Intestinal epithelial-like cells and method for producing them

Abstract

To provide excellent intestinal epithelial-like cells expressing drug-metabolizing enzymes and drug transporters applicable for pharmacokinetic assessment, more specifically, intestinal epithelial-like cells having properties closer to those of primary cultured human intestinal epithelial cells that are difficult to obtain, and to provide production methods thereof.SOLUTION: The present invention is characterized by the monolayer culturing of organoid-derived cells. As a result of analyzing the obtained monolayer film, drug-metabolizing enzymes (each CYP) and drug transporters (PEPT1, MDR1), and the like possessed by small intestinal epithelial cells were expressed and excellent intestinal epithelial-like cells that also have a tight junction function were obtained. From the above, intestinal epithelial-like cells that can be used for pharmacokinetic evaluation and / or drug toxicity evaluation can be efficiently produced.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to intestinal epithelial-like cells derived from intestinal organoids, which express drug-metabolizing enzymes and drug transporters and possess tight junction function. Furthermore, the invention relates to a method for producing intestinal epithelial-like cells derived from intestinal organoids. [Background technology]

[0002] Drugs administered orally are first absorbed, metabolized, and excreted in the small intestine. These pharmacokinetic processes, including absorption, metabolism, and excretion, are largely controlled by absorption transporters such as PEPT1 (Peptide transporter 1), drug-metabolizing enzymes such as CYP3A4 (Cytochrome P450 family 3 subfamily A member 4), and efflux transporters such as P-gp (P-glycoprotein) and BCRP (Breast cancer resistance protein). The gene encoding P-gp is called MDR1 (ABCB1), and the gene encoding BCRP is called ABCG2. P-gp is a phosphorylated protein with a molecular weight of approximately 180,000, located on the cell membrane, and is responsible for the extracellular efflux of cytotoxic compounds. In vitro evaluation of pharmacokinetics is crucial for the effective development of safe pharmaceuticals.

[0003] Obtaining primary cultured human intestinal epithelial cells is difficult, and even when cells are obtained, individual differences in their characteristics pose a problem. Furthermore, maintaining the function of the obtained cells while culturing them over the long term is also difficult. Current in vitro intestinal pharmacokinetic studies commonly utilize methods such as intestinal inversion using intestinal tissue from small animals like rats, tests using artificial lipid membranes, and evaluation systems using cell lines including Caco-2 cells. However, these evaluation systems have problems such as species differences compared to humans, low expression levels of drug transporters and drug-metabolizing enzymes, and the accumulation of genetic mutations specific to cancer cell lines. For these reasons, it has been difficult to obtain superior cells that can stably test drug metabolism and permeability in the small intestine.

[0004] There are reports on small intestinal epithelial-like cells derived from pluripotent stem cells. Non-patent document 1 reports the world's first creation of small intestinal-like tissue from human pluripotent stem cells. Non-patent document 2 reports that long-term self-renewing small intestinal stem cells can be created from human pluripotent stem cells. Non-patent document 3 reports that differentiation induction from mouse and human pluripotent stem cells into small intestinal lineage cells can be promoted using GSK-3 inhibitor IX, BIO(6-Bromoindirubin-3'-oxime), and γ-secretase inhibitor, DAPT(N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester), etc. Non-patent document 4 reports an attempt to induce differentiation from human pluripotent stem cells into small intestinal epithelial-like cells. Furthermore, there is a disclosure of superior intestinal epithelial-like cells expressing drug-metabolizing enzymes and drug transporters derived from pluripotent stem cells, as well as a method for selectively inducing differentiation from pluripotent stem cells into intestinal epithelial-like cells using a next-generation gene therapy vector system (Patent Document 1).

[0005] LGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5) positive stem cells contained in intestinal epithelial cells are processed into MatrigelTM (Matrix TM There are reports on intestinal organoids prepared by embedding them in a culture medium and adding several humoral factors called niche factors. Intestinal organoids are three-dimensional cultured organisms that have the function and structure of intestinal epithelium and can be maintained through long-term passage. The preparation of intestinal organoids derived from human biopsy tissue is reported in Non-Patent Literature 5. There are also reports on a method of preparing single cells by dispersing intestinal organoids and culturing these single cells in a monolayer on an extracellular matrix (Patent Literature 2, Non-Patent Literature 6). However, the cells shown in Patent Literature 2 are for infection and proliferation of human diarrheal virus, and the cells shown in Non-Patent Literature 6 are for confirming the effect of intestinal bacteria (Klebsiella pneumoniae) on the barrier function of the large intestine. Neither of these are cells used for pharmacokinetic evaluation, and there is no mention of the expression of drug-metabolizing enzymes or drug transporters. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Nature, 2011 Feb 3;470(7332):105-9 [Non-Patent Document 2] Stem Cell Reports, 2014 Jun 3;2(6):838-52 [Non-Patent Document 3] Stem Cells, 2013 Jun;31(6):1086-96 [Non-Patent Document 4] Drug Metab Pharmacokinet, 2014;29(1):44-51 [Non-Patent Document 5] Gastroenterology, 2011;141(5):1762-72 [Non-Patent Document 6] Nature Microbiology, 2019 March; Vol.4: 492-503 [Patent Documents]

[0007] [Patent Document 1] International Publication WO2016 / 147975 [Patent Document 2] International Publication WO2018 / 038042 [Summary of the Invention] [Problems to be Solved by the Invention]

