Inflammatory bowel disease model production using patient-derived intestinal organoids, and use thereof

Abstract

The present invention relates to inflammatory bowel disease model production using patient-derived intestinal organoids, and use thereof, wherein the inflammatory bowel disease model produced by an optimized production method for inflammatory bowel disease model production can be used as a patient-customized bowel disease model by constructing an evaluation method for inflammatory bowel disease drugs.

Description

Creation of an inflammatory bowel disease model using patient-derived intestinal organoids and its use

[0001] The present invention relates to the production of an inflammatory bowel disease model using patient-derived intestinal organoids and their use, and more specifically, to the establishment of an optimized production method for producing an inflammatory bowel disease model and an inflammatory bowel disease drug evaluation method using the same, for use as a patient-tailored disease model.

[0002] Organoids are organ-specific cell aggregates created by culturing or recombining cells isolated from stem cells or organ cells. They have a structure and function very similar to each organ, and are characterized by the ability to differentiate as closely as possible to actual tissues. They are also called 'mini-organs' or 'pseudo-organs'.

[0003] Specifically, organoid culture is not simply a spheroid, but a small organ that implements the function of an actual organ, and when established as a disease model, it exhibits various symptoms caused by the disease, so it is being used in various ways as a research model for new drug development (Kondo J, Inoue M. Cells. 2019;8(5):470). Therefore, if an appropriate disease model is established and the efficacy of clinical drugs is verified in the established in vitro model, 3D organoids can be utilized for drug screening for new drug development.

[0004] Inflammatory bowel disease (IBD) is a complex condition caused by genetic and environmental factors. It is a rare, chronic, and incurable disorder that causes abnormal inflammation and ulceration in the intestines. While the prevalence of IBD is rapidly increasing worldwide, a lack of patient specificity and understanding of the disease mechanism limits successful treatment. IBD is broadly classified into ulcerative colitis (UC) and Crohn's disease (CD), and these two types differ in their onset sites, symptoms, and histological characteristics.

[0005] The types of intestinal organoids for creating in vitro IBD models with high human mimicry can be broadly classified according to their origin into (1) human adult stem cell-derived intestinal organoids and (2) human induced pluripotent stem cell (hiPSC)-derived intestinal organoids.

[0006] Human adult stem cell-derived intestinal organoids are models that are differentiated into structures similar to the intestinal mucosa through three-dimensional culture using stem cells isolated from the patient's intestinal tissue (primary tissue). They are capable of self-renewal and differentiation in vitro, allowing for long-term storage and reuse in biobanks.

[0007] Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids are known to preserve genetic and epigenetic mutations from the time the tissue was isolated from the patient, making them useful as in vitro disease models that can reproduce the pathological characteristics of each patient. They also have the advantage of enabling drug response analysis in patient groups with diverse clinical information, and can be produced through stepwise differentiation techniques. However, compared to adult stem cell-derived intestinal organoids, they require high cost and time, and have limitations in terms of differentiation maturity. Therefore, advanced technologies are needed to overcome these issues in the future.

[0008] To date, various prior studies have been conducted on the creation of inflammation models and drug evaluation methods using patient-derived intestinal organoids. However, when creating models by inducing inflammation in normal or mouse-derived organoids, it is difficult to reproduce the characteristics of IBD patients and reflect clinical diversity. In addition, since the lesion characteristics of UC and CD patient tissues appear differently, studies limited to one patient group are difficult to apply to all IBD patients. Furthermore, when organoids that have not been sufficiently differentiated and matured are used, their biomimeticity is low, making it difficult to apply them to the creation of disease models and drug responsiveness. Inflammation models using patient-derived intestinal organoids reported to date have limitations in that they are developed for transcriptome, genome, and phenotype analysis of patient organoids, or for studies limited to specific inflammatory mechanisms.

[0009] Accordingly, the inventors of the present invention were researching the introduction of an inflammatory model optimized for efficacy evaluation, which could serve as a platform for evaluating IBD drugs that reflects patient-specific characteristics. They developed a method for constructing an in vitro inflammatory model by establishing a biobank of intestinal organoids derived from patients with various inflammatory bowel diseases. This model can be used to analyze patient-specific IBD-related genetic mutations, and by establishing a linkage between the evaluation of various IBD reference drugs and clinical information, the inventors completed an invention that can be utilized as a method for providing information for patient-tailored drug selection.

