WO2024112170A1 - Method for non-xenogeneic culturing of intestinal stem cells on surface coated with thin film - Google Patents

Abstract

The present invention relates to a culture method for non-xenogeneic culturing of intestinal stem cells on a culture dish coated with a polymeric thin film, which does not include xenogeneic components such as Matrigel, and a use thereof. The method for culturing intestinal stem cells according to the present invention enables easy and rapid cultivation of homogeneous cells, enables mass-cultivation of cells at a low cost, and eliminates the risk of xenogeneic infection by culturing cells in a non-xenogeneic culture environment, thereby enabling the development of cell therapeutics that can actually be transplanted in practice. Another advantage is that cells can be cultured in a state in which cellular characteristics are maintained even during organ subculture and cryopreservation. Furthermore, the method for producing intestinal epithelial cells, according to the present invention, enables intestinal organoid-derived intestinal stem cells cultured in a non-xenogeneic culture environment to differentiate into intestinal epithelial cells in the same non-xenogeneic culture environment.

Description

Method for xenogeneic culture of intestinal stem cells on thin film-coated surfaces

The present invention relates to a culture method for xenogeneic culture of intestinal stem cells on a culture dish coated with a polymer thin film without containing xenogeneic components such as Matrigel, and its use.

Intestinal organoids are used as cell therapy and are attracting attention as a regenerative treatment that can regenerate the affected area by transplanting organoids into the intestines that are damaged or underdeveloped due to intestinal disease. Intestinal organoids are composed of various cells, including intestinal stem cells, which are a key component of regeneration, and have been found to engraft in the affected area and efficiently regenerate damaged tissue, so the potential for development as a therapeutic agent continues to increase.

Recently, several groups have repeatedly confirmed the phenomenon of regeneration of damaged intestinal tissue by transplanting human intestinal organoids into models with damaged intestinal tissue, and the transplanted intestinal organoids can regenerate the structure and function of actual intestinal tissue at a high level. It has been confirmed that it is possible.

However, in order for intestinal organoids to be used as actual cell therapy products, they must contain intestinal stem cells with homogeneous performance, and it must be possible to mass-culture intestinal organoids with a constant ratio of intestinal stem cells. To reduce the risk of cross-infection between species, the development of a culture system capable of heterogeneous cell culture is necessary.

In addition, in addition to the utilization of intestinal stem cells in vivo, the above culture system was developed to secure an intestinal epithelial cell model containing various intestinal cell types through a differentiation process from intestinal stem cells with excellent performance at the laboratory level. Needs are continuously growing.

Currently, intestinal organoids are cultured through a three-dimensional culture method using Matrigel, which is produced by separating the extracellular matrix produced from sarcomas transplanted to animals. The differences between batches are severe, so homogeneity is low and mass culture is difficult. , it has the disadvantage of high cultivation costs. In addition, since it is extracted from a heterogeneous species, there is a risk of causing heterogeneous infection, which is acting as an obstacle to the development of cell therapy.

Accordingly, there is a need to develop an intestinal stem cell culture method that has no risk of heterogeneous infection, can mass culture homogeneous intestinal stem cells, and can reduce the cost of therapeutic agents through low production costs.

Against this background, the present inventors completed the invention by confirming a culture method that can easily and quickly cultivate xenogeneic intestinal stem cells derived from intestinal organoids in a two-dimensional culture environment by coating the surface of a culture dish with a polymer and various uses related thereto. did.

The purpose of the present invention is to provide a method of culturing intestinal stem cells on a culture substrate containing a polymer thin film manufactured through an iCVD process.

Another object of the present invention is to provide a method of producing intestinal epithelial cells from intestinal stem cells cultured on a culture substrate containing a polymer thin film produced through the iCVD process.

In the following, in order to prevent confusion with overlapping content, description of overlapping content will be omitted. In other words, the content of the invention is not limited only to the following content, and the content of the invention should be interpreted according to the overall content of the invention.

The present invention provides a method of culturing intestinal stem cells on a culture substrate containing a polymer thin film manufactured through an iCVD process.

The method of culturing the intestinal stem cells is carried out through the following steps:

(1) providing a culture substrate containing a polymer thin film manufactured through an iCVD process; and

(2) Adding a culture medium containing a Wnt signaling pathway activator, a Wnt agonist, a TGF-beta inhibitor, a BMP inhibitor, and a receptor tyrosine kinase ligand to the culture substrate, and cultivating intestinal stem cells in two dimensions.

In the present invention, “iCVD (initiated chemical vapor deposition) process” refers to a process of coating a polymer thin film by vaporizing an initiator and a monomer to cause a polymer reaction in the gas phase. Specifically, in this process, the initiator is decomposed by the high-temperature filament to form free radicals, which activates the monomer and causes a chain polymerization reaction. Through this, it is possible to coat a polymer thin film while preserving the functional groups of the monomer without unnecessary side reactions.

In other words, the iCVD process is a chemical vapor deposition process, so it is possible to deposit the desired polymer thin film using monomers and initiators under gaseous conditions without using solvents, especially organic solvents, and even if it includes a substrate underneath, the substrate due to solvent The risk of damage can be eliminated, and a high-purity thin film can be obtained as no residue remains.

The initiator is a material that induces the activation of the first reaction so that the monomer can form a polymer in the iCVD process. A material that can thermally decompose at a temperature lower than the temperature at which the monomer thermally decomposes to form free radicals is preferable. When free radicals are formed by thermal decomposition of the initiator, the free radicals activate the monomers and subsequently induce polymerization of surrounding monomers, and this reaction continues to form an organic polymer thin film. For example, the initiator may be tert-butyl peroxide (TBPO) or benzophenone, but is not limited thereto. TBPO is a volatile substance with a boiling point of about 110°C and is a substance that thermally decomposes around 150°C. Meanwhile, the amount of initiator added is the amount required for a normal polymerization reaction and can be added in an amount known in the art, for example, 0.5 to 5 mol%, but is not limited to the above range and may be more than the above range. You can write it down.

In the present invention, “monomer” refers to a unit that can be used to form an organic polymer thin film, and is a material that is volatile in a chemical vapor deposition process and can be activated by an initiator. The monomer may be vaporized under reduced pressure and elevated temperature.

The monomers include DMAEMA (2-(Dimethylamino)ethyl methacrylate), DMAPMA (Dimethylaminopropyl methacrylamide), DEAEMA (2-(Diethylamino)ethyl methacrylate), TBAEMA (2-tert-Butylaminoethyl methacrylate), and DMAEA (2-(Dimethylamino )ethyl acrylate), GMA (Glycidyl methacrylate), EGDMA (Ethylene glycol dimethacrylate), VBC (Vinyl benzyl chloride), CHMA (Cyclohexyl methacrylate), V4D4 (2,4,6,8-Tetramethyl-2,4,6,8 It may be any one or more compounds selected from the group consisting of -tetravinylcyclotetra siloxane) and BMA (Benzyl methacrylate). Preferably, it may be EGDMA (Ethylene glycol dimethacrylate) or CHMA (Cyclohexyl methacrylate).

The polymer thin film produced through the iCVD process may be, for example, any one selected from the following:

pDMAEMA(poly(2-(Dimethylamino)ethyl methacrylate)), pDMAPMA(poly(Dimethylaminopropyl methacrylamide)), pDEAEMA(poly(2-(Diethylamino)ethyl methacrylate)), pTBAEMA(poly(2-tert-Butylaminoethyl methacrylate)), pDMAEA(poly(2-(Dimethylamino)ethyl acrylate)), pGMA(poly(Glycidyl methacrylate)), p(GMA-co-DMAEMA)(poly(Glycidyl methacrylate-co-(2-(dimethylamino)ethyl methacrylate))) , p(GMA-co-DMAPMA)(poly(Glycidyl methacrylate-co-dimethylaminopropyl methacrylamide)), p(GMA-co-DEAEMA)(poly(Glycidyl methacrylate-co-(diethylamino)ethyl methacrylate))), p(GMA -co-TBAEMA)(poly(Glycidyl methacrylate-co-(2-tert-butylaminoethyl methacrylate))), p(GMA-co-DMAEA)(poly(Glycidyl methacrylate-co-(2-(dimethylamino)ethyl acrylate)) ), pEGDMA(poly(Ethylene glycol dimethacrylate)), p(EGDMA-co-DMAEMA)(poly(Ethylene glycol dimethacrylate-co-(2-(dimethylamino)ethyl methacrylate))), p(EGDMA-co-DMAPMA)( poly(Ethylene glycol dimethacrylate-co-dimethylaminopropyl methacrylamide)), p(EGDMA-co-DEAEMA)(poly(Ethylene glycol dimethacrylate-co-(diethylamino)ethyl methacrylate))), p(EGDMA-co-TBAEMA)(poly( Ethylene glycol dimethacrylate-co-(2-tert-butylaminoethyl methacrylate))), p(EGDMA-co-DMAEA)(poly(Ethylene glycol dimethacrylate-co-(2-(dimethylamino)ethyl acrylate))), pVBC(poly( Vinyl benzyl chloride)), p(VBC-co-DMAEMA)(poly(Vinyl benzyl chloride-co-(2-(dimethylamino)ethyl methacrylate))), p(VBC-co-DMAPMA)(poly(Vinyl benzyl chloride- co-dimethylaminopropyl methacrylamide)), p(VBC-co-DEAEMA)(poly(Vinyl benzyl chloride-co-(diethylamino)ethyl methacrylate))), p(VBC-co-TBAEMA)(poly(Vinyl benzyl chloride-co- (2-tert-butylaminoethyl methacrylate))), p(VBC-co-DMAEA)(poly(Vinyl benzyl chloride-co-(2-(dimethylamino)ethyl acrylate)))), pCHMA(poly(Cyclohexyl methacrylate)), pV4D4 (poly(2,4,6,8-Tetramethyl-2,4,6,8-tetravinylcyclotetra siloxane)) and pBMA (poly(Benzyl methacrylate)).

More preferably, it may be pEGDMA (poly(Ethylene glycol dimethacrylate)) or pCHMA (poly(Cyclohexyl methacrylate)). The pEGDMA (poly(Ethylene glycol dimethacrylate)) or pCHMA (poly(Cyclohexyl methacrylate)) has the advantage of exhibiting excellent surface contact angle level and surface homogeneity through the iCVD process.

For example, the polymer thin film manufactured through the iCVD process may have a water contact angle in the range of 10 to 45°, more preferably in the range of 15 to 35°.

In addition, it has an additional advantage in that it can provide culture conditions without endotoxin components.

Accordingly, it increases the engraftment rate of intestinal stem cells in intestinal stem cell culture and provides culture conditions for intestinal stem cells that exhibit characteristics of intestinal stem cells at a level similar to that of the Matrigel environment.

The polymer thin film produced through this iCVD process may be coated on a culture substrate.

A method of providing a culture substrate including a polymer thin film manufactured through an iCVD process includes supplying a monomer and an initiator to the culture substrate; Injecting heat under constant pressure and thermally decomposing the initiator to form free radicals; And it may be characterized by including the step of activating the monomers using the free radicals to carry out a chain polymerization reaction of the monomers and depositing the polymer formed as a biomimetic thin film on the substrate.

More preferably, plasma treatment may be further included after the iCVD process.

For example, after the polymer thin film is coated through the iCVD process, plasma treatment may be provided to the culture substrate for 1 to 200 seconds under 5 to 100 W, more preferably for 5 to 20 seconds under 10 to 20 W. .

Through this, it is possible to provide a more preferable culture substrate for culturing intestinal stem cells by controlling the surface contact angle level and surface homogeneity of the culture substrate.

The culture substrate may be selected from the group consisting of glass, metal, metal oxide, fiber, paper, and plastic, and the plastic is polyethylene (PE), polypropylene (PP), and polystyrene. , PS), polyethylene terephthalate (PET), polyamides (PA), polyester (PES), polyvinyl chloride (PVC), polyurethanes (PU), polycarbonate (polycarbonate, PC), polyvinylidene chloride (PVDC), polytetrafluoroethylene (PTFE), polyetheretherketone (PEEK), and polyetherimide (PEI). It can be characterized as being.

