Method for producing small intestine tissue

Abstract

The present invention provides a method for producing small intestine epithelial cells or small intestine tissue from midgut cells, the method comprising: (1) a step for culturing midgut cells in a medium containing a Wnt agonist, a BMP inhibitor, and a growth factor to obtain intestinal progenitor cells; and (2) a step for culturing the intestinal progenitor cells obtained in step (1) in a medium containing a growth factor, an adenylate cyclase activator, an MEK inhibitor, a DNA methylation inhibitor, a TGF-β inhibitor, a GSK3β inhibitor, and a γ-secretase inhibitor to obtain small intestine epithelial cells or small intestine tissue.

Description

Method for producing small intestine tissue

[0001] The present invention relates to a method for producing small intestinal epithelial cells or small intestinal tissue, more specifically, to a method for producing small intestinal epithelial cells or small intestinal tissue, which includes a step of culturing midgut cells under specific conditions, and uses of the small intestinal epithelial cells or small intestinal tissue obtained by the method.

[0002] The intestine is one of the main parts of the digestive tract, an organ where numerous digestive glands open and perform digestion and absorption. The intestine can be broadly divided into the small intestine and large intestine, with the small intestine further divided into the duodenum, jejunum, and ileum (from the oral side). Small intestinal dysfunction has no fundamental treatment, and many cases are treated only with symptomatic measures such as restricting oral intake and administering total parenteral nutrition. When complications make it difficult to continue total parenteral nutrition, small intestine transplantation becomes an option. However, because the small intestine used in small intestine transplantation is an organ that directly contacts the outside world and performs digestion and absorption, it possesses a complex immune defense mechanism. Therefore, compared to other organ transplants, rejection reactions are more difficult to control, and there are many post-transplant infectious complications.

[0003] Furthermore, orally administered drugs are first absorbed, metabolized, and excreted in the small intestine. Therefore, evaluating pharmacokinetics in small intestinal epithelial cells and small intestinal tissue is important for the effective development of safe pharmaceuticals. However, obtaining primary cultures of human intestinal epithelial cells is difficult, and the resulting cells also have individual differences in their properties. Furthermore, maintaining the function of the resulting cells over the long term while culturing them has been difficult. Therefore, attention has been focused on developing methods to induce small intestinal epithelial cells or small intestinal tissue from pluripotent stem cells.

[0004] For example, a method for inducing intestinal epithelial cells has been reported by culturing definitive endoderm cells derived from human ES cells in a medium containing the GSK inhibitor BIO (6-bromoindirubin-3'-oxime) and the γ-secretase inhibitor DAPT (N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester) (Non-Patent Document 1). Another method for inducing small intestinal epithelial cells has been reported by culturing human iPS cell-derived intestinal stem cells in a medium containing forskolin, followed by the addition of EGF, PD98059, 5-aza-2'-dC, and A-83-01 (Non-Patent Document 2). Furthermore, it has been reported that organoids containing more than 40 crypt domains surrounding a central lumen lined with villus-like epithelium can be generated by isolating crypts from mouse small intestine and culturing the crypt preparation on Matrigel (Non-Patent Document 3). It has also been reported that small intestinal tissue chips can be produced by obtaining epithelial cells from human intestinal organoids derived from induced pluripotent stem cells and then incorporating the cells into a microengineered chip (Non-Patent Document 4). However, these small intestinal models are limited to small intestinal epithelial cells as constituent cells, and do not contain mesenchymal cells that support the small intestinal epithelial cells, which means that they differ greatly from in vivo small intestinal tissue.

[0005] Ogaki et al., Stem Cells. 31(6):1086-1096 (2013)Kabeya et al., Drug Metab Pharmacokinet. 35(4):374-382 (2020)Sato et al., Nature. 459(7244):262-265 (2009)Workman et al., Cell Mol Gastroenterol Hepatol. 5(4):669-677 (2017)

[0006] As described above, in vitro small intestine models prepared by conventional methods contain only small intestinal epithelial cells. Furthermore, there have been no successful examples of constructing a small intestinal tissue model that combines small intestinal epithelial cells with crypts and villi-like structures and supporting mesenchymal cells (particularly fibroblasts). Therefore, an object of the present invention is to provide a novel method for producing small intestinal epithelial cells or small intestinal tissue, particularly a method for producing a novel small intestinal tissue model that combines small intestinal epithelial cells and supporting mesenchymal cells.

[0007] The present inventors first attempted to develop a novel method for inducing small intestinal epithelial cells by improving the method described in Non-Patent Document 2. After extensive research, they discovered that small intestinal epithelial cells can be obtained by a two-step process: inducing midgut cells into intestinal progenitor cells and inducing the intestinal progenitor cells into small intestinal epithelial cells. Specifically, in the first step, midgut cells are cultured in the presence of a Wnt agonist and a BMP inhibitor. In the second step, the intestinal progenitor cells obtained in the first step are cultured in the presence of a GSK inhibitor, a γ-secretase inhibitor, or the like. Wnt agonists and BMP inhibitors are substances often used for replicating intestinal progenitor cells, while in existing methods, GSK inhibitors and γ-secretase inhibitors are substances used immediately after inducing definitive endoderm cells. However, the method of the present invention surprisingly produced small intestinal epithelial cells despite using these substances at a completely different timing than conventional methods.

[0008] The present inventors have conducted further research and found that small intestinal tissue containing fibroblasts can be obtained by carrying out the above-mentioned culture method in a microfluidic device, and that perfusion culture can produce small intestinal tissue that is closer to the morphology of a living organism than that obtained by conventional methods. Based on these findings, the present inventors have conducted further research and have completed the present invention.

[0009] That is, the present invention is as follows: [1] A method for producing small intestinal epithelial cells or small intestinal tissue from midgut cells, comprising: (1) culturing midgut cells in a medium containing a Wnt agonist, a BMP inhibitor, and a growth factor to obtain intestinal progenitor cells; and (2) culturing the intestinal progenitor cells obtained in step (1) in a medium containing a growth factor, an adenylate cyclase activator, a MEK inhibitor, a DNA methylation inhibitor, a TGF-β inhibitor, a GSK3β inhibitor, and a γ-secretase inhibitor to obtain small intestinal epithelial cells or small intestinal tissue. [2-1] The method according to [1], wherein the Wnt agonist comprises a Wnt protein and R-spondin. [2-2] The method according to [2-1], wherein at least one of the Wnt proteins is Wnt3A. [2-3] The method according to [2-1] or [2-2], wherein at least one of the R-spondins is R-spondin3. [2-4] The method according to any one of [2-1] to [2-3], wherein at least one of the BMP inhibitors is noggin. [3] The method according to any one of [1] to [2-4], wherein the Wnt agonist and the BMP inhibitor are obtained from cells expressing them. [4-1] The method according to any one of [1] to [3], wherein the growth factor in steps (1) and (2) comprises EGF. [4-2] The method according to [4-1], wherein the growth factor in step (1) comprises IGF. [4-3] The method according to any one of [1] to [4-2], wherein at least one of the adenylate cyclase activators is forskolin. [4-4] The method according to any one of [1] to [4-3], wherein at least one of the MEK inhibitors is PD98059. [4-5] The method according to any one of [1] to [4-4], wherein at least one of the DNA methylation inhibitors is 5-aza-2'-deoxycytidine. [4-6] The method according to any one of [1] to [4-5], wherein at least one of the TGF-β inhibitors is A-83-01. [4-7] The method according to any one of [1] to [4-6], wherein at least one of the GSK-3β inhibitors is BIO. [4-8] The method according to any one of [1] to [4-7], wherein at least one of the γ-secretase inhibitors is DAPT.[5] The method according to any one of [1] to [4-8], wherein steps (1) and (2) are carried out in a microfluidic device. [6] The method according to [5], wherein steps (1) and (2) comprise a step of performing perfusion culture. [7] The method according to [6], wherein perfusion culture is carried out using a peristaltic pump or a shaker. [8] The method according to any one of [1] to [7], comprising a step of inducing differentiation of definitive endoderm cells into midgut cells prior to step (1). [9-1] The method according to [8], further comprising a step of inducing differentiation of pluripotent stem cells into definitive endoderm cells. [9-2] The method according to [9-1], wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells. [9-3] The method according to [9-1] or [9-2], wherein the pluripotent stem cells are human pluripotent stem cells.

[10] Small intestinal epithelial cells, small intestinal tissue, or a microfluidic device carrying them, obtained by the method according to any one of [1] to [9-3]. [11-1] Small intestinal tissue or a microfluidic device carrying said small intestinal tissue, having all of the following characteristics (A) to (F): (A) having absorptive epithelial cells (B) having fibroblasts (C) having collagen fibers (D) expressing one or more (preferably all) genes selected from the group consisting of TGFB1, Wnt5A, and IGF2 (E) expressing one or more (preferably all) genes selected from the group consisting of MMP1, COL3A1, and TIMP1 (F) having drug-metabolizing enzyme activity [11-2] The small intestinal tissue or a microfluidic device carrying said small intestinal tissue according to [11-1], having at least one (preferably all) of the following characteristics (G) to (K): (G) derived from pluripotent stem cells (H) having goblet cells (I) having Paneth cells (J) having enteroendocrine cells (K) having crypt-like structures and villi-like structures [11-3] (L) expressing a transporter gene, or a microfluidic device carrying the small intestinal tissue according to [11-1] or [11-2]. [11-4] (M) having tight junctions (TJs), adherens junctions (AJs), and desmosomes (DSs), or a microfluidic device carrying the small intestinal tissue according to any one of [11-1] to [11-3].[12-1] A transplantation therapy agent comprising the small intestinal epithelial cells or small intestinal tissue described in any one of

[10] to [11-4]. [12-2] The transplantation therapy agent described in [12-1] for treating or preventing a small intestinal disease.

[13] A method for screening for a therapeutic or prophylactic agent for a viral infection, comprising: (I) contacting the small intestinal tissue described in any one of

[10] to [11-4] with a virus in the presence or absence of a test substance and culturing the tissue; (II) assessing the level of infection of the virus in the small intestinal tissue; and (III) selecting the test substance as a candidate for a therapeutic or prophylactic agent for a viral infection when the level of viral infection is assessed to be lower in the presence of the test substance compared to in the absence of the test substance in step (II).

[14] The method described in

[13] , wherein the virus is a virus belonging to the Coronaviridae family.

[15] A method for evaluating the degree of metabolism of a test substance, comprising: (i) contacting the test substance with the small intestinal tissue described in any one of

[10] to [11-4], (ii) measuring the activity level of a drug-metabolizing enzyme, and (iii) evaluating the degree of metabolism of the test substance by the drug-metabolizing enzyme based on the activity level of the drug-metabolizing enzyme in step (ii). [16-1] The method described in

[15] , wherein the drug-metabolizing enzyme is at least one selected from the group consisting of CYP3A4, UGT1A1, and CYP2C9. [16-2] The method described in

[15] or [16-1], wherein at least one of the drug-metabolizing enzymes is CYP3A4.

[17] A method for treating or preventing a small intestinal disease, comprising administering or transplanting an effective amount of the small intestinal epithelial cells or small intestinal tissue described in any one of

[10] to [11-4] into a mammal.

[18] The small intestinal epithelial cells or small intestinal tissue according to any one of

[10] to [11-4] for use in the treatment or prevention of a small intestinal disease.

[19] Use of the small intestinal epithelial cells or small intestinal tissue according to any one of

[10] to [11-4] in the manufacture of a drug for the treatment or prevention of a small intestinal disease.

[0010] According to the present invention, small intestinal epithelial cells can be produced from midgut cells by a method different from conventional methods. Furthermore, by using a microfluidic device, small intestinal epithelial cells and mesenchymal cells such as fibroblasts can be simultaneously induced to differentiate from pluripotent stem cells within a single microfluidic device. The mesenchymal cells produce extracellular matrix such as collagen and niche factors, allowing the small intestinal epithelial cells to exhibit polarity and functions similar to those in vivo.