[0010] The present invention aims to provide a method for producing an inflammatory bowel disease model using a patient-derived intestinal organoid, an inflammatory bowel disease model produced by the method, a method for screening an inflammatory bowel disease prevention or treatment agent using the inflammatory bowel disease model, and a method for providing information for selecting a patient-tailored inflammatory bowel disease prevention or treatment method.

[0011] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0012] According to one embodiment of the present invention, a method for producing an inflammatory bowel disease model is provided, comprising: 1) culturing a patient-derived intestinal organoid; 2) inducing differentiation of the cultured intestinal organoid of 1) in a medium (WRN-conditioned medium, WRN-CM) containing Wnt3a, R-spondin, and Noggin, thereby culturing the organoid to maturity; and 3) inducing inflammation in the intestinal organoid matured according to 2).

[0013] In the present invention, the patient in step 1) may be a patient with inflammatory bowel disease (IBD) including at least one selected from the group consisting of intestinal lesions accompanying Behcet's disease, ulcerative colitis, Crohn's disease, hemorrhagic rectal ulcer, and ileal pouchitis.

[0014] In the present invention, the patient-derived intestinal organoid of step 1) may be a method of culturing intestinal organoids from stem cells derived from a normal part or a lesion part of the patient's colon.

[0015] In the present invention, the stem cell may be a human adult stem cell or a human induced pluripotent stem cell.

[0016] In the present invention, the maturation culture of step 2) may be performed by including WRN-CM at a concentration of 20 to 40% (v / v) in the intestinal organoid maintenance medium.

[0017] In the present invention, the maturation culture of step 2) may be performed for 15 to 22 days.

[0018] In the present invention, the step of inducing inflammation in step 3) may be a method of inducing inflammation for 30 to 60 hours with one or more inflammation-inducing factors selected from the group consisting of TNF-α (Tumor necrosis factor-α), IL-1b (Interleukin 1 beta), and Flagellin.

[0019] According to another embodiment of the present invention, an inflammatory bowel disease model manufactured by a method for manufacturing an inflammatory bowel disease model is provided.

[0020] According to another embodiment of the present invention, a method for screening for an agent for preventing or treating inflammatory bowel disease is provided, comprising the steps of treating a candidate substance in the inflammatory bowel disease model; and comparing the reduction in the level of inflammatory factor expression or the level of cell viability in the inflammatory bowel disease model treated with the candidate substance with a positive control model.

[0021] In the present invention, the inflammatory bowel disease model treated with the above candidate substance can increase the expression level of E-cadherin protein.

[0022] In the present invention, if the level of expression of the inflammatory factor is reduced or the level of cell viability is increased compared to the positive control model, it can be determined that the candidate substance is an agent for preventing or treating inflammatory bowel disease in the inflammatory bowel disease model.

[0023] According to another embodiment of the present invention, a method for providing information for selecting a method for preventing or treating inflammatory bowel disease tailored to a patient is provided, comprising the steps of treating a candidate substance in the inflammatory bowel disease model, and comparing the decrease in the level of inflammatory factor expression or the level of cell viability by the candidate substance in the inflammatory bowel disease model treated with the candidate substance with a positive control model, thereby selecting the candidate substance as a method for preventing or treating inflammatory bowel disease in a patient of interest.

[0024] An inflammatory bowel disease model using a patient-derived intestinal organoid according to one embodiment of the present invention and a drug evaluation method using the same can be used to predict the presence or absence of drug responsiveness in a patient group with various clinical information and to study the mechanism thereof, and can be used to predict clinical results of new drug candidates as an alternative model to animal testing.

[0025] Figure 1 is a schematic diagram illustrating a method for screening or providing information based on the responsiveness of a drug using an intestinal organoid inflammation model derived from an IBD patient, taking into account the clinical diversity aimed at by the present invention.

[0026] Figure 2a is a diagram showing the results of confirming changes in the composition of Wnt3a, R-spondin, and Noggin components of organoid medium according to L-WRN cell culture conditions.

[0027] Figure 2b is a diagram showing the results of confirming Wnt signaling pathway activity according to L-WRN cell culture conditions.

[0028] Figure 2c is a diagram showing the results of intestinal organoid culture according to WRN-CM content.

[0029] Figure 3a is a diagram showing the results of intestinal organoid culture according to the culture period.