In the present invention, “stem cells” refer to cells in an undifferentiated state that have the ability to continue proliferation, that is, have the ability to self-replicate, and have the ability to differentiate into various types of specific cells that make up tissues or organs. . More specifically, “intestinal stem cells” are undifferentiated cells derived from intestinal epithelial tissue that have the ability to self-replicate and refer to cells that have the ability to differentiate into various types of specific cells present in the intestinal epithelium.

Differentiation from intestinal stem cells into, for example, intestinal stem cells, intestinal progenitor cells, intestinal epithelial cells, intestinal goblet cells, intestinal endocrine cells, Paneth cells, etc. is possible.

If necessary, the culture conditions according to the present invention are also suitable for culturing intestinal stem cells, intestinal progenitor cells, intestinal epithelial cells, intestinal goblet cells, enteroendocrine cells, and Paneth cells and can be used for their application.

Intestinal stem cells more specifically exhibit LGR5 positive characteristics.

The intestinal stem cells may be adult-derived intestinal stem cells or intestinal organoid-derived intestinal stem cells derived from hPSC (human pluripotent stem cells).

More specifically, it is characterized by further exhibiting positive expression characteristics of EPHB3, CD44, SOX9 and MKI67.

More specifically, it may be intestinal stem cells derived from intestinal organoids derived from human pluripotent stem cells (hPSC), including human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC).

The method for culturing intestinal stem cells according to the present invention provides preferable culture conditions for culturing cells exhibiting the characteristics of intestinal stem cells. More specifically, it provides a preferable environment for culturing intestinal stem cells derived from intestinal organoids derived from hPSC (human pluripotent stem cells), including human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC).

In the present invention, “culture medium” refers to a medium for maintaining or cultivating a cell population containing nutrients that maintain cell viability and support proliferation.

Culture media used in the present invention include basic media. The basic medium is any basic medium suitable for culturing animal or human cells.

The basal medium typically contains a number of components necessary to support the maintenance of cultured cells. A combination of suitable ingredients can be easily formulated by a skilled person by considering the following. It also includes a nutrient solution containing common standard cell culture ingredients such as amino acids, vitamins, lipid supplements, mineral salts, carbon energy sources, and buffers.

The basal media can be obtained commercially, including but not limited to Dulbecco's Modified Eagles Media (DMEM), Minimum Essential Media (MEM), KnockOut-DMEM (KO-DMEM), Glasgow's Minimum Essential Media (G-MEM), Basal Medium. Eagle (BME), DMEM/Ham's F12, Advanced DMEM/Ham's F12, Iscove's Modified Dulbecco's Media and Minimum Essential Media (MEM), Ham's F-10, Ham's F-12, Medium 199, RPMI 1640 Medium, and KnockOut Serum replacement XenoFree Includes medium. For example, the basic medium may be DMEM/F12, Advanced DMEM/F12, and/or RPMI 1640 medium.

If necessary, Advanced DMEM/F12 or Advanced RPMI, which are optimized for serum-free culture and already contain insulin, are used.

The culture medium used in the present invention is provided for culturing intestinal stem cells and essentially includes a Wnt signaling pathway activator, a Wnt agonist, a TGF-beta inhibitor, a BMP inhibitor, and a receptor tyrosine kinase ligand.

The Wnt signaling pathway activator refers to a substance that activates the Wnt/beta-catenin pathway to increase the target of Wnt/beta-catenin, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, It may be one of Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt16. Preferably it may be Wnt3a. More preferably, about 50 to 300 ng/ml Wnt3a may be used.

The Wnt agonist may preferably be R-spondin 1, R-spondin 2, R-spondin 3 or R-spondin 4 or a derivative thereof. The culture medium contained 50 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, 1 It may be included at a concentration of ug/ml, 1.5 ug/ml, or 2 ug/ml or more. Preferably it may be R-spondin 1. More preferably, it may contain approximately 50 to 800 ng/ml of R-spondin 1.

The TGF-beta inhibitor is any substance that inhibits the function of the TGF-beta receptor, such as a protein, peptide, or small molecule, such as A83-01, SB-431542, SB-505124, SB-525334, SD-208, It may be LY-36494 and SJN-2511. Preferably it may be A83-01. More preferably, it may be 100 nM to 1,000 nM of A83-01.

The BMP inhibitor is an agent that binds to BMP molecules to form a complex. The inhibitor may be an agent that binds to the BMP receptor and prevents binding of the BMP ligand to the receptor, for example, an antibody that binds to the receptor. BMP inhibitors may be proteins or small molecules and may be natural, modified and/or partially or fully synthetic. The BMP inhibitor may be Noggin, Dorsomorphin, DMH1, or LDN-193189. Preferably it may be a noggin. More preferably, it may contain about 10 ng/ml to 150 ng/ml of noggin.

The receptor tyrosine kinase ligands include epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), and hepatocyte growth factor (HGF). ), a mitogenic growth factor selected from growth factors consisting of neuregulin 1 (NRG1) and keratinocyte growth factor (KGF). Preferably it is EGF. EGF is a potent mitogen for a variety of cultured ectodermal and mesodermal cells and has sufficient effects on the differentiation of certain cells in vivo and in vitro and some fibroblasts in cell cultures. Preferred concentrations are 10, 20, 25, 30, 40, 45, or 50 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml. That's it. A more preferable concentration is 50 ng/ml or more and 300 ng/ml or less.

In addition to the above-described essential composition, the culture medium of the present invention may further include one or more additional components selected from the group consisting of p38 kinase inhibitor, Prostaglandin E2, N-acetylcysteine, Gastrin, B27, and Nicotinamide. Additionally, a ROCK inhibitor, a Notch activator, or both may be further included in the culture medium at the beginning of the culture.

The p38 kinase inhibitor inhibits the activity of p38 kinase, which plays a role in phosphorylating and activating transcription factors and other kinases, for example, SB202190, SB203580, SB239063, SB706504, BIR796, JX401, EO1428, RWJ67657, SCIO469, VX745, It may be TAK715, ML3403, DBM1285, or PH797804. Preferably it may be SB202190. More preferably, it may be 5 uM to 50 uM of SB202190.

Prostaglandin E2, N-acetylcysteine, Gastrin, B27, and Nicotinamide can be added as needed to improve culture efficiency and lifespan, control cell proliferation, and help with DNA stability.

The B27 may be replaced by a generic formulation containing one or more of the following ingredients: biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, tri-iodothyronine. (T3), DL-alpha-tocopherol (vitamin E), albumin, insulin and transferrin.

The ROCK (Rho-associated protein kinase) inhibitor serves to inhibit the activity of serine/threonine kinases that act as target proteins for Rho (Rho A, Rho B, and Rho C), and is preferably R-(+ )-trans-4-(1-Aminoethyl)-N-(4-pyridyl)cyclohexane carboxamide dihydrochloride monohydrate (Y-27632).

The Notch activator refers to a protein or small molecule compound that activates the Notch pathway function, and is preferably Jagged-1 (JAG 1).

More specifically, (2) according to the present invention, a culture medium containing a Wnt signaling pathway activator, a Wnt agonist, a TGF-beta inhibitor, a BMP inhibitor, and a receptor tyrosine kinase ligand is added to the culture substrate to form intestinal stem cells. The two-dimensional culture step may be two-dimensional culture of intestinal stem cells in a culture medium containing Wnt3a, R-spondin 1, A-83-01, Noggin, and EGF.

More specifically, it may further include one or more additional ingredients selected from the group consisting of SB202190, Prostaglandin E2, N-acetylcysteine, Gastrin, B27, and Nicotinamide.

In addition, at the beginning of the culture, for example, immediately after treating the intestinal stem cells on the stomach culture substrate, ROCK inhibitor, Notch activator, or all of them are added to the culture medium for 1, 2, 3, 4, or 5 days. It can be included.

The culture of intestinal stem cells according to the present invention is not limited to this, but is cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, 2 weeks, 3 days. It can proceed for a week or more, and subculture can be performed as needed.

Additionally, cells can be frozen or thawed under freezing conditions as needed.

Cell characteristics are maintained and culture can proceed even under subculture conditions and/or freezing and thawing conditions for these organs.

The present invention provides a method of producing intestinal epithelial cells from intestinal stem cells cultured on a culture substrate containing a polymer thin film produced through an iCVD process.

The method for producing the intestinal epithelial cells is carried out through the following steps:

(1) providing a culture substrate containing a polymer thin film manufactured through an iCVD process;

(2) adding a culture medium containing a Wnt signaling pathway activator, a Wnt agonist, a TGF-beta inhibitor, a BMP inhibitor, and a receptor tyrosine kinase ligand to the culture substrate and cultivating intestinal stem cells in two dimensions; and

(3) The intestinal stem cells cultured through step (2) above were placed on the culture substrate with differentiation medium containing a Wnt agonist, p38 kinase inhibitor, receptor tyrosine kinase ligand, Prostaglandin E2, and Nicotinamide, and placed at the air-liquid interface ( Differentiation step through the Air-Liquid Interface (Air-Liquid Interface) culture method.

Since the culture medium in step (2) includes description of the above-described Wnt signaling pathway activator, Wnt agonist, TGF-beta inhibitor, BMP inhibitor, p38 kinase inhibitor, and receptor tyrosine kinase ligand, duplicate content is provided herein. To avoid excessive complexity of the specification, its description is omitted.

In addition, matters regarding the polymer thin film, iCVD process, and plasma treatment mentioned in step (1) above include the above-mentioned description, and duplicate description is omitted to avoid excessive complexity of the present specification.

In the present invention, “differentiation medium” refers to a cell growth medium that allows undifferentiated stem cells to develop into cells with some or all of the characteristics of differentiated cells when cultured in the medium, and includes basic medium.

In the present invention, “air-liquid interface culture method” may mean culturing in a partially open culture vessel or a culture vessel partially filled with a medium, but is not limited thereto. For example, it may be exposing the surface of cells or organoids to air. Although referred to as “air” for convenience, the present invention is not limited to compositions and mixtures of gases found in the ambient environment. Specifically, the present invention contemplates and encompasses gas mixtures that have a different composition than the surrounding environment, for example, mixtures enriched for certain components or mixtures depleted or eliminated of certain components.

When culturing cells at an air-liquid interface, the cells can be cultured on the porous substrate by contacting the air on the top side of the porous substrate and contacting the cell culture medium on the bottom side. For example, a sufficient volume of medium containing a porous substrate (e.g., a filter insert) such that the medium contacts the bottom surface of the cells present on the porous substrate but does not encapsulate or submerge the cells. It can be added to the bottom of the culture vessel. A suitable porous substrate can be formed from any material that does not adversely affect the growth and differentiation of cells. Exemplary porous substrates are made from polymers such as polyethylene terephthalate (PET), polyester, or polycarbonate. Suitable porous substrates may be coated or uncoated. Examples of commercially available extracellular matrices include extracellular matrix proteins (Invitrogen) and basement membrane preparations from Angelbreath-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex®). Basement membrane extract (Trevigen, Inc., type I collagen (Invitrogen), Vitrogel (registered trademark) (TheWell Bioscience Inc.) or Matrigel (trademark) ( BD Biosciences), etc. Preferably, in one embodiment of the present invention, the porosity of the substrate is sufficient to maintain cell viability and promote differentiation of cells. Suitable substrates should be about 0.3 to about 3.0 μm, about 0.3 to about 2.0 μm, about 0.3 to about 1.0 μm, about 0.3 to about 0.8 μm, about 0.3 to about 0.6 μm, about 0.3 to about 0.5 μm, about 0.5 μm. pore sizes of about 3.0 μm, about 0.6 to about 3.0 μm, about 0.8 to about 3.0 μm, about 1.0 to about 3.0 μm, about 2.0 μm to about 3.0 μm, preferably about 0.4 μm, and about 50 million to about 50 million. 120 million pores/cm2, about 60 million to about 110 million pores/cm2, about 70 million to about 100 million pores/cm2, preferably about 80 million to about 100 million pores/cm2, about 90 million pores. and a filter insert having a pore density of from about 100 million pores/cm2, more preferably about 100 million pores/cm2.

It may be advantageous to replace or regenerate the medium daily or every other day. Cells grown on top of a porous substrate are generally not single cells; rather, these cells exist in the form of sheets or aggregate clusters of cells. Cells cultured at the air-liquid interface can experience much higher oxygen tension compared to cells immersed in medium.