[0011] Overview of the protocol for inducing differentiation of pluripotent stem cells into small intestinal epithelial cells or small intestinal tissue. Cells were seeded onto 24-well plates or microfluidic devices on Day 3 and cultured until Day 19 or Day 24. Schematic diagram of the microfluidic device. Induction of differentiation into small intestinal epithelial cells using a 24-well plate. Data on Villin (also referred to as Villin 1 (VIL1)), MUC2, LGR5, OSTa, ASBT, and CYP3A4 mRNA expression levels demonstrated that differentiation into small intestinal epithelial cells was possible under all conditions in Protocols 1–4 (Figure 1 shows an outline of Protocols 1–4; the same applies below). n=5, error bars indicate standard deviation. Induction of differentiation into small intestinal tissue using a microfluidic device. Brightfield images on Days 19 and 24 for each protocol are shown. In Protocols 1 and 2, cells died during the culture period, making it impossible to continue. In Protocols 3 and 4, viable cells were observed even on Days 19 and 24. Induction of differentiation into small intestinal tissue using a microfluidic device. The amount of RNA extracted from cells recovered after differentiation induction in each protocol is shown. Cells were recovered on day 19 for protocols 1 and 2, and on day 24 for protocols 3 and 4. n=5, error bars: standard deviation. Induction of differentiation into small intestinal tissue using a microfluidic device. Measurement of the mRNA expression levels of small intestinal markers in each protocol is shown. n=5, error bars: standard deviation. Induction of differentiation into small intestinal tissue using a microfluidic device. Measurement of the mRNA expression levels of small intestinal markers in each protocol is shown. The data on SI, CHGA, REG4, LGR5, ASBT, and CYP3A4 mRNA expression indicate that protocol 4 is superior to protocol 3. Furthermore, compared to cells prepared using protocol 1 in a 24-well plate, more genes for small intestinal markers were highly expressed in cells prepared using protocol 4. Therefore, protocol 4 was used in the following experiments. n=5, error bars: standard deviation. The effect of medium perfusion using small intestinal tissue chips. The mRNA expression levels of small intestinal markers were compared between static and perfusion culture conditions. n=3, error bars: standard deviation. The effect of medium perfusion using small intestinal tissue chips.We compared the mRNA expression levels of small intestinal markers under static and perfusion culture conditions. No significant differences were observed between the two conditions for any of the markers. Furthermore, perfusion culture did not significantly affect the expression levels of small intestinal markers. n=3, error bars: standard deviation. The effect of medium perfusion using small intestinal tissue chips. Bright-field images are shown on days 10, 17, and 25. Static and perfusion cultures were compared. Perfusion culture demonstrated the formation of three-dimensional small intestinal-like tissue. It was demonstrated that perfusion using a shaker, as well as perfusion into the lower channel, can also produce three-dimensional small intestinal-like tissue. The effect of medium perfusion on small intestinal tissue chips. HE-stained images are shown for static and perfusion cultures. A single layer of small intestinal epithelial cells was formed during static culture. Perfusion culture demonstrated small intestinal crypt and villi-like structures, as well as underlying lamina propria-like and extracellular matrix-like structures. Effect of medium perfusion on small intestinal tissue chips. Gene expression analysis (left) and protein expression analysis (right) of villin under static and perfusion culture conditions. Villin gene expression levels were unchanged by perfusion, but villin protein expression levels increased with perfusion. CYP3A4 protein expression levels were unchanged with or without perfusion. n=3, error bars: standard deviation, *: P<0.05, **: P<0.01. CYP3A4 activity assay results for small intestinal tissue chips. Small intestinal tissue exhibited high CYP3A4 activity in both static and perfusion cultures, but there was no significant difference between the two. This indicates that CYP3A4 activity was not affected by perfusion, consistent with the gene expression and immunostaining results. n=3, error bars: standard deviation. Drug-metabolizing enzyme activity measurements for small intestinal tissue chips. Drug-metabolizing enzyme activity was measured using UGT1A1 activity, CYP2C9 activity, and CYP3A4 activity. Small intestinal tissue showed high drug-metabolizing enzyme activity in both static and perfusion cultures. n=6, error bars: standard deviation. Effect of medium perfusion in small intestinal tissue chips. Immunostaining results for Villin, MUC2, CHGA, CYP3A4, Ki67, and Lysozyme under static or perfusion culture conditions.Perfusion confirmed the presence of numerous MUC2-positive goblet cells, lysozyme-positive Paneth cells, CHGA-positive endocrine cells, and Ki67-positive proliferating cells in the small intestinal tissue chip. Villin was also localized to the apical side of the small intestinal tissue. Perfusion did not affect CYP3A4 expression levels. Effect of medium perfusion on small intestinal tissue chips. Results of Alcian blue staining (mucin staining) under static or perfusion culture conditions. Perfusion confirmed the presence of numerous Alcian blue-positive mucin-producing cells (goblet cells). A thin mucin layer was observed on the apical side of small intestinal epithelial cells (black triangles). Analysis of the effect of medium perfusion rate on various small intestinal markers. Results of fully automated capillary electrophoresis immunoassays for villin, CYP3A4, ANPEP, and β-actin. n=3, error bars: standard deviation. Effect of medium perfusion on small intestinal tissue chips. Results of Masson's trichrome staining under static or perfusion culture conditions. Masson's trichrome staining stains collagen fibers and their main component, collagen, blue. Perfusion confirmed the formation of a Masson's trichrome-positive collagen layer aligned on the basement membrane side. The small intestinal epithelial layer is supported by the collagen layer. Since fibroblasts are expected to be the primary collagen-producing cells, we next conducted experiments to confirm their presence. Fibroblast localization in small intestinal tissue chips. Results of automated capillary electrophoresis immunoassays for vimentin, α-SMA, and β-actin (left) and immunostaining for villin, vimentin, and α-SMA (right). Expression of vimentin, a marker for intestinal fibroblasts, was maintained and localized to the basolateral side by perfusion. Expression of α-SMA, a marker for myofibroblasts, was also reduced by perfusion. n=3, error bars: standard deviation, **: P<0.01. Fibroblast localization in small intestinal tissue chips. Immunostaining for COL1A1 and vimentin. COL1A1 was confirmed to co-localize with Vimentin. Effect of medium perfusion on small intestinal tissue chips (RNA-seq analysis). Perfusion increased the expression of genes controlling extracellular structure and secretion, which may be factors controlling the mucin and collagen layers adjacent to small intestinal epithelial cells.Expression analysis of the viral receptor ANPEP in small intestinal tissue chips. Results of automated capillary electrophoresis immunoassays for β-actin and ANPEP, one of the infection receptors for human coronavirus 229E, are shown. n=3, error bars: standard deviation, *: P<0.05. Viral infection experiments using small intestinal tissue chips. Human coronavirus (HCoV-229E) infection experiments using small intestinal tissue chips. Virus infection was performed from either the upper (apical) or lower (basolateral) channel, and infection efficiency and viral responses in host cells were assessed. Viral genome load (HCoV-229E mRNA) and mRNA levels of genes involved in immune responses to viral infection (IL-8, IL-1β) were measured. Results showed that apical infection resulted in efficient viral replication and host responses. n=4, error bars: standard deviation, *: P<0.05, **: P<0.01. Comparison of the developed small intestinal tissue chip with in vivo human small intestine (small intestinal total RNA) (RNA-seq analysis). Gene expression profiles were compared between perfusion-cultured small intestinal tissue chips and small intestinal total RNA. 4,510 genes were more than twice as expressed in the small intestinal tissue chip, while 5,462 genes were less expressed than in the small intestinal tissue chip. Comparison of the developed small intestinal tissue chip with in vivo human small intestine. Increased expression of genes related to collagen fibrillogenesis, negative regulation of apoptotic pathways, and ECM formation was demonstrated in the small intestinal tissue chip. Comparison of the developed small intestinal tissue chip with in vivo human small intestine. Gene expression levels of extracellular matrix (ECM) and ECM degradation regulators (MMP1, COL3A1, TIMP1), as well as growth factors (TGFB1, WNT5A, IGF2) (WNT5A is essential for small intestinal regeneration, and IGF2 is essential for the maintenance of intestinal stem cells) were measured. In small intestinal tissue chips, expression of ECM and ECM degradation regulators, as well as growth factors, was elevated. n=3, error bars: standard deviation. Comparison of protein expression changes in small intestinal tissue chips cultured statically (Static) or perfusionally (Flow) (proteome analysis) (left), and results of cell type analysis using the human protein atlas (right).Heatmap of genes highly expressed in the small intestine (left) and in small intestinal absorptive epithelial cells (enterocytes) (right). Heatmap showing protein expression changes for drug-metabolizing enzymes cytochrome P450 (CYP), uridine diphosphate glucuronosyltransferase (UGT), sulfotransferase (SULT), and carboxylesterase (CES) (left). Comparison of expression levels of CES2, which is highly expressed in the human small intestine, and CES1, which is highly expressed in the liver (right). n=3, error bars: standard deviation, **: P<0.01). Comparison of mRNA and protein expression changes in small intestinal tissue chips cultured by static and perfusion culture (left), and analysis results using the human protein atlas (right). Overview of the protocol for inducing differentiation of pluripotent stem cells into small intestinal tissue by varying the duration of perfusion culture. Brightfield image of a small intestinal tissue chip. Analysis of the effect of the duration of perfusion culture (flow) on the function of the small intestinal tissue chip. Results of fully automated capillary electrophoresis immunoassay for vimentin, CYP3A4, ANPEP, and β-actin. n=3, error bars: standard deviation. *: P<0.05, **: P<0.01. Brightfield image of small intestinal tissue chip. 3D small intestinal tissue was generated without the use of HCM. 3D small intestinal tissue was also generated using William's E medium instead of HCM. Immunostaining for vimentin (upper right) and the degree of vimentin orientation calculated using FiberOri8single03 (lower) under static and perfusion culture conditions. Proteomic analysis of MMP-1 expression levels (top) and brightfield image at Day 18 (intestinal progenitor cells) (bottom). Use of the MMP inhibitor GM 6001 prevented the small intestinal tissue from becoming three-dimensional. n=3, error bars: standard deviation. *: P<0.05. Time-dependent gene expression in small intestinal tissue chip (RT-qPCR analysis).Relative mRNA expression levels of the undifferentiated cell marker POU class 5 homeobox 1 (POU5F1) (A), the mesendodermal cell marker Mix paired-like homeobox (MIXL1) (B), the endodermal cell marker forkhead box A2 (FOXA2), the absorptive epithelial cell markers villin 1 (VIL1) and sucrase-isomaltase (SI), the mesodermal cell marker T-box transcription factor T (TBXT), and the mesenchymal cell markers vimentin (VIM) and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) (C). n=3, error bars: standard deviation. RT-qPCR analysis of gene expression in small intestinal tissue chips (prepared under static and perfusion culture conditions) compared with intact human small intestine and Caco-2 cells.The absorptive epithelial cell markers VIL1 and fatty acid-binding protein 2 (FABP2), the goblet cell marker mucin 2 (MUC2), the Paneth cell marker lysozyme (LYZ), the neuroendocrine cell marker regenerating family member 4 (REG4), the M cell marker glycoprotein 2 (GP2), the tuft cell marker doublecortin-like kinase 1 (DCLK1) (A), the drug-metabolizing enzymes cytochrome P450 family 3 subfamily A member 4 (CYP3A4), CYP family 2 subfamily C member 9 (CYP2C9), UDP glucuronosyltransferase family 1 member A1 (UGT1A1), and sulfotransferase family 1B member 1 (SULT1B1) (B), and the transporters ATP binding cassette subfamily B member 1 (ABCB1 / MDR1), ABC subfamily C member 1 (ABCC1 / MRP1), and ABC subfamily C member Measurement of relative mRNA expression levels of ABCC2 / MRP2, ABC subfamily G member 2 (ABCG2 / BCRP), solute carrier organic anion transporter family member 2B1 (SLCO2B1 / OATP2B1), and SLC family 15 member 1 (SLC15A1 / PEPT1) (C). n=4 (small intestinal tissue chips and Caco-2 cells) or n=1 (living human small intestine). Error bars: standard deviation. Morphological analysis of small intestinal tissue chips (electron microscopy). The morphology of small intestinal tissue chips prepared under static culture conditions was compared with that of small intestinal tissue chips prepared under perfusion culture conditions. TJ (tight junction): tight junction; AJ (adherence junction): adherens junction; DS: desmosome. Scale bar: 6 μm (A), 600 nm (B, C).In situ gene expression profiling in small intestinal tissue chips. A shows an H&E stained image of the small intestinal tissue chip used for analysis. B shows an image of cell morphology inferred from the position of cell nuclei. C and D show the results of expression analysis in small intestinal tissue chips of the absorptive epithelial cell markers solute carrier family 26 member 2 (SLC26A2) and solute carrier family 26 member 3 (SLC26A3), the intestinal progenitor cell marker SRY-box transcription factor 9 (SOX9), the stem cell markers leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) and Bestrophin 4 (BEST4), and the neuroendocrine cell marker CHGA. In situ gene expression profiling in small intestinal tissue chips. A shows an H&E stained image of the small intestinal tissue chip used for analysis. B shows an image of cell morphology inferred from the position of cell nuclei. C and D show transcriptome analysis results for the intestinal fibroblast marker vimentin (VIM), telocyte or trophocyte markers platelet-derived growth factor receptor alpha (PDGFRA), and frizzled-related protein (FRZB, a Wnt antagonist) in small intestinal tissue chips. Gene expression analysis (scRNA-seq analysis) of fibroblasts comprising the small intestinal tissue chip. A and B show the results of subcluster analysis of interstitial cell clusters. Interstitial cells were classified into three subclusters (A). 0, 1, and 2 in the figure represent subclusters 0, 1, and 2, respectively. Results of virus (HCoV-229E) infection experiments using small intestinal tissue chips (RNA-seq analysis). A shows the results of infection with HCoV-229E from the apical side (Mock vs. Apical) and the basolateral side (Mock vs. Basolateral) of small intestinal tissue chips perfused with medium.B and C show changes in the expression of host genes associated with viral infection in small intestinal tissue chips infected apically with HCoV-229E. Verification was performed using small intestinal tissue chips prepared under static or perfusion culture conditions using a human ES cell line (KhES-3). A shows a bright-field image on day 24. Scale bar: 500 μm. B shows an HE-stained image. Scale bar: 50 μm. C shows an Alcian blue-stained image. Scale bar: 50 μm. D shows the results of automated capillary electrophoresis immunoassays for ANPEP, VIL1, and β-actin. n=3, error bars: standard deviation, *: P<0.05. E shows an immunostained image for VIL1 and VIM. Nuclei were stained with DAPI. Scale bar: 50 μm. F shows a Masson's trichrome-stained image. Scale bar: 50 μm. Validation was performed using a small intestinal tissue chip prepared using a human ES cell line (H9) under static or perfusion culture conditions. A shows a bright-field image on day 20. Scale bar: 500 μm. B shows an HE-stained image. Scale bar: 50 μm. C shows an Alcian blue-stained image. Scale bar: 50 μm. D shows the results of a fully automated capillary electrophoresis immunoassay for ANPEP, VIL1, and β-actin. n=4, error bars: standard deviation, *: P<0.05, **: P<0.01. E shows an immunostained image for VIL1 and VIM. Nuclei were stained with DAPI. Scale bar: 50 μm. F shows an image stained with Masson's trichrome. Scale bar: 50 μm.

[0012] 1. Method for Producing Small Intestinal Epithelial Cells or Small Intestinal Tissue The present invention provides a method for producing small intestinal epithelial cells or small intestinal tissue from midgut cells. Specifically, the method comprises the steps of: (1) culturing midgut cells in a medium containing a Wnt agonist, a BMP inhibitor, and a growth factor to obtain intestinal progenitor cells; and (2) culturing the intestinal progenitor cells obtained in step (1) in a medium containing a growth factor, an adenylate cyclase activator, a MEK inhibitor, a DNA methylation inhibitor, a TGF-β inhibitor, a GSK3β inhibitor, and a γ-secretase inhibitor to obtain small intestinal epithelial cells or small intestinal tissue (hereinafter, sometimes referred to as the "production method of the present invention").

[0013] Midgut cells are cells that constitute the midgut, which forms the basis of the small intestine during vertebrate development. As used herein, "midgut cells" refers to cells that express CDX2 and have the ability to differentiate into intestinal progenitor cells. Small intestinal epithelial cells are cells that constitute the small intestinal epithelium in vivo. Specific examples of small intestinal epithelial cells obtained by cell culture include absorptive epithelial cells that express Villin, goblet cells that express MUC2, intestinal stem cells that express LGR5, Paneth cells that express LYZ, enteroendocrine cells that express CHGA, M cells that express GP2, and tuft cells that express DCLK1. As used herein, "small intestinal tissue" refers to a structure that contains at least absorptive epithelial cells as small intestinal epithelial cells and has a layered structure composed of cells. Absorptive epithelial cells express Villin, and preferably also express SI and / or FABP2.

[0014] As used herein, unless otherwise specified, the term "cells" includes "cell populations." Furthermore, unless otherwise specified, "cells" refers to cells obtained by cell culture. A cell population may be composed of one type of cell, or two or more types of cells. Thus, the term "small intestinal epithelial cells" includes not only one type of small intestinal epithelial cells (e.g., absorptive epithelial cells), but also cell populations containing two or more types of small intestinal epithelial cells (e.g., a combination of absorptive epithelial cells, goblet cells, and intestinal stem cells).

[0015] In step (1) of the method of the present invention, intestinal progenitor cells can be obtained by culturing midgut cells in a medium containing a Wnt agonist, a BMP inhibitor, and a growth factor. Intestinal progenitor cells are cells that arise from intestinal stem cells during the developmental stage of vertebrates and can differentiate into cells that constitute the small intestine, which are cells that have the ability to differentiate into small intestinal epithelial cells. As used herein, "intestinal progenitor cells" refers to cells that are induced from midgut cells or intestinal stem cells and have the ability to differentiate into small intestinal epithelial cells. Furthermore, "intestinal progenitor cells" includes not only cells that do not have the ability to self-proliferate, but also cells that have the ability to self-proliferate (i.e., intestinal stem cells). Intestinal progenitor cells are typically cells that weakly express Villin, MUC2, LYZ, and CHGA.

[0016] As used herein, the term "Wnt agonist" refers to a substance that activates T-cell factor (TCF) / lymphoid enhancer actor (LEF)-mediated transcription in cells. Therefore, Wnt agonists are not limited to Wnt family proteins, but also include Wnt agonists that bind to and activate Frizzled receptor family members, inhibitors of intracellular β-catenin degradation, and TCF / LEF activators. Examples of Wnt agonists used in step (1) of the production method of the present invention include Wnt proteins and R-spondins. It is preferable to use a combination of a Wnt protein (e.g., Wnt3A) and an R-spondin (e.g., R-spondin3).

[0017] Examples of Wnt proteins used in step (1) of the production method of the present invention include Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, and Wnt16. Of these, Wnt3A is preferred. Multiple Wnt proteins may be used in combination.

[0018] Examples of R-spondins used in step (1) of the production method of the present invention include R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. Among these, R-spondin 3 is preferred. Multiple types of R-spondins may be used in combination.

[0019] Bone morphogenetic proteins (BMPs) bind as dimeric ligands to a receptor complex consisting of two distinct receptor serine / threonine kinases, type I and type II receptors. The type II receptor phosphorylates the type I receptor, activating the receptor kinase. The type I receptor then phosphorylates specific receptor substrates (SMADs), resulting in transcriptional activation via a signal transduction pathway. Generally, BMP inhibitors are substances that bind to BMP molecules to form complexes that neutralize BMP activity, e.g., inhibit the binding of BMP molecules to BMP receptors. BMP inhibitors are also substances that bind to BMP receptors and inhibit the binding of BMP molecules to the receptor, acting as antagonists or inverse agonists.

[0020] Examples of BMP inhibitors used in step (1) of the production method of the present invention include chordin-like proteins such as noggin, chordin, and chordin domains; follistatin-related proteins such as follistatin and follistatin domains; DAN-like proteins such as DAN and DAN cysteine-knot domains; sclerostin / SOST, decorin, and α2-macroglobulin. Among these, chordin-like proteins and DAN-like proteins are preferred, with chordin-like proteins (particularly noggin) being more preferred. Chordin-like proteins and DAN-like proteins are diffusible proteins that bind to BMP molecules with varying affinities and can inhibit the BMP molecules from approaching signaling receptors.