[0030] Figure 3b is a diagram showing the results of qRT-PCR analysis performed on intestinal organoids.

[0031] Figure 3c is a diagram showing the results of analyzing intestinal organoids using immunofluorescence staining.

[0032] Figure 4a is a diagram showing the results of 11 patient organoids and common genetic mutations related to IBD through whole exome sequencing (WES) analysis.

[0033] Figure 4b is a diagram showing the top 29 gene mutations among common genes related to IBD.

[0034] Figure 4c is a diagram showing the KEGG pathway database for patient organoid gene mutations.

[0035] Figure 5 is a diagram showing the establishment of inflammation-inducing conditions according to Experimental Example 2-1, and comparing the cytokine and chemokine secretion patterns of each patient organoid before and after treatment with inflammation-inducing factors (TNF-α, IL-1b, Flagellin).

[0036] Figure 6a is a diagram analyzing cell viability according to the combination and concentration of various inflammation-inducing factors.

[0037] Figure 6b is a diagram analyzing the level of inflammation-related gene expression (IL-8, IL-1β, TNF-α) according to the combination and concentration of various inflammation-inducing factors.

[0038] Figure 6c is a diagram analyzing IL-8 levels according to the combination and concentration of various inflammatory inducing factors.

[0039] Figure 6d is a diagram analyzing cell viability according to the combination and concentration of various inflammatory inducing factors and the inflammatory treatment time.

[0040] Figure 6e is a diagram analyzing IL-8 levels according to the combination and concentration of various inflammatory inducing factors and the time of inflammation treatment.

[0041] Figure 7a is a diagram analyzing cell viability according to inflammation-inducing conditions in the intestinal organoids of the affected and normal parts of each patient.

[0042] Figure 7b is a diagram analyzing IL-8 levels according to inflammation-inducing conditions in the intestinal organoids of the affected and normal parts of each patient.

[0043] Figure 7c is a diagram showing the expression level of intestinal cell-related markers according to inflammation-inducing conditions analyzed using a fluorescence microscope.

[0044] Figure 7d is a diagram showing the expression level of intestinal cell-related markers according to inflammation-inducing conditions analyzed by qRT-PCR.

[0045] Figure 8a is a diagram showing clinical information of an intestinal organoid inflammation model.

[0046] Figure 8b is a diagram showing the results of transcriptome analysis of the intestinal organoid inflammation model.

[0047] Figure 8c is a diagram showing differentially expressed genes in an intestinal organoid inflammation model.

[0048] Figure 8d is a diagram showing the results of Gene ontology (GO) term analysis of the intestinal organoid inflammation model.

[0049] Figure 9a is a diagram showing the results of analyzing cell viability according to drug and culture period in an intestinal organoid inflammation model derived from an IBD patient.

[0050] Figure 9b is a diagram showing the results of analyzing the amount of IL-8 secreted according to drug and culture period in an intestinal organoid inflammation model derived from an IBD patient.

[0051] Figure 10a is a diagram showing the results of analyzing the differences in drug response by patient based on cell viability.

[0052] Figure 10b is a diagram showing the results of analyzing the differences in drug response by patient based on the amount of IL-8 secreted.

[0053] Figure 11 is a diagram showing the IBD drug effect profiling according to the patient-specific inflammation model in Experimental Example 5, and the IBD drug effect according to the patient's normal area and lesion area.

[0054] Figure 12a is a diagram showing the results of analyzing changes in intestinal cell wall markers using a fluorescence microscope after IBD drug treatment in a patient-specific inflammation model.

[0055] Figure 12b is a diagram analyzing changes in intestinal cell wall markers after IBD drug treatment in a patient-specific inflammation model.

[0056] Hereinafter, examples will be given to provide a more specific explanation. However, the following examples are illustrative only and the scope of the present invention is not limited thereto.

[0057] According to one embodiment of the present invention, there is provided a method comprising: 1) culturing a patient-derived intestinal organoid;

[0058] 2) A step of culturing the cultured intestinal organoids of the above 1) to induce differentiation in a medium containing Wnt3a, R-spondin, and Noggin (WRN-conditioned medium, WRN-CM) and maturation; and

[0059] 3) A method for producing an inflammatory bowel disease model, comprising: a step of inducing inflammation in an intestinal organoid matured according to 2) above;

[0060] As used herein, the term "organoid" refers to a cell mass with a three-dimensional structure, and refers to a reduced and simplified version of an organ produced through an artificial culture process rather than being collected or acquired from animals or the like. The origin of the cells constituting the organoid is not limited. Furthermore, due to their self-renewal and differentiation capabilities, they can be cultured in three dimensions, and the organoid can have an environment that allows interaction with the surrounding environment during cell growth.