Intestinal epithelial cells prepared according to this method of producing intestinal epithelial cells can exhibit LGR5 positive characteristics, which are characteristics of intestinal stem cells, and can exhibit positive characteristics of maturation markers including OLFM4 and ASCL2 according to stem cell maturation. . It can show positive characteristics of VIL1, KRT20, FABP1, LCT, and SI, and expresses MUC2 as a characteristic of intestinal goblet cells, CHGA as a characteristic of enteroendocrine cells, and LYZ as a characteristic of Paneth cells.

The preparation of intestinal epithelial cells according to the present invention is not limited thereto, but may be carried out for 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, 2 weeks, 3 weeks or longer.

The intestinal stem cells or intestinal epithelial cells according to the present invention have the potential to be used as therapeutic agents.

Accordingly, the present invention provides a pharmaceutical composition containing the intestinal stem cells and/or intestinal epithelial cells.

The present invention provides a pharmaceutical composition for preventing or treating intestinal diseases containing the intestinal stem cells and/or intestinal epithelial cells.

The present invention provides a pharmaceutical composition for intestinal transplantation assistance containing the intestinal stem cells and/or intestinal epithelial cells. In the present invention, the intestinal stem cells can be used alone or in combination with other ingredients for various diseases that require administration. In particular, it can be used for therapeutic purposes as a bioengineering technology to restore the function of cells or tissues. For example, the pharmaceutical composition can be used as a transplant material and can be applied to the treatment of various intestinal diseases. In particular, it can be considered for use as a material for regeneration and reconstruction of damaged (including dysfunctional) intestinal tissue. Accordingly, the pharmaceutical composition according to the present invention may be a tissue therapeutic agent.

The present invention provides a method of treating patients suffering from intestinal disease or at risk of developing intestinal disease. The intestinal disease may be any one or more selected from the group consisting of leaky gut syndrome, short bowel syndrome, irritable bowel syndrome, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, infectious enteritis, ischemic bowel disease, and radiation enteritis.

The invention also provides a cell or cell population obtainable or obtained by a method of the invention for use in a method of treatment. In particular, the present invention provides a cell or cell population obtainable or obtained by the method of the present invention for use in a method of treating a patient suffering from or at risk of developing an enteric disease. In one embodiment, the method of treatment comprises transplanting cells obtainable or obtained by the method of the invention into the patient. In one embodiment, the method of treatment includes preparing pluripotent stem cells into intestinal organoids, e.g., as described herein, and then transplanting the resulting intestinal stem cells and/or intestinal epithelial cells into a patient. do.

The patient is a mammal, preferably a human. In one embodiment, the cells may be implanted as dispersed cells, provided in a biocompatible degradable polymer support, a porous non-degradable device, or encapsulated to protect them from host immune responses. The cells can be transplanted to an appropriate site in the recipient.

To enhance further differentiation, survival, or activity of transplanted cells in vivo, additional factors, such as growth factors, antioxidants, or anti-inflammatory agents, can be administered prior to, concurrently with, or after cell administration.

The amount of cells used for transplantation depends on a variety of factors, including the patient's condition and response to therapy, and can be determined by one of ordinary skill in the art. In one embodiment, the method of treatment further comprises incorporating the cells into the three-dimensional scaffold prior to implantation. Cells can be maintained on such scaffolds in vitro before transplantation into a patient. Alternatively, scaffolds containing cells can be implanted directly in the patient without further in vitro culture. The scaffold may optionally be incorporated with at least one pharmaceutical agent that promotes survival and function of the transplanted cells.

In these transplants, intestinal stem cells include fibrin, laminin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, poly(glycolic acid) (PGA), and poly(lactic acid-co-glycolic acid). )(poly(lacticcoglycolic acid), PLGA), poly-ε-(caprolactone), polyanhydride, polyorthoester, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide , poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymer, copolymers thereof, and mixtures thereof can be used for transplantation with any one or more biodegradable supports selected from the group consisting of there is

In other words, the cells that are the target of the above transplant material can be used for transplantation as is or embedded in the aforementioned support. In addition, dimethyl sulfoxide (DMSO), etc. can be added for the purpose of protecting cells, antibiotics, etc. can be added for the purpose of preventing the incorporation of bacteria, and inducing cell activation, proliferation, or differentiation, etc. For this purpose, various components (vitamins, cytokines, growth factors, steroids, etc.) may be added to the transplant material of the present invention.

In the present invention, prevention refers to all actions that suppress or delay the onset of intestinal disease by administering the composition, and treatment refers to all actions that improve or benefit symptoms of intestinal disease by administering the composition.

The composition of the present invention may contain 1.0×10 ⁵ to 1.0×10 ¹⁰ cells per ml, preferably 1.0×10 ⁶ to 1.0×10 ⁹ cells.

The pharmaceutical composition of the present invention can be formulated into various dosage forms such as liquid and suspension according to conventional methods.

The pharmaceutical composition of the present invention can be formulated and administered as a unit dosage pharmaceutical preparation suitable for administration into the patient's body according to a conventional method in the pharmaceutical field, and the preparation can be effectively administered by single or multiple administrations. Includes quantity. Formulations suitable for this purpose include parenteral preparations such as injections, infusions, and implants. Additionally, the pharmaceutical composition may include a conventional pharmaceutically acceptable inert carrier and diluent. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically friendly to the mesenchymal stromal cells and the recipient to whom they are transplanted. Diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and/or dispersion media. In addition, for example, in the case of injections, a preservative, analgesic agent, solubilizer or stabilizer may be added, and in the case of preparations for topical administration, a base, excipient, lubricant or preservative may be additionally included.

Compositions of the present invention may be used unfrozen or frozen for later use. If freezing is to be performed, standard cryopreservation agents (e.g. DMSO, glycerol, Epilife® Cell Freezing Medium (Cascade Biologics)) can be added to the cell population prior to freezing.

In addition, it can be implanted and administered using administration methods commonly used in the art, and preferably can be engraftment or transplantation directly into the diseased area of a patient requiring treatment, but is not limited thereto. In addition, the above administration can be done both non-surgically using a catheter and surgical administration such as injection or transplantation after incision of the diseased area. The dosage can be 5 x 10 ⁵ ~10 ⁸ /60 kg per adult or 5 x 10 ⁵ ~10 ⁸ /time. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and gender. Therefore, the dosage should be determined in any way. It does not limit the scope of the present invention in any way.

The present invention also provides a pharmaceutical composition comprising the intestinal stem cells and/or intestinal epithelial cells for use in preventing or treating intestinal diseases.

The present invention also provides the use of the intestinal stem cells and/or intestinal epithelial cells in the manufacture of a medicament for use in preventing or treating intestinal diseases.

A method of treating intestinal disease is provided, comprising administering the intestinal stem cells and/or intestinal epithelial cells to a subject in need thereof.

Through the intestinal stem cell culture method according to the present invention, homogeneous cells can be cultured easily and quickly, large quantities of cells can be cultured at a low price, and the risk of heterogeneous infection is eliminated by culturing the cells in a xenogeneic culture environment. It is possible to develop cell therapy that can actually be transplanted. In addition, it has the advantage of being able to be cultured while maintaining cell characteristics even during organ subculture and cryopreservation.

In addition, through the method for producing intestinal epithelial cells according to the present invention, intestinal stem cells derived from intestinal organoids cultured in a xenogeneic culture environment can be differentiated into intestinal epithelial cells in the same xenogeneic culture environment.

Figure 1 is a schematic diagram of a polymer screening method conducted to find the optimal thin film coating that can replace Matrigel.

Figure 2 shows the chemical structures of polymers used in polymer screening to find optimal thin film coatings.

Figure 3 is a result showing that when the engraftment rate of intestinal stem cells was confirmed on a culture dish coated with a thin film of various polymers, no polymer showed better performance than a culture dish coated with Matrigel.

Figure 4 is a diagram showing the results of controlling the physical/chemical properties and wettability of the surface of a culture dish coated with a thin film of high molecular weight polymer by treating it with plasma to increase the adhesion ability and engraftment rate of intestinal stem cells.

(a) This diagram shows the results of confirming the terminal functional groups of the plasma-treated polymer thin film through FT-IR spectrum analysis.

(b) This is a diagram showing the wettability of a plasma-treated culture dish coated with a polymer thin film through the difference in water contact angle.

Figure 5 is a result showing that the adhesion ability and engraftment rate of intestinal stem cells increase when the wettability is increased by treating a culture dish coated with a thin film of high molecular weight polymer with plasma.

(a) A diagram showing the morphology of intestinal stem cells engrafted on a culture dish coated with a plasma-treated polymer thin film.

(b) This is a diagram showing the size of colonies of intestinal stem cells engrafted on a plasma-treated, high-molecular polymer thin film-coated culture dish using crystal violet (CV) staining.

(c) This is a graph showing the results of calculating the surface area of the colony size confirmed through crystal violet (CV) staining in (b) using the Image J program.

Figure 6 shows the results showing that subculture and cell proliferation of intestinal stem cells are possible on a culture dish coated with a thin film of the two types of polymers that showed the best performance in Figure 5.

(a) A diagram showing the cell morphology of intestinal stem cells cultured in a culture dish coated with a thin film of two types of polymers (pEGDMA, pCHMA) at passage 0, 1, or 2.

(b) This is a graph showing the proliferation rate of cells according to passage when intestinal stem cells were cultured in a culture dish coated with a thin film of two types of polymers (pEGDMA, pCHMA).

Figure 7 shows the results showing changes in the adhesion ability and engraftment rate of intestinal stem cells according to changes in surface energy and thickness when a culture dish coated with a polymer polymer (pEGDMA) thin film was treated with plasma at an intensity of 15W over time.

(a) This is a graph showing the change in the surface energy of the culture dish and the thickness of the thin film according to the treatment of plasma at 15W intensity over time on a culture dish coated with a polymer polymer (pEGDMA) thin film.

(b) This is a diagram showing the cell morphology of intestinal stem cells engrafted when a culture dish coated with a thin film of high molecular weight polymer (pEGDMA) was treated with plasma at an intensity of 15W over time.

(c) This is a diagram showing the colony size of intestinal stem cells engrafted when a culture dish coated with a thin film of high molecular weight polymer (pEGDMA) was treated with plasma at an intensity of 15W over time through Crystal violet (CV) staining.

(d) This is a graph showing the results of calculating the surface area of the colony size confirmed through crystal violet (CV) staining in (c) using the Image J program.

Figure 8 shows the results showing changes in the adhesion ability and engraftment rate of intestinal stem cells according to changes in surface energy and thickness when a culture dish coated with a polymer polymer (pEGDMA) thin film was treated with plasma of different intensities for 10 seconds.

(a) This is a graph showing the change in surface energy and thickness of the thin film according to plasma treatment of 0, 3, 5, 15, 50, and 100 W intensities for 10 seconds on a culture dish coated with a polymer polymer (pEGDMA) thin film.

(b) A diagram showing the cell morphology of intestinal stem cells engrafted when a high-molecular polymer (pEGDMA) thin film-coated culture dish was treated with plasma at intensities of 0, 3, 5, 15, 50, and 100 W for 10 seconds.

(c) Crystal violet (CV) staining of the colony size of intestinal stem cells engrafted when a culture dish coated with a thin film of high molecular weight polymer (pEGDMA) was treated with plasma at intensities of 0, 3, 5, 15, 50, and 100 W for 10 seconds. This is the way shown through.

(d) This is a graph showing the results of calculating the surface area of the colony size confirmed through crystal violet (CV) staining in (c) using the Image J program.

Figure 9 shows no change in physical properties of a general culture dish (TCPS or bare wafer), a culture dish coated with a polymer (pEGDMA) thin film, and a culture dish treated with plasma under optimized conditions after coating with a polymer (pEGDMA) thin film. This result shows that there is no difference depending on the size and that storage for 2 years is possible.

(a) Showing that there is no difference in appearance and transparency between a regular culture dish (TCPS), a culture dish coated with a polymer (pEGDMA) thin film, and a culture dish treated with plasma under optimized conditions after coating with a polymer (EGDMA) thin film. It is a result.

(b) This is a result showing the water contact angle of a general culture dish (Bare wafer), a culture dish coated with a polymer (pEGDMA) thin film, and a culture dish coated with a polymer (pEGDMA) thin film and then treated with plasma under optimized conditions.