[0021] Examples of growth factors used in step (1) of the production method of the present invention include epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor-α (TGF-α), fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF), and keratinocyte growth factor (KGF). Examples of FGFs include 23 types of proteins, FGF1 to FGF23. IGF may be either IGF1 or IGF2. Preferred growth factors used in step (1) of the production method of the present invention are EGF and IGF, and a combination of EGF and IGF (particularly IGF1) is more preferred. These growth factors may also be used in combination.

[0022] The concentration of EGF used in step (1) of the production method of the present invention in the medium is typically 5 ng / mL to 500 ng / mL, preferably 10 ng / mL to 300 ng / mL, more preferably 30 ng / mL to 100 ng / mL (particularly 50 ng / mL). The concentration of IGF1 used in step (1) of the production method of the present invention in the medium is typically 1 ng / mL to 200 ng / mL, preferably 2 ng / mL to 100 ng / mL, more preferably 10 ng / mL to 50 ng / mL (particularly 20 ng / mL). When a growth factor other than EGF or IGF1 is used, the concentration of the growth factor in the medium is selected appropriately.

[0023] The culture period in step (1) of the production method of the present invention is typically 3 to 13 days, preferably 4 to 10 days, more preferably 5 to 7 days (particularly 6 days).

[0024] In step (2) of the production method of the present invention, small intestinal epithelial cells or small intestinal tissue can be obtained by culturing the intestinal progenitor cells obtained in step (1) in a medium containing a growth factor, an adenylate cyclase activator, a MEK inhibitor, a DNA methylation inhibitor, a TGF-β inhibitor, a GSK3 inhibitor, and a γ-secretase inhibitor.

[0025] The growth factors used in step (2) of the production method of the present invention include those similar to those used in step (1). The growth factor used in step (2) of the production method of the present invention is preferably EGF. These growth factors may be used in combination.

[0026] The concentration of EGF in the medium used in step (2) of the production method of the present invention is typically 0.5 ng / mL to 100 ng / mL, preferably 1 ng / mL to 50 ng / mL, more preferably 5 ng / mL to 30 ng / mL (particularly 10 ng / mL). When a growth factor other than EGF is used, the concentration of the growth factor in the medium is appropriately selected.

[0027] The adenylate cyclase activator used in step (2) of the production method of the present invention may be any that increases intracellular cAMP concentration, such as forskolin, HJC0350, 8-Br-cAMP, adenosine 3',5'-cyclic monophosphate (cAMP), dibutyryl-cAMP (bucladesine), etc. Among these, forskolin is preferred. These adenylate cyclase activators may be used in combination.

[0028] The concentration of forskolin used in step (2) of the production method of the present invention in the medium is typically 1 μM to 200 μM, preferably 3 μM to 100 μM, more preferably 5 μM to 50 μM (particularly 15 μM). When an adenylate cyclase activator other than forskolin is used, the concentration of the adenylate cyclase activator in the medium is appropriately selected.

[0029] Examples of MEK inhibitors used in step (2) of the production method of the present invention include PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one), PD184352 (2-(2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide), PD184161 (2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-5-bromobenzamide), PD0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide), U0126, MEK inhibitor I, MEK inhibitor II, MEK1 / 2 inhibitor II, and SL327. Of these, PD98059 is preferred. These MEK inhibitors may be used in combination.

[0030] The concentration of PD98059 in the medium used in step (2) of the production method of the present invention is typically 0.5 μM to 100 μM, preferably 1 μM to 50 μM, and more preferably 5 μM to 30 μM (particularly 10 μM). When a MEK inhibitor other than PD98059 is used, the concentration of the MEK inhibitor in the medium is appropriately selected.

[0031] Examples of DNA methylation inhibitors used in step (2) of the production method of the present invention include 5-aza-2'-deoxycytidine (5-aza-2'-dC), 5-azacytidine, RG108, and Zebularine. Among these, 5-aza-2'-dC is preferred. These DNA methylation inhibitors may be used in combination.

[0032] The concentration of 5-aza-2'-dC used in step (2) of the production method of the present invention in the medium is typically 0.1 μM to 100 μM, preferably 0.5 μM to 50 μM, more preferably 1 μM to 5 μM (particularly 2.5 μM). When a DNA methylation inhibitor other than 5-aza-2'-dC is used, the concentration of the DNA methylation inhibitor in the medium is appropriately selected.

[0033] Examples of the TGF-β inhibitor used in step (2) of the production method of the present invention include SB431542 (4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, 4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide), A-83-01 (3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole), LDN193189 (4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), G W788388 (4-[4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl]-N-(tetrahydro-2H-pyran-4-yl)benzamide), SM16 (4-[4-(1,3-benzodioxol-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-2-yl]-bicyclo[2.2.2]octane-1-carboxamide), IN-113 0 (3-[[5-(6-methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]-benzamide), GW6604 (2-phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine), SB505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methyl Examples of TGF-β inhibitors include SB525334 (6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), SD-208 (2-(5-chloro-2-fluorophenyl)pteridin-4-yl)pyridin-4-ylamine), and LY-364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]quinoline). Among these, A-83-01 is preferred. These TGF-β inhibitors may be used in combination.

[0034] The concentration of A-83-01 in the medium used in step (2) of the production method of the present invention is typically 10 nM to 10 μM, preferably 50 nM to 5 μM, and more preferably 100 nM to 500 nM (particularly 250 nM). When a TGF-β inhibitor other than A-83-01 is used, the concentration of the TGF-β inhibitor in the medium is appropriately selected.

[0035] As used herein, the term "GSK3β inhibitor" refers to a substance having inhibitory activity against GSK3β (glycogen synthase kinase 3β). GSK3 (glycogen synthase kinase 3) is a type of serine / threonine protein kinase and is involved in many signal pathways related to glycogen production, apoptosis, stem cell maintenance, and the like. GSK3 exists in two isoforms, α and β. The "GSK3β inhibitor" used in the present invention is not particularly limited as long as it has GSK3β inhibitory activity, and may be a substance that has both GSK3β inhibitory activity and GSK3α inhibitory activity.

[0036] Examples of GSK3β inhibitors used in step (2) of the production method of the present invention include CHIR98014 (2-[[2-[(5-nitro-6-aminopyridin-2-yl)amino]ethyl]amino]-4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidine), CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile), CP21R7 (3-(3-aminopyridin-2-yl)pyrimidinyl)amino]ethyl]amino]nicotinonitrile), and CP21R8 (3-(3-aminopyridin-2-yl)pyrimidinyl)amino]ethyl]amino). (amino-phenyl)-4-(1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione), LY2090314 (3-[9-Fluoro-1,2,3,4-tetrahydro-2-(1-piperidinylcarbonyl)pyrrolo[3,2,1-jk][1,4]benzodiazepin-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1h-pyrrole-2,5-dione), TDZD-8 (4-benzyl-2-methyl-1,2,4 -thiadiazolidine-3,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), TWS-119 (3-[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy]phenol), Kenpaullone, 1-Azakenpaullone, SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-( Examples of GSK3 inhibitors include (2-nitrophenyl)-1H-pyrrole-2,5-dione), AR-AO144-18 (1-[(4-methoxyphenyl)methyl]-3-(5-nitro-1,3-thiazol-2-yl)urea), CT99021, CT20026, BIO ((2'Z,3'E)-6-bromoindirubin-3'-oxime), BIO-acetoxime, pyridocarbazole-cyclopentadienyl ruthenium complex, OTDZT, alpha-4-dibromoacetophenone, and lithium. Among these, BIO is preferred. These GSK3 inhibitors may be used in combination.

[0037] The concentration of BIO in the medium used in step (2) of the production method of the present invention is typically 0.1 μM to 100 μM, preferably 0.5 μM to 50 μM, and more preferably 1 μM to 5 μM (particularly 2.5 μM). When a GSK3β inhibitor other than BIO is used, the concentration of the GSK3β inhibitor in the medium is selected appropriately.

[0038] Examples of γ-secretase inhibitors used in step (2) of the production method of the present invention include DAPT, DBZ, LY450139, BMS-708163, NIC5-15, GSI-953, ELND006, and CHF-5074. Among these, DAPT is preferred. These γ-secretase inhibitors may be used in combination.

[0039] The concentration of DAPT used in step (2) of the production method of the present invention in the medium is typically 0.2 μM to 200 μM, preferably 1 μM to 100 μM, more preferably 2 μM to 10 μM (particularly 5 μM). When a γ-secretase inhibitor other than DAPT is used, the concentration of the γ-secretase inhibitor in the medium is appropriately selected.

[0040] Each substance used in the production method of the present invention may be synthesized by a known method, or a commercially available product may be used. Furthermore, when the compound is a protein or peptide such as a Wnt protein, the target protein or peptide may be obtained from a cell that expresses it. Cells that express the target protein or peptide can be produced by inserting DNA encoding the target protein or peptide into a known expression vector and introducing the resulting expression vector into an appropriate host cell.

[0041] Established cells expressing the desired protein or peptide may also be used. Examples of Wnt protein-expressing cells include L cells (ATCC CRL-2647) stably expressing mouse Wnt3a and L cells (ATCC CRL-2814) stably expressing mouse Wnt5a. Cells expressing multiple proteins or peptides may also be used, specifically, L-WRN cells (ATCC CRL-3276) expressing Wnt-3A, R-spondin 3, and noggin. Therefore, in one embodiment, the Wnt agonist (e.g., a combination of a Wnt protein and R-spondin) and BMP inhibitor (e.g., chordin-like protein) used in step (1) of the method of the present invention are obtained from cells expressing these proteins.

[0042] When the substances used in the production methods of the present invention are proteins or peptides, their origin is not particularly limited, but is preferably mammalian (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.). Furthermore, the proteins or peptides used in the production methods of the present invention include not only wild-type proteins or peptides but also their mutants having similar functions. Examples of mutants include proteins or peptides that have a high degree of identity (e.g., 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence of a specific wild-type protein or peptide.

[0043] The culture period in step (2) of the production method of the present invention is not particularly limited, but is 5 days or more (e.g., 6, 7, 8, 9, 10 days or more). Because small intestinal epithelial cells and small intestinal tissue can be maintained by step (2), there is no particular upper limit, but the period is typically 20 days or less (e.g., 19, 18, 17, 16 days or less). In one embodiment, the culture period in step (2) of the production method of the present invention is 5 to 15 days, or 7 to 13 days.

[0044] The basal medium used in the production method of the present invention is not particularly limited, but includes RPMI-1640 medium, Eagle MEM (EMEM), Dulbecco's modified MEM, Glasgow's MEM (GMEM), α-MEM, 199 medium, IMDM, DMEM, Hybridoma Serum-free medium, KnockOut TM DMEM, Advanced TM medium (e.g. Advanced MEM, Advanced RPMI, Advanced DMEM / F-12), Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, DMEM / F-12, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's Medium (e.g. Waymouth's MB752 / 1), CMRL medium (e.g. CMRL-1066), Williams' medium E, Brinster's BMOC-3 Medium, E8 Medium, StemPro 34, MesenPRO RS (Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (ReproCELL Co., Ltd.), ProculAD (Rohto Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (Lonza), EX-CELL302 medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMed TM Examples include, but are not limited to, iPSC Medium (ReproCELL Corporation) and mixtures thereof. Among these, Advanced DMEM / F-12 is preferred.

[0045] In particular, for culture under feeder-free and xeno-free conditions, StemFit (registered trademark) AK02 medium (Ajinomoto Co., Inc.), StemFit (registered trademark) AK03 medium (Ajinomoto Co., Inc.), StemFit (registered trademark) Basic03 medium, CTS (registered trademark) KnockOut SR XenoFree Medium (Gibco), mTeSR1 medium, TeSR1 medium (Stem Cell Technologies), Iscove's modified Dulbecco's medium (GE Healthcare), Improved MEM (Thermo Fisher Scientific), and the like can also be used.

[0046] In particular, the medium used in step (2) of the present invention includes the above-mentioned basal medium (preferably Advanced DMEM / F-12) and a medium obtained by mixing the basal medium with a hepatocyte culture medium, such as HCM (LONZA), William's E Medium (Thermo Fisher Scientific), or Leibovitz's L-15 Medium (Thermo Fisher Scientific).

[0047] If necessary, the medium may contain serum, such as fetal bovine serum, or one or more serum substitutes such as Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol, and may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone, and putrescine.

[0048] Examples of culture vessels used in the present invention include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates (e.g., 24-well plates, 96-well microplates, etc.), chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles.

[0049] As shown in the examples below, small intestinal tissue can be efficiently produced by using a microfluidic device as a culture vessel. Therefore, steps (1) and (2) of the present invention are preferably performed in a microfluidic device. As used herein, "microfluidic device" refers to a cell culture device having microchannels, and the width and depth of the microchannels can be freely designed. The width and height of each microchannel in the microfluidic device used in the present invention are typically 10 μm to 10 mm, and preferably 100 μm to 1 mm.

[0050] The microfluidic device used in the present invention preferably has two upper and lower flow channels (upper and lower channels) separated by a semipermeable membrane. The substrate of a microfluidic device having two upper and lower flow channels is typically made of PDMS (polydimethylsiloxane), but may also be made of, for example, cycloolefin polymer, polymethyl methacrylate, or cycloolefin copolymer. The membrane of a microfluidic device having two upper and lower flow channels is typically made of polyethylene terephthalate (PET), but may also be made of plastics such as polycarbonate or polytetrafluoroethylene. Microfluidic devices can be fabricated by known methods. The flow channels can be fabricated using, for example, soft lithography (D.C. Duffy, J.C. McDonald, O.J.A. Schueller, G.M. Whitesides, Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Analytical Chemistry 70, 4974-4984 (1998)).

[0051] The culture in the production method of the present invention is preferably adhesion culture. In this specification, "adhesion culture" refers to culture under conditions that allow strong cell-substrate bonds to be formed between the cells and the culture vessel or the like.

[0052] Typically, cells are cultured in a microfluidic device by seeding the cells in the upper channel and filling the lower channel with culture medium. The culture medium in the lower channel may be perfused. Perfusion allows the production of small intestinal tissue that is more similar to that of a living organism. Thus, in one aspect of the present invention, perfusion culture is performed for at least a portion (preferably the entire period) of the culture period in steps (1) and / or (2) of the production method of the present invention. In a preferred embodiment, perfusion culture is performed for the entire period in steps (1) and (2) of the production method of the present invention.

[0053] Perfusion culture is typically performed using a peristaltic pump or shaker (a shaking device). A peristaltic pump, also known as a tube pump, pumps fluid by squeezing a soft tube made of silicone or other materials with a roller. The lower channel of the microfluidic device is connected to the soft tube, and the medium in the lower channel is perfused by rotating the roller. As shown in the examples below, small intestinal tissue can be produced at perfusion rates of 10 μL / h, 30 μL / h, and 100 μL / h. Therefore, the perfusion rate is not particularly limited, but is preferably between 10 μL / h and 100 μL / h. Furthermore, it has been shown that the expression level of certain genes varies depending on the perfusion rate, so it is also preferable to adjust the perfusion rate appropriately depending on the purpose.

[0054] Furthermore, by placing the microfluidic device on a shaker, it is also possible to generate perfusion of the medium. The shaker used in the present invention may be an orbital shaker, a reciprocating shaker, a figure-of-eight shaker, or a shaker with the ability to switch between these shaking modes as appropriate. The shaking direction may be either horizontal or vertical, but is typically horizontal. The shaking speed is not particularly limited and is typically in the range of 50 to 450 rpm, preferably 100 to 200 rpm.

[0055] Culture vessels used in adherent culture include those in which the surface of the vessel or the flow channels of a microfluidic device have been artificially treated to improve adhesion to cells (e.g., coating with basement membrane preparations, extracellular matrices such as fibronectin, laminin or fragments thereof, entactin, collagen, gelatin, synthemax, vitronectin, etc., or polymers such as polylysine and polyornithine, or surface treatments such as positive charge treatment). Commercially available basement membrane preparations include, for example, Matrigel and Geltrex. Among these, culture vessels coated with Matrigel are preferred.