[0061] Accordingly, the intestinal organoid of the present invention can be an excellent model for observing the development of disease treatments and the like, as it almost perfectly mimics the intestinal processes that actually interact in vivo.

[0062] In the present invention, the term "intestinal organoid" may mean "human intestinal organoids (hIO)", and means an organoid that includes intestinal gland cells, villus-like structures, and four major specialized cells (absorptive enterocytes, Paneth cells, enteroendocrine cells, and goblet cells), and reproduces the diversity and structure of epithelial cells of the large or small intestine, and can perform basic physiological functions.

[0063] In the present invention, the patient of step 1) is an inflammatory bowel disease (IBD) patient including at least one selected from the group consisting of intestinal lesions accompanying Behcet's disease, ulcerative colitis, Crohn's disease, hemorrhagic rectal ulcer, and ileal pouchitis, most preferably including ulcerative colitis or Crohn's disease, or both ulcerative colitis and Crohn's disease, but is not limited thereto.

[0064] In the present invention, the patient-derived intestinal organoid of step 1) is cultured from stem cells derived from a normal part or a lesion part of the patient's colon. For example, tissue from a normal part or a lesion part (affected part) of the colon of a patient with inflammatory bowel disease can be isolated through a biopsy and cultured.

[0065] In the present invention, the stem cell may be a human adult stem cell or a human induced pluripotent stem cell, and most preferably, a human adult stem cell.

[0066] In the present invention, the maturation culture of step 2) can be performed in an intestinal organoid maintenance medium containing WRN-CM at a concentration of 20 to 40%, 22 to 37%, or 25 to 35% (v / v), and most suitably, in an intestinal organoid maintenance medium containing WRN-CM at a concentration of 27 to 33% (v / v), for example, in an intestinal organoid maintenance medium containing WRN-CM at a concentration of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% (v / v).

[0067] For example, if the concentration of WRN-CM is less than 20% (v / v), a problem of cell death may occur, and if the concentration of WRN-CM exceeds 40% (v / v), a problem of going beyond the range of the intended Wnt signaling pathway activation may occur.

[0068] The above WRN-conditioned medium, WRN-CM, is a medium containing Wnt3a, R-spondin, and Noggin. WRN-CM manufactured using the L-WRN cell line (CRL-3276, ATCC) is used, and a commonly used medium commonly used in the relevant technical field can be used.

[0069] In the present invention, the maturation culture of step 2) may be performed for 15 to 22 days, 16 to 21 days, or 17 to 20 days, and most suitably may be performed for 18 to 20 days, and for example, may be performed for 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or 22 days.

[0070] For example, if the above maturation culture is performed for less than 15 days, a problem may arise in which the shape of the organoid is spherical and thus unsuitable for conducting experiments, and if the above maturation culture is performed for more than 22 days, a problem may arise in which cell necrosis occurs due to insufficient supply of nutrients and oxygen within the cells.

[0071] In the present invention, the step of inducing inflammation in step 3) may be induced for 30 to 60 hours, 35 to 55 hours, or 40 to 53 hours by at least one inflammation-inducing factor selected from the group consisting of TNF-α (Tumor necrosis factor-α), IL-1b (Interleukin 1 beta), and Flagellin, and most suitably may be induced for 45 to 50 hours in terms of maintaining the viability of the intestinal organoids at 80% or more, for example, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, It can be induced for 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, and 60 hours.

[0072] For example, if the time for inducing the inflammation is less than 30 hours, the viability of the intestinal organoids manufactured according to the present invention may increase, resulting in a problem in which the desired inflammation expression is not sufficiently expressed. If the time for inducing the inflammation is more than 60 hours, the viability of the intestinal organoids may decrease significantly, resulting in a problem in which it is difficult to predict recovery in response to the drug.

[0073] According to another embodiment of the present invention, the present invention relates to an inflammatory bowel disease model manufactured by the method for manufacturing the inflammatory bowel disease model described above.