[0008] Intestinal organoids are excellent cells that can be passaged and have the functions of the intestinal epithelium. However, intestinal organoids form a three-dimensional structure in a matrix containing a solubilized basement membrane extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma rich in ECM proteins including laminin C (main component), type IV collagen, heparan sulfate proteoglycan, entactin / nidogen, and various growth factors, and such a structure is considered unsuitable for pharmacokinetic studies. An object is to provide excellent intestinal epithelial-like cells expressing drug-metabolizing enzymes and drug transporters that are applicable to the evaluation of pharmacokinetics. More specifically, an object is to provide intestinal epithelial-like cells having properties closer to those of primary cultured human intestinal epithelial cells, which are difficult to obtain, and a method for producing the same. [Means for Solving the Problems] <00​​​​​​​​​​​​​​​​​​​​​ A process of culturing under specific conditions to produce a single-layer membrane. 2. Step 2) to produce the single layer film However, culture the intestinal organoid-derived cells for 2 to 14 days after seeding. The process is as described in item 1 above. As described Method for producing intestinal epithelial-like cells. 3. The method for producing intestinal epithelial-like cells as described in paragraph 1 above, wherein the step of producing a single-layer membrane as in 2) above is a step of culturing intestinal organoid-derived cells for 3 to 7 days after seeding. 4. A method for producing intestinal epithelial-like cells as described in item 1 above, wherein the intestinal organoids are intestinal organoids derived from the small intestine. 5. A method for producing intestinal epithelial-like cells as described in item 4 above, wherein the intestinal organoid derived from the small intestine is an intestinal organoid derived from the duodenum, jejunum, or ileum. 6. Intestinal epithelial-like cells prepared by the method described in item 1 above. 7. Intestinal epithelial-like cells as described in paragraph 6 above, comprising either of the following characteristics 1) and / or 2): 1) The expression of drug-metabolizing enzyme genes is at least 10 times higher than in organoid-derived cells at the start of seeding; 2) The expression of drug efflux transporter genes is at least five times higher than that of organoid-derived cells at the start of seeding. . 8 The drug-metabolizing enzyme gene is the CYP3A4 gene, as mentioned above. 7 Intestinal epithelial-like cells as described above. 9 The drug efflux transporter gene is the MDR1 gene, as mentioned above. 7 Intestinal epithelial-like cells as described above. 10 Furthermore, the transepithelial membrane resistance (TEER) of intestinal epithelial-like cells was 100-1500 Ω·cm. 2 The preceding paragraph is a cooperation of being 7 Intestinal epithelial-like cells as described above. 11 Intestinal organoids are intestinal organoids derived from the small intestine, as mentioned above. 6 Intestinal epithelial-like cells as described above. 12 .Intestinal organoids derived from the small intestine are intestinal organoids derived from the duodenum, jejunum, or ileum, as stated in the previous paragraph. 6 Intestinal epithelial-like cells as described above. 13 Previous section 6 A method for using the intestinal epithelial-like cells described above for pharmacokinetic and / or pharmacotoxicity evaluation. [Effects of the Invention]

[0011] The intestinal epithelial-like cells obtained by the method of the present invention exhibit high gene expression levels of drug-metabolizing enzymes (e.g., CYP3A4) and transporters (e.g., PEPT1, MDR1), which play important roles in drug metabolism in the small intestine, and also possess tight junction function, thus having properties similar to intestinal epithelial cells, particularly small intestinal epithelial cells. The intestinal epithelial-like cells of the present invention are superior to conventionally used Caco-2 cells in terms of the expression levels of drug-metabolizing enzymes and transporters. As a result, it has become possible to efficiently produce intestinal epithelial-like cells that can simultaneously evaluate drug metabolism and drug absorption.

[0012] Conventionally, obtaining primary cultured human intestinal epithelial cells has been difficult, and there has been no excellent cell model that accurately reflects normal human intestinal epithelial cells and can be reliably tested. However, we have been able to create intestinal epithelial-like cells that have properties closer to those of human intestinal epithelial cells. Furthermore, the intestinal epithelial-like cells of the present invention can be produced in a shorter period of time compared to human iPS cell-derived small intestinal epithelial cells or conventionally used Caco-2 cells, making them convenient and reducing the costs required for production. This will enable stable testing of pharmacokinetics such as absorption, metabolism, and excretion in the small intestine, and is expected to be very useful in the analysis and development of pharmaceuticals and food products. [Brief explanation of the drawing]

[0013] [Figure 1] The following shows the results of measuring the expression levels of various genes and TEER (transepithelial electrical resistance) when organoid-derived cells were cultured for various time periods. (A) LGR5, (B) CYP3A4, (C) MDR1 (Multidrug resistance 1, P-gp), (D) Villin, (E) CLDN3 (Claudin 3), and (F) TEER (n=3, mean ± standard deviation). In the figure, "days of monolayer culture" refers to the number of days of culture after seeding of organoid-derived cells. (Experimental Example 1) [Figure 2]This figure shows the morphological analysis results of monolayer membranes prepared from organoid-derived cells. (A) Phase-contrast microscopy image, (B) Transmission electron microscopy image, (C) Immunostaining image, (D) Alkaline phosphatase staining image, and (E) TEER measurement results (n=3, mean ± standard deviation). In the figure, "Organoid-monolayer" refers to monolayer membranes prepared from organoid-derived cells. (Experimental Example 2) [Figure 3] This figure shows the results of comparing the expression levels of genes involved in drug metabolism, transport, or regulation in monolayers prepared from organoid-derived cells and Caco-2 cells. (A) Various CYP (Cytochrome P450 family), (B) Various CES (Carboxylesterase), (C) Various UGT (Uridine diphosphate glucuronosyltransferase), (D) Various nuclear receptors, (E) Various transcription factors, (F) Apical membrane transporters, (G) Basement membrane transporters. (Each gene expression is a relative ratio with n=3, mean ± standard deviation, and Caco-2 cell value set to 1). In the figure, "Organoid-monolayer" refers to a monolayer prepared from organoid-derived cells. (Comparative Example 1) [Figure 4] The results of testing drug-metabolizing enzyme activity (CYP3A4 activity) in monolayers prepared from organoid-derived cells and Caco-2 cells are shown. (A) shows a schematic diagram of the reaction, and (B) shows the measurement results (n=3, mean ± standard deviation). In the figure, "Organoid-monolayer" refers to a monolayer prepared from organoid-derived cells. (Comparative Example 2) [Figure 5] The results of confirming the CES2 (Carboxylesterase 2) activity contributing to prodrug metabolism in monolayers prepared from organoid-derived cells and Caco-2 cells are shown. (A) shows a schematic diagram of the reaction, and (B) shows the measurement results (n=3, mean ± standard deviation). In the figure, "Organoid-monolayer" refers to a monolayer prepared from organoid-derived cells. (Comparative Example 3) [Figure 6] The results of testing the enzymatic activity of CYP3A4 and the ability to induce CYP3A4 activity in monolayers prepared from organoid-derived cells and intestinal-like epithelial cells derived from human iPS cells are shown. (A) shows the results for intestinal-like epithelial cells derived from human iPS cells (n=3, mean), and (B) shows the results for monolayers prepared from organoid-derived cells (n=3, mean). (Comparative Example 4) [Figure 7] The results of examining the P-gp transport activity and P-gp activity induction ability of monolayers prepared from organoid-derived cells and human iPS cell-derived intestinal-like epithelial cells are shown. (A) shows the results for human iPS cell-derived intestinal-like epithelial cells (n=3, mean), and (B) shows the results for monolayers prepared from organoid-derived cells (n=3, mean). (Comparative Example 5) [Modes for carrying out the invention]

