltic motility obtained in Example B, and the cell structure with peristaltic motility obtained in Comparative Example A. In Example A, the PCNA positivity rate was 1% or less among the E-cadherin-positive small intestinal epithelial cells. In Comparative Example A, the PCNA positivity rate was in the range of 6.0 to 16.3% among the E-cadherin-positive small intestinal epithelial cells. On the other hand, in Example B, the PCNA positivity rate was increased to 9.6%, suggesting that the inhibition of epithelial dividing cells in Example A was induced by immersion in a medium containing N-2 supplement.

Figure 10 shows images of the pouch-shaped cell structures of Example A and Comparative Example A observed in culture medium. The pouch-shaped cell structures are characterized by the villus layer having small intestinal epithelial cells facing outward, and as such, cells that turn over during suspension culture are released into the culture medium. The number of cells released into the culture medium tended to be far fewer in Example A than in Comparative Example A. This result also suggests that there is a difference in the number of dividing cells.

<Experiment 4>
To examine the effects of differences in cell types, the following analysis was performed.

Using human ES cells, SEES2 cells, pattern culture and suspension culture were performed under the same conditions as in Example A of Experiment 1, and a pouch-shaped cell structure containing small intestinal epithelial cells was obtained. For suspension culture, the cells were cultured for about one month in a medium containing 1/100 diluted N-2 supplement. This is referred to as Example C.

On the other hand, human ES cells, SEES2 cells, were used to carry out pattern culture and suspension culture under the same conditions as Comparative Example A of Experiment 1, and a pouch-shaped cell structure containing small intestinal epithelial cells was obtained. This is referred to as Comparative Example D.

As in Experiment 1, when 10 μM serotonin-creatinine sulfate monohydrate (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the culture medium containing the cell structure of Example C, peristalsis-like movement was observed in all six cell structures tested.
In contrast, when the cell constructs of Comparative Example D were stimulated in the same manner, none of the eight cell constructs tested exhibited peristaltic movement.

Furthermore, immunostaining of tissue sections of the cell structure of Example C was performed using the same procedure as in Experiment 3. CDX2, Villin, Ki67, E-cadherin, and AFP were immunostained as markers.

The results of immunostaining are shown in FIG. 11. It was confirmed that the cell structure of Example C has a small intestinal epithelial structure with the villus layer facing outward, as in the cell structure of Example A. In addition, the proportion of CDX2-positive cells positive for Ki67, a dividing cell marker, in the cell structure of Example C was only 0.038% of the CDX2-positive cells, suggesting that the proportion of dividing cells was reduced by the addition of N-2 supplement, as in the cell structure of Example A. Furthermore, although not shown, the presence of AFP-positive small intestinal epithelial cells was not observed in the cell structure of Example C. That is, in the cell structure of Example C, a reduction in AFP expression was observed, as in the cell structures of Examples A and B.
The above results indicate that the promotion of peristalsis and the suppression of AFP expression by N-2 supplement are not limited to the type of pluripotent stem cells used.

<Experiment 5>
The following investigation was carried out regarding limitations on the nutritional factors and medium type to be added.

The medium used for the suspension culture in Example A of Experiment 1 (XF32 medium with N-2 supplement) contains basal medium KDMEM, 15% by volume of XF-KSR, bFGF, IGF-I, Hereglin, 1/100 diluted N-2 supplement, and other components. The concentrations of each component are as described in Experiment 1. The medium used in Comparative Example A of Experiment 1 is the same as that in Example A, except for the absence of N-2 supplement.

In this experiment, media 1 to 8 were prepared with different contents or types of serum replacement components, nutritional factors (bFGF, IGF-I, Hereglin), and candidate components expected to promote peristalsis. The table below shows the differences between media 1 to 8 and the XF32 medium supplemented with N-2 used in Example A. Components not shown in the table in media 1 to 8 have the same substances and contents as the XF32 medium supplemented with N-2 used in Example A.

Medium 1 has a composition similar to that of XF32 medium supplemented with N-2 supplement, except that bFGF, IGF-I, and Hereglin have been removed.

Medium 2 is an N-2 supplement-added XF32 medium, but with B-27 (registered trademark) supplement (Invitrogen) added instead of N-2 supplement. B-27 supplement contains all the components of N-2 supplement (WO2013/061608).

Medium 3 is an N-2 supplement-added XF32 medium, but with 15 volume % KSR (Knockout Serum Replacement) (Life Technologies) added as a serum replacement component in place of XF-KSR.
Medium 4 is an N-2 supplemented XF32 medium in which the concentration of XF-KSR was reduced to 1% by volume.
Medium 5 is an N-2 supplement-added XF32 medium from which XF-KSR has been removed.

Medium 6 is an N-2 supplement-added XF32 medium that contains 15% by volume of FBS (fetal bovine serum) (Life Technologies) instead of XF-KSR as a serum replacement component.