(c) When plasma was treated on a culture dish coated with a polymer (pEGDMA) thin film under optimal conditions (15 W, 10 seconds), surface homogeneity and average height (Rq) were measured using atomic force microscopy (AFM). This is the result of measurement.

(d) This is the result of measuring surface uniformity and average height (Rq) using atomic force microscopy (AFM) when polymer polymer (pEGDMA) thin films were produced in three batches.

(e) This is a diagram showing the colonies of intestinal stem cells engrafted in three batches of culture dishes coated with a high molecular weight polymer (pEGDMA) thin film prepared in (d) through Crystal violet (CV) staining.

(f) This is a graph showing the results of calculating the surface area of the colony size confirmed through crystal violet (CV) staining in (e) using the Image J program.

(g) This diagram shows the cell morphology of intestinal stem cells engrafted in a culture dish coated with Matrigel, a culture dish coated with a polymer (pEGDMA) thin film, and a culture coated with a polymer (pEGDMA) thin film stored for 2 years.

(h) WST-1 assay was performed on the intestinal stem cells cultured in (g), and the cell proliferation of intestinal stem cells was confirmed even on a culture dish coated with a pEGDMA thin film stored for 2 years in a Matrigel-coated culture dish. The results show that the results are similar to those of a culture dish coated with a culture dish and polymer (pEGDMA) thin film.

Figure 10 shows the results of chemical characterization of EGDMA monomer, EGDMA polymer (pEGDMA), and plasma-treated EGDMA polymer thin film using FT-IR.

(a) As a result of analyzing the chemical properties of the surface of EGDMA monomer and EGDMA polymer (pEGDMA), it was confirmed that the amount of vinyl group (C=C, 1650-1620 cm ^-1 ) decreases when polymer is formed.

(b) As a result of analyzing the chemical properties before and after plasma treatment on the surface of a culture dish coated with a pEGDMA thin film, it was confirmed that there was no difference in chemical bonding.

Figure 11 shows the results of chemical characterization of EGDMA monomer, EGDMA polymer (pEGDMA), and plasma-treated EGDMA polymer thin film using XPS.

(a) This is the result of analyzing the types of atoms constituting the surface coated with the pEGDMA thin film using X-ray photoelectron spectroscopy (XPS).

(b) This is the result of analyzing the types of atoms constituting the surface of the pEGDMA thin film after plasma treatment using X-ray photoelectron spectroscopy (XPS).

(c) This is a chart summarizing the composition of carbon, oxygen, and nitrogen atoms detected in (a) and (b).

(d) The results analyzed in (a) are the results of analyzing the type and composition of chemical bonds on the surface through a deconvoluted high resolution scan of C1s.

(e) The results analyzed in (b) are the results of analyzing the type and composition of chemical bonds on the surface through a deconvoluted high resolution scan of C1s.

(f) This is a chart summarizing the types and compositions of chemical bonds analyzed in (d) and (e).

Figure 12 is a result showing that culturing intestinal stem cells on a culture dish coated with an optimized pEGDMA thin film is superior to a regular culture dish (Bare) and can be cultured with similar efficiency as a culture dish coated with Matrigel.

(a) Schematic diagram of the process of coating an optimized pEGDMA thin film on a culture dish and culturing intestinal stem cells using a chemical vapor deposition (iCVD) process.

(b) Results showing through cell morphology analysis that when culturing intestinal stem cells on a culture dish coated with an optimized pEGDMA thin film, cell attachment and growth are superior to those on a regular culture dish (bare) and are comparable to those on a Matrigel-coated culture dish. am.

(c) Using the WST-1 assay, it is shown that the cell proliferation of intestinal stem cells on a culture dish coated with a pEGDMA thin film is superior to that of a regular culture dish (Bare) and is at a similar level to that of a culture dish coated with Matrigel. It is a result.

(d) This result shows that the efficiency of culturing intestinal stem cells on a culture dish coated with a pEGDMA thin film using crystal violet (CV) staining is superior to that on a regular culture dish (Bare) and is at a similar level to that of a culture dish coated with Matrigel.

(e) When the colony area in (d) was quantified and measured, the intestinal stem cell colony area on the pEGDMA thin film-coated culture dish was superior to that of the regular culture dish (Bare) and was similar to that of the Matrigel-coated culture dish. It is a result.

(f) This is the result of confirming the survival rate of intestinal stem cells cultured in a culture dish coated with a pEGDMA thin film and a culture dish coated with Matrigel using LIVE/DEAD staining.

(g) As a result of quantitative analysis of the results confirmed in (f), it was confirmed that the survival rate was 100% in both conditions.

Figure 13 is a diagram showing that high proliferation rate, long-term subculture, and frozen storage are possible when culturing intestinal stem cells on a culture dish coated with an optimized pEGDMA thin film.

(a) A schematic diagram showing that high proliferation rate, long-term subculture, and frozen storage of intestinal stem cells are possible on a culture dish coated with an optimized pEGDMA thin film.

(b) This is the result confirming that when intestinal stem cells were cultured on a Matrigel-coated surface, the number of cells increased during subculture and the proliferation was similar to the theoretical value.

(c) This is the result confirming that when intestinal stem cells were cultured on a surface coated with an optimized pEGDMA thin film, the number of cells increased during subculture and proliferation was similar to the theoretical value.

(d) This is the result of confirming through cell shape analysis that intestinal stem cells can be cultured up to passage 30 during continuous subculture on a surface coated with an optimized pEGDMA thin film.

(e) Cell morphology analysis showed that when frozen intestinal stem cells were thawed and injected into a culture dish coated with an optimized pEGDMA thin film, engraftment and growth of cells were possible with similar efficiency as in a Matrigel-coated culture dish. This is the confirmed result.

(f) This result shows that there is no difference in the efficiency of the intestinal stem cells attached and grown when thawed intestinal stem cells are injected into a culture dish coated with a pEGDMA thin film optimized using cell count analysis and a culture dish coated with Matrigel. .

(g) This result shows that there is no difference in the efficiency of the intestinal stem cells attached and grown when thawed intestinal stem cells are injected into a culture dish coated with a pEGDMA thin film optimized using cell growth ability analysis and a culture dish coated with Matrigel. .

(h) The efficiency of intestinal stem cells grown by attaching when thawed intestinal stem cells were injected after long-term storage for more than 3 years in a culture dish coated with a pEGDMA thin film optimized using cell count analysis and a culture dish coated with Matrigel. This result shows that there is no difference.

(i) When thawed intestinal stem cells were injected into a culture dish coated with a pEGDMA thin film optimized using cell growth ability analysis and a culture dish coated with Matrigel after long-term storage for more than 3 years, the efficiency of the intestinal stem cells grown by attaching was determined. This result shows that there is no difference.

Figure 14 is a diagram showing the endotoxin level of the optimized pEGDMA thin film and the small amount of TNF-α secretion by macrophages.

(a) This diagram shows that the amount of endotoxin on the surface of a culture dish coated with an optimized pEGDMA thin film is lower than the FDA standard amount.

(b) A diagram showing that the amount of TNF-α secreted by macrophages is low on the surface of a culture dish coated with an optimized pEGDMA thin film.

Figure 15 is a diagram showing that intestinal stem cells can be cultured without differences in genetic characteristics when cultured on a culture dish coated with an optimized pEGDMA thin film and a culture dish coated with Matrigel.

(a) This is a volcano plot showing that there is no difference in expression at the transcriptome level when culturing intestinal stem cells in a culture dish coated with an optimized pEGDMA thin film and a culture dish coated with Matrigel.

(b) When the results of (a) are shown in an MDS (Multi-dimensional scaling) graph, the intestinal stem cells cultured on the optimized pEGDMA thin film-coated culture dish and the Matrigel-coated culture dish are well positioned in similar positions. This is a result showing binding.

(c) Volcano plot showing that there is no difference in expression at the intestinal stem cell proteome level when culturing intestinal stem cells on a culture dish coated with an optimized pEGDMA thin film and a culture dish coated with Matrigel. am.

(d) This is the result of confirming through qPCR gene expression analysis that there was no change in the expression of intestinal stem cell marker genes when culturing intestinal stem cells in a culture dish coated with an optimized pEGDMA thin film and a culture dish coated with Matrigel.

(e) This is the result of confirming through qPCR gene expression analysis that there was no change in the expression of intestinal stem cell marker genes when subculturing intestinal stem cells in a culture dish coated with an optimized pEGDMA thin film and a culture dish coated with Matrigel.

(f) When culturing intestinal stem cells in a culture dish coated with an optimized pEGDMA thin film and a culture dish coated with Matrigel, the expression of intestinal stem cell marker proteins exceeded 90%. Fluorescence activated cell sorter; This is a result confirmed through FACS).

(g) This is the result of immunofluorescence staining confirming that there was no change in the expression of intestinal stem cell marker proteins when culturing intestinal stem cells in a culture dish coated with an optimized pEGDMA thin film and a culture dish coated with Matrigel.

Figure 16 shows that when culturing human embryonic stem cell-derived intestinal stem cells in a culture dish coated with an optimized pEGDMA thin film, it is possible to culture them with the same efficiency as in a Matrigel-coated culture dish, like pluripotent stem cell-derived intestinal stem cells. It is also a degree.

(a) Results showing through cell shape analysis that when culturing embryonic stem cell-derived intestinal stem cells on a culture dish coated with an optimized pEGDMA thin film, cell attachment, growth, and subculture are possible at a level similar to that of a Matrigel-coated culture dish. am.

(b) This is the result of confirming the survival rate of embryonic stem cell-derived intestinal stem cells cultured on a culture dish coated with an optimized pEGDMA thin film and a culture dish coated with Matrigel using LIVE/DEAD staining.

(c) This is a graphical quantification of the LIVE/DEAD staining results in (b).

(d) When culturing embryonic stem cell-derived intestinal stem cells in a culture dish coated with an optimized pEGDMA thin film and a culture dish coated with Matrigel, there was no change in the expression of intestinal stem cell marker genes for passages 2 and 8. This is a result confirmed through expression analysis.

Figure 17 is a diagram showing that intestinal stem cells can be differentiated into intestinal epithelial cells in a Transwell plate coated with an optimized pEGDMA thin film with similar efficiency to a Transwell plate coated with Matrigel.

(a) Schematic diagram of the differentiation method for differentiating intestinal stem cells into intestinal epithelial cells in a transwell plate coated with an optimized pEGDMA thin film.

(b) Day 0 after differentiating intestinal stem cells into intestinal epithelial cells using an air-liquid interface culture method on a Transwell plate coated with an optimized pEGDMA thin film and a Transwell plate coated with Matrigel. , This is the result of analyzing the morphology of cells on days 2, 4, 6, and 8.

(c) Day 0 after differentiating intestinal stem cells into intestinal epithelial cells using an air-liquid interface culture method on a Transwell plate coated with an optimized pEGDMA thin film and a Transwell plate coated with Matrigel. , This is the result of analyzing marker gene expression in 4- and 8-day-old cells and human small intestine using qPCR.

(d) Day 0 after differentiating intestinal stem cells into intestinal epithelial cells using an air-liquid interface culture method on a Transwell plate coated with an optimized pEGDMA thin film and a Transwell plate coated with Matrigel. , This is the result of analyzing the cross-sectional shape of 4- and 8-day-old cells and the expression of marker proteins through H&E staining and immunofluorescence staining.

(e) Two days after differentiating intestinal stem cells into intestinal epithelial cells using an air-liquid interface culture method on a Transwell plate coated with an optimized pEGDMA thin film and a Transwell plate coated with Matrigel. , This is the result of analyzing the functionality of intestinal epithelial cells by measuring the transepithelial electric resistance (TEER) of cells aged 4, 6, and 8 days.

Figure 18 is a diagram showing that human embryonic stem cell-derived intestinal stem cells can be differentiated into intestinal epithelial cells in a Transwell plate coated with an optimized pEGDMA thin film with similar efficiency to a Transwell plate coated with Matrigel.

(a) Differentiation of embryonic stem cell-derived intestinal stem cells into intestinal epithelial cells using an air-liquid interface culture method on a Transwell plate coated with an optimized pEGDMA thin film and a Transwell plate coated with Matrigel. This is the result of analyzing the shape of cells on the 2nd, 6th, 8th, and 10th day after treatment.