[0014] This invention relates to intestinal epithelial-like cells derived from intestinal organoids, which express drug-metabolizing enzymes and drug efflux transporters and possess tight junction function. Furthermore, the invention relates to a method for producing intestinal epithelial-like cells derived from intestinal organoids.

[0015] Predicting drug absorption in the small intestine is crucial for forecasting drug pharmacokinetics, but obtaining primary cultured human intestinal epithelial cells is extremely difficult. Caco-2 cells are a cell line derived from human colon cancer and can form strong tight junctions, making them widely used as an in vitro absorption evaluation system to predict drug permeability in the small intestine. On the other hand, the main drug-metabolizing enzyme in intestinal epithelial cells, especially small intestinal epithelial cells, is CYP3A4, but unlike the human small intestine, Caco-2 cells express very little of this enzyme and cannot be used to evaluate drug metabolism capacity. Currently, human iPS cell-derived small intestinal-like epithelial cells are the only experimental system that can simultaneously evaluate drug metabolism and drug absorption in the small intestine, but there is still room for improvement in terms of function, and superior intestinal epithelial-like cells are needed.

[0016] It is known that the expression levels of certain types of drug-metabolizing enzymes (CYP3A4) increase several times or more in response to inducible drugs; this phenomenon is called CYP induction. CYP induction significantly alters the rate of drug metabolism compared to the uninduced state. CYP3A4, the main CYP isoform in the small intestine, is induced by drugs such as vitamin D3 and rifampicin.

[0017] In this specification, "intestinal organoid" refers to a three-dimensional tissue structure in which multiple cells aggregate to form a hollow portion with the luminal side facing inward within the intestinal tract, mimicking the villous-crypt structure that exists in living organisms, and forming a sphere or spheroid as a whole. Uh. Book Intestinal organoids used in this specification are particularly preferably those derived from biopsies.

[0018] In this specification, "intestinal organoids" refer to intestinal organoids derived from the small intestine (duodenum, jejunum, ileum, etc.) or the large intestine (cecum, colon, rectum, etc.), and are not particularly limited. However, organoids derived from the small intestine such as the duodenum, jejunum, or ileum are preferred, and organoids derived from the duodenum or jejunum are particularly preferred.

[0019] Intestinal organoids can be cultured by methods already known or methods to be developed in the future. The extracellular matrix used for embedding intestinal organoids is preferably one containing hydrogel, laminin (main component), type IV collagen, heparin sulfate proteoglycan, enteractin / nidogen, and a solubilized basement membrane extracted from EHS mouse sarcoma rich in ECM proteins containing various growth factors, such as Matrigel. TM (Corning) basement membranes and the like are preferred.

[0020] The medium that can be used for maintaining and culturing intestinal organoids is not particularly limited as long as it can culture small intestine tissue-derived cells. As the main medium, Advanced DMEM / F12 is used as the main component, and for example, a medium containing the medium components described in Jung et al., Nat. Med. 17, 1225-1227, 2011 and Sato et al., Gastroenterology 141, 1762-1772, 2011 can be used. Specifically, IntestiCult TM Organoid Growth Medium (Human) (STEMCELL Technologies) can be used. In the initial culture, antibiotics such as penicillin G sodium salt, streptomycin sulfate, and amphotericin B, for example, 1×Antibiotic-Antimycotic (Thermo Fisher Scientific), etc., and Rho-associated kinase inhibitors, for example, Y-27632, etc., can be appropriately included in the above medium components and used.

[0021] The split ratio of intestinal organoids can be 1:3 to 1:10. The subculture protocol can be applied by improving existing methods, for example, the report by Miyoshi et al. (Miyoshi and Stappenbeck, Nat. Protoc. 8, 2471-2482, 2013). Intestinal organoids, for subculture, for example, proteolytic enzymes such as trypsin, collagenase, dispase I, etc., and a solution containing at least any one of EDTA, EGTA, etc., for example, TrypLE Select TMThe pellet is suspended in (Thermo Fisher Scientific) and incubated at 37°C to decompose the matrix. Multiple pipetting and centrifugation are performed, the supernatant is discarded, and the pellet is resuspended (embedded) in the matrix to a concentration appropriate to the subculturing ratio. This is then dropped onto a culture substrate and allowed to solidify at 37°C. After subculturing, for example, up to day 2 of culture, the above culture medium components can be appropriately supplemented with a Rho-binding kinase inhibitor, such as Y-27632. Furthermore, throughout the entire culture period, antibiotics such as penicillin G sodium salt, streptomycin sulfate, and amphotericin B, such as 1×Antibiotic-Antimycotic, can be appropriately supplemented with the above culture medium components.