[0056] Alternatively, from the viewpoint of xeno-free, laminin or a fragment thereof is also preferred. Examples of laminin or a fragment thereof include laminin-111 or a fragment thereof comprising its E8 region, laminin-211 or a fragment thereof comprising its E8 region (e.g., iMatrix-211), laminin-121 or a fragment thereof comprising its E8 region, laminin-221 or a fragment thereof comprising its E8 region, laminin-332 or a fragment thereof comprising its E8 region, laminin-3A11 or a fragment thereof comprising its E8 region, laminin-411 or a fragment thereof comprising its E8 region (e.g., iMatrix-411), laminin-421 or a fragment thereof comprising its E8 region, and laminin-511 or a fragment thereof comprising its E8 region (e.g., iMatrix-511, iMatrix-511). silk), laminin-521 or a fragment thereof containing its E8 region, laminin-213 or a fragment thereof containing its E8 region, laminin-423 or a fragment thereof containing its E8 region, laminin-523 or a fragment thereof containing its E8 region, laminin-212 / 222 or a fragment thereof containing its E8 region, and laminin-522 or a fragment thereof containing its E8 region.

[0057] In at least one of steps (1) and (2) of the production method of the present invention, cells may be cultured under feeder-free conditions and / or xeno-free conditions. In the production method of the present invention, all steps may be performed under feeder-free and xeno-free conditions. Furthermore, in any step of the production method of the present invention other than steps (1) and (2), cells may be cultured under feeder-free and / or xeno-free conditions. As used herein, "feeder-free" refers to a medium or culture conditions that do not contain other cell types (i.e., feeder cells) that play a supporting role and are used to establish culture conditions for the cells to be cultured. Furthermore, "xeno-free" refers to a medium or culture conditions that do not contain components derived from organisms other than the organism species of the cells to be cultured.

[0058] The culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being about 2 to 5%.

[0059] The seeding density of cells is not particularly limited as long as the cells can grow. Typically, the seeding density is 1.0 × 10 2 ~1.0×10 7 cells / cm 2 , preferably 1.0 x 10 3 ~1.0×10 6 cells / cm 2 , more preferably 1.0 × 10 4 ~1.0×10 5 cells / cm 2 is.

[0060] The midgut cells used in the present invention can be obtained by known methods. Examples include methods of isolating midgut cells from biological tissue (e.g., intestinal tissue, etc.) using known techniques, methods of inducing differentiation of definitive endoderm cells, and methods of obtaining midgut cells from companies such as ATCC. Midgut cells can be isolated from biological tissue using, for example, methods using flow cytometry or mass cytometry with expressed antigens as an indicator, magnetic cell separation, affinity columns on which desired antigens are immobilized, etc. The midgut cells used in the present invention are preferably obtained by methods of inducing differentiation of definitive endoderm cells.

[0061] As used herein, "definitive endoderm cells" refers to cells that express SOX17 and FOXA2 and have the potential to differentiate into endodermal cells, such as those of the digestive tract. Definitive endoderm cells used in the present invention can be obtained by known methods. Examples include isolation from biological tissues (e.g., intestinal tissue, etc.) using known techniques, methods that induce differentiation of pluripotent stem cells, and methods of obtaining cells from companies such as ATCC. Midgut cells can be isolated from biological tissues, for example, using expression antigens (e.g., E-cadherin, CXCR4, CD55, etc.) as indicators, using flow cytometry or mass cytometry, magnetic cell separation, affinity columns immobilized with the desired antigen, and the like. The definitive endoderm cells used in the present invention are preferably obtained by methods that induce differentiation of pluripotent stem cells.

[0062] The term "pluripotent stem cells" refers to stem cells that can differentiate into various tissues and cells with different morphologies and functions in the body and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), multipotent germline stem cells (mGS cells), and embryonic germ stem cells (EG cells). Preferably, iPS cells (more preferably, human iPS cells) are used. When the pluripotent stem cells are ES cells or any cells derived from human embryos, they may be produced by or without the destruction of the embryo. However, from an ethical standpoint, cells produced without the destruction of the embryo are preferred.

[0063] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by isolating the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from a developmentally arrested embryo (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).

[0064] nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same properties as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). Specifically, nt ES (nuclear transfer ES) cells are established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To generate nt ES cells, nuclear transfer technology (Cibelli JB et al. (1998), Nature Biotechnol., 16:642-646) is combined with ES cell generation technology (mentioned above) (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, and the egg is then cultured for several hours to reprogram the embryo.

[0065] Examples of ES cell lines that can be used in the present invention include mouse ES cell lines established by, for example, inGenious targeting laboratory, Inc., RIKEN (Riken), etc., and human ES cell lines established by, for example, the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, and Cellartis, Inc. Specific examples of human ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 strains distributed by ESI Bio, H1 and H9 strains distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 strains distributed by RIKEN.

[0066] iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of iPS cells, including iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPSCs established by selecting using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPSCs created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.), etc. can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells created by Sakurada et al. (JP Patent Publication No. 2008-307007) can also be used.In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7, 795-797), or patent publications (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008-2336610 A, US 2009-047263 A, WO 2007-069666 A, WO 2008-118220 A, WO 2008-124133 A, WO 2008-151058 A, WO 2009-006930 A, WO 2009-006997 A, WO 2009-007852 A) and known in the art can be used.

[0067] Available induced pluripotent stem cell lines include various iPSC lines established by the NIH, RIKEN, Kyoto University, etc. Examples of human iPSC lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 lines, and Kyoto University's 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, and FfI-01s04 lines.

[0068] The induced pluripotent stem cells used in the production methods of the present invention may be cells derived from patients with hereditary diseases (e.g., patients with hereditary small intestinal diseases). Cells induced to differentiate from pluripotent stem cells derived from patients with hereditary small intestinal diseases can serve as disease models that reflect the pathology of the disease, and are therefore suitable for screening therapeutic or preventive drugs for the disease. Alternatively, pluripotent stem cells derived from patients with hereditary small intestinal diseases can be genetically repaired by genome editing using the CRISPR-Cas system or the like, and then differentiated into the desired cells or tissues, making it possible to use the cells or tissues as therapeutic agents for the disease.

[0069] mGS cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various cell lineages, e.g., when transplanted into mouse blastocysts, chimeric mice can be generated (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004) Cell, 119:1001-1012). They are capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, germline stem cells can be obtained by repeated passage under similar culture conditions to ES cells (Takebayashi M. et al. (2008) Experimental Medicine, Vol. 26, No. 5 (Supplementary Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0070] EG cells are derived from embryonic primordial germ cells (PGCs) and have pluripotency similar to that of ES cells. They can be established by culturing PGCs in the presence of LIF, bFGF, stem cell factor, and other substances (Matsui Y. et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551).

[0071] The species from which the pluripotent stem cells are derived is not particularly limited, and may be cells from, for example, rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. The preferred species is human.

[0072] Pluripotent stem cells can be differentiated into midgut cells by known methods, such as those described in Non-Patent Documents 1 to 4, Toyoda T, et al. Stem Cell Reports 2017, and WO 2017 / 047797. Specifically, pluripotent stem cells can be differentiated into midgut cells by a method comprising, for example, the steps of: A) inducing the differentiation of pluripotent stem cells into definitive endoderm cells; and B) inducing the differentiation of definitive endoderm cells into midgut cells. Therefore, the production method of the present invention may include the above-described steps A) and / or B) before step (1).

[0073] The basal medium and medium additives used in steps A) and B) can be the same as those used in the production method of the present invention. RPMI1640 medium is preferred as the basal medium used in step A). ​​Advanced DMEM / F12 is preferred as the basal medium used in step B). The culture temperature in steps A) and B) is typically about 30-40°C, preferably about 37°C, and the culture is performed in a CO2-containing air atmosphere, with a CO2 concentration of about 2-5%. Adherent culture is preferably used in steps A) and B). The definition and method of adherent culture are as described above. In a preferred embodiment, step B) is performed in a microfluidic device.

[0074] The differentiation of pluripotent stem cells into definitive endoderm cells in step A) can be carried out, for example, by culturing pluripotent stem cells in a medium containing a low dose of activin A. The medium may further contain a ROCK inhibitor or a GSK3β inhibitor. The culture period is typically 2 to 8 days, preferably 2 to 4 days (particularly 3 days).

[0075] The concentration of activin A in the medium used in step A) is, for example, 5 to 1000 ng / mL, preferably 20 to 500 ng / mL, more preferably 50 to 150 ng / mL.

[0076] Examples of the GSK3β inhibitor used in step A) include those listed in step (2) of the production method of the present invention. Among them, CHIR99021 is preferred. When CHIR99021 is used as the GSK3β inhibitor, its concentration in the medium is typically 0.5 to 5 μM, preferably 1 to 4 μM.

[0077] Examples of the ROCK inhibitor used in step A) include Y-27632 (see, e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), fasudil / HA1077 (see, e.g., Uenata et al., Nature 389: 990-994 (1997)), SR3677 (see, e.g., Feng Y et al., J. Med. Chem. 51: 6642-6645 (2008)), GSK269962 (see, e.g., Stavenger RA et al., J. Med. Chem. 50: 2-5 (2007) or WO2005 / 037197), GSK429286A, H1152 (see, e.g., Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (see, e.g., Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)), thiazovivin, and salts or derivatives thereof. Among these, Y-27632 is preferred. When Y-27632 is used as a ROCK inhibitor, the concentration in the medium is typically 1 to 20 μM, preferably 5 to 15 μM.

[0078] The medium may further contain insulin. The concentration of insulin in the medium is typically 0.01 to 20 μM, preferably 0.1 to 10 μM, and more preferably 0.5 to 5 μM. The concentration of insulin in the medium may be, but is not limited to, the concentration of insulin contained in the added B-27 supplement.

[0079] The differentiation into midgut cells in step B) can be carried out, for example, by culturing definitive endoderm cells in a medium containing growth factors. The culture period is typically 2 to 8 days, preferably 3 to 5 days (particularly 4 days).

[0080] In one embodiment of the present invention, perfusion culture is performed for at least a part of the culture period (preferably the entire period) in step B). In a preferred embodiment, perfusion culture is performed for the entire period in step B). Specific methods for perfusion culture are as described above.

[0081] Growth factors used in step B) include those listed for step (1) of the production method of the present invention. Among them, EGF, KGF, FGF2, and FGF10 are preferred, with FGF2 being more preferred. The concentration of the growth factor in the medium is set appropriately depending on the type of growth factor used, but is typically about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / ml (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / ml (i.e., about 0.8 to 160 nM), and more preferably about 10 to 1000 ng / ml (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / ml (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM), and more preferably about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM). For example, when KGF is used as a growth factor, the concentration is typically 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably about 50 ng / mL.

[0082] The above steps A) and / or B) may be cultured under feeder-free conditions and / or xeno-free conditions.

[0083] The production method of the present invention may include a step of recovering the target cells or tissues obtained in each step. The recovered cells may be cryopreserved using a cell cryopreservation solution. The recovered cells may be counted using a cell counter, or may be labeled with an antibody against a cell surface marker and selected by flow cytometry, mass cytometry, magnetic cell sorting, or the like.

[0084] 2. Small Intestinal Epithelial Cells and Small Intestinal Tissue Step (2) of the production method of the present invention provides small intestinal epithelial cells, small intestinal tissue, or a microfluidic device carrying the same. Therefore, in another aspect of the present invention, small intestinal epithelial cells, small intestinal tissue, or a microfluidic device carrying the same are also provided, which are obtained by the production method of the present invention ("obtained" can be read as "obtained" as appropriate). Small intestinal tissue obtained by the production method of the present invention (hereinafter, sometimes referred to as "small intestinal tissue of the present invention") typically has all of the following characteristics (A) to (F): (A) Contains absorptive epithelial cells; (B) Contains fibroblasts; (C) Contains collagen fibers; (D) Expresses one or more (preferably all) genes selected from the group consisting of TGFB1, Wnt5A, and IGF2; (E) Expresses one or more (preferably all) genes selected from the group consisting of MMP1, COL3A1, and TIMP1; and (F) Has drug-metabolizing enzyme activity.

[0085] Furthermore, the small intestinal tissue of the present invention further has at least one (preferably all) of the following characteristics (G) to (K): (G) derived from pluripotent stem cells; (H) having goblet cells; (I) having Paneth cells; (J) having enteroendocrine cells; and (K) having crypt-like structures and villi-like structures.

[0086] Furthermore, the small intestinal tissue of the present invention may be characterized by expressing (L) a transporter gene. Examples of transporter genes include ABCB1 / MDR1, ABCC1 / MRP1, ABCC2 / MRP2, ABCG2 / BCRP, SLCO2B1 / OATP2B1, and SLC15A1 / PEPT1. In one embodiment, the small intestinal tissue of the present invention expresses ABCB1 / MDR1, ABCC1 / MRP1, ABCC2 / MRP2, ABCG2 / BCRP, SLCO2B1 / OATP2B1, and SLC15A1 / PEPT1.

[0087] In one embodiment, the small intestinal tissue of the present invention is supported in a microfluidic device, in which case the fibroblasts in the small intestinal tissue can be arranged in parallel along the flow path of the microfluidic device.

[0088] The small intestinal tissue of the present invention may be composed of uniform columnar epithelial cells and have microvilli arranged in an orderly fashion on their surface. The small intestinal tissue of the present invention may also have tight junctions (TJs), adherens junctions (AJs), and desmosomes (DSs), and is preferably a tissue in which TJs, AJs, and DSs are aligned. In another embodiment, the small intestinal tissue of the present invention is a tissue in which absorptive epithelial cells expressing SLC26A2 or SLC26A3 are mainly present on the apical side, and intestinal progenitor cells expressing SOX9 and intestinal stem cells expressing LGR5 are mainly present on the basal side.

[0089] In another aspect, the present invention also provides small intestinal tissue having all of the above characteristics (A) to (F). Also provided is small intestinal tissue having at least any (preferably all) of the above characteristics (G) to (K) and / or the above characteristic (L). Such small intestinal tissue may be obtained by the production method of the present invention, or may be obtained by another method. Hereinafter, the small intestinal tissue may also be referred to as "small intestinal tissue of the present invention."

[0090] As used herein, unless otherwise specified, the terms "expressing" or "positively expressing" a gene are used to mean at least "production of mRNA encoded by the gene," but preferably also "production of a protein encoded by the mRNA." Therefore, if production of mRNA encoded by the gene is detected at least by the methods described in the Examples below (RT-qPCR or RNA-seq), the gene can be said to be expressed.

[0091] As used herein, "positive" includes both strong expression (also referred to as "strong positive") and weak expression (also referred to as "weak positive"). Strong positive is sometimes indicated by the superscript "high" (e.g., PDGFRA high ), and weak positives may be indicated by the superscript "low" (e.g., PDGFRA low Strong positivity and weak positivity can be determined based on the chart obtained by flow cytometry. The positions appearing on the chart may vary depending on the voltage settings, sensitivity settings, antibody clones used, staining conditions, dyes used, etc., but those skilled in the art can draw appropriate lines on the chart obtained so as not to separate cell populations recognized as a single group.

[0092] The fibroblasts contained in the small intestinal tissue of the present invention are positive for Vimentin, an intestinal fibroblast marker, and have the ability to produce collagen. The fibroblasts are typically PDGFRA-positive cells. As used herein, "collagenous fiber" refers to fibers formed by the aggregation of multiple collagen molecules. In one embodiment, the small intestinal tissue of the present invention contains multiple types of fibroblasts, and in a preferred embodiment, the small intestinal tissue of the present invention contains fibroblasts that express R-spondin 3 (RSPO3) and bone morphogenetic protein 5 (BMP5).

[0093] The small intestinal tissue of the present invention expresses one or more (preferably all) genes selected from the group consisting of TGFB1, Wnt5A, and IGF2. These genes encode growth factors. Furthermore, the small intestinal tissue of the present invention typically expresses one or more (preferably all) of these genes at higher levels than normal small intestine of a human adult.

[0094] The small intestinal tissue of the present invention expresses one or more (preferably all) genes selected from the group consisting of MMP1, COL3A1, and TIMP1. These genes encode extracellular matrix (ECM) and factors regulating its degradation. Furthermore, the small intestinal tissue of the present invention typically expresses one or more (preferably all) of these genes at higher levels than normal small intestine of a human adult.