(b) Differentiation of embryonic stem cell-derived intestinal stem cells into intestinal epithelial cells using an air-liquid interface culture method on a Transwell plate coated with an optimized pEGDMA thin film and a Transwell plate coated with Matrigel. This is the result of analyzing the shape of the cross section of cells 8 days after treatment and the expression of marker proteins through H&E staining and immunofluorescence staining.

(c) Differentiation of embryonic stem cell-derived intestinal stem cells into intestinal epithelial cells using an air-liquid interface culture method on a Transwell plate coated with an optimized pEGDMA thin film and a Transwell plate coated with Matrigel. This is the result of analyzing marker gene expression in cells 8 days after treatment using qPCR.

(d) Differentiation of embryonic stem cell-derived intestinal stem cells into intestinal epithelial cells using an air-liquid interface culture method on a Transwell plate coated with an optimized pEGDMA thin film and a Transwell plate coated with Matrigel. This is the result of analyzing the functionality of intestinal epithelial cells by measuring the transepithelial electric resistance (TEER) of cells aged 8 days.

Figure 19 shows the results of transplanting intestinal stem cells cultured in a culture dish coated with an optimized pEGDMA thin film into the colon of a mouse model of EDTA-induced intestinal epithelial damage.

(a) A schematic diagram showing the process of transplanting, extracting, and analyzing intestinal stem cells cultured on a fibrin implant or pEGDMA thin film into an immunodeficient mouse-based EDTA-induced intestinal epithelial damage model.

(b) A diagram showing the morphology of intestinal stem cells cultured on a pEGDMA thin film before transplantation.

(c) This is the result of colonoscopic observation before transplantation of an immunodeficient mouse-based EDTA-induced intestinal epithelial damage model (Day 0) and on day 14 after transplantation with intestinal stem cells cultured in a fibrin implant or pEGDMA thin film.

(d) After transplantation into an immunodeficient mouse-based EDTA-induced intestinal epithelial damage model with intestinal stem cells cultured in Fibrin implants or pEGDMA thin films, major organs (stomach, liver, kidney, spleen, This result shows that no abnormal tissue or form appeared in the small intestine.

(e) A diagram showing the histological analysis of a colonic tissue section removed on the 14th day after transplantation with intestinal stem cells cultured on a fibrin implant or pEGDMA thin film. This result confirmed that the crypt depth of the intestinal stem cell transplant group was increased through histological analysis through H&E, and that the mucin secretion ability of goblet cells was restored through AB-PAS staining.

(f) Graph quantitatively evaluating the crypt depth of the fibrin-only transplant group (n=246 crypts) or the group transplanted with intestinal stem cells cultured on pEGDMA thin films (n=303 crypts) using the Image J program (n≥3) mice of each group).

(g) Immunofluorescence staining with human-specific ECAD antibody (hECAD) in colonic tissue sections extracted 14 days after transplantation with intestinal stem cells cultured on fibrin implants or pEGDMA thin films showed that human intestinal stem cells were present in mice. This result shows that it has taken root in the large intestine.

Figure 20 shows the results of transplanting intestinal stem cells cultured in a culture dish coated with an optimized pEGDMA thin film into the colon of a mouse with DSS-induced inflammatory bowel disease.

(a) Schematic diagram of transplanting, extracting, and analyzing intestinal stem cells cultured on a Fibrin implant or pEGDMA thin film into an immunodeficient mouse-based DSS-induced colitis model.

(b) A diagram showing the morphology of intestinal stem cells cultured on a pEGDMA thin film before transplantation.

(c) This is the result of colonoscopy observed before transplantation of the immunodeficient mouse-based DSS-induced colitis model (Day 0) and on days 14 and 28 after transplantation with intestinal stem cells cultured in a fibrin implant or pEGDMA thin film.

(d) A diagram showing the histological analysis of a colonic tissue section removed on the 28th day after transplantation with intestinal stem cells cultured on a fibrin implant or pEGDMA thin film. This result confirmed that the epithelial recovery and crypt depth of the intestinal stem cell transplant group were increased through histological analysis through H&E, and that the mucin secretion ability of goblet cells was restored through AB-PAS staining.

(e) Graph quantitatively evaluating the crypt depth of the group transplanted with fibrin alone (n=228 crypts) or the group transplanted with intestinal stem cells cultured on pEGDMA thin film (n=714 crypts) using the Image J program (n≥3) mice of each group).

(f) Immunofluorescence staining with human-specific ECAD antibody (hECAD) in colonic tissue sections extracted 28 days after transplantation with intestinal stem cells cultured on fibrin implants or pEGDMA thin films showed that human intestinal stem cells were in mice. This result shows that it has taken root in the large intestine.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

Experimental Example 1. Polymer thin film coating using chemical vapor deposition (iCVD)

A cell culture dish was placed in an iCVD reactor (Daeki Hi-tech Co. Ltd.), and polymer (pEGDMA) monomer and initiator (tert-Butyl peroxide, TBPO) were injected into the iCVD reactor under vacuum. When the injected initiator meets a filament at 140°C, it is activated and promotes the binding of monomers located on the surface of the culture dish, forming a thin film on the surface of the culture dish. A culture dish was placed in the plasma chamber, the pressure was lowered to less than 10 mTorr, and 5 sccm N ₂ gas was injected to control the flow of molecules. Afterwards, to optimize the surface, plasma was processed for 10 seconds at a power of 15W in the chamber of a low-pressure radio frequency (RF) plasma system (Daeki Hi-tech Co. Ltd.).

In the same manner as the above method, polymer thin film production was performed through the iCVD process while only changing the type of polymer.

Experimental Example 2. Analyzing the physicochemical properties of the surface coated with a polymer thin film

To measure the surface energy of the surface coated with a polymer thin film, a 2μl deionized (DI) water droplet was dropped and the contact angle was measured using a contact angle analyzer (phoenix 150, Surface Electro Optics, Inc.), and the roughness of the surface was measured. was measured using an atomic force microscope (AFM), NX-10 (Park Systems). The pEGDMA polymer was analyzed by FT-IR spectroscopy using an Alpha Fourier-transform infrared red spectrometer (Bruker). The chemical composition of the pEGDMA thin film was analyzed using X-ray photoelectron spectroscopy (XPS, K-alpha, Thermo VG Scientific, Inc.).

Experimental Example 3. Cell culture and iPSC production

Human pluripotent stem cells (hPSC), including human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), were purified using known methods (Molecular carcinogenesis 55, 387-396 (2016), Proteomics 15, 2220-2229 (2015). )). Non-inserted-hiPSCs were reprogrammed by transfection by electroporation using an Episomal iPSC reprogramming vector (Cat. No. A14703. Invitrogen, Carlsbad, CA, USA) according to a known method.

Five days after electroporation, fibroblasts were plated at ¹ , Vancouver, Canada). After 3 weeks, hiPSC colonies were selected and cell numbers were expanded for subculture and subsequent characterization.

Experimental Example 4. Differentiation of hPSCs into intestinal organoids (hIO) for preparation of immature intestinal organoids (Cont-hIO)

Human intestinal organoids (hIOs) were prepared using a known method (Nature 470, 105-109 (2011)). To induce intact endoderm, hPSCs were plated on Matrigel- or ECMatrix™-511-coated dishes and incubated with purified fetal bovine serum (dFBS, HyClone, Thermo Fisher Scientific Inc.) at concentrations of 0%, 0.2%, and 2%. , Waltham, MA, USA) and treated with 100 ng/ml Activin A (R&D Systems, Minneapolis, MN, USA) for 3 days in RPMI 1640 medium. Additionally, to differentiate into 3D hindgut spheroids, 500 ng/ml FGF4 (R&D Systems) and 3 μM CHIR99021 (TOCRIS) were treated with RPMI 1640 medium containing 2% dFBS for 4 to 6 days. From day 4 of induction into the hindgut, spheroids were inserted into Matrigel (BD Biosciences) and supplemented with 1 ) and 40~50ng/ml Noggin (R&D Systems) were cultured in hIO medium (2mM L-glutamine, 1% Penicillin-Streptomycin, and 15mM HEPES buffer in Advanced DMEM F12), and cultured for 10~14 days. Subculturing was performed each time.

Experimental Example 5. Method for isolating and culturing intestinal stem cells from intestinal organoids

After hIO was collected from the Matrigel dome, it was washed 2-3 times using physiological saline. Afterwards, all of the PBS was removed, and the hIO pellet was treated with trypsin-EDTA (TE) in a 37°C water bath for about 5 minutes to separate into single cells, which were then diluted by adding hIO basal medium. Cells supplied by centrifugation were placed on a pEGDMA-coated culture dish with 1 10nM [Leu15]-Gastrin I (Sigma-aldrich), 100ng/ml human recombinant WNT3A (R&D Systems), 500nM A-83-01 (Tocris), 10uM SB202190 (Sigma-aldrich), 2.5μM Prostaglandin E2 (Sigma-aldrich) ), 1mM N-acetylcysteine (Sigma-aldrich), 10mM Nicotinamide (Sigma-aldrich), and intestinal stem cell culture medium (2mM L-glutamine, 1% Penicillin-Streptomycin, and 15mM HEPES buffer in Advanced DMEM F12). and subcultured once every 7 to 10 days. During subculture, 1 μM Jagged-1 (Anaspec) and 2.5 μM Y-27632 (Tocris) were added to the intestinal stem cell culture medium for the first 2 days.

Experimental Example 6. Intestinal Epithelial Cell Differentiation Method Using Air-Liquid Interface Culture

Intestinal stem cells proliferated to a density of 7-80% were washed 1-2 times with PBS and then treated with TE for 5-7 minutes in an incubator at 37°C. Intestinal stem cells separated into single cells were collected and diluted using hIO basic medium. After collecting the cells using centrifugation, the supernatant was removed, intestinal stem cell culture medium was added, mixed thoroughly, and the number of cells was measured using a CountessIII cell counter (Thermo Scientific. Inc.). 2.5-3.5X10 ⁵ cells were added to the insert of a 12-Transwell plate (Corning) coated with 1% Matrigel and cultured in an incubator. After the cell density reached 100%, all of the culture medium in the upper layer was removed, and the medium in the lower layer was supplemented with 200ng/ml R-Spondin 1 (R&D Systems), 100ng/ml EGF (R&D Systems), and 2.5μM Prostaglandin E2 (Sigma- aldrich), 10μM SB202190 (Sigma-aldrich), and 10mM Nicotinamide (Sigma-aldrich). Afterwards, the surface of the upper layer was washed with PBS or hIO basic medium every two days, and the lower layer was replaced with new differentiation medium and cultured for about 8-12 days.

Experimental Example 7. Cell viability measurement method

To measure the survival rate of intestinal stem cells cultured on a culture dish coated with 1% Matrigel or a polymer (pEGDMA) thin film, a kit (LIVE/DEAD Viability/Cytotoxicity Kit, Invitrogen) can be used to distinguish between viable and dead cells and stain them. ) was used. Surviving cells were stained with calcein-AM, and dead cells were stained with ethidium homodimer-1. Stained cells were observed through a fluorescence microscope (Nikon).

Experimental Example 8. Cell growth rate measurement method

10X10 ³ cells were plated on a culture dish coated with 1% Matrigel or polymer (pEGDMA) thin film and cultured for 7 days. After removing the culture medium, 100 μl of new medium containing 10 μl of WST-1 cell proliferation reagent (Roche) was added per well. After culturing for about 3 hours, the absorbance was measured at 440 nm using a microplate reader (Molecular Devices).

Experimental Example 9. Crystal Violet (CV) staining method

Intestinal stem cells and differentiated intestinal epithelial cells were fixed with 4% paraformaldehyde (PFA) and stained with 0.02% crystal violet solution (Sigma-aldrich) for 10 minutes at room temperature. Afterwards, images were acquired after washing three times with sterile water. Intestinal stem cell colony size was analyzed using Image J software (National Institute of Health).

Experimental Example 10. Live Cell Imaging

To confirm the surface adhesion and colony forming ability of intestinal stem cells, real-time images were recorded using an optical microscope (Lumascope 620, Etaluma). The recorded images were produced as videos using the Adobe Premiere Pro 2020 program.

Experimental Example 11. Quantitative real-time RT-PCR (qRT-PCR)

Total RNA was extracted from cells using the RNeasy kit (Qiagen) and reverse transcribed using the Superscript III cDNA synthesis kit (Invitrogen). qRT-PCR was performed on a 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA) using a known method (Cho et al., Oncotarget 6, 23837-23844, 2015). All experiments were repeated three times, and the CT value of each target gene was calculated using software provided by the manufacturer. The base sequences of the primers used are shown in Table 1.