[0022] In this specification, "intestinal epithelial-like cells" are obtained by culturing intestinal organoid-derived cells separated into single cells, and are used separately from intestinal epithelial cells directly collected from living organisms.

[0023] (Generation of intestinal epithelial-like cells) The intestinal epithelial-like cells of the present invention can be produced by a method comprising the following steps. 1) The process of separating intestinal organoids into single cells; 2) A step in which intestinal organoid-derived cells, isolated into single cells, are seeded onto a culture substrate and cultured to produce a monolayer membrane.

[0024] 1) The process of separating intestinal organoids into single cells. The method for separating intestinal organoids into single cells can be any known method and is not particularly limited, but the intestinal organoids can be separated into a liquid containing, for example, a proteolytic enzyme such as trypsin, collagenase, or dispase I, or at least one of EDTA, EGTA, etc., for example, TrypLE Select TM The cells can be separated by means of, for example, filtration, centrifugation, or pipetting. Hereinafter, in this specification, cells derived from intestinal organoids that have been separated into single cells will be referred to as "intestinal organoid-derived cells."

[0025] 2) Process for fabricating a single layer film The intestinal organoid-derived cells contained in the cell suspension prepared above were divided into 1 cm 2 5.0 per unit x 10 5 ~5.0×10 6 10 units, preferably 5.0 × 10 5 ~1.0×10 6 Seed the cells onto the culture substrate to a seeding density of 1, and culture at 37±1°C under 5% CO2 conditions. After seeding the intestinal organoid-derived cells, for example, until day 2 of culture, add a Rho-binding kinase inhibitor, such as Y-27632, to the above culture medium components. TM Organoid Growth Medium (Human), etc., can be used. Intestinal organoid-derived cells reached confluence approximately 3-7 days after seeding and formed a monolayer membrane. Throughout the entire monolayer membrane culture period, antibiotics such as penicillin G sodium salt, streptomycin sulfate, and amphotericin B, for example, IntestiCult containing 1× Antibiotic-Antimycotic, were used as maintenance media. TM Organoid Growth Medium (Human), etc., can be used. In maintenance culture, the culture medium can be changed once every 1 to 7 days, preferably once every 1 to 3 days, and may be changed daily depending on the intended use of the cells. The culture substrate used is preferably coated in advance with a matrix containing hydrogel, laminin (main component), type IV collagen, heparin sulfate proteoglycan, entactin / nidogen, and a solubilized basement membrane extracted from EHS mouse sarcoma rich in ECM proteins containing various growth factors, such as Matrigel. TM It is preferable to coat the substrate with Corning. The coating procedure can be carried out by a known method, and it is also possible to use commercially available culture substrates that have been pre-coated.

[0026] (Function and properties of intestinal epithelial-like cells) The monolayer membrane of intestinal organoid-derived cells produced by the method described above expresses various genes characteristic of intestinal epithelial cells, particularly small intestinal epithelial cells, and is therefore referred to as "intestinal epithelial-like cells" below. The intestinal epithelial-like cells produced by this invention express genes related to various factors characteristic of intestinal epithelial cells, particularly small intestinal epithelial cells, such as absorptive epithelial markers of human intestinal epithelial cells, drug-metabolizing enzyme genes, drug transporters, and factors that play an important role in tight junction formation in the intestinal epithelium. In the intestinal epithelial-like cells of this invention, for example, the expression of drug-metabolizing enzyme genes is at least 10 times higher than that of organoid-derived cells at the start of seeding, and the expression of drug efflux transporter genes is at least 5 times higher than that of organoid-derived cells at the start of seeding. Examples of drug-metabolizing enzymes that play an important role in drug metabolism include CYP3A4, and examples of transporters include PEPT1 and MDR1. Furthermore, the expression of the CLDN3 gene, which plays an important role in tight junction formation in the intestinal epithelium, and TEER, an indicator of tight junction formation, are elevated. The expression level of the drug efflux transporter (MDR1) gene was significantly higher than that of Caco-2 cells, which are commonly used as a model of intestinal epithelial cells, and was close to that of the human small intestine. In vivo, human small intestinal epithelial cells are known to be tightly bound to each other, forming tight junctions. The intestinal epithelial-like cells of the present invention also showed an increasing trend in TEER over time, compared to the TEER (100-1500 Ω·cm) of Caco-2 cells. 2 They showed comparable values ​​in barrier function. Intestinal organoid-derived cells used to create intestinal epithelial-like cells tended to show a decrease in the stem cell marker gene (LGR5) upon culture, but they expressed various genes characteristic of small intestinal epithelial cells and exhibited transepithelial membrane barrier function, suggesting that they lost stem cell characteristics over time and differentiated into absorptive epithelial-like cells.

[0027] (Uses and applications of intestinal epithelial-like cells) The intestinal epithelial-like cells of the present invention can be used for pharmacokinetic evaluation, such as drug metabolism and drug absorption, and / or drug toxicity evaluation, by adding a candidate drug compound. For use in pharmacokinetic evaluation, it is preferable to use cells derived from intestinal organoids that have been cultured for 3 to 7 days, preferably within 14 days, after seeding.