[0074] According to another embodiment of the present invention, a method for screening for an agent for preventing or treating inflammatory bowel disease is provided, comprising: treating a candidate substance in an inflammatory bowel disease model manufactured according to the present invention; and comparing the reduction in the level of inflammatory factor expression or the level of cell viability caused by the candidate substance in the inflammatory bowel disease model treated with the candidate substance with a positive control model.

[0075] In the present invention, the candidate substance is a substance for preventing or treating inflammatory bowel disease, and may be a commonly used therapeutic substance or a new substance.

[0076] For example, among the above candidates, commonly used therapeutic agents may be 5-ASA: 5-Aminosalicylic acid (anti-inflammatory); SSZ: Sulfasalazine (anti-inflammatory); DEX: Dexamethasone (anti-inflammatory, corticosteroids); AZA: Azathioprine (immune suppressor); 6-MP: 6-Mercaptopurine (immune suppressor); MTZ: Metronidazole (antibiotic); or INF: Infliximab (anti-TNFa).

[0077] In the present invention, the inflammatory bowel disease model treated with the above candidate substance can increase the expression level of E-cadherin protein.

[0078] The above E-cadherin protein is a protein that induces intercellular bonding, and an increase in the expression of the E-cadherin protein may indicate the therapeutic effect of the candidate substance by repairing damaged junction proteins and improving tight junctions between intestinal epithelial cells.

[0079] In the present invention, if the level of expression of the inflammatory factor is reduced or the level of cell viability is increased compared to the positive control model, the candidate substance may be judged to be an agent for preventing or treating inflammatory bowel disease in the inflammatory bowel disease model, which may be a method for screening an agent for preventing or treating inflammatory bowel disease.

[0080] The above inflammatory factor refers to an inflammatory cytokine, and may be any one or more inflammatory factors selected from the group consisting of CCL1, CCL 2, CCL 5, CXCL1, CXCL10, CXCL11, CXCL12, G-CSF, GM-CSF, ICAM-1, IL-1β, IL-1ra, IL-2, IL-8, IL-18, IL-21, MIF, Serpin E1, and TNF-α, and most preferably, IL-8, but is not limited thereto.

[0081] The above positive control model refers to a state in which inflammation is induced by treating a patient organoid with an inflammatory factor, but only inflammation is induced without treating a drug or candidate substance, and refers to a control group for confirming the effect of a candidate substance from an inflammatory bowel disease model manufactured according to the present invention.

[0082] More specifically, the term negative control (NC) used in the present invention refers to intestinal organoids derived from a normal or lesioned area of ​​a patient's colon, positive control (PC) refers to intestinal organoids prepared by inducing inflammation in the negative control group, and experimental group refers to intestinal organoids treated with a drug or candidate substance in the positive control group.

[0083] The inflammatory bowel disease model manufactured according to the present invention may most suitably mean a positive control.

[0084] According to another embodiment of the present invention, there is provided a method for providing information for selecting a method for preventing or treating inflammatory bowel disease tailored to a patient, comprising the steps of treating a candidate substance in an inflammatory bowel disease model, and comparing the decrease in the level of inflammatory factor expression or the level of cell viability in the inflammatory bowel disease model treated with the candidate substance with a positive control model that only induces inflammation, thereby selecting the candidate substance as a method for preventing or treating inflammatory bowel disease in a patient of interest.

[0085] Accordingly, it is possible to construct intestinal organoids or intestinal organoid biobanks derived from patients with various inflammatory bowel diseases to produce in vitro inflammation models, and analyze patient-specific IBD-related gene mutations through genetic profiling (genome, transcriptome) using these, and establish a link between evaluations of various drug effects and clinical information to construct an optimized analysis system or screening method, and provide information for selecting patient-tailored drugs using this.

[0086] Hereinafter, the present invention will be described in detail through the following examples and experimental examples.

[0087] However, the following examples and experimental examples are only illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

[0088] Example

[0089] Example 1. Real clinical profile of IBD patients

[0090] The present invention was conducted on 11 IBD patients, and first, a clinical profile was created by receiving specific clinical information of the IBD patients, such as the patient's IBD type (UC, CD), age, sex, severity of lesion, lesion site, and medication history.

[0091] Example 2. Production of intestinal organoids derived from IBD patients

[0092] 1. Cultivation and freezing

[0093] In the same IBD patient as Example 1, normal and lesion tissues of the colon were isolated through biopsy, and in vitro expansion was performed with reference to existing literature to culture intestinal organoids of the normal and lesion regions, respectively (Nature Medicine 2011, 17:1225-27).