[0095] In this specification, the expression level of a specific gene can be evaluated by the method described in the Examples below, specifically, by RNA-seq analysis using total RNA isolated from the small intestinal tissue of the present invention and total RNA from normal adult human small intestine (BioChain Institute, R1234226-50) as samples. If the TPM (transcripts per million) value for a specific gene obtained as a result of such RNA-seq analysis is higher in the small intestinal tissue of the present invention than in the normal adult human small intestine, it can be said that the small intestinal tissue of the present invention has a higher expression level of the gene than the normal adult human small intestine.

[0096] The drug-metabolizing enzyme activity of the above characteristic (F) includes CYP3A4 activity, UGT1A1 activity, and CYP2C9 activity, and the small intestinal tissue of the present invention preferably has at least CYP3A4 activity. "Having drug-metabolizing enzyme activity" means exhibiting drug-metabolizing enzyme activity at least by the method described in the Examples below.

[0097] Crypts are tubular depressions located deep within the villi in the small intestine, and herein, "crypt-like structures" refer to structures similar in appearance to the crypts of the small intestine. Villi are protrusions present in the circular folds of the inner wall of the small intestine, and herein, "villi-like structures" refer to structures similar in appearance to the villi of the small intestine.

[0098] 3. Uses of Small Intestinal Epithelial Cells and Small Intestinal Tissue The small intestinal epithelial cells and small intestinal tissue of the present invention can be used in medical treatments such as regenerative medicine. Therefore, in another aspect, a transplantation therapeutic agent (hereinafter, sometimes referred to as the "transplantation therapeutic agent of the present invention") containing the small intestinal epithelial cells or small intestinal tissue of the present invention is provided. The present invention also encompasses a method for treating or preventing small intestinal diseases, in which an effective amount of the small intestinal epithelial cells or small intestinal tissue of the present invention is administered or transplanted into a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) that is the target of treatment or prevention. Unless otherwise specified, the term "disease therapeutic or preventive agent (or treatment or prevention method)" used herein also encompasses a pharmaceutical agent (or method) that can both treat and prevent the disease.

[0099] Examples of small intestinal diseases include Crohn's disease, gastrointestinal polyposis, small intestinal tumors, protein-losing gastroenteropathy, malabsorption syndrome, intestinal diverticular disease, Meckel's diverticulum, etc. The small intestinal epithelial cells and small intestinal tissues of the present invention exhibit therapeutic or preventive effects against small intestinal diseases when transplanted into an affected area or into a site resected for disease treatment.

[0100] The transplantation therapy agent of the present invention can be administered or transplanted into the body of a subject in need thereof. The small intestinal epithelial cells or small intestinal tissue to be transplanted may be administered in a therapeutically or prophylactically effective amount, which may vary depending on factors such as the age, weight, size of the transplant site, and severity of the disease of the recipient, and is not particularly limited. For example, the number of cells may be 10 × 10 4 Cell ~10×10 11 It can be as small as a cell.

[0101] When the small intestinal epithelial cells or small intestinal tissue of the present invention are used as a transplantation therapy agent, it is desirable to use small intestinal epithelial cells or small intestinal tissue derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, in order to avoid rejection. Here, "substantially the same" means that the HLA genotype is identical to that of the transplanted cells to an extent that immune responses can be suppressed with immunosuppressants, e.g., somatic cells with an HLA type that matches the three HLA loci (HLA-A, HLA-B, and HLA-DR) or the four HLA loci (HLA-C). If sufficient cells cannot be obtained due to age, constitution, or other reasons, they can be transplanted in a state that avoids rejection by embedding them in capsules or porous containers made of polyethylene glycol or silicone.

[0102] The small intestinal epithelial cells or small intestinal tissue of the present invention are prepared as parenteral preparations such as injections, suspensions, or infusions by mixing with a pharmaceutically acceptable carrier according to conventional methods. Therefore, in one embodiment, a method for producing a transplantation therapy agent is also provided, which includes a step of formulating the small intestinal epithelial cells or small intestinal tissue of the present invention. Such a method may also include a step of preparing the small intestinal epithelial cells or small intestinal tissue of the present invention. Furthermore, it may also include a step of preserving the small intestinal epithelial cells or small intestinal tissue of the present invention.

[0103] Pharmaceutically acceptable carriers that can be contained in such parenteral formulations include, for example, aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), etc. The small intestinal epithelial cells and small intestinal tissues of the present invention may be formulated with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0104] The transplantation therapy agent of the present invention is provided in a cryopreserved state under conditions typically used for cryopreserving cells and can be thawed immediately before use. In this case, it may further contain serum or a serum substitute, an organic solvent (e.g., DMSO), etc. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0105] The small intestinal epithelial cells or small intestinal tissue of the present invention can also be used in methods for screening candidate drugs that are useful for treating or preventing small intestinal diseases. Thus, in yet another aspect of the present invention, there is provided a method for screening therapeutic or preventive drugs for diseases, comprising culturing the small intestinal epithelial cells or small intestinal tissue of the present invention in the presence or absence of a test substance. Such small intestinal diseases include those similar to those described above that are the target of treatment or prevention using the transplantation therapy agent of the present invention.

[0106] The small intestinal tissue of the present invention typically expresses ANPEP (Alanyl aminopeptidase, membrane). ANPEP is known as an infection receptor for human coronavirus 229E (HCoV-229E). The small intestinal tissue of the present invention may also express ACE2. ACE2 is known as an important infection receptor for several viruses belonging to the coronavirus family, such as SARS-CoV and SARS-CoV-2. Therefore, the small intestinal epithelial cells and small intestinal tissue of the present invention may be useful tools for research into the infection mechanisms of viruses that infect cells via ANPEP and / or ACE2, particularly viruses belonging to the coronavirus family, and for drug development.

[0107] Therefore, in yet another aspect of the present invention, there is provided a method for screening for a therapeutic or preventive agent for a viral infection (hereinafter also referred to as the "screening method of the present invention"), comprising: (I) a step of contacting the small intestinal tissue of the present invention with a virus and culturing it in the presence or absence of a test substance; (II) a step of evaluating the degree of infection of the virus in the small intestinal tissue; and (III) a step of selecting the test substance as a candidate substance for a therapeutic or preventive agent for a viral infection when the degree of infection of the virus is evaluated to be lower in the presence of the test substance compared to in the absence of the test substance in step (II).

[0108] The target viruses in the screening method of the present invention are not limited as long as they have the ability to infect the small intestinal tissue of the present invention via cell surface receptors such as ANPEP and ACE2. Examples of viruses include viruses belonging to the Coronaviridae family (hereinafter sometimes referred to as "coronaviruses"). Among these, viruses belonging to the Sarbecovirus subgenus that infect cells via ANPEP, ACE2, etc. are preferred, and viruses belonging to the Coronaviridae family (hereinafter sometimes referred to as "coronaviruses") (e.g., HCoV-229E, etc.), novel coronavirus (SARS-CoV-2), and severe acute respiratory syndrome coronavirus (SARS-CoV) are particularly preferred.

[0109] When cells are infected with a virus, an immune response to the infection (e.g., increased expression and secretion of cytokines such as IL-1β, IL-6, IL-8, IFN-α, IFN-β, and IFN-γ) occurs, and the degree of infection of small intestinal tissue with the virus can be evaluated by assessing the level of this immune response. Thus, one embodiment of step (II) of the screening method of the present invention is a step of measuring the expression and / or secretion levels of cytokines in small intestinal tissue and / or culture medium supernatant.

[0110] When cells are infected with a virus such as coronavirus and the virus begins to replicate within the cells, characteristic changes in the appearance of small intestinal tissue (i.e., cytopathic effect (CPE)) occur. This phenomenon is an indicator of intracellular viral replication. Examples of CPE include the formation of syncytia, where multiple cells fuse together, and detachment from the bottom of the culture vessel. These phenomena can be analyzed by microscopy, immunostaining, and other techniques to assess the extent of viral infection.

[0111] Furthermore, a substance that can inhibit the binding of, for example, viral spike proteins or envelopes to cellular ANPEP and / or ACE2 can be used as a therapeutic or preventive agent for viral infection. In the presence of such a substance, a higher amount of virus is detected in the culture supernatant than in its absence, and therefore the amount of virus in the culture supernatant serves as an indicator of the degree of viral infection. Therefore, another aspect of step (II) of the screening method of the present invention is a step of measuring the amount of virus in the culture supernatant.

[0112] Alternatively, step (II) of the screening method of the present invention can be performed by isolating the virus from the small intestinal tissue of the present invention that has been contacted with the virus and comparing the amount of the virus. In this case, if the amount of virus in the small intestinal tissue is lower in the presence of the test substance than in its absence, the test substance is a candidate for a drug for treating or preventing a viral infection.

[0113] The amount of virus can be measured, for example, by quantitative PCR, such as competitive PCR or real-time PCR, using viral genomic RNA. Alternatively, it can be measured by immunological assays (e.g., CLEIA, ELISA, FIA, RIA, Western blot, etc.) using antibodies that specifically recognize viral peptides (e.g., spike proteins). Commercially available virus quantification kits can also be used. Cytokine expression and secretion levels can also be measured in a similar manner.

[0114] Furthermore, the small intestinal tissue of the present invention can be used to evaluate the pharmacokinetics and toxicity of drugs. Specifically, the small intestinal tissue of the present invention can be used to test, for example, the absorbability or membrane permeability of a test substance, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, toxicity, etc.

[0115] The small intestinal tissue of the present invention typically expresses high levels of drug-metabolizing enzymes, making it possible to evaluate the degree of drug metabolism using the activity of these enzymes as an indicator. Furthermore, by analyzing (e.g., identifying) the metabolites of a drug produced as a result of an enzymatic reaction using the drug as a substrate by the drug-metabolizing enzyme, it is also possible to evaluate the toxicity of the drug. Therefore, in yet another aspect of the present invention, there is provided a method for evaluating the degree of metabolism of a test substance, comprising: (i) contacting a test substance with the small intestinal tissue of the present invention; (ii) measuring the activity level of the drug-metabolizing enzyme; and (iii) evaluating the degree of metabolism of the test substance by the drug-metabolizing enzyme based on the activity level of the drug-metabolizing enzyme in step (ii).

[0116] Step (i) can typically be carried out by adding a test substance to a medium in which the small intestinal tissue of the present invention is cultured, or by transferring the small intestinal tissue of the present invention to a medium to which the test substance has been added in advance.

[0117] Examples of drug-metabolizing enzymes used in the present invention include cytochrome P450 (e.g., CYP3A4, CYP2C9, etc.), uridine diphosphate-glucuronosyltransferase (e.g., UGT1A1, UGT1A8, UGT1A10, etc.), and sulfotransferase (e.g., SULT1A3, etc.). Among these, CYP3A4, UGT1A1, and CYP2C9 are preferred, with CYP3A4 being particularly preferred. Multiple types of drug-metabolizing enzymes may also be used in combination.

[0118] Step (ii) is typically a step of measuring the expression level of a drug-metabolizing enzyme. The expression level of a drug-metabolizing enzyme can be evaluated at the mRNA level or protein level. For example, if the mRNA level of the drug-metabolizing enzyme increases compared to before contact with the test substance or compared to contact with a negative control, it can be determined that at least a portion of the test substance has been metabolized. In addition, the expression level can also be used to evaluate the degree of drug metabolism (e.g., the proportion of drug metabolized, metabolic rate, etc.).

[0119] Furthermore, in step (i), the small intestinal tissue of the present invention can also be contacted with a substance known to be metabolized by a drug-metabolizing enzyme (e.g., triazolam (metabolite; α-hydroxy-triazolam), diclofenac (metabolite; 4-hydroxydiclofenac), 7-hydroxycoumarin (metabolite; 7-hydroxycoumarin glucuronide), etc.), and the amount of the metabolite of the substance can be measured. Alternatively, the measurement can be performed using a commercially available kit for measuring the activity of drug-metabolizing enzymes (e.g., P450-Glo TM It can also be measured using CYP3A4 assays.

[0120] To evaluate the absorption of a test substance, for example, the amount of the test substance remaining in the culture medium can be measured, and the degree of absorption of the test substance can be evaluated based on this remaining amount.

[0121] Methods for measuring the expression level of metabolic enzymes, the amount of metabolites, and the remaining amount of test substance include, but are not limited to, mass spectrometry, liquid chromatography, immunological assays, and the like.

[0122] The duration of the above steps (I) and (i) is not particularly limited, but is typically 1 hour to 10 days.

[0123] Test substances used in the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low molecular weight compounds, and natural compounds.

[0124] The test substance can also be obtained using any of the many approaches to combinatorial library technology known in the art, including (1) biological libraries, (2) synthetic library technology using deconvolution, (3) "one-bead one-compound" library technology, and (4) synthetic library technology using affinity chromatography selection. While the biological library technology using affinity chromatography selection is limited to peptide libraries, the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12:145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al. (1994) J. Med. Chem. 37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al. (1994) J. Med. Chem. 37:1233-51).Compound libraries can be stored in solution (see Houghten (1992) Bio / Techniques 13:412-21) or on beads (Lam (1991) Nature 354:82-4), chips (Fodor (1993) Nature 364:555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-9), or phage (Scott and Smith (1990) Science 249:386-90; Devlin (1990) Science 249:404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-82; Felici (1991) J. Mol. Biol. 222:301-10; U.S. Patent Application No. 2002103360).

[0125] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0126] Materials and Methods <Fabrication of Microfluidic Device> The microfluidic device consists of two layers of microchannels separated by a semipermeable membrane (Figure 2). The microchannel layer was fabricated from polydimethylsiloxane (PDMS) using soft lithography (DC Duffy, JC McDonald, OJA Schueller, GM Whitesides, Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Analytical Chemistry 70, 4974-4984 (1998).). PDMS prepolymer (Sylgard 184, Dow Corning) was cast in a 10:1 ratio of base to curing agent onto a mold consisting of SU-8 2150 (MicroChem) patterned on a silicon wafer. The microchannel dimensions were 1 mm wide and approximately 300 μm high. To introduce cell culture medium into the microchannel, an access hole was drilled in the PDMS using a biopsy trephine (Kai Corporation) with a 6 mm tip diameter. A semipermeable polyethylene terephthalate (PET) membrane with 3.0 μm pores (Corning, Catalog No. 353091) was attached to the two PDMS layers using a thin layer of liquid PDMS prepolymer as an adhesive. (Chueh BH, Huh D, Kyrtsos CR, Houssin T, Futai N, Takayama S. Leakage-free bonding of porous membranes into layered microfluidic array systems. Anal Chem. 2007 May 1;79(9):3504-8. doi: 10.1021 / ac062118p. Epub 2007 Mar 28. PMID: 17388566; PMCID: PMC2517097.) The PDMS prepolymer was spin-coated onto a glass slide at 4,000 rpm for 60 seconds.Next, both the top and bottom channel layers were placed on a glass slide, and a thin layer of PDMS prepolymer was transferred onto the embossed PDMS surface. A PET membrane was placed on the top layer and then adhered to the bottom layer. The combined layers were left at room temperature for 1 day to remove air bubbles and then placed in a 60 °C oven overnight to cure the PDMS prepolymer. The PDMS-based microfluidic device was sterilized by placing it under ultraviolet (UV) light for 1 hour before cell culture.

[0127] <Maintenance of human pluripotent stem cells> The induced pluripotent stem (iPS) cell line 1383D6 (provided by Dr. Masato Nakagawa, Kyoto University), and the embryonic stem (ES) cell lines KhES-3 (Kyoto University) and H9 (WA09, WiCell Research Institute) were maintained in StemFit AK02N medium (catalog number RCAK02N; Ajinomoto) at a concentration of 0.5 μg / cm 2 The cells were maintained on recombinant human laminin 511 E8 fragment (iMatrix-511; Catalog No. 892012; Nippi). Cells were passaged every 6 days. For cell passage, iPS cell colonies were treated with TrypLE Select Enzyme (Cat. No. 12563011; Thermo Fisher Scientific) for 10 minutes at 37°C and plated in StemFit AK02N medium containing 10 μM Y-27632 (Cat. No. 034-24024; Fujifilm Wako Pure Chemical Industries). Human ES cells were used in accordance with the Guidelines for the Establishment and Use of Human Embryonic Stem Cells of the Ministry of Education, Culture, Sports, Science and Technology of Japan, and this study was approved by an independent ethics committee.