[0028] As a second application, the intestinal epithelial-like cells of the present invention can be used as an active ingredient in a cell preparation. The cell preparation of the present invention is applicable to the treatment of various intestinal diseases. In particular, it is envisioned to be used as a material for the regeneration and reconstruction of damaged intestinal epithelial tissue, including dysfunction. The cell preparation may contain dimethyl sulfoxide (DMSO) or serum albumin for the purpose of protecting cells, antibiotics for the purpose of preventing bacterial contamination, and various components (vitamins, cytokines, growth factors, steroids, etc.) for the purpose of cell activation, proliferation, or differentiation induction. Furthermore, other pharmaceutically acceptable components (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, preservatives, antiseptics, physiological saline, etc.) may be contained in the cell preparation of the present invention. [Examples]

[0029] The present invention will be described in detail below with reference examples, examples, experimental examples, and comparative examples to deepen understanding of the present invention, but it goes without saying that these do not limit the scope of the present invention.

[0030] (Reference Example 1) Establishment and subculturing of human duodenal organoids This reference example describes the establishment, maintenance, and subculturing of human duodenal organoids used in the examples.

[0031] 1. Establishment of human duodenal organoids Human duodenal organoids were established using duodenal biopsy tissue from patients (6 individuals) who had given their consent and whose ethics committees at Sapporo Medical University (a public university corporation in Hokkaido), Osaka University (a national university corporation), and the National Institute of Biomedical Innovation, Health and Nutrition (a national research and development agency) had approved the research.

[0032] The human duodenal organoids prepared in this example were prepared based on previously reported methods (Sugimoto and Sato, Methods Mol. Biol. 1612, 97-105, 2017). Biopsy tissue, including crypts, obtained within 24 hours was placed in a 15 mL low-protein-adsorption tube (Sumitomo Bakelite, MS-90150) and gently washed 3-5 times with ice-cold PBS containing 1× Antibiotic-Antimycotic (Thermo Fisher Scientific). Then, 10 mL of 2.5 mM ethylenediaminetetraacetic acid (EDTA, Thermo Fisher Scientific, 15575020) PBS solution was added, and the tube was incubated at 4°C for 30 minutes with gentle shaking. The tube was left upright for 5 minutes to allow the biopsy tissue to settle, and after removing the supernatant, 5 mL of ice-cold PBS containing 1× Antibiotic-Antimycotic was added and vigorously pipetted using a 5 mL pipette. The tube was allowed to stand vertically for 1 minute to allow the biopsy tissue to settle, and the supernatant containing the crypts was collected in a 50 mL low-protein-adsorption tube (Sumitomo Bakelite, MS-52550) through a 70 μm cell strainer (Corning, 352350). This crypt separation process was repeated at least 5 times. The 50 mL tubes were centrifuged at 300-400 g for 3-5 minutes to separate the crypts (pellet) from the single cells (supernatant), and the supernatant was removed. The crypts were counted under a microscope and then denatured with Matrigel. TM (Corning, 354230) was suspended to a concentration of 10-30 crypts / μL. Next, 25-40 μL of crypt suspension Matrigel was added. TM The solution was applied to the center of each well of a 24-well plate (Thermo Fisher Scientific, 142475) using a pre-chilled pipette. The plate was placed in a CO2 incubator (5% CO2, 37°C) for 10-15 minutes to allow the Matrigel to polymerize completely. After the polymerization of the Matrigel, IntestiCult containing 1×Antibiotic-Antimycotic was added. TM500 μL of Organoid Growth Medium (Human) was added to each well, and the plate was placed in a CO2 incubator. For the first two days of culture, IntestiCult TM Organoids were cultured in Organoid Growth Medium (Human) with 10 μM Y-27632 (FUJIFILM Wako Pure Chemical, 036-24023) added.

[0033] 2. Maintenance and passage of human duodenal organoids The culture medium for human duodenal organoids was changed every 2-3 days. The passage interval was 5-7 days, and the split ratio for passage cells was between 1:3 and 1:10. The passage protocol for human duodenal organoids was primarily based on the report by Miyoshi et al. (Miyoshi and Stappenbeck, Nat. Protoc. 8, 2471-2482, 2013), with a few modifications. First, the medium was removed, and the wells were washed with 500 μL of 0.5 mM EDTA in PBS. Next, 500 μL of TrypLE Select was added. TM Add (Thermo Fisher Scientific, 12563029) to each well and place the organoids in Matrigel TM Use a 1000 μL pipette to perform TrypLE Select TM The solution was suspended in the solution and collected in a 15 mL tube (Sumitomo Bakelite, MS-90150 or Corning, 430791). The 15 mL tube was immersed in a water bath and incubated at 37°C for 57 minutes, then immersed in Matrigel. TM The material was decomposed. After incubation, 1 mL of PBS containing 1× Antibiotic-Antimycotic was added to the tube and pipetted 2-5 times using a 1000 μL pipette. The tube was then centrifuged at 400 g for 5 minutes at 4°C, the supernatant was discarded, and the pellet was transferred to Matrigel. TM The organoid suspension was then resuspended and adjusted to a concentration corresponding to the passage ratio. TMUsing a pre-cooled pipette, 25-40 μL of the solution was applied to the center of each well of a 24-well plate. The 24-well plate was placed in a CO2 incubator for 10-15 minutes, and then Matrigel was applied. TM It was completely polymerized. Matrigel TM After polymerization, IntestiCult containing 1×Antibiotic-Antimycotic TM 500 μL of Organoid Growth Medium (Human) (STEMCELL Technologies) was added to each well, and the 24-well plate was placed in a CO2 incubator. After subculturing, IntestiCult was used for the first two days of culture. TM The cells were cultured in Organoid Growth Medium (Human) with 10 μM Y-27632 added.