[0094] Specifically, maintenance medium for intestinal organoids was Advanced DMEM / F12 medium (12634-010, Thermo), supplemented with 100 units penicillin / streptomycin (15140-122, Thermo), 1X GlutaMax (35050-061, Thermo), 1X B-27 supplement (12587-010, Thermo), 1X N2 Supplement (17502-048, Thermo), 1 mM N-Acetyl-L-cysteine ​​(A9165, Sigma), 1 mM Nicotinamide (N0636, Sigma), 50% WRN-CM (Wnt-3a / R-spondin / Noggin conditioned medium), 10 nM Gastrin 1 (G9020, Sigma), 500 nM MA-83-01 (SML0788, Sigma), and 50 ng / ml EGF. (236-EG, R&D systems), and 100 μg / ml primocin (Ant-pm-05, Invivogen) were prepared (see Gastroenterology 2011, 141(5):1762-72).

[0095] At this time, WRN-CM was manufactured using L-WRN cell line (CRL-3276, ATCC), and L-WRN was cultured at 10 x 10 6 cells / 150 cm 2Intestinal organoids were cultured by seeding at a density of (150 pi dish), culturing for 3 days, and then collecting and filtering the culture solution every 24 hours for 4 days, and then frozen and stored.

[0096] 2. Thawing

[0097] The intestinal organoids cryopreserved using the method of Example 2-1 were rapidly thawed in a 37°C water bath, washed, and centrifuged. The cell pellet was mixed with Matrigel (356231, Corning) kept cold on ice and placed in a 12-well or 14-well culture plate in the form of a dome of 20 μl each. After solidifying for more than 15 minutes in a 37°C incubator, intestinal organoid maintenance medium was added and cultured (500 μl of medium per 20 μl dome). 10 μM Y-27632 (1254, Tocris) was added to the maintenance medium for the first 2 days after seeding, and the medium was replaced once every 2-3 days. Organoid passage was performed every 5–7 days after seeding. Organoids contained in Matrigel were collected by pipetting with cold phosphate-buffered saline (PBS), centrifuged, and dissociated into single cells using TrypLE (12604-013, Thermo). The dissociated organoid single-cell pellet was diluted 1:4, mixed with fresh Matrigel, solidified into a dome shape, and then cultured in maintenance medium.

[0098] When patient-derived intestinal organoids were cultured in maintenance medium, the number of organoids increased and they grew into sphere-shaped structures within 3D Matrigel.

[0099] Example 3. Establishment of culture conditions for maturation of intestinal organoids derived from IBD patients.

[0100] 1. Determination of medium composition according to culture conditions

[0101] WRN-CM contains Wnt-3a, R-spondin, and Noggin, which are necessary for stem cell maintenance and proliferation. These components were confirmed through ELISA assay and Wnt signaling pathway activation analysis using a Wnt3a reporter cell line (TCF / LEF Reporter-HEK293).

[0102] Based on this, the content of WRN-CM was adjusted to establish conditions optimized for the survival and differentiation of patient-derived organoids.

[0103] Specifically, to reduce the content of Wnt3a, R-spondin, and Noggin in WRN-CM, the seeding density of L-WRN cell line was 0.25, 0.5, 1, and 10 × 10 6 cells / 150 cm 2 (150pi dish) was varied, and the content ratio of Wnt3a, R-spondin, and Noggin in WRN-CM of each condition was investigated through ELISA analysis, and the results are shown in Figures 2a and 2b.

[0104] According to Figure 2a, 1 x 10 6 cells / 150 cm 2 In the composition of WRN-CM harvested by seeding, the contents of Wnt3a, R-spondin, and Noggin were each higher than those of the conventional method (10 x 10 6 cells / 150 cm 2 ) is about 30% less than that of the reference strain, and is 0.25 or 0.5 x 10 6 cells / 150 cm 2 It was confirmed that there was no significant difference compared to the case where seeding was performed.

[0105] According to Fig. 2b, activity was also observed in the Wnt signaling pathway activation analysis using the Wnt3a reporter cell line, but 1 x 10 6 cells / 150 cm2 In the case of the existing method (10 x 10 6 cells / 150 cm 2 ) was confirmed to be reduced by approximately 30%.