Gene Primer (Forward) sequence number Primer (Reverse) sequence number GAPDH GAAGGTGAAGGTCGGAGTC One GAAGATGGTGATGGGATTTC 2 LGR5 CCTGCTTGACTTTGAGGAAGACC 3 CCAGCCATCAAGCAGGGTGTTCA 4 EPHB3 TCGTGGTCATCGCTATCGTCT 5 AAACTCCCGAACAGCCTCATT 6 MKI67 TGACCCTGATGAGAAAGCTCAA 7 CCCTGAGCAACACTGTCTTTT 8 SOX9 GTACCCGCACTTGCACAAC 9 TCTCGCTCCTCGTTCAGAAGTC 10 CD44 CCAGAAGGAACAGTGTTTGGC 11 ACTGTCCTTGGGCTTGGTGTT 12 LYZ AAAACCCCAGGAGCAGTTAAT 13 CAACCCTCTTTGCACAAGCT 14 VIL1 AGCCAGATCACTGCTGAGGT 15 TGGACAGGTGTTCCTCCTTC 16 MUC2 TGTAGGCATCGCTTCTTCTCA 17 GACACCATCTACCTCACCCG 18 CHGA TGACCTCAACGATGCATTTC 19 CTGTCCTGGCTCTTCTGCTC 20

Experimental Example 12. EDTA-induced intestinal epithelial injury model

The EDTA-induced intestinal epithelial damage model was created using a known method ( Cell Stem Cell 22(2):171-176 (2018)). NSG or NIG mice (male, 6-12 weeks old) were fed a standard diet without fasting and maintained at a constant temperature (20-22°C) under a 12-hour day/night cycle. 250-500 mg/kg Tribromoethanol (Avertin). The mouse was anesthetized by intraperitoneal injection, and a thin catheter was inserted into the colon of the mouse and washed with PBS for 2 minutes at 50°C to prevent damage to the anus and rectum. After 2 minutes of EDTA/PBS exposure, the lumen was washed with PBS for 1 minute using an electric toothbrush equipped with a soft interdental brush (EW-DL22, EW0945, Panasonic Holdings Corp., Japan). At this time, the brush head was inserted 1.5 cm into the colon and the intestinal epithelial surface was scraped in a circular manner. Success could be confirmed when the separated crypts were observed when the brush was washed in PBS after 1 minute. Afterwards, the intestines were washed again with PBS.

Experimental Example 13. Inflammatory bowel disease model (DSS-induced colitis model)

NSG or NIG mice (male, 6-12 weeks old) were not fasted, fed a standard diet, and maintained at a constant temperature (20-22°C) with a 12:00 day/night cycle. The DSS-induced colitis model was incubated with 5% CO for 5-7 days. Drinking water containing (w/v) DSS (36-50 kDa; MP Biomedicals, Hampton, NH, USA) was administered for 3 days to prepare for DSS transplantation. In the inflammatory bowel model, body weight, stool consistency, and bleeding parameters were measured daily to evaluate the disease activity index (DAI) to determine whether symptoms occurred.

Experimental Example 14. Colonic transplantation

For xenografting, intestinal stem cells were dissociated with TrypLE before transplantation, washed with Advanced DMEM/F12, and suspended in Fibrin. A 100 ul suspension contains 1-2 x 10 ⁶ cells. Before transplantation, the animals were not fasted, fed a standard diet, and anesthetized with 250-500 mg/kg Tribromoethanol (Avertin) by intraperitoneal injection. To remove intestinal contents, a thin catheter was inserted into the mouse's colon and washed with PBS. Fibrin or intestinal stem cells were injected into the colon lumen of disease-induced mice using a 200 ul pipette, soft catheter, or endoscopic catheter. At each observation time, mice were humanely euthanized and xenografts were isolated for analysis. To confirm the xenograft of colon tissue, it was observed using an inverted fluorescence dissecting microscope (SZX16, Olympus) or Axiovert 200M microscope (Carl Zeiss, Gottingen, Germany).

Experimental Example 15. Colonoscopy

To check the level of inflammation and environment in the intestines, the patient was anesthetized with an intraperitoneal injection of Avertin before endoscopy. To remove intestinal contents, the mouse colon was washed with PBS by inserting a thin catheter. Endoscopic images were recorded by injecting medical gas using an air pump and carefully inserting the endoscope of a sturdy HOPKINS telescope (Karl Storz) into the rectum.

Experimental Example 16. Cell and tissue immunofluorescence test

Immunofluorescence test was performed according to a known method (Kwak et al., Biochemical and biophysical research communications 457, 554-560, 2015). Specifically, intestinal stem cells, differentiated intestinal epithelial cells, and colon tissue extracted after transplantation were fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS containing 0.1% Triton X-100.

After cryoprotecting differentiated intestinal epithelial cells or extracted colon tissue with sucrose, the membrane of the insert well was cut, placed vertically in an optimal cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan), and then frozen. Then, frozen sections were cut into 10 μm sections using a cryostat microtome at -20°C and permeabilized with PBS containing 0.1% Triton X-100 for immunofluorescence.

After blocking with 4% BSA, the cells were reacted with primary antibodies overnight at 4°C. Then, it was reacted with secondary antibody for 1 hour at room temperature. The primary antibodies used are listed in Table 2. DAPI was added to visualize nuclei. Slides were observed using an EVOS FL Auto2 (ThermoFisher) and Axiovert 200M microscope (Carl Zeiss, Gottingen, Germany) or a fluorescence microscope (IX51, Olympus, Japan).

antibody Catalog. No. company dilution anti-Ki67 MAB9260 Millipore 1:100 anti-CD44 ab6124 abcam 5 μg/mL anti-SOX9 AF3075 R&D systems 5 μg/ml Anti-Lysozyme ab76784 abcam 1:200 anti-Villin sc-58897 Santa Cruz 1:50 anti-Mucin2 sc-7314 Santa Cruz 1:50 Anti-Chromogranin A MA5-14536 Thermo Scientific 1:100 anti-E-cadherin AF648 R&D systems 1:200 anti-E-Cadherin Ab1416 abcam 1:100

Experimental Example 17. RNA sequencing and RNA quantification

For RNA base sequencing and quantification, RNA samples were first prepared with an RNA Integrity Number (RIN) value of 7.5 or higher using an Agilent 2100 Bioanalyzer system (Agilent Biotechnologies, Palo Alto, USA), and mRNA libraries were prepared using an Illumina TruSeq kit. It has been done. Sequencing was performed using Illumina HiSeq2500 machines (Illumina, San Diego, CA, USA). Sequencing quality was determined using the FastQC package, and trimmed read lengths of 50 bases or less were excluded. Afterwards, mapping was performed using HISAT2 (v2.0.5), and hg19 was used for human genome information. Differentially expressed genes (DEGs) between samples were analyzed using Cuffquant and Cuffnorm (Cufflinks v2.2.1).

Experimental Example 18. Bioinformatic analysis

Bioinformatic analyzes were performed using IPA analysis software (Ingenuity systems, Redwood City, CA, USA), the PANTHER (Protein ANalysis THRough Evolutionary Relationships, http:/www.pantherdb.org) database, and DAVID Bioinformatics Resource 6.7 (http:/david. It was conducted using abcc.ncifcrf.gov). Functionally grouped gene ontology (GO)/pathways were generated using the Cytoscape software platform (version 3.3.0) with the ClueGO plug-in (Version 2.2.5, http:/apps.cytoscape.org/apps/cluego). , http:/www.cytoscape.org/what_is_cytoscape.html) was used to analyze it.

Experimental Example 19. Histological (Hematoxylin&Eosin, H&E) staining experiment

For histopathological analysis, intestinal epithelial cells were cryoprotected with sucrose, cut into the membrane of the insert well, placed vertically in optimal cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan), and then frozen. Alternatively, colon tissue removed after transplantation was cryoprotected in sucrose in the same manner and then frozen in OCT compound. Afterwards, the frozen section was cut into 10 μm using a cryostat microtome at -20°C, adhered to a glass slide, and H&E staining was performed according to a known method. Slides were observed through an optical microscope (BX53F, Olympus, Japan). Crypt depth of colon tissue was analyzed using Image J software (National Institute of Health).

Experimental Example 20. Statistical analysis

All results are expressed as mean ± standard error of the mean (s.e.m.), and all experiments were repeated at least three times. P values were determined using two-tailed t-test or one-tailed ANOVA. Length analysis of crypt depth was determined by Welch's t-test. All analyzes of statistical significance were calculated relative to the control group, unless otherwise specified.

Experimental Example 21. Inflammatory cytokine (TNF-α) secretion analysis experiment

Immunogenicity was evaluated by measuring inflammatory cytokine (TNF-α) secretion from RAW 264.7 mouse macrophages purchased from the Korean Cell Line Bank. Mouse TNF-alpha quantikine ELISA kit (R&D systems) was used and the procedure suggested by the manufacturer was followed. Briefly, RAW264.6 macrophages were cultured at a density of 3.5x10 ⁶ cells/mL, and after 12 hours, the medium was harvested and tested using an ELISA kit. Macrophages to which 5 μg/mL lipopolysaccharide (LPS) was added to the medium were used as a positive control.

Example 1. Screening to discover the optimal polymer that can replace Matrigel

In order to find a polymer that can replace Matrigel for intestinal stem cell culture, a screening method was established to find the optimal thin film using a polymer library (Figure 1). For this purpose, the surface of the culture dish was coated with a thin film using polymers with different terminal functional groups (Figure 2). When intestinal stem cells were injected onto a culture dish coated with a thin film of various polymers and cultured for 4 days, engraftment of cells was checked. To find a polymer that showed better performance compared to the number of cells engrafted on a Matrigel-coated culture dish. It was difficult (Figure 3). Afterwards, in order to increase the surface adhesion ability and engraftment rate of intestinal stem cells, plasma was treated on a culture dish coated with a polymer thin film, and the change in the characteristics of the surface of the culture dish as a result was confirmed through changes in wettability according to water contact angle measurement. (Figure 4). Afterwards, when intestinal stem cells were injected onto the plasma-treated polymer thin film surface, it was confirmed that the adhesion ability and engraftment rate of the cells increased (Figure 5).

Example 2. Subculture and cell proliferation rate of intestinal stem cells in a culture dish coated with a polymer thin film

It was confirmed that engraftment of intestinal stem cells was possible on the surface of a plasma-treated polymer thin film, and among various polymer thin films, the engraftment rate of intestinal stem cells was confirmed to be the highest when the thin film was coated with pEGDMA and pCHMA and then treated with plasma. Afterwards, it was confirmed whether subculture and cell proliferation of intestinal stem cells were possible on culture dishes coated with two types of polymer thin films. As a result, it was confirmed that stable subculture of intestinal stem cells and rapid cell proliferation were possible, and pEGDMA It was also confirmed that the culture efficiency of intestinal stem cells was higher in the thin film-coated culture dish (FIG. 6).

Example 3. Verification of plasma processing conditions for pEGDMA thin film coating optimization

To maximize the engraftment of intestinal stem cells on a culture dish coated with a pEGDMA thin film, we sought to optimize surface modification following plasma treatment. First, as a result of checking the engraftment rate of intestinal stem cells according to the treatment time of plasma of the same intensity (15W), it was confirmed that the engraftment of intestinal stem cells was maximized when treated for more than 10 seconds (FIG. 7). In addition, when the pEGDMA thin film surface was treated with different intensities of plasma for the same time (10 seconds), it was confirmed that the intestinal stem cell engraftment rate was maximized on the pEGDMA thin film surface treated with an intensity of about 15 W (FIG. 8). Through this, it was confirmed that the optimal plasma processing conditions for pEGDMA thin film surface modification are 15W and 10 seconds.