Medium 7 is a medium in which 10 μM L-carnosine (Fujifilm Wako Pure Chemical Industries, Ltd.) is added instead of N-2 supplement in the XF32 medium supplemented with N-2 supplement. According to Fujii et al., Cytotechnology Vol. 69, pp. 523-527, 2017 and Sugihara et al., Plos One Vol. 14, e0217394, 2019, it has been reported that L-carnosine induces the neurotrophic factor BDNF in Caco-2 cells used in intestinal epithelial cell culture, and neural induction may occur in the presence of L-carnosine. Similarly, the above literature has also reported that exosomes released from Caco-2 cells supplemented with L-carnosine promote the growth of neural cells.

Medium 8 is an N-2 supplement-added XF32 medium, but with 10 ng/mL BMP4 (R&D System) added instead of the N-2 supplement. Regarding BMP4, WO2018/199142 discloses a technique for inducing sympathetic nerve dominance by culturing neurospheres in a medium containing BMP4. Since sympathetic nerves have the effect of enhancing peristalsis, BMP4 may be able to activate peristalsis.

Pattern culture and suspension culture from Edom iPS cells were performed under the same conditions as in Example A, except that one of the above media 1 to 8 was used as the medium for suspension culture and suspension culture was performed for one month, and a bag-shaped cell structure was obtained. After one month of suspension culture, 10 μM serotonin-creatinine sulfate monohydrate was added to the bag-shaped cell structures obtained under each condition, as in Example 1, and the presence or absence of peristalsis was observed. Six or more cell structures were used as samples for each condition and observations were performed.

The results are shown in the "Peristaltic Movement" column in the table above. If peristaltic movement was observed in more than half of the six or more cell structures observed, it was marked "Yes," and if peristaltic movement was observed in less than half, it was marked "No." For comparison, Example A and Comparative Example A are also shown in the table.

Even when medium 1, from which the nutritional factors bFGF, heleglin, and IGF-I were removed, was used for suspension culture, cell structures with significant peristaltic motility were obtained, as in Example A, suggesting that these nutritional factors are not essential for inducing peristaltic motility.

Furthermore, when medium 3 containing KSR as a serum replacement component and medium 4 containing 1% by volume of XF-KSR were used for suspension culture, cell structures with significant peristaltic motility were obtained, as in Example A, whereas when medium 5 from which the serum replacement component was removed and medium 6 containing serum were used for suspension culture, no cell structures with peristaltic motility were obtained. These results suggest that serum replacement components are desirable. It is also suggested that the concentration of serum replacement components in suspension culture media should be 1% by volume or more as a guideline.

When medium 2, which was prepared by adding B-27 (registered trademark) supplement (containing components of N-2 supplement) instead of N-2 supplement in XF32 medium supplemented with N-2 supplement, was used for suspension culture, cell structures capable of peristaltic motility were obtained. On the other hand, when medium 7, which was prepared by adding L-carnosine instead of N-2 supplement in XF32 medium supplemented with N-2 supplement, and medium 8, which was prepared by adding BMP4, were used for suspension culture in XF32 medium supplemented with N-2 supplement, cell structures capable of peristaltic motility were not obtained. This suggests that it is desirable to add components including N-2 supplement to the medium used for suspension culture.

<Experiment 6>
The following study was carried out to examine whether the cell structures in which peristaltic motility was induced by suspension culture in a medium containing N-2 supplement could be used in toxicity detection tests.

10 μM serotonin-creatinine monohydrate was added to the bag-shaped cell structure obtained in Example A of Experiment 1 to confirm that the cell structure had peristaltic motility, and then 25 μM tetrodotoxin (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the cell structure and left for 5 to 10 minutes, after which changes in peristaltic motility were observed. After the observation, the same cell structure was placed in new XF32 medium supplemented with serotonin-creatinine monohydrate to confirm whether peristaltic motility occurred again.

When tetrodotoxin was added to the cell structure that was undergoing peristaltic movement, it was observed that the movement of the cell structure stopped. Furthermore, when the cell structure was added to fresh XF32 medium containing serotonin-creatinine monohydrate after the observation, peristalsis resumed. This suggested that the toxicity of tetrodotoxin did not cause the tissue to die, causing the movement to stop.

In addition, when 100 ng/ml palytoxin (Fujifilm Wako Pure Chemical Industries) was added to the culture medium instead of tetrodotoxin and changes in the peristaltic movement of the cell structures were observed in the same manner as above, the peristaltic movement stopped 5 to 10 minutes after the addition of palytoxin, as above. It was confirmed that this method can be used in toxicity detection tests regardless of the type of poison.

The above tests confirmed that cell structures in which peristaltic motility was induced by suspension culture in a medium containing N-2 supplements can be used in toxicity detection tests.

<Experiment 7>
In Experiment 1, tissue sections were prepared from the cell structure with peristaltic motility obtained in Example A and the cell structure without peristaltic motility obtained in Comparative Example A, and immunostaining was performed using the procedure described in Experiment 3.