[0106] Next, to compare the results of intestinal organoid culture according to WRN-CM content, intestinal organoids cultured in maintenance medium for 5 days were additionally cultured for 7 days in WRN-CM at concentrations of 0%, 30%, and 50% (v / v), and the results confirmed by optical microscopy are shown in Fig. 2c.

[0107] At this time, WRN-CM with a concentration of 50% (v / v) was prepared using the existing method (10 x 10 6 cells / 150 cm 2 ) is included in the maintenance medium for intestinal organoids at a concentration of 50% (v / v).

[0108] In addition, WRN-CM at a concentration of 30% (v / v) was produced by the conventional method (10 x 10 6 cells / 150 cm 2 ) was maintained at only 30% (v / v) in the medium, or the WRN content was reduced by adjusting the L-WRN seeding density (1 x 10 6 cells / 150 cm 2 ) refers to the case where WRN-CM produced by the method is included in the maintenance medium at 50% (v / v).

[0109] Specifically, the maintenance medium refers to a medium manufactured according to Example 2-1.

[0110] According to Fig. 2c, no cell death was observed in intestinal organoids derived from normal induced pluripotent stem cells (iPSCs), but cell death was observed in patient-derived intestinal organoids cultured at a concentration of 0% (v / v) of WRN-CM. In addition, no cell death was observed at concentrations of 30% and 50% (v / v) of WRN-CM.

[0111] Accordingly, in the case of patient-derived intestinal organoids that underwent the maturation culture step under the condition of 30% (v / v) WRN-CM, it was confirmed that differentiation was induced through the reduction of the desired Wnt signaling pathway activity without the occurrence of apoptosis.

[0112] This experiment was performed using patient-derived intestinal organoids cultured to maturity under conditions of 30% (v / v) WRN-CM.

[0113] 2. Maturation conditions according to incubation period

[0114] Intestinal organoids were cultured for a long period of time without passage in a three-dimensional Matrigel according to the medium conditions of Example 3-1 above, and confirmed with an optical microscope. As a result, it was confirmed that the size of the organoids increased with the culture period, and that they differentiated from a sphere-shaped structure into an irregular (budding) structure similar to the shape of the intestine. The results are shown in Fig. 3a.

[0115] According to Figure 3a, intestinal organoids cultured for a short period of time maintained a spherical shape with a size of 50 to 200 μm, whereas intestinal organoids matured for a long period of time were observed to have an irregular shape with a size of 300 to 500 μm. This suggests that the size, shape, and number of budding of intestinal organoids before and after maturation culture may vary from patient to patient.

[0116] Specifically, when cultured for more than 24 days, the size of organoid cells increased significantly, and necrosis occurred due to insufficient nutrient and oxygen supply within the organoid cells. Accordingly, the maturation culture period was optimized to 18 to 20 days.

[0117] Next, intestinal organoids were cultured in Matrigel for 6, 14, and 20 days and qRT-PCR analysis was performed, and the results are shown in Fig. 3b.

[0118] According to Figure 3b, it was confirmed that the expression of cell-specific markers constituting the intestinal organoids was highest at day 20.

[0119] Specifically, a significant increase in markers expressed in differentiated intestinal cells, such as CDX2 (intestine progenitor), VIL1 (enterocyte), and MUC2 (goblet cell), was observed, and the expression of LGR5 stem cells, which may be involved in the regeneration of intestinal organoids, and EPHB2 markers related to proliferation were also observed at high levels. In addition, a significant increase in Occludin and ZO-1, which are tight junction markers important for the formation of the epithelial barrier of differentiated intestinal cells, was also observed.

[0120] Next, the intestinal organoids after 6 days of culture and after 20 days of culture were compared by immunofluorescence staining, as shown in Figure 3c.

[0121] According to Fig. 3c, it was confirmed that the expression of stem cells (LGR5, CD44), differentiated enterocytes (KRT20, Villin), goblet cells (MUC2), enteroendocrine cells (CHGA), and epithelial junctions (OCLN, F-actin) that constitute the intestinal organoids was high after 20 days of culture.

[0122] Accordingly, in the case of patient-derived intestinal organoids that underwent a maturation culture step for 18 to 20 days, differentiation occurred in an atypical form similar to the appearance of the intestine, and it was confirmed that the expression of stem cells (LGR5, CD44), differentiated enterocytes (KRT20 and Villin), goblet cells (MUC2), enteroendocrine cells (CHGA), and epithelial junctions (OCLN and F-actin) constituting the intestinal organoids was excellent.