Example 4. Verification of properties of plasma-treated pEGDMA thin film surface

Previously, it was confirmed that the engraftment rate of intestinal stem cells was maximized when the pEGDMA thin film was treated with 15W plasma for 10 seconds, so this time, an experiment was conducted to determine how the properties of the pEGDMA surface change under these conditions. After coating the pEGDMA thin film, it was confirmed that there was no difference in the shape and transparency of the culture dish when the pEGDMA thin film was treated with plasma (Figure 9(a)), and when measuring the water contact angle to measure the wettability of the surface, the pEGDMA thin film coating The water contact angle was 62.34° before, 68.54° after pEGDMA thin film coating, and 27.07° for the plasma-treated pEGDMA thin film (Figure 9(b)). In addition, when surface homogeneity was measured using atomic force microscopy (AFM), it was confirmed that the surface was homogeneous both before and after plasma treatment on the pEGDMA thin film-coated culture dish (Figure 9 (c)). When the surface homogeneity of the pEGDMA thin films produced in three batches was measured using atomic force microscopy (AFM), it was confirmed that all surfaces were homogeneous (Figure 9(d)). In addition, it was confirmed through quantitative evaluation through crystal violet analysis and imageJ that intestinal stem cell engraftment was uniformly good in the three batches (Figures 9(e) and 9(f)). Additionally, it was confirmed through images and WST-1 assay that intestinal stem cells engrafted well and cell growth did not deteriorate even in pEGDMA thin film-coated culture dishes stored for a long period of time for 2 years (Figure 9(g) and Figure 9() h)).

Example 5. Verification of chemical properties of pEGDMA thin film surface according to plasma treatment using FT-IR

Since it was confirmed that there was no difference in physical properties after plasma treatment on a culture dish coated with a pEGDMA thin film, an experiment was performed to determine whether there was a difference in chemical properties. As a result of analyzing the chemical characteristics of the surface using Fourier-transform infrared spectrodcopy (FT-IR), it was confirmed that the vinyl peak (C=C, 1650-1620 cm ^-1 ) decreased when forming a polymer (pEGDMA) compared to the EGDMA monomer. (FIG. 10(a)), it was confirmed that there was no significant change in the chemical bond on the surface of the pEGDMA thin film when treated with plasma (FIG. 10(b)).

Example 6. Verification of chemical properties of pEGDMA thin film surface following plasma treatment using XPS

X-ray photoelectron spectroscopy (XPS) was used to analyze the chemical properties of the surface of a culture dish coated with a pEGDMA thin film before and after plasma treatment. As a result, oxygen atoms on the surface increased during plasma treatment, while carbon atoms decreased, and nitrogen atoms decreased during plasma treatment. It was confirmed that the amount of nitrogen atoms increased (Figures 11(a) to 11(c)). In addition, through deconvoluted high resolution scan of C1s, the bond between carbon atoms (-C ^* H ₃ -, -CC ^* H ₂ -) is reduced and the bond with oxygen atom (-C ^* (CH ₃ )-CO The results of increased -O-, -CH ₂ -C ^* HO-, -OC ^* =O) were confirmed (Figures 11(d) to 11(f)).

Example 7. Intestinal stem cell culture and characterization in culture dishes coated with optimized pEGDMA thin film

An experiment was performed to compare the culture efficiency when culturing intestinal stem cells on a culture dish coated with Matrigel and when culturing intestinal stem cells on a culture dish coated with an optimized pEGDMA thin film (Figure 12(a)). did. First, intestinal stem cells were injected into an untreated culture dish (Bare), a culture dish coated with 1% Matrigel, or a culture dish coated with a pEGDMA thin film, and cultured for 1 week, and then the engraftment and growth rates of intestinal stem cells were compared. analyzed. When intestinal stem cells were cultured on a culture dish coated with a pEGDMA thin film, there was no difference from the engraftment and growth rates on a Matrigel-coated culture dish, but the engraftment and growth rates were higher than those on an untreated culture dish (Figure 12 (Figure 12) b) and Figure 12(c)). In addition, when crystal violet (CV) staining was performed to compare and analyze the colony size of engrafted cells, there was no difference in colony size compared to the Matrigel-coated culture dish, but the size was much larger than that of the untreated culture dish. It was confirmed that colonies were growing (Figures 12(d) and 12(e)). Finally, in order to check the survival rate of the intestinal stem cells growing attached on the culture dish, the survival rate of the cells was checked through LIVE/DEAD staining. When the intestinal stem cells cultured on the Matrigel-coated culture dish and the pEGDMA thin film-coated culture dish were both 100%. It was confirmed that the survival rate was 10% (Figure 12(f) and Figure 12(g))

Example 8. Confirmation of long-term culture and cryopreservation of intestinal stem cells in a culture dish coated with an optimized pEGDMA thin film

It was confirmed that when culturing intestinal stem cells on a culture dish coated with an optimized pEGDMA thin film, it was possible to culture intestinal stem cells with similar efficiency as when culturing them on a culture dish coated with Matrigel. Therefore, in order to confirm whether intestinal stem cells can be stably cultured for a long period of time, subculture was performed to check the growth rate of the cells, and it was also confirmed whether frozen storage of intestinal stem cells was possible (FIG. 13(a)). To confirm this, continuous subculture was performed on Matrigel-coated culture dishes and pEGDMA thin film-coated culture dishes, and the cell morphology and growth rate were compared and analyzed. It was confirmed that there was no difference in the morphology and growth rate of intestinal stem cells cultured in the two culture dishes when subcultured more than 30 times (>6 months), and it was confirmed that the survival rate did not decrease even if subcultured more than 30 times. (Figures 13(b) to 13(d)). In addition, when frozen stored intestinal stem cells were thawed and then injected into a pEGDMA thin film-coated culture dish and a Matrigel-coated culture dish, respectively, the cells stably attached and grew, and there was no difference in the size of the formed colonies. By confirming this, it was confirmed that intestinal stem cells cultured on a culture dish coated with a pEGDMA thin film can be frozen and stored (FIGS. 13(e) to 13(g)). Intestinal stem cells stored for more than 3 years were thawed and cultured in a culture dish coated with a pEGDMA thin film, and cell adhesion efficiency and growth ability were confirmed (Figures 13(h) and 13(i)). Additionally, when the amount of endotoxin on the surface of the culture dish coated with the pEGDMA thin film was measured, it was found to be lower than the FDA standard amount, confirming that cells could be safely cultured (FIG. 14(a)). Finally, when we measured whether RAW 264.7 macrophage cells secreted inflammatory cytokines on the surface of a culture dish coated with a pEGDMA thin film, it was confirmed that the levels were low, similar to TCPS (Figure 14(b)). Therefore, it was confirmed that the culture dish coated with the pEGDMA thin film can replace Matrigel coating and is a suitable material for culturing intestinal stem cells.

Example 9. Analysis of genetic characteristics of intestinal stem cells cultured in a culture dish coated with an optimized pEGDMA thin film

We examined whether there were differences in expression levels and characteristics at the gene level when intestinal stem cells were cultured on a culture dish coated with an optimized pEGDMA thin film compared to when cultured on a Matrigel-coated culture dish. When analyzing the transcriptome and proteome using RNA-seq and mass spectrometry technology, there were almost no genes that showed differences in expression at the transcript and proteome levels. A culture dish coated with an optimized pEGDMA thin film and Matrigel coated was found. It was confirmed that the characteristics of intestinal stem cells cultured in the culture dish were similar (FIGS. 15(a) to 15(c)). In addition, as a result of confirming the expression of intestinal stem cell-specific marker genes through qPCR, immunofluorescence staining, and automatic cell analysis separation device, it was verified that there was no difference in the expression of marker genes (Figure 15(d), Figure 15(f) ) and Figure 15(g)). Additionally, even when subcultured, the expression of intestinal stem cell-specific marker genes was confirmed by qPCR, confirming once again that there was no difference in the expression of the marker genes (FIG. 15(e)).

Example 10. Analysis of genetic characteristics of human embryonic stem cell-derived intestinal stem cells cultured in a culture dish coated with an optimized pEGDMA thin film

To confirm the versatility of this technology, human embryonic stem cell-derived intestinal stem cells were cultured in a culture dish coated with an optimized pEGDMA thin film, and culture stability and gene expression characteristics were analyzed. As a result, it was confirmed that stable subculture of human embryonic stem cell-derived intestinal stem cells was possible in a culture dish coated with an optimized pEGDMA thin film (Figure 16(a)), and the corresponding intestinal stem cells were grown in a culture dish coated with Matrigel. It was confirmed that, like cultured intestinal stem cells, a survival rate of 100% was observed (Figures 16(b) and 16(c)). In addition, as a result of confirming the expression of intestinal stem cell marker genes through qPCR, it was confirmed that there was no difference in the expression of marker genes across several passages (Figure 16(d)).

Example 11. Development of intestinal epithelial cell differentiation method of intestinal stem cells using optimized pEGDMA thin film-coated transwell plate

To verify the differentiation ability of intestinal stem cells, intestinal stem cells were differentiated into intestinal epithelial cells using an air-liquid interface culture method on a transwell plate coated with optimized pEGDMA (Figure 17(a)). When differentiation was induced by exposing only the upper part of the transwell plate to the air, it was confirmed that intestinal stem cells were differentiated into intestinal epithelial cells and that structures similar to villus were generated over time. When compared with intestinal epithelial cells differentiated in transwells, it was confirmed that there was no difference in cell morphology (FIG. 17(b)). As a result of marker gene expression, immunofluorescence staining, and tissue staining analysis, it was confirmed that there was no difference in the expression levels of marker genes and proteins compared to when differentiated on Matrigel-coated Transwell plates (Figures 17(c) and Figure 17(d)). Finally, to verify the functionality of differentiated intestinal epithelial cells, the transepithelial electric resistance (TEER) was measured and showed a value of about 290 ohm x cm ² , and differentiation was performed on Matrigel-coated transwell plates. It was confirmed that there was no difference compared to when ordered (Figure 17(e)). Therefore, it was confirmed that the culture dish coated with the optimized pEGDMA thin film can replace Matrigel coating and is a suitable material not only for culturing intestinal stem cells but also for differentiating them into intestinal epithelial cells.

Example 12. Development of an intestinal epithelial cell differentiation method of human embryonic stem cell-derived intestinal stem cells using a transwell plate coated with an optimized pEGDMA thin film

To confirm the versatility of this technology, human embryonic stem cell-derived intestinal stem cells were differentiated into intestinal epithelial cells using an air-liquid interface culture method on an optimized pEGDMA-coated transwell plate. When differentiation was induced by exposing only the upper part of the transwell plate to the air, it was confirmed that human embryonic stem cell-derived intestinal stem cells were also differentiated into intestinal epithelial cells to generate villus-like structures, and Matrigel-coated cells were also differentiated into intestinal epithelial cells. When compared with intestinal epithelial cells differentiated in transwell, it was confirmed that there was no difference in cell morphology (Figure 18(a)). In addition, as a result of marker gene expression, immunofluorescence staining, and tissue staining analysis, it was confirmed that there was no difference in the expression levels of marker genes and proteins compared to when differentiated on Matrigel-coated Transwell plates (Figure 18 (Figure 18) b) and Figure 18(c)). Finally, in order to verify the functionality of differentiated intestinal epithelial cells, the transepithelial electric resistance (TEER) was measured, and the result showed a value of about 125 ohm x cm ² , and differentiation was performed on Matrigel-coated transwell plates. It was confirmed that there was no difference compared to when ordered (Figure 18(d)).

Example 13. Confirmation of transplantation and engraftment ability of intestinal stem cells cultured in optimized pEGDMA thin film-coated culture dishes in various intestinal injury models

To verify the transplantation and engraftment ability of intestinal stem cells (FIG. 19(b)) cultured in a culture dish coated with a pEGDMA thin film in mice, EDTA-induced intestinal epithelial damage immunodeficient mice were prepared. Fibrin was adopted as a transplant agent, and the fibrin transplant group (control group) and intestinal stem cell transplant group (pEGDMA-ISC) were transplanted into the intestinal epithelial damage model (Figure 19(a)). When the degree of epithelial damage was confirmed immediately after epithelial damage through a colonoscope for experimental animals and transplantation was performed, it was confirmed that there was no tissue damage or redness in the intestinal stem cell transplant group 14 days after transplantation (Figures 19(c) and 19 (d)). Histological staining (H&E) of the xenograft colon area also showed that compared to the fibrin transplant group, the crypt depth and formation (restoration) rate of the group transplanted with intestinal stem cells cultured on pEGDMA thin film coating was significantly higher, especially functionally. It was confirmed that secretion of mucin was active (Figures 19(e) and 19(f)). In addition, histological staining of longitudinal sections was performed to confirm the overall pattern within the colon of transplanted mice, and it was re-verified that transplantation and reconstruction occurred in the xenograft of the intestinal stem cell transplant group compared to the fibrin transplant group (Figure 19(e) )). Human-specific antibody (hECAD) showed that the xenograft intestinal stem cells were still located and engrafted in the colon (FIG. 19(g)).