To confirm the presence of smooth muscle cells, the following antibodies were used as primary antibodies.
Rabbit IgG-labeled anti-Desmin antibody (Abeam, dilution ratio 1/500)
Mouse IgG1-labeled anti-Vimentin antibody (DAKO, undiluted)
Mouse IgG2a-labeled anti-Smooth Muscle Actin antibody (SMA; Sigma, dilution ratio 1/500)

The following antibodies were used as secondary antibodies.
Alexa488-labeled anti-rabbit IgG antibody Alexa488-labeled anti-mouse IgG1 antibody Alexa543-labeled anti-mouse IgG antibody Alexa568-labeled anti-mouse IgG2a antibody

The results of immunostaining for Desmin and SMA for the cell structure sample with peristaltic motility obtained in Example A are shown in Figure 12, and the results of immunostaining for Vimentin and SMA are shown in Figure 13. The results of immunostaining for Desmin and SMA for the cell structure sample without peristaltic motility obtained in Comparative Example A are shown in Figure 14, and the results of immunostaining for Vimentin and SMA are shown in Figure 15. The scale bars in Figures 12 to 15 are 100 μm.

Figures 12 and 13 suggest that in the cell structure with peristaltic motility obtained in Example A, SMA-positive cells include both Vimentin-positive cells and Desmin-positive cells.

Figures 14 and 15 suggest that in the cell structure without peristalsis obtained in Comparative Example A, most SMA-positive cells were Vimentin-negative, and the proportion of smooth muscle cells was extremely low.

Claims (15)

Small intestinal epithelial cell layer, and
tissues including Cajal cells, nerve cells and muscle cells,
The small intestinal epithelial cell layer contains α-fetoprotein-negative small intestinal epithelial cells.
A cell structure comprising:
maintained in a medium containing serum replacement components, as well as transferrin, insulin, progesterone, putrescine and selenite;
The cell structure. The cell structure is sac-shaped;
The small intestinal epithelial cell layer is present on the outer periphery of the cell structure with the villus layer facing outward.
The cell structure of claim 1. The cell structure according to claim 1 or 2, wherein the proportion of α-fetoprotein-positive small intestinal epithelial cells among the small intestinal epithelial cells contained in the small intestinal epithelial cell layer is 8% or less. The cell structure according to any one of claims 1 to 3, wherein the muscle cells include desmin-positive and smooth muscle actin-positive smooth muscle cells. The cell structure according to any one of claims 1 to 4, which has the ability to spontaneously undergo peristaltic movement in the presence of one or more compounds selected from histamine compounds and serotonin compounds. The cell structure according to any one of claims 1 to 5, which is induced to differentiate from a pluripotent stem cell. The cell structure according to claim 6, wherein the pluripotent stem cells are derived from humans. A method for evaluating an effect of a test substance on the small intestine, comprising the steps of:
A step 11 of observing the cell structure according to any one of claims 1 to 7 in the presence of a test substance; and a step 12 of evaluating the effect of the test substance on the small intestine based on the observation results.
The method includes: The method according to claim 8, wherein step 11 is carried out in the presence of one or more compounds selected from histamine compounds and serotonin compounds. After step 11, the method further comprises step 13 of washing the cell structure with a liquid medium not containing the test substance,
After step 13, using the cell structure again in step 11;
10. The method of claim 9, comprising: Small intestinal epithelial cell layer, and
tissues including Cajal cells, nerve cells and muscle cells,
The small intestinal epithelial cell layer contains α-fetoprotein-negative small intestinal epithelial cells.
A method for producing a cell structure, comprising the steps of:
21. Seeding stem cells onto a cell culture substrate having a pattern of cell adhesion sites on its surface;
Step 22: culturing the stem cells to induce differentiation into immature cell structures including a small intestinal epithelial cell layer;
and step 23 of culturing the immature cell structure in suspension in a medium containing a serum replacement component, as well as transferrin, insulin, progesterone, putrescine and selenite, to induce differentiation into the cell structure. A kit for evaluating the effect of a test substance on the small intestine, comprising the cell structure according to any one of claims 1 to 7. Serum replacement components,
Transferrin,
Insulin,
Progesterone and/or progestin,
The agent for activating the peristaltic movement of a cell structure according to any one of claims 1 to 7 , comprising putrescine and selenite as active ingredients. Serum replacement components,
Transferrin,
Insulin,
Progesterone and/or progestin,
8. An inhibitor of small intestinal epithelial cell proliferation , comprising the cell structure according to claim 1, and comprising putrescine and selenite as active ingredients. Serum replacement components,
Transferrin,
Insulin,
Progesterone and/or progestin,
A maturation promoter for small intestinal epithelial cells , which is contained in the cell structure according to any one of claims 1 to 7 and comprises putrescine and selenite as active ingredients.

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