[0034] (Example 1) Preparation of a monolayer membrane derived from human duodenal organoid cells In this example, a human duodenal organoid-derived monolayer membrane was prepared using human duodenal organoids established, maintained, and subcultured in Reference Example 1. Although the human duodenal organoids prepared for this example can be maintained and cultured for more than 30 subcultures, those with 10 subcultures or less were used in this example.

[0035] 1. Pretreatment for the preparation of monolayer membranes from human duodenal organoid-derived cells Before culturing human duodenal organoid-derived cells into a monolayer, apply Matrigel to a 48-well plate (Thermo Fisher Scientific, 150687, medium volume: 300 μL) or a 24-well cell culture insert (Corning, 353095, medium volume: 250 μL). TM The cells were coated with Matrigel. In the 24-well cell culture insert, culture medium was filled only in the apical membrane compartment. First, as part of the coating procedure, Matrigel was used. TM Dilute with ice-cold Advanced DMEM / F-12 (Thermo Fisher Scientific, 12634010) and Matrigel TM The amount is 5-6 μg / cm³2 The mixture was added to each culture surface, and the plates were placed in a CO2 incubator and incubated for 1 hour to overnight.

[0036] 2. Preparation of human duodenal organoid-derived cells, cell seeding, and preparation of a monolayer membrane. The culture medium was removed from the wells containing the human duodenal organoids established, maintained, and subcultured in Reference Example 1 above, and the wells were washed with 500 μL of PBS containing 0.5 mM EDTA. Next, 500 μL of TrypLE Select was added. TM Add to each well, and place the organoids in Matrigel TM Use a 1000 μL pipette to perform TrypLE Select TM The solution was suspended and collected in a 15 mL tube. The 15 mL tube was immersed in a water bath and incubated at 37°C for 5-7 minutes, then removed from the Matrigel. TM I disassembled it.

[0037] After incubation, 1 mL of PBS containing 1× Antibiotic-Antimycotic was added to the tube, and the organoids were dissociated into single cells by pipetting 10-20 times using a 1000 μL pipette to produce organoid-derived cells. The tube was centrifuged at 400 g at room temperature for 5 minutes, the supernatant was discarded, and the pellet was treated with IntestiCult containing 1× Antibiotic-Antimycotic and 10 μM Y-27632. TM The organoid single-cell suspension was resuspended in Organoid Growth Medium (Human). This suspension was passed through a 70 μm cell strainer and counted using a Countess automated cell counter (Thermo Fisher Scientific, C10227) with default parameter settings, and then processed using Matrigel. TM 5.0 x 10 on a coated plate 5 ~1.0×10 6 cells / cm 2 The seeds were seeded in this manner. The culture medium after monolayering was the same as during maintenance culture, containing 1×Antibiotic-Antimycotic IntestiCultTM Organoid Growth Medium (Human) was used, and the medium was changed daily until each assay performed in the following experimental examples. 10 μM Y-27632 was added only at the time of seeding.

[0038] The seeded organoid-derived cells reached confluence approximately 3 days after seeding and formed a monolayer membrane. For the assay, monolayer membranes cultured for 3 to 7 days after seeding of organoid-derived cells were used.

[0039] (Experimental Example 1) Optimization of the post-seeding culture period of organoid-derived cells We investigated the optimization of the culture period after seeding of organoid-derived cells isolated into single cells as prepared in Example 1. After seeding organoid-derived cells, the expression of each gene encoding a stem cell marker (LGR5), a major drug-metabolizing enzyme (CYP3A4), a drug efflux transporter (MDR1), an absorptive epithelial marker (Villin), and a factor important for tight junction formation (CLDN3) was measured over time by qRT-PCR. The expression of each gene is shown as a relative ratio with n=3, mean ± standard deviation, and the value of human duodenal organoids in maintenance culture state set to 1. Furthermore, the TEER value (n=3, mean ± standard deviation), an indicator of membrane barrier function, was measured over time after cell seeding.

[0040] LGR5 levels decreased from day 0 to day 2 after cell seeding, while CYP3A4, MDR1, and Villin levels increased. This suggests that cells forming a monolayer membrane after organoid-derived cell seeding lose their stem cell properties over time and differentiate into absorptive epithelial-like cells. Additionally, CLDN3 expression and TEER levels increased over time. The TEER value at day 3 after seeding was the value typically shown for currently used Caco-2 cells (100-1500 Ω·cm). 2The results reached the specified range. This suggests that the monolayer membrane produced after organoid-derived cell seeding strengthens the epithelial barrier over time. Since Villin expression tends to decrease after 8 days post-seeding, it is considered desirable to use monolayer membranes prepared from organoid-derived cells from 3 to 7 days post-seeding when using them in pharmacokinetic studies (Figure 1).

[0041] Considering that Caco-2 cells, commonly used in pharmacokinetic studies, require 21 days of culture, and that generating intestinal-like epithelial cells from human iPS cells requires 20-30 days of culture, monolayer membranes produced from organoid-derived cells can be considered a tool that can be produced and used in a short period of time.

[0042] (Experimental Example 2) Morphological analysis of monolayer membranes prepared from organoid-derived cells To morphologically evaluate the monolayer membranes prepared in Example 1, monolayer membranes prepared 3 to 7 days after organoid-derived cell seeding were observed using phase-contrast microscopy, transmission electron microscopy, immunostaining, fluorescence microscopy or confocal laser microscopy, alkaline phosphatase staining, and phase-contrast microscopy. TEER was also measured (n=3, mean ± standard deviation).