[0128] Preparation of small intestinal tissue chips: Human iPS cells were seeded onto cell culture plates coated with Matrigel Growth Factor Reduced Basement Membrane (Corning, Catalog No. 354230). To induce definitive endoderm differentiation, human iPS cells were treated with 100 ng / mL activin A (Cat. No. 338-AC-01M; R&D Systems) for 3 days in RPMI 1640 medium (Sigma-Aldrich, Catalog No. R8758-500) supplemented with 1X B-27 Supplement Minus Vitamin A (Cat. No. 12587001; Thermo Fisher Scientific), 1X GlutaMAX (Cat. No. 35050-079; Thermo Fisher Scientific), and 1X penicillin-streptomycin. Next, human iPS cell-derived definitive endoderm cells were detached and cultured at 5 × 10 in intestinal differentiation medium (advanced DMEM / F12 (catalog number 12634028; Thermo Fisher Scientific) supplemented with 2% FBS and 1X GlutaMAX) supplemented with 100 ng / mL FGF2 and 10 μM Y-27632 (catalog number 034-24024; Fujifilm Wako Pure Chemical Industries, Ltd.). 6 The cells were suspended at 100 cells / mL. The suspension medium (10 μL) was then injected into the Matrigel-coated upper channel of the microfluidic device. After 1 hour, intestinal differentiation medium containing 100 ng / mL FGF2 and 10 μM Y-27632 was added to the upper channel of the microfluidic device and cultured for 24 hours. Human iPS cell-derived definitive endoderm cells were then treated with 100 ng / mL FGF2 (catalog number 160-0010-3; Katayama Chemical Industry) in intestinal differentiation medium for 3 days to induce midgut cell differentiation.

[0129] To induce small intestinal progenitor cell differentiation, cells were treated for 5 days with 50 ng / mL EGF (catalog no. AF-100-15; PeproTech) and 20 ng / mL IGF-1 (catalog no. AF-100-11; PeproTech) in intestinal differentiation medium containing 25% Wnt-3A, R-spondin3, and Noggin-conditioned medium prepared by L-WRN cells (catalog no. CRL-3276; American Type Culture Collection (ATCC)). To induce differentiation of small intestinal tissue, cells were cultured in intestinal differentiation medium containing 50% Hepatocyte Culture Media BulletKit (catalog no. CC-3198; LONZA) supplemented with 10 ng / mL EGF, 15 μM forskolin (catalog no. 063-02193; Fujifilm Wako Pure Chemical Industries), 10 μM PD98059 (catalog no. S1177; Selleck), 2.5 μM 5-aza-2′-dC (catalog no. 018-20941; Fujifilm Wako Pure Chemical Industries), 0.25 μM A-83-01 (catalog no. 035-24113; Fujifilm Wako Pure Chemical Industries), 2.5 μM (2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO) (catalog no. 361550; Sigma-Aldrich), and 5 μM HCl. (3,5-Difluorophenylacetyl)-Ala-Phg-OBu t The cells were treated with DAPT (Cat. No. 3219-v; Peptide Institute) for 12 days. Medium flow was maintained at a flow rate of 30 μL / h using a peristaltic pump (Programmable Motor Controller, Cat. No. RE-C200 / C201 and Ring Pump, Cat. No. RP-TXP5F-P03A-DC3VS, Aqua Tech) or a seesaw rocking shaker (Infinity Rocker Mini, Next Advance) for 20 days (days 4 to 24).

[0130] To differentiate human iPS cells into small intestinal epithelial cells in 24-well plates, human iPS cell-derived definitive endoderm cells were dissociated and placed in intestinal differentiation medium at a concentration of 0.3 × 10 6 The cells were suspended at 0.05 cells / mL. The suspension medium was then seeded onto Matrigel-coated 24-well plates (500 μL / well). The growth factors or compounds used for intestinal differentiation in the 24-well plates were the same as those used in the microfluidic device.

[0131] <RT-qPCR> Total RNA from small intestinal tissue chips was isolated using ISOGENE (catalog number 319-90211; Nippon Gene). Human small intestinal total RNA was purchased from Takara Bio USA, Inc. (catalog number 636539). cDNA was synthesized using the Superscript VILO cDNA Synthesis Kit (catalog number 11754250; Thermo Fisher Scientific). RT-qPCR was performed using the StepOnePlus real-time PCR system, QuantStudio 1, or QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) with SYBR Green PCR Master Mix (catalog number 4385614; Thermo Fisher Scientific). Relative quantification of target mRNA levels was performed using 2 -ΔΔCt The assay was performed using the ELISA method. Values ​​were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences are summarized in Tables 1-1 and 1-2. Note that for GAPDH, primers with sequences in Table 1-1 were used in all experiments except for Example 10, and only in Example 10 was the primer with sequences in Table 1-2 used.

[0132]

[0133]

[0134] <Immunofluorescence Staining> For immunofluorescence staining of small intestinal tissue chips, they were fixed in 4% paraformaldehyde for 15 minutes. The small intestinal tissue chips were harvested and used to prepare frozen sections (5 μm). For antigen retrieval, sections were treated with HistoVT One (catalog no. 06380-05; Nacalai Tesque) at 70°C for 30 minutes. Sections were permeabilized for 10 minutes using Tris-buffered saline containing 0.1% Tween 20 surfactant (catalog no. 34874-91; Nacalai Tesque) and blocked for 45 minutes using Blocking One Histo (catalog no. 06349-64; Nacalai Tesque). Sections were incubated in Tris-buffered saline containing 0.1% Tween 20 surfactant at room temperature for 2 hours in the presence or absence of primary antibodies. Sections were washed with PBS and incubated for 45 minutes at room temperature in Tris-buffered saline containing 0.1% Tween 20 surfactant with Alexa Fluor 488- or 594-conjugated secondary antibodies. Finally, sections were washed, mounted with Fluoro-KEEPER Antifade Reagent, Non-hardening Type with DAPI (Cat. No. 12593-64; Nacalai Tesque), and analyzed using a BZ-X700 (Keyence Corporation) or FV3000 confocal microscope (Evident). Antibodies are summarized in Table 2.

[0135]

[0136] <Quantification of Protein Expression; Automated Capillary Electrophoresis Immunoassay> Small intestinal tissue chips were lysed in RIPA buffer (catalog no. 89900; Thermo Fisher Scientific) containing a protease inhibitor mixture (catalog no. P8340; Sigma-Aldrich). After centrifugation, the supernatant was collected. Protein expression quantification was performed using Jess (ProteinSimple) equipped with a 12-230 kDa Separation Module (catalog no. SM-W001; ProteinSimple) according to the manufacturer's instructions. The antibodies used in this analysis are summarized in Table 3. Data were analyzed and visualized using Compass for Simple Western software (ProteinSimple). Protein images from Jess are used throughout the specification.

[0137]

[0138] RNA-seq analysis: RNA was isolated from small intestinal tissue chips cultured under static or perfusion conditions. Human small intestinal total RNA was purchased from BioChain Institute (catalog number R1234226-50). RNA integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies). Library preparation was performed using the TruSeq stranded mRNA sample prep kit (Illumina) according to the manufacturer's instructions. Sequencing was performed on an Illumina NextSeq500. Fastq files were generated using bcl2fastq-2.20. Adapter sequences and low-quality bases were trimmed from raw reads using Cutadapt v3.4 (M. Martin, Cutadapt removes adapter sequences from high-throughput sequencing reads. 2011 17, 3 (2011).). Trimmed reads were mapped to the human reference genome sequence (hg38) using STAR v2.7.9a (A. Dobin et al., STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 (2013)) with the gtf file from GENCODE (release 36, GRCh38.p13) (A. Frankish et al., GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Research 47, D766-D773 (2019)). The raw number of protein-coding genes was calculated using htseq-count v0.13.5 (S. Anders, P.T. Pyl, W. Huber, HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166-169 (2015)) with the GENCODE gtf file.Differentially expressed genes were identified using DEseq2 v1.30.1 (M.I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15 (2014).), and gene expression levels were determined as transcripts per million (TPM).

[0139] <H&E staining> Small intestinal tissue chips were fixed in 4% paraformaldehyde for 15 minutes. The small intestinal tissue on the chip was collected and used to prepare paraffin sections. Paraffin-embedded tissue sections and H&E staining were performed by Applied Medical Research.

[0140] <Alcian Blue Staining> Small intestinal tissue chips were fixed in 4% paraformaldehyde for 15 minutes. The small intestinal tissue on the chip was collected and used to prepare paraffin sections. Paraffin-embedded tissue sections and Alcian Blue staining were performed by Applied Medical Research.

[0141] <Masson's Trichrome Staining> Small intestinal tissue chips were fixed in 4% paraformaldehyde for 15 minutes. The small intestinal tissue on the chip was collected and used to prepare paraffin sections. Paraffin-embedded tissue sections and Masson's Trichrome staining were performed by Applied Medical Research.

[0142] <Transmission electron microscopy observation> The microintestinal system was fixed with 2% glutaraldehyde in 0.1 M phosphate buffer at 4°C. After fixation, ultrathin sections, resin embedding, and electron microscopy observation were performed by Hanaichi Electron Microscopy Technology Institute.

[0143] <scRNA-seq Analysis> Single-cell cDNA libraries were prepared using the 10X Genomics Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics) according to the manufacturer's instructions. Briefly, microintestinal systems were trypsinized to dissociate single cells. These cells were mixed, adjusted to a cell concentration of 1,000 cells / μL, and then applied to the Chromium 10X Single Cell System (10X Genomics) to generate gel beads-in-emulsion (GEM). cDNA libraries were prepared using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10X Genomics) according to the manufacturer's instructions. Sequencing was performed using a NovaSeq 6000 (Illumina). The 10X Genomics Cell Ranger pipeline (version 7.1.0) was used to demultiplex samples, align them to the hg38 human reference genome, process barcodes / UMIs, and count genes in each cell. The gene count matrix data was processed as follows. Briefly, low-quality cells were excluded based on the following criteria: (I) the number of detected genes was 200 or less, (II) the number of detected UMI molecules in the cell was 100,000 or more, and (III) the proportion of mitochondrial genes was 12.5% ​​or more. The filtered unique molecular identifier (UMI) functional barcode matrix was processed using ICARUS v.2.0 (Jiang, AD, et al., Nar Genomics and Bioinformatics. 5:lqad032 (2023)). The gene count matrix was normalized and scaled 10,000 times.Dimensionality reduction by principal component analysis was performed using a set of 2,000 top variable genes (Seurat::FindVariableFeatures) (Hao, YH, et al., Nature Biotechnology. 42(2):293-304 (2023)). Clustering was performed using the Louvain clustering algorithm with 15 principal components and a k-nearest neighbor value of 20. Cell type annotation was performed using scType (Ianevski, A., et al., Nature Communications. 13(1):1246 (2022)). The accession number for the scRNA-seq data reported in this study is GSE244013. The scRNA-seq expression dataset for human fetal small intestine was obtained from a public database (ArrayExpress accession number E-MTAB-9536) (Elmentaite, R., et al., Nature. 597(7875):250-255 (2021)).

[0144] In situ gene expression analysis. In situ RNA expression analysis at the single-cell level was performed using Xenium (10x Genomics). The microintestinal system was fixed in 4% paraformaldehyde for 15 minutes. FFPE tissue sections (5 μm thick) were prepared on Xenium slides (10x Genomics) with the support of the Tokushima Institute of Molecular Pathology. Fixation and tissue permeabilization were performed according to the manufacturer's protocol (Cat. No. CG000581, Rev. A, 10x Genomics). Probe hybridization mixes were prepared using redesigned panels (Xenium Human Colon Gene Expression and Xenium Human Skin Gene Expression panels) according to the user guide (Cat. No. CG000582, Rev. A, 10x Genomics). The prepared probes were hybridized overnight at 50°C. Post-hybridization washes (30 min at 37°C), ligation (2 h at 37°C), and amplification (2 h at 30°C) were performed according to the user guide. Autofluorescence quenching and nuclear staining were performed in the dark. Using the prepared slides, fluorescent probe hybridization and imaging were performed using the Xenium Analyzer (onboard analysis: version 1.8.2.1, software: version 1.7.1.0, 10x Genomics). Output images and expression profiles were evaluated using Xenium Explorer (version 2.0.0, 10x Genomics). After Xenium was run, H&E staining was performed on the Xenium slides. For quality control of the Xenium analysis, the output summary HTML file from the Xenium Analyzer was checked to ensure no errors were reported. In situ gene expression profiling was supported by KOTAI Biotechnologies.

[0145] HCoV-229E Infection: Human coronavirus 229E (HCoV-229E) (catalog number VR-740) was purchased from the American Type Culture Collection (ATCC). HCoV-229E was produced in MRC-5 (catalog number CCL-171; ATCC) cells and stored at -80°C until use. MRC-5 cells (catalog number CCL-171; ATCC) were cultured in RPMI-1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Medium containing HCoV-229E (0.1 MOI) was injected into the upper or lower channel of the small intestinal tissue on the chip. One day postinfection (1 dpi), the medium was replaced with fresh medium and cultured for three days.

[0146] RNA-seq analysis: RNA was isolated from a microintestinal system infected with HCoV-229E. RNA integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies). RNA-seq libraries were constructed using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the manufacturer's instructions. Sequencing was performed on an Illumina NextSeq 2000 in paired-end mode. FASTQ files were generated using bcl2fastq-2.20. Adapter sequences and low-quality bases were trimmed from raw reads using cutadapt ver v4.1 (Kechin, A., et al., Journal of Computational Biology. 24(11):1138-1143 (2017)). Trimmed reads were mapped to the human reference genome sequence (hg38) using STAR ver. 2.7.10a (Dobin, A., et al., Bioinformatics 29(1):15-21 (2019)) and GENCODE (release 32, GRCh38.p13) (Frankish, A., et al., Nucleic Acids Research. 47(D1):D766-D773 (2019)) GTF files. Unique and well-mapped reads were used for further analysis. Raw read counts were calculated using htseq-count ver. 2.0.2 (Anders, S., Pyl, PT, and Huber, W. Bioinformatics. 31(2):166-169 (2015)) and GENCODE GTF files. Differential expression analysis was performed using DESeq2 v1.34.0 (Love, MI, Huber, W., and Anders, S. Genome Biology. 15 (12):550 (2014)) with the Wald test. p-values ​​were corrected for multiple testing using the Benjamini and Hochberg method and expressed as false discovery rates (FDR).The raw data of this study were submitted to Gene Expression Omnibus (GEO) under accession number GSE263691.

[0147] <Drug Metabolizing Enzyme Activity Assay> Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis was performed to examine cytochrome P450 (CYP) activity. LC-MS / MS analysis was performed using an LCMS-8060NX (Shimadzu Corporation). Before CYP substrate treatment, small intestinal tissue on the chip was incubated with HBSS for 30 minutes. The small intestinal tissue chips were then cultured in HBSS containing 10 μM triazolam (catalog no. 205-14221; Fujifilm Wako Pure Chemical Industries) (metabolite: α-hydroxytriazolam, catalog no. H2529; Sigma-Aldrich), diclofenac (catalog no. D2508; Tokyo Chemical Industry Co., Ltd.) (metabolite: 4-hydroxydiclofenac, catalog no. Cay10008518; Cayman Chemical), or 7-hydroxycoumarin (catalog no. H0236; Tokyo Chemical Industry Co., Ltd.) (metabolite: 7-hydroxycoumarin glucuronide, catalog no. Cay19882; Cayman Chemical). Half of the medium was collected at 2, 4, and 6 hours after substrate treatment. When the supernatant was collected, an equal volume of medium containing the substrate was added. The collected supernatant was mixed with an equal volume of acetonitrile. The samples were filtered through a 0.45 μm Cosmonice Filter W and then analyzed by LC-MS / MS to measure the concentrations of the substrate and its metabolites according to a standard curve. Details of the mass spectrometer ionization mode and multiple reaction monitoring (MRM) transitions are summarized in Table 4. The dwell time of each MRM transition was set to 100 ms. LC separation was performed at 40 °C using a Cosmocore® 2.6C18 packed column, 2.1 mm I.D. × 50 mm (catalog number 12631-41; Nacalai Tesque). The mobile phase conditions are summarized in Table 5.