In addition, to confirm whether intestinal stem cells grown in a culture dish coated with a pEGDMA thin film (FIG. 20(b)) can be transplanted in an inflammatory bowel disease model, immunodeficient mice with DSS-induced colitis were prepared. In the same way as applied to the intestinal epithelial damage model, fibrin was adopted as a transplant agent, and fibrin or intestinal stem cells were transplanted into a mouse model showing a colitis phenotype through disease activity measurement (Figure 20(a)). The level of inflammation before transplantation was confirmed through colonoscopy, and the environment in the colon was observed 14 and 28 days after transplantation. On the 14th day after transplantation, it was confirmed that inflammation was rapidly reduced in the intestinal stem cell transplant group compared to the fibrin-only transplant group, and on the 28th day after transplantation, the intestinal epithelium was confirmed to be restored (Figure 20(c)). Histological analysis of the xenograft colon performed with a longitudinal section on the 28th day after transplantation showed that the recovery of the crypt structure of the intestinal epithelium in the intestinal stem cell transplant group was faster than that of fibrin, and the crypt depth was also significantly increased (Figure 20 (d) and Figure 20(e)). Through AB-PAS staining, it was confirmed that the mucin secretion capacity was increased compared to the fibrin transplant group (Figure 20(d)). It was verified through human-specific antibody (hECAD) that the xenograft intestinal stem cells engrafted in the colon and formed a structure (FIG. 20(f)).

Claims (25)

(1) providing a culture substrate containing a polymer thin film manufactured through an iCVD process; and (2) adding a culture medium containing a Wnt signaling pathway activator, a Wnt agonist, a TGF-beta inhibitor, a BMP inhibitor, and a receptor tyrosine kinase ligand to the culture substrate and cultivating intestinal stem cells in two dimensions; A method of culturing intestinal stem cells comprising. According to claim 1, the polymer thin film manufactured through the iCVD process is pDMAEMA (poly(2-(Dimethylamino)ethyl methacrylate)), pDMAPMA(poly(Dimethylaminopropyl methacrylamide)), pDEAEMA(poly(2-(Diethylamino)ethyl methacrylate)) ), pTBAEMA(poly(2-tert-Butylaminoethyl methacrylate)), pDMAEA(poly(2-(Dimethylamino)ethyl acrylate)), pGMA(poly(Glycidyl methacrylate)), p(GMA-co-DMAEMA)(poly(Glycidyl methacrylate-co-(2-(dimethylamino)ethyl methacrylate))), p(GMA-co-DMAPMA)(poly(Glycidyl methacrylate-co-dimethylaminopropyl methacrylamide)), p(GMA-co-DEAEMA)(poly(Glycidyl methacrylate -co-(diethylamino)ethyl methacrylate))), p(GMA-co-TBAEMA)(poly(Glycidyl methacrylate-co-(2-tert-butylaminoethyl methacrylate))), p(GMA-co-DMAEA)(poly( Glycidyl methacrylate-co-(2-(dimethylamino)ethyl acrylate))), pEGDMA(poly(Ethylene glycol dimethacrylate)), p(EGDMA-co-DMAEMA)(poly(Ethylene glycol dimethacrylate-co-(2-(dimethylamino)) ethyl methacrylate))), p(EGDMA-co-DMAPMA)(poly(Ethylene glycol dimethacrylate-co-dimethylaminopropyl methacrylamide)), p(EGDMA-co-DEAEMA)(poly(Ethylene glycol dimethacrylate-co-(diethylamino)ethyl methacrylate ))), p(EGDMA-co-TBAEMA)(poly(Ethylene glycol dimethacrylate-co-(2-tert-butylaminoethyl methacrylate))), p(EGDMA-co-DMAEA)(poly(Ethylene glycol dimethacrylate-co-( 2-(dimethylamino)ethyl acrylate))), pVBC(poly(Vinyl benzyl chloride)), p(VBC-co-DMAEMA)(poly(Vinyl benzyl chloride-co-(2-(dimethylamino)ethyl methacrylate))), p(VBC-co-DMAPMA)(poly(Vinyl benzyl chloride-co-dimethylaminopropyl methacrylamide)), p(VBC-co-DEAEMA)(poly(Vinyl benzyl chloride-co-(diethylamino)ethyl methacrylate))), p( VBC-co-TBAEMA)(poly(Vinyl benzyl chloride-co-(2-tert-butylaminoethyl methacrylate))), p(VBC-co-DMAEA)(poly(Vinyl benzyl chloride-co-(2-(dimethylamino)ethyl acrylate))), pCHMA (poly(Cyclohexyl methacrylate)), pV4D4 (poly(2,4,6,8-Tetramethyl-2,4,6,8-tetravinylcyclotetra siloxane)), and pBMA (poly(Benzyl methacrylate)). A method of cultivating intestinal stem cells, which consists of any one. The method of claim 2, wherein the polymer thin film produced through the iCVD process is pEGDMA (poly(Ethylene glycol dimethacrylate)) or pCHMA (poly(Cyclohexyl methacrylate)). The method of culturing intestinal stem cells according to claim 1, wherein the polymer thin film produced through the iCVD process has a contact angle with water in the range of 10 to 45°. The method of culturing intestinal stem cells according to claim 1, wherein the culture substrate is prepared through plasma treatment under 10 to 20 W for 5 to 20 seconds. The method of claim 1, wherein the Wnt signaling pathway activator is Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt16. A method of cultivating intestinal stem cells, which is any one selected from the group consisting of. The method of culturing intestinal stem cells according to claim 1, wherein the Wnt agonist is R-spondin 1, R-spondin 2, R-spondin 3, or R-spondin 4. The method of claim 1, wherein the TGF-beta inhibitor is any one selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-2511. Method for cultivating cells. The method of culturing intestinal stem cells according to claim 1, wherein the BMP inhibitor is Noggin, Dorsomorphin, DMH1, or LDN-193189. The method of claim 1, wherein the receptor tyrosine kinase ligand is epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), A method of cultivating intestinal stem cells, which is any one selected from the group consisting of neuregulin 1 (NRG1), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF). The method of culturing intestinal stem cells according to claim 1, wherein the culture medium further contains one or more additional components selected from the group consisting of p38 kinase inhibitor, Prostaglandin E2, N-acetylcysteine, Gastrin, B27, and Nicotinamide. . The method of culturing intestinal stem cells according to claim 1, wherein the culture medium further contains a ROCK inhibitor, a Notch activator, or both at the beginning of the culture. (1) providing a culture substrate containing a polymer thin film manufactured through an iCVD process; (2) adding a culture medium containing a Wnt signaling pathway activator, a Wnt agonist, a TGF-beta inhibitor, a BMP inhibitor, and a receptor tyrosine kinase ligand to the culture substrate and cultivating intestinal stem cells in two dimensions; and (3) The intestinal stem cells cultured through step (2) above were placed on the culture substrate with differentiation medium containing a Wnt agonist, p38 kinase inhibitor, receptor tyrosine kinase ligand, Prostaglandin E2, and Nicotinamide, and placed at the air-liquid interface ( Differentiating through the Air-Liquid Interface (Air-Liquid Interface) culture method; A method for producing intestinal epithelial cells comprising. The method of claim 13, wherein the polymer thin film manufactured through the iCVD process is pDMAEMA (poly(2-(Dimethylamino)ethyl methacrylate)), pDMAPMA(poly(Dimethylaminopropyl methacrylamide)), pDEAEMA(poly(2-(Diethylamino)ethyl methacrylate)) ), pTBAEMA(poly(2-tert-Butylaminoethyl methacrylate)), pDMAEA(poly(2-(Dimethylamino)ethyl acrylate)), pGMA(poly(Glycidyl methacrylate)), p(GMA-co-DMAEMA)(poly(Glycidyl methacrylate-co-(2-(dimethylamino)ethyl methacrylate))), p(GMA-co-DMAPMA)(poly(Glycidyl methacrylate-co-dimethylaminopropyl methacrylamide)), p(GMA-co-DEAEMA)(poly(Glycidyl methacrylate -co-(diethylamino)ethyl methacrylate))), p(GMA-co-TBAEMA)(poly(Glycidyl methacrylate-co-(2-tert-butylaminoethyl methacrylate))), p(GMA-co-DMAEA)(poly( Glycidyl methacrylate-co-(2-(dimethylamino)ethyl acrylate))), pEGDMA(poly(Ethylene glycol dimethacrylate)), p(EGDMA-co-DMAEMA)(poly(Ethylene glycol dimethacrylate-co-(2-(dimethylamino)) ethyl methacrylate))), p(EGDMA-co-DMAPMA)(poly(Ethylene glycol dimethacrylate-co-dimethylaminopropyl methacrylamide)), p(EGDMA-co-DEAEMA)(poly(Ethylene glycol dimethacrylate-co-(diethylamino)ethyl methacrylate ))), p(EGDMA-co-TBAEMA)(poly(Ethylene glycol dimethacrylate-co-(2-tert-butylaminoethyl methacrylate))), p(EGDMA-co-DMAEA)(poly(Ethylene glycol dimethacrylate-co-( 2-(dimethylamino)ethyl acrylate))), pVBC(poly(Vinyl benzyl chloride)), p(VBC-co-DMAEMA)(poly(Vinyl benzyl chloride-co-(2-(dimethylamino)ethyl methacrylate))), p(VBC-co-DMAPMA)(poly(Vinyl benzyl chloride-co-dimethylaminopropyl methacrylamide)), p(VBC-co-DEAEMA)(poly(Vinyl benzyl chloride-co-(diethylamino)ethyl methacrylate))), p( VBC-co-TBAEMA)(poly(Vinyl benzyl chloride-co-(2-tert-butylaminoethyl methacrylate))), p(VBC-co-DMAEA)(poly(Vinyl benzyl chloride-co-(2-(dimethylamino)ethyl acrylate))), pCHMA (poly(Cyclohexyl methacrylate)), pV4D4 (poly(2,4,6,8-Tetramethyl-2,4,6,8-tetravinylcyclotetra siloxane)), and pBMA (poly(Benzyl methacrylate)). A method for producing intestinal epithelial cells, comprising any one. The method of claim 14, wherein the polymer thin film produced through the iCVD process is pEGDMA (poly(Ethylene glycol dimethacrylate)) or pCHMA (poly(Cyclohexyl methacrylate)). The method of claim 13, wherein the polymer thin film produced through the iCVD process has a contact angle with water in the range of 10 to 45°. The method of claim 13, wherein the culture substrate is prepared through plasma treatment under 10 to 20 W for 5 to 20 seconds. The method of claim 13, wherein the Wnt signaling pathway activator in step (2) is Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a. , any one selected from the group consisting of Wnt10b, Wnt11, and Wnt16. The method of claim 13, wherein the Wnt agonist is R-spondin 1, R-spondin 2, R-spondin 3, or R-spondin 4. The method of claim 13, wherein the TGF-beta inhibitor in step (2) is selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-2511. Any one, a method of producing intestinal epithelial cells. The method of claim 13, wherein the BMP inhibitor in step (2) is Noggin, Dorsomorphin, DMH1, or LDN-193189. The method of claim 13, wherein the p38 kinase inhibitor is selected from the group consisting of SB202190, SB203580, SB239063, SB706504, BIR796, JX401, EO1428, RWJ67657, SCIO469, VX745, TAK715, ML3403, DBM1285, or PH797804 Intestinal epithelium, which is selected Method for producing cells. 14. The method of claim 13, wherein the receptor tyrosine kinase ligand is epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), A method for producing intestinal epithelial cells, which are any one selected from the group consisting of neuregulin 1 (NRG1), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF). The method of claim 13, wherein the culture medium in step (2) further contains one or more additional components selected from the group consisting of Prostaglandin E2, N-acetylcysteine, Gastrin, B27, and Nicotinamide. method. The method of claim 13, wherein the culture medium in step (2) further contains a ROCK inhibitor, a Notch activator, or both at the beginning of the culture.

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