[0043] As a result of the above, (A) a dense columnar epithelial monolayer was observed in the phase-contrast microscope image, (B) a brush border with microvilli structure ((B) left panel) and a tight junction structure ((B) right panel, black arrow) was observed in the transmission electron microscope image, and (C) immunohistochemistry confirmed the uniform expression of epithelial markers (E-cadherin; Epithelial cadherin), tight junction markers (ZO-1; Zonula occludens 1), and drug-metabolizing enzymes (CYP3A4), and the expression of Villin protein was also confirmed on the apical membrane side. Furthermore, (D) alkaline phosphatase staining (positive for small intestinal epithelium in living organisms) showed positivity in most of the cells constituting the prepared monolayer. (E) TEER was measured in the presence / absence of the absorption enhancer capric acid (C10, 10 mM, 30 minutes). TEER was lower in the group treated with C10 compared to the group treated with solvent only (DMSO). Therefore, it was concluded that the fabricated monolayer film possessed normal barrier function.

[0044] Based on the above, the monolayer membrane prepared in Example 1 is a polar, absorptive epithelial monolayer membrane with sufficient barrier function and is therefore considered applicable to pharmacokinetic studies (Figure 2). In the following comparative examples, the monolayer membrane prepared in Example 1 will be referred to as the "intestinal epithelial-like cell" of the present invention.

[0045] (Comparative Example 1) Comparison of various gene expression levels The expression levels of genes involved in drug metabolism, transport, or regulation were compared between the intestinal epithelial-like cells of the present invention (7 days of culture) and currently widely used Caco-2 cells (derived from colorectal cancer, cultured for 21 days after confluence to form a monolayer membrane). The expression of each gene encoding the factors shown in (A) to (G) was measured over time by qRT-PCR. The expression of each gene is shown as a relative ratio with n=3, mean ± standard deviation, and the value for Caco-2 cells set to 1 (see Figure 3).

[0046] (A) Various CYPs (Cytochrome P450 family) (B) Various CES (Carboxylesterase) (C) Various UGTs (Uridine diphosphate glucuronosyltransferase) (D) Various nuclear receptors (E) Various transcription factors (F) Apex membrane transporter (G) Gene expression levels of basement membrane transporters

[0047] Regarding various CYPs, which are responsible for the majority of drug metabolism, all isoforms were highly expressed in the intestinal epithelial-like cells of this invention compared to Caco-2 cells. In particular, CYP3A4, which is known to contribute to the metabolism of a great many pharmaceuticals, was expressed approximately 5700 times more than in Caco-2 cells. Regarding CES, which is known to be responsible for the metabolism of many prodrugs, the expression level of CES1, which is highly expressed in the large intestine, was lower than in Caco-2 cells, while the expression level of CES2, which is highly expressed in the small intestine, was higher than in Caco-2 cells, as previously reported. Furthermore, regarding UGTs, which contribute to drug conjugation reactions, the trends differed among isoforms. As for nuclear receptors and transcription factors, PXR (Pregnane X Receptor) and VDR (Vitamin D receptor) were highly expressed compared to Caco-2 cells, suggesting a high possibility that rifampicin and activated vitamin D3 induce CYP3A4 expression. Regarding apical membrane transporters, in addition to the major drug efflux transporters BCRP (Breast cancer resistant protein) and MDR1, the peptide absorption transporter PEPT1 (Peptide transporter 1) was expressed at a higher level than in Caco-2 cells. Regarding basement membrane transporters, all transporters except OSTα (Organic solute transporter α) showed lower expression compared to Caco-2 cells. Considering that OSTα forms a heterodimer with OSTβ on the membrane and possesses transport activity, it is expected that the transport capacity on the basement membrane side of the intestinal epithelial-like cells of the present invention is lower than that of Caco-2 cells. From the above, it is considered that the gene expression levels of major drug metabolism and transport-related molecules and their regulators are higher in the intestinal epithelial-like cells of the present invention than in Caco-2 cells, and that they can be applied to various pharmacokinetic studies.

[0048] (Comparative Example 2) Comparison of CYP3A4 activity CYP3A4 activity was confirmed in Comparative Example 1, the intestinal epithelial-like cells of the present invention, and Caco-2 cells using a luciferase precursor compound. CYP3A4 activity was measured using the P450-Glo CYP3A4 Assay with Luciferin-IPA (Promega), and the luminescence intensity was measured using Lumat LB9507 (Berthold Technologies) (see Figure 4(A)). The activity was also confirmed in systems with and without 10 μM Ketoconazole as a CYP3A4 inhibitor. The obtained CYP3A4 activity (luminescence intensity) was corrected for total protein content. The Pierce BCA Protein Assay (Thermo Fisher Scientific) was used to measure total protein content.

[0049] The intestinal epithelial-like cells of the present invention showed higher CYP3A4 activity compared to Caco-2 cells, and this correlated with gene expression (see Figure 4(B)).

[0050] (Comparative Example 3) Comparison of CES2 activity Similar to Comparative Examples 1 and 2, fluorescein diacetate (FD) hydrolysis assays were performed on intestinal epithelial-like cells and Caco-2 cells of the present invention to compare the enzymatic activity of CES2 contributing to the metabolism of the prodrug. S9 fractions were prepared from each cell type, and these S9 fractions were reacted with FD. The fluorescence intensity of fluorescein produced by CES2 cleaving FD (a precursor of fluorescein) at two sites indicated by arrows was measured using TriStar LB941 (Berthold Technologies) (see Figure 5(A)). Systems containing and without 1 mM loperamide as a CES2 inhibitor were also examined. The obtained CES2 activity was corrected for total protein content. Pierce BCA Protein Assay (Thermo Fisher Scientific) was used to measure protein content.

[0051] The intestinal epithelial-like cells of the present invention showed higher CES2 activity compared to Caco-2 cells, and this correlated with gene expression (see Figure 5(B)).