[0148]

[0149]

[0150] <P450-GloTM CYP3A4 assay: CYP3A4 activity was measured using the P450-Glo™ CYP3A4 Assay Kit (Cat. No. V9001; Promega). Small intestinal tissue on the chip was treated with luciferin-IPA, and luciferase intensity was measured using a luminometer (Lumat LB 9508; Berthold Technologies) according to the manufacturer's instructions.

[0151] Statistical analysis: Statistical analysis was evaluated by unpaired two-tailed Student's t-test. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test.

[0152] <Proteome analysis> Small intestinal tissue chips were lysed in PTS buffer (12 mM SDC, 12 mM SLS, 100 mM Tris-HCl (pH 9.0)) containing a protease inhibitor mixture (catalog number P8340; Sigma-Aldrich). After sonication, the cell lysate was centrifuged and the supernatant was collected. The supernatant was used as a protein sample and processed using a modified protein aggregate capture method (TS Batth et al., Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation. Mol Cell Proteomics 18, 1027-1035 (2019)). Equal amounts of two types of Sera-Mag SpeedBead carboxylate-modified magnetic particles (hydrophilic particles, catalog no. 45152105050250; hydrophobic particles, catalog no. 65152105050250; Cytiva, Marlborough, MA, USA) were mixed and washed three times with distilled water. The beads were redissolved in distilled water to a concentration of 15 μg beads / μL. Protein samples were reduced and alkylated with final concentrations of 20 mM tris(2-carboxyethyl)phosphine (TCEP) and 36 mM 2-iodoacetamide (IAA), respectively. The alkylated protein sample was mixed with 20 μL of the redissolved beads and a final concentration of 75% (v / v) ethyl alcohol. The mixed sample was washed twice with 80% ethyl alcohol, and the supernatant was discarded. The sample was then suspended in 100 μL of 50 mM Tris-HCl (pH 8.0) containing 2 μL of trypsin / Lys-C Mix (catalog no. V5072, Promega), and protein digestion was carried out overnight at 37°C.The digested sample was acidified with 20 μL of 5% trifluoroacetic acid (TFA) and desalted using an SDB-StageTip (J. Rappsilber et al., Stop and go extraction tips for matrix-assisted laser desorption / ionization, nanoelectrospray, and LC / MS sample pretreatment in proteomics. Anal Chem 75, 663-670 (2003)). Peptide concentrations were measured using a Quantitative peptide assay kit (catalog no. 23290, Thermo Fisher Scientific). A total of 200 ng of desalted peptides was loaded onto an Aurora column (250 mm long, 75 mm inner diameter, IonOpticks) using a nanoElute (Bruker) and separated. The peptides were then analyzed using a timsTOF Pro2 (Bruker). The mobile phases were 0.1% formic acid (solution A) and 0.1% formic acid acetonitrile (solution B). The flow rate was 400 nL / min for 60 min at 2-17% solution B, 30 min at 17-25% solution B, 10 min at 25-37% solution B, 10 min at 37-80% solution B, and 10 min at 80% solution B (total 120 min). The spray voltage was 1400 V, and the interface heater temperature was 180 °C. To obtain MS and MS / MS spectra, we used the parallel accumulation serial fragmentation (PASEF) acquisition method combined with data-independent acquisition (DIA) (diaPASEF) (F. Meier et al., diaPASEF: parallel accumulation-serial fragmentation combined with data-independent acquisition. Nat Methods 17, 1229-1236 (2020)).The diaPASEF settings were precursor ion scan with a cycle time of 1.7 seconds, 16 diaPASEF scans, MS / MS isolation width of 25 m / z, precursor ion ranges of 400-1200 m / z, and ion mobility range of 0.57-1.47 V·s·cm. -2 The DIA data were then searched against the human data from UniProt / Swiss-Prot release 2022_03 using DIA-NN (v1.81) (PMID: 31768060, Demichev et al., 2020), with cysteine ​​carbamidomethylation as a fixed modification and protein N-terminal acetylation and methionine oxidation as variable modifications. Other DIA-NN parameters included Trypsin / P protease, missed cleavage of 1, peptide length of 7-30, precursor m / z range of 300-1800, precursor charge of 1-4, fragment ion m / z range of 200-1800, and a 1% precursor FDR. "PG.MaxLFQ" was used as a representative protein area value for comparison of protein expression levels.

[0153] <Measurement of orientation> Vimentin-stained small intestine tissue chips were observed using a confocal laser scanning microscope FV3000 (Olympus Life Science). The captured images were binarized using ImageJ software and subjected to fast Fourier transform using FiberOri8single03 to calculate the orientation and orientation intensity.

[0154] Example 1: Preparation of small intestinal tissue chip To prepare a small intestinal tissue chip, a protocol for inducing differentiation from human iPS cells into small intestinal epithelial cells was investigated. Differentiation induction from human iPS cells into small intestinal epithelial cells was carried out on a 24-well plate. Previously reported differentiation induction protocols using Activin A, FGF2, EGF, Forskolin, PD98059, 5-aza-2'-dC, and A-83-01 (Kabeya T, Mima S, Imakura Y, Miyashita T, Ogura I, Yamada T, Yasujima T, Yuasa H, Iwao T, Matsunaga T. Pharmacokinetic functions of human induced pluripotent stem cell-derived small intestinal epithelial cells. Drug Metab Pharmacokinet. 2020 Aug;35(4):374-382. doi: 10.1016 / j.dmpk.2020.04.334. Epub 2020 May 16. PMID: Differentiation was induced using four protocols: Condition 1 (Protocol 1) was a mixture of 32651148. and HCM as the basal medium in Condition 2 (Protocol 2); Condition 3 (Protocol 3) was a mixture of Condition 1 and Wnt-3A, R-spondin 3, Noggin, BIO, and DAPT; and Condition 4 (Protocol 4) was a mixture of Condition 3 and HCM. An overview of each protocol is shown in Figure 1. The gene expression level of the small intestine marker Villin (measured by RT-qPCR) was equal to or greater than that of Condition 1 under all four conditions (Figure 3).

[0155] Human iPS cells were induced to differentiate into small intestinal tissue in a microfluidic device. Human iPS cells were cultured on a 12-well plate until they were definitive endoderm cells, then detached and seeded into the upper channel of the microfluidic device. Differentiation into small intestinal tissue was induced. Figure 4 shows bright-field images of cells cultured on the microfluidic device, and Figure 5 shows the amount of RNA extracted from cells recovered after differentiation induction. When differentiation was induced under conditions 1 and 2, almost no cells survived in the microfluidic device, and only small amounts of RNA were recovered. In contrast, when differentiation was induced under conditions 3 and 4, cells survived in the microfluidic device. Gene expression analysis (measured by RT-qPCR) was performed on cells cultured on a 24-well plate under condition 1 and on small intestinal tissue chips cultured on the microfluidic device under conditions 3 and 4 (Figure 6). Villin gene expression levels were comparable in all three groups. Gene expression levels of SI and the drug-metabolizing enzyme CYP3A4, which are known to be expressed in mature small intestinal epithelial cells, were higher in cells cultured on the microfluidic device under condition 4. These results suggest that condition 4 is optimal for producing human iPS cell-derived small intestinal tissue on a microfluidic device.

[0156] Example 2: Study of Medium Perfusion Using Small Intestine Tissue Chips. The effects of medium perfusion were investigated using small intestine tissue chips. A peristaltic pump was connected to the lower channel of the microfluidic device, and medium was perfused. Gene expression analysis (measured by RT-qPCR analysis) revealed no differences in the gene expression levels of the absorptive epithelial cell marker (Villin), goblet cell marker (MUC2), and intestinal stem cell marker (LGR5), which constitute small intestine epithelial cells (Figure 7). Figure 8 shows a bright-field image of the small intestine tissue chip. While cells cultured in static conditions did not undergo three-dimensional organization, cells formed raised structures in the small intestine tissue chips perfused with medium. H&E staining of cross-sections of the small intestine tissue chips confirmed the formation of protruding raised structures, and villi-like structures were observed due to medium perfusion (Figure 9). The results of gene expression analysis in Figure 7 showed that perfusion did not affect the gene expression level of Villin, but the protein expression level of Villin (measured by Jess) significantly increased with perfusion (Figure 10). On the other hand, the protein expression level of CYP3A4 (measured by Jess) did not change with perfusion. TM CYP3A4 assays demonstrated high activity regardless of medium perfusion (Figure 11). Furthermore, to examine the activity of drug-metabolizing enzymes CYP3A4, CYP2C9, and UGT1A1, substrates for each drug-metabolizing enzyme were applied to the small intestinal tissue chips, and the amount of metabolites produced was measured (LC-MS / MS) (Figure 12). The production levels of CYP3A4 and UGT1A1 metabolites were comparable between the static culture and perfusion groups, while the production level of CYP2C9 metabolites was higher in the static culture group than in the perfusion group. These results suggest that small intestinal tissue chips have high CYP3A4, CYP2C9, and UGT1A1 activities regardless of medium perfusion.

[0157] Immunofluorescence staining was performed for the absorptive epithelial cell marker (Villin), goblet cell marker (MUC2), Paneth cell marker (lysozyme), neuroendocrine cell marker (CHGA), and proliferating cell marker (Ki67) that compose the small intestinal epithelial cells (Figure 13). Villin expression increased with medium perfusion and was localized to the apical side. Large numbers of MUC2-positive goblet cells, LYZ-positive Paneth cells, CHGA-positive neuroendocrine cells, and Ki67-positive proliferating cells were confirmed with medium perfusion (Figure 13). CYP3A4 expression was high in both statically cultured and perfused small intestinal tissue chips (Figure 13). Next, Alcian blue staining (to stain mucin) was performed (Figure 14). In the perfused small intestinal tissue chip, blue-stained goblet cells, which are mucin-producing cells, and the mucus layer (black triangles) were confirmed. These findings suggest that in small intestinal tissue chips perfused with culture medium, multiple types of cells that make up the small intestinal epithelium are present, and that the small intestinal tissue takes on a morphology similar to that of living tissue.

[0158] We investigated whether small intestinal tissue chips could be fabricated similarly by changing the perfusion rate (Figure 15). Villin protein expression (measured by Jess) was high even at a slower flow rate (10 μL / h) than the above-mentioned perfusion rate (30 μL / h), but expression levels decreased at a faster flow rate (100 μL / h). CYP3A4 protein expression (measured by Jess) was high at a faster flow rate (100 μL / h). Alanyl aminopeptidase, membrane (ANPEP) protein expression (measured by Jess) was high at the above-mentioned perfusion rate (30 μL / h). These studies demonstrated that differentiation of small intestinal tissue chips was possible at any medium perfusion rate (10, 30, or 100 μL / h), but suggested that a slow rate (10 μL / h) was preferable for inducing the expression of the small intestinal marker Villin, and a fast rate (100 μL / h) was preferable for inducing the expression of the drug-metabolizing enzyme CYP3A4.

[0159] Example 3: Exploring the Cause of Medium Perfusion Promoting Differentiation in Small Intestinal Tissue Chips Further analysis was performed to explore the cause of the promotion of differentiation by medium perfusion in small intestinal tissue chips. Masson's trichrome staining (primarily staining collagen) was performed to examine the localization of collagen fibers (Figure 16). Collagen fibers, which support epithelial cells, aligned on the basolateral side of the epithelial cell layer after medium perfusion, exhibiting a localization similar to that found in the in vivo small intestine. Because collagen, the main component of collagen fibers, is produced by fibroblasts, the presence of fibroblasts in the small intestinal tissue chips was confirmed. The protein expression level of the fibroblast marker Vimentin (measured by Jess) did not change with or without medium perfusion. However, immunofluorescence staining revealed that while Vimentin-positive cells were present in a disordered manner in the static culture group, they were localized to the basolateral side of the epithelial cell layer after medium perfusion (Figure 17). The expression level of the myofibroblast marker α-SMA was reduced by medium perfusion (immunofluorescence staining) (Figure 17). Furthermore, immunofluorescence staining of COL1A1 revealed that it colocalized with Vimentin (Figure 18), suggesting that Vimentin-positive fibroblasts localized on the basolateral side of the epithelial cell layer produce COL1A1. GO enrichment analysis revealed that medium perfusion increased the expression of genes containing terms that control extracellular secretions and structures in the small intestinal tissue chip (Figure 19, right, underlined). These results suggest that medium perfusion in the small intestinal tissue chip promoted differentiation into small intestinal tissue by aligning fibroblasts secreting collagen fibers to the basolateral side.

[0160] Example 4: Viral Infection Experiment Using Small Intestine Tissue Chip Viral infection experiments were conducted using small intestine tissue chips. Human coronavirus 229E (HCoV-229E) uses aminopeptidase N (APN / CD13 / ANPEP), which is highly expressed in the small intestine, as an infection receptor. The protein expression level of ANPEP in small intestine tissue chips was examined and found to increase with medium perfusion (measured by Jess) (Figure 20). Small intestine tissue chips perfused with medium were infected with HCoV-229E through either the upper channel (apical side) or the lower channel (basolateral side) (Figure 21). The results showed that the amount of viral genome (HCoV-229E mRNA) detected intracellularly was higher in the apical side than in the basolateral side. The immune response to viral infection (increased gene expression of IL-8 and IL-1β) was also higher in the apical side-infected group than in the basolateral side-infected group. These results demonstrate that the small intestine tissue chip can also be used as a system for evaluating infection with human coronavirus 229E (HCoV-229E).

[0161] Example 5: Comparison of Small Intestinal Tissue Chips with Indigenous Small Intestine. Perfused small intestinal tissue chips and indigenous small intestinal total RNA were compared using RNA-seq analysis. The results showed that the expression of 4,510 genes was elevated in the small intestinal tissue chip compared to indigenous small intestinal total RNA (Figure 22). Enrichment analysis of the elevated genes revealed elevated expression of genes related to collagen fibrils, extracellular matrix (ECM), and negative regulation of apoptosis (Figure 23). Furthermore, the small intestinal tissue chips showed elevated expression of growth factors (TGFB1, WNT5A, IGF2), ECM, and factors regulating its degradation (Figure 24). These findings suggest that the small intestinal tissue chips tend to have higher expression levels of genes related to ECM regulation than indigenous small intestinal total RNA.

[0162] Example 6: Proteome Analysis Proteome analysis was performed on small intestinal tissue chips that had undergone static or perfusion culture. The results showed that perfusion increased the protein expression levels of 469 genes and decreased the protein expression levels of 344 genes (Figure 25, left). Next, the Human Protein Atlas was used to investigate the organs in which genes whose protein expression levels increased with perfusion culture (Figure 25, right). The results revealed that the genes whose protein expression levels increased with perfusion culture (Flow) were highly expressed in cell types present in the intestinal tract (gray columns).

[0163] Next, 3097 genes highly expressed in the normal small intestine were extracted from the Human Protein Atlas. The expression changes of these 3097 genes in small intestine tissue chips are shown as a heat map (Figure 26, left). Perfusion culture (Flow) increased the protein expression of genes characteristic of the normal small intestine. 321 genes highly expressed in absorptive epithelial cells (enterocytes) of the normal small intestine were extracted from the Human Protein Atlas. The expression changes of these 321 genes in small intestine tissue chips are shown as a heat map (Figure 26, right). Perfusion culture (Flow) increased the protein expression of many genes characteristic of absorptive epithelial cells.

[0164] The changes in protein expression of drug-metabolizing enzymes, cytochrome P450 (CYP), uridine diphosphate glucuronosyltransferase (UGT), sulfotransferase (SULT), and carboxylesterase (CES), are shown in a heat map (Figure 27, left). Perfusion culture (Flow) increased the protein expression levels of many drug-metabolizing enzymes. The expression ratio of CES2, which is highly expressed in the small intestine, to CES1, which is highly expressed in the liver, is shown (Figure 27, right). Perfusion culture (Flow) increased the protein expression level of CES2, a small intestine-specific carboxylesterase.

[0165] A diagram comparing the mRNA and protein expression changes in small intestinal tissue chips that underwent static culture (Static) and perfusion culture (Flow) (Figure 28, left). The amount of mRNA expression change was calculated from the results of RNA-seq analysis, and the protein expression change was calculated from the results of proteome analysis. The perfusion culture (Flow) resulted in small changes in mRNA expression, while large changes in protein expression. The correlation coefficient between mRNA expression change and protein expression change is R 2 = 0.0794, suggesting a low correlation between the two. There were 703 genes (gray) whose protein expression level increased but not their mRNA expression level with perfusion culture (Flow), while there were 401 genes (dark gray) whose protein expression level decreased but not their mRNA expression level with perfusion culture (Flow).

[0166] To identify the genes (703, gray) whose protein expression levels increased with perfusion culture (Flow) but whose mRNA expression levels did not, we performed analysis using the human protein atlas (Figure 28, right). As a result, we found that the group of genes whose protein expression levels increased with perfusion culture (Flow) were highly expressed in cell types present in the intestinal tract (gray columns).

[0167] Example 7: Verification of Optimized Culture Conditions. We investigated the effect of the duration of perfusion culture (Flow) on the structure and function of small intestinal tissue chips. Figure 29 shows an overview of the protocol for inducing differentiation of pluripotent stem cells into small intestinal epithelial cells. We shortened the perfusion culture (Flow) period of small intestinal tissue chips from the usual 20 days to 3, 7, or 12 days to examine whether this affected the structure and function of the small intestinal tissue chips. Figure 30 shows bright-field images of small intestinal tissue chips. Protruding three-dimensional structures were observed in small intestinal tissue chips that underwent 20 days of perfusion culture (Flow), but such three-dimensional structures were not fully observed in perfusion cultures for 3, 7, or 12 days. This indicates that culture for more than 12 days is necessary to develop crypt- and villi-like three-dimensional structures, and that perfusion culture (Flow) for at least 20 days is preferable. We also investigated the protein expression levels of Villin, ANPEP, and CYP3A4, which are highly expressed in small intestinal epithelial cells. The results are shown in Figure 31. In particular, the protein expression levels of Villin and ANPEP increased after 20 days of perfusion, whereas the protein expression level of CYP3A4 remained almost unchanged after perfusion culture.

[0168] Next, we investigated the medium conditions for inducing small intestinal tissue from intestinal progenitor cells. Three-dimensional small intestinal tissue chips were confirmed by medium perfusion under all conditions: "normal medium with HCM (+HCM)," "medium without HCM (-HCM)," and "medium with William's E medium instead of HCM (+William's E Medium)" (Figure 32). Therefore, to construct a 3D small intestinal tissue chip, it is not necessary to use hepatocyte culture medium (HCM or William's E medium) in combination with the basal medium used to culture intestinal progenitor cells.

[0169] Example 8: Verification of the construction mechanism of small intestine tissue chips. Small intestine tissue chips were stained with the fibroblast marker Vimentin (Figure 33, upper right). In the static culture group, Vimentin-positive cells were present randomly, but in the perfusion culture group, Vimentin-positive cells were oriented along the medium flow (white arrow). Therefore, the degree of Vimentin orientation was calculated using FiberOri8single03 (Figure 33, lower). The orientation intensity was 1.05 in the static culture group and 1.21 in the perfusion culture group, with perfusion culture showing a higher value. These results demonstrate that medium perfusion (Flow) causes Vimentin-positive fibroblasts in the small intestine tissue chip to orient along the medium flow.

[0170] Matrix metalloproteinases (MMPs) are enzymes that degrade extracellular matrix components such as collagen. Previous studies have shown that MMPs promote fibroblast migration. Proteomic analysis revealed that the protein expression level of MMP-1, which degrades type I collagen, increased with medium perfusion in small intestinal tissue chips (Figure 34, top panel). To investigate the role of MMPs in the differentiation induction process of small intestinal tissue chips, we used the MMP inhibitor GM 6001 (Figure 34, bottom panel). Three-dimensional small intestinal tissue was constructed in a microfluidic device by medium perfusion, but the construction of 3D small intestinal tissue was inhibited by the use of medium containing GM 6001. These findings suggest that MMPs play an important role in the construction of 3D small intestinal tissue by medium perfusion. These findings suggest that perfusion culture (flow) increases MMP expression in small intestinal tissue chips, promotes fibroblast migration, and orients fibroblasts along the medium flow, thereby promoting the construction of 3D small intestinal tissue.

[0171] Example 9: Gene Expression Analysis in Small Intestinal Tissue-Chips Perfused with Medium To examine temporal changes in gene expression in small intestinal tissue-chips, RT-qPCR analysis was performed (Figure 35). Expression of the undifferentiated cell marker POU class 5 homeobox 1 (POU5F1) and the mesendodermal cell marker Mix paired-like homeobox (MIXL1) was high on days 0 and 4 of differentiation induction and gradually decreased thereafter (Figures 35A and 35B). Differentiation into endoderm and mesoderm cells was then induced by adding medium containing 1-2% fetal bovine serum (FBS). Expression of the endodermal cell marker forkhead box A2 (FOXA2) transiently increased around day 8 of differentiation induction, followed by increased expression of the absorptive epithelial cell markers villin 1 (VIL1) and sucrase-isomaltase (SI) (Figure 35C). Similarly, the mesodermal cell marker T-box transcription factor T (TBXT) transiently increased around day 8 of differentiation induction, followed by increased gene expression of mesenchymal cell markers vimentin (VIM) and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) (Fig. 35D). These results demonstrate that the medium-perfused small intestinal tissue-in-a-chip induced the simultaneous differentiation of human iPS cells into both small intestinal epithelial cells and mesenchymal cells.

[0172] Example 10: Comparative Analysis of Small Intestinal Tissue Chips, In Vivo Human Small Intestine, and Caco-2 Cells Gene expression was compared between Caco-2 cells, a commonly used small intestinal epithelial cell model, and in vivo human small intestine by RT-qPCR analysis (Figure 36). Gene expression of absorptive epithelial cell markers, VIL1 and fatty acid-binding protein 2 (FABP2), was higher in small intestinal tissue chips than in Caco-2 cells (Figure 36A). Furthermore, the goblet cell marker mucin 2 (MUC2), the Paneth cell marker lysozyme (LYZ), the neuroendocrine cell marker regenerating family member 4 (REG4), the M cell marker glycoprotein 2 (GP2), and the tuft cell marker doublecortin-like kinase 1 (DCLK1), were barely expressed in Caco-2 cells but were expressed in small intestinal tissue chips. Furthermore, the gene expression levels of the drug-metabolizing enzymes cytochrome P450 family 3 subfamily A member 4 (CYP3A4), CYP family 2 subfamily C member 9 (CYP2C9), UDP glucuronosyltransferase family 1 member A1 (UGT1A1), and sulfotransferase family 1B member 1 (SULT1B1) were low in Caco-2 cells but high in the small intestinal tissue chips (Figure 36B).The gene expression of transporters ATP binding cassette subfamily B member 1 (ABCB1 / MDR1), ABC subfamily C member 1 (ABCC1 / MRP1), ABC subfamily C member 2 (ABCC2 / MRP2), ABC subfamily G member 2 (ABCG2 / BCRP), solute carrier organic anion transporter family member 2B1 (SLCO2B1 / OATP2B1), and SLC family 15 member 1 (SLC15A1 / PEPT1) in small intestinal tissue chips was comparable to that in Caco-2 cells (Figure 36C). These results suggest that small intestinal tissue chips are more functional than the existing intestinal model, Caco-2 cells.

[0173] Example 11: Morphological Analysis of Small Intestinal Tissue Chips. The effects of medium perfusion on the structure of absorptive epithelial cells were evaluated in more detail using electron microscopy. In the medium-perfused small intestinal tissue chips, the small intestinal epithelial cell layer was composed of uniform columnar epithelial cells, with well-ordered microvilli on their surface (Figures 37A and 37B). Such mature epithelial cells were not observed in the static culture group. Furthermore, tight junctions (TJs), adherens junctions (AJs), and desmosomes (DSs) were aligned in the medium-perfused small intestinal tissue chips, whereas they remained immature in the static culture group (Figure 37C). These results suggest that medium perfusion in the microfluidic device promoted the maturation of small intestinal epithelial cells.

[0174] Example 12: In situ gene expression profiling in small intestinal tissue chips. In situ gene expression profiling at the single-cell level was used to examine the localization of various small intestinal epithelial cell markers in small intestinal tissue chips after medium perfusion (Figures 38A and 38B). The absorptive epithelial cell markers solute carrier family 26 member 2 (SLC26A2) and solute carrier family 26 member 3 (SLC26A3) were expressed on the apical side of the small intestinal tissue chips, while the intestinal progenitor cell marker SRY-box transcription factor 9 (SOX9) and the stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) were expressed on the basal side (Figures 38C and 38D). Bestrophin 4 (BEST4) and the neuroendocrine cell marker CHGA were also expressed. These results demonstrate that medium perfusion successfully established a polarized small intestinal epithelial layer. Furthermore, we investigated the localization of interstitial cell markers in the medium-perfused small intestinal tissue chips by in situ gene expression profiling at the single-cell level (Fig. 39A, Fig. 39B). Vimentin (VIM), a marker for intestinal fibroblasts, was localized to the basal side of the small intestinal tissue chips (Fig. 39C, Fig. 39D). Furthermore, platelet-derived growth factor receptor alpha (PDGFRA), a telocyte marker, was also expressed in the small intestinal tissue chips. high Cell and trophocyte marker PDGFRA low There were also frizzled-related protein (FRZB, a Wnt antagonist)-positive cells.

[0175] Example 13: Gene Expression Analysis of Fibroblasts Constituting Small Intestine Tissue Chips We investigated the cell types present in small intestine tissue chips among the various fibroblasts that make up the intestine. scRNA-seq analysis of medium-perfused small intestine tissue chips was performed, and 10 clusters, including epithelial cells and interstitial cells, were identified. Subcluster analysis of the interstitial cell clusters was performed among the 10 clusters. Interstitial cells were classified into three subclusters (Figure 40A). All subclusters expressed the fibroblast marker PDGFRA (Figure 40B). Subcluster 0 expressed R-spondin 3 (RSPO3), which is important for the maintenance of intestinal stem cells. Subclusters 1 and 2 expressed bone morphogenetic protein 5 (BMP5), which is important for the differentiation of small intestinal epithelial cells. These results demonstrate that small intestine tissue chips contain fibroblasts that express growth factors important for the maintenance and differentiation of small intestinal epithelial cells.

[0176] Example 14: Viral Infection Experiment Using Small Intestine Tissue Chip (HCoV-229E Infection Experiment) A human coronavirus 229E (HCoV-229E) infection experiment was performed using a small intestine tissue chip, and RNA-seq analysis of the infected small intestine tissue chip was performed (Figure 41). When HCoV-229E was infected from the apical side of the medium-perfused small intestine tissue chip, the expression of 182 genes changed, but when infection was from the basolateral side, gene expression did not change (Figure 41A). Host genes associated with viral infection were elevated in small intestine tissue chips infected from the apical side with HCoV-229E (Figures 41B and 41C). These results demonstrate that the small intestine tissue chip of the present invention can be used to evaluate viral infection.

[0177] Example 15: Verification Using Various Human Pluripotent Stem (ES) Cells We investigated whether small intestinal tissue chips with equivalent functionality could be fabricated using human ES cells, in addition to human iPS cells (Figures 42 and 43). Using human ES cells, KhES-3 cells, we were able to construct small intestinal tissue chips with a villi-like raised three-dimensional structure (Figures 42A and 42B) through the action of interstitial flow. Small intestinal tissue chips induced to differentiate from KhES-3 cells demonstrated high mucus production and expression of absorptive epithelial cell markers (Figures 42C and 42D). In small intestinal tissue chips induced to differentiate from KhES-3 cells, a fibroblast layer and collagen fibers formed beneath the epithelial cell layer (Figures 42E and 42F). Furthermore, using human ES cells, H9 cells, we were able to construct small intestinal tissue chips with a villi-like raised three-dimensional structure (Figures 43A and 43B) through the action of interstitial flow. Small intestinal tissue chips induced to differentiate from H9 cells showed high mucus production and expression of absorptive epithelial cell markers (Figures 43C and 43D). In small intestinal tissue chips induced to differentiate from H9 cells, a fibroblast layer and collagen fibers were formed beneath the epithelial cell layer (Figures 43E and 43F). These results demonstrate that the small intestinal tissue chips of the present invention can be produced using not only human iPS cells but also human ES cells.

[0178] According to the present invention, small intestinal epithelial cells can be produced from midgut cells using a method different from conventional methods. Furthermore, a method using a microfluidic device can simultaneously induce differentiation of small intestinal epithelial cells and mesenchymal cells such as fibroblasts from pluripotent stem cells within a single microfluidic device. The production of extracellular matrix (e.g., collagen) and niche factors by mesenchymal cells allows small intestinal epithelial cells to exhibit polarity and function similar to that of living organisms. The use of small intestinal tissue, which may possess functions comparable to or even superior to those of living organisms, enables more accurate drug discovery studies (e.g., pharmacokinetic studies, drug toxicity studies, infection experiments) than conventional small intestinal models. Drug discovery studies with high clinical predictive potential are expected to lead to the efficiency and simplification of animal experiments and clinical trials. Small intestinal tissue also has potential as a resource for regenerative medicine.

[0179] This application is based on patent application No. 2023-132372 filed in Japan (filing date: August 15, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing small intestinal epithelial cells or small intestinal tissue from midgut cells, comprising: (1) culturing midgut cells in a medium containing a Wnt agonist, a BMP inhibitor and a growth factor to obtain intestinal progenitor cells; and (2) culturing the intestinal progenitor cells obtained in step (1) in a medium containing a growth factor, an adenylate cyclase activator, a MEK inhibitor, a DNA methylation inhibitor, a TGF-β inhibitor, a GSK3β inhibitor and a γ-secretase inhibitor to obtain small intestinal epithelial cells or small intestinal tissue.

2. The method of claim 1, wherein the Wnt agonist comprises a Wnt protein and R-spondin.

3. The method of claim 1 or 2, wherein the Wnt agonist and BMP inhibitor are obtained from cells which express them.

4. The method according to any one of claims 1 to 3, wherein the growth factors in steps (1) and (2) include EGF.

5. The method according to any one of claims 1 to 4, wherein steps (1) and (2) are carried out in a microfluidic device.

6. The method according to claim 5, wherein steps (1) and (2) include a step of performing perfusion culture.

7. The method according to claim 6, wherein the perfusion culture is carried out using a peristaltic pump or a shaker.

8. The method according to any one of claims 1 to 7, comprising a step of inducing differentiation of definitive endoderm cells into midgut cells prior to step (1).

9. The method according to claim 8, further comprising the step of inducing differentiation of pluripotent stem cells into definitive endoderm cells.

10. Small intestinal epithelial cells, small intestinal tissue, or a microfluidic device carrying them, obtained by the method according to any one of claims 1 to 9.

11. Small intestinal tissue or a microfluidic device carrying said small intestinal tissue, having all of the following characteristics (A) to (F): (A) having absorptive epithelial cells; (B) having fibroblasts; (C) having collagen fibers; (D) expressing one or more genes selected from the group consisting of TGFB1, Wnt5A, and IGF2; (E) expressing one or more genes selected from the group consisting of MMP1, COL3A1, and TIMP1; and (F) having drug metabolizing enzyme activity.

12. A transplantation therapy comprising the small intestinal epithelial cells or small intestinal tissue according to claim 10 or 11.

13. A method for screening for a therapeutic or preventive drug for a viral infection, comprising: (I) contacting the small intestinal tissue described in claim 10 or 11 with a virus in the presence or absence of a test substance and culturing the virus; (II) evaluating the degree of infection of the small intestinal tissue with the virus; and (III) selecting the test substance as a candidate for a therapeutic or preventive drug for a viral infection when the degree of infection of the virus is evaluated to be lower in the presence of the test substance compared to the absence of the test substance in step (II).

14. The method of claim 13, wherein the virus is a member of the Coronaviridae family.

15. A method for evaluating the degree of metabolism of a test substance, comprising: (i) contacting the test substance with the small intestine tissue described in claim 10 or 11; (ii) measuring the activity level of a drug-metabolizing enzyme; and (iii) evaluating the degree of metabolism of the test substance by the drug-metabolizing enzyme based on the activity level of the drug-metabolizing enzyme in step (ii).

16. The method according to claim 15, wherein the drug metabolizing enzyme is at least one selected from the group consisting of CYP3A4, UGT1A1 and CYP2C9.

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