[0052] (Comparative Example 4) Comparison of CYP3A4 activity and CYP3A4 activity induction ability The enzymatic activity and inducibility of CYP3A4 were confirmed in the intestinal epithelial-like cells of the present invention and intestinal-like small intestine cells derived from human iPS cells developed and produced in the laboratory to which the inventor belongs (Takayama, K., et al., Cell. Mol. Gastroenterol. Hepatol. 8, 513-526, 2019). Testosterone was used as a substrate, and CYP3A4 activity was quantified by measuring the amount of the metabolite, the 6β-hydroxylated product, over time using LC-MS. 100 nM active vitamin D3 and 20 μM rifampicin were treated as CYP3A4 inducers for 48 hours at the end of the culture period of each cell, after which the substrate was added. The substrate treatment was performed without the inducers.

[0053] Human iPS cell-derived intestinal epithelial cells also showed CYP3A4 activity and CYP3A4 induction ability, but the intestinal epithelial-like cells of the present invention showed more than 40 times the CYP3A4 activity and CYP3A4 induction ability compared to human iPS cell-derived intestinal epithelial cells, confirming that they have higher metabolic enzyme activity (see Figure 6(A)(B)). Each result is the average value for n=3.

[0054] (Comparative Example 5) Comparison of P-gp transport activity and P-gp activity induction ability Similar to Comparative Example 4, the P-gp transport activity and P-gp activity induction ability were confirmed for the intestinal epithelial-like cells of the present invention and human iPS cell-derived small intestinal-like epithelial cells developed and produced in the laboratory to which the inventor belongs (Takayama, K., et al., Cell. Mol. Gastroenterol. Hepatol. 8, 513-526, 2019). Transcellular transport tests were performed using digoxin as a substrate in both the absorption direction (permeation from the apical membrane side to the basement membrane side: a to b) and the excretion direction (permeation from the basement membrane side to the apical membrane side: b to a). After seeding the cells in a culture insert, the intestinal epithelial-like cells were cultured for 3 to 7 days, and the human iPS cell-derived small intestinal-like epithelial cells were cultured for 32 days, after which the culture medium was removed. After adding a buffer solution without the substrate to the receiving chamber and initiating the transport test, samples were taken from the receiving chamber at time, and the amount of digoxin that moved was measured by LC-MS to calculate the P-gp transport rate in each direction. 100 nM active vitamin D3 and 20 μM rifampicin were treated as P-gp activity inducers for 48 hours at the end of the culture period for each cell, after which the substrate was added. The substrate treatment was performed without the inducers.

[0055] In human iPS cell-derived intestinal epithelial cells, both the absorption direction (a to b) and the excretion direction (b to a) were around 1, regardless of the presence or absence of an inducer. In contrast, the intestinal epithelial-like cells of the present invention showed low values ​​in the absorption direction (a to b) and high values ​​in the excretion direction, regardless of the presence or absence of an inducer (see Figures 7(A) and 7(B)). Each result represents the average value for n=3. The numbers indicated above the bars in Figures 7(A) and 7(B) represent the ER (Efflux Ratio) value. The ER (Efflux Ratio) is calculated as (b to a) / (a to b). A larger value indicates higher excretory transport activity. P-gp is a protein present on the cell membrane that facilitates the extracellular excretion of cytotoxic compounds and other substances. Evaluating pharmacokinetics and / or drug toxicity in vitro from the dynamics of P-gp is important for the effective development of safe pharmaceuticals, and the intestinal epithelial-like cells of the present invention have been confirmed to be useful for drug evaluation. [Industrial applicability]

[0056] As detailed above, the intestinal epithelial-like cells of the present invention, which consist of a single membrane prepared from organoid-derived cells, are superior intestinal epithelial-like cells because they express markers expressed by intestinal epithelial cells, particularly small intestinal epithelial cells, express drug-metabolizing enzymes and transporters, and possess tight junction function. In particular, the expression levels of drug-metabolizing enzymes and transporters are superior to those of conventionally used Caco-2 cells.

[0057] Primary cultured human small intestinal epithelial cells are difficult to obtain, and individual differences in their properties are also a problem. In contrast, the intestinal epithelial-like cells of the present invention, which consist of a single membrane produced from organoid-derived cells, can be easily produced. Furthermore, the intestinal epithelial-like cells of the present invention can be produced in a shorter period of time compared to human iPS cell-derived small intestinal-like epithelial cells and conventionally used Caco-2 cells, making them convenient and reducing the costs required for production. This enables stable testing of pharmacokinetics such as absorption, metabolism, and excretion in the small intestine. By adding candidate compounds such as pharmaceuticals, they can be used for pharmacokinetic and / or drug toxicity evaluation, and are expected to be very useful in the analysis and development of pharmaceuticals and food products.

Claims

1. A method for producing human intestinal epithelial-like cells, including the following steps: 1) The process of separating human intestinal organoids derived from biopsy into single cells; 2) The human intestinal organoid-derived cells isolated into single cells are cultured on a 1 cm culture medium. 2 5.0 per unit x 10 5 ~5.0 x 10 6 Human intestinal organoid-derived cells were seeded on a culture substrate to achieve a cell seeding density of 1, and cultured at 37±1°C in 5% CO2. 2 A process of culturing under specific conditions to produce a single-layer membrane.

2. The method for producing human intestinal epithelial cells according to claim 1, wherein the step of producing a single-layer membrane in 2) above is a step of culturing human intestinal organoid-derived cells for 2 to 14 days after seeding.

3. The method for producing human intestinal epithelial cells according to claim 1, wherein the step of producing the monolayer membrane in 2) above is a step of culturing human intestinal organoid-derived cells for 3 to 7 days after seeding.

4. A method for producing human intestinal epithelial cells according to claim 1, wherein the human intestinal organoid is a human intestinal organoid derived from the small intestine.

5. A method for producing human intestinal epithelial-like cells according to claim 4, wherein the human small intestine-derived intestinal organoid is a human intestinal organoid derived from the duodenum, jejunum, or ileum.

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