[0123] In summary, it was found that intestinal organoids derived from IBD patients that were cultured for 18 to 20 days at a concentration of 30% (v / v) of WRN-CM were most suitable for producing an inflammatory bowel disease model as intended in the present invention.

[0124] Experimental Example 1: Genome-based Profiling of Patient-Derived Intestinal Organoids

[0125] Whole exome sequencing (WES) analysis of IBD patient-derived intestinal organoids prepared according to the above Example 3 was used to identify common genetic mutations related to IBD in 11 patients, and patient-specific mutation diversity and drug efficacy mechanism-related profiling analysis was performed, and the results are shown in Fig. 4.

[0126] Experimental Example 2: Creation of an inflammatory bowel disease model using patient-derived intestinal organoids.

[0127] 1. Establishment of conditions for inducing inflammation

[0128] The results of cytokine / chemokine profiling analysis performed on patient-derived intestinal organoids prepared according to Example 3 above by treating them with inflammatory inducers TNF-α (Tumor necrosis factor-α), IL-1β (Interleukin 1β), and Flagellin are shown in Figure 5.

[0129] Here, #11, #13, #19, and #27 represent the intestinal organoids of each patient who underwent the experiment.

[0130] According to Figure 5, it was confirmed that the types and amounts of inflammatory factors secreted by each patient's intestinal organoid were different, and since it was confirmed that the expression of IL-8 (Interleukin 8) was high among several factors in common, IL-8 was used as an indicator of inflammation induction.

[0131] Next, in order to confirm the responsiveness of the patient-derived intestinal organoids manufactured according to Example 3 according to the inflammation-inducing factor, the mixture 0~4 (M0~4) conditions were presented with various combinations and concentrations of inflammation-inducing factors, and the viability of the intestinal organoids according to each condition, the change in the expression of inflammation-related genes (IL-8, IL-1β, TNF-α), the change in the IL-8 level (ELISA), and the change in the viability and inflammation level (IL-8 expression) of the intestinal organoids according to the inflammation treatment time (24, 48, 72 h) were confirmed, and the results are shown in Fig. 6.

[0132] According to Figures 6a, 6b, and 6c, it was confirmed that as the combination and concentration of inflammation-inducing factors increased, viability decreased, and the expression of inflammation-related genes and IL-8 levels increased in a similar trend.

[0133] According to Fig. 6d, it was confirmed that when inflammation was induced for 24 hours, viability increased, indicating that inflammation was not sufficient, and when inflammation was induced for 72 hours, viability dropped to less than 80%. According to Fig. 6e, it was confirmed that the IL-8 level was significantly reduced for 72 hours compared to 24 and 48 hours.

[0134] Accordingly, it was found that when inflammation was induced for 48 hours, inflammation was sufficiently induced and cell viability was also maintained, and thus it was found to be the most suitable model for producing the inflammatory bowel disease model aimed at in the present invention.

[0135] In addition, among the various mixture conditions, the M0 condition was excluded because it was expected to be difficult to predict recovery of drug responsiveness as organoid viability was significantly reduced to less than 80%.

[0136] 2. Patient-specific differences in inflammation induction

[0137] The results of confirming the differences in inflammation induction and changes in intestinal cell-related markers according to inflammation-inducing conditions (M1, M2, and M3) in the intestinal organoids of the affected and normal regions for each patient are shown in Figure 7.

[0138] According to Figures 7a and 7b, although there were differences in the degree of inflammation induction among patients, it was confirmed that the viability and IL-8 change trends of intestinal organoids in the inflamed and normal areas were similar.

[0139] According to Figures 7c and 7d, the expression of markers related to tight junctions between intestinal cells and cells was confirmed, and it was confirmed that the expression of tight junction-related markers was reduced before and after the induction of inflammation.

[0140] Accordingly, the analysis of inflammation induction through IL-8 quantification was significant, and the viability of intestinal organoids was maintained at more than 80%, while the expression of tight junction-related markers was somewhat reduced, and 48 hours and M2 (IL-1b: Flagellin = 1:5) were established as inflammation induction conditions.

[0141] At this time, in order to observe the responsiveness of anti-TNFα drugs in the future, the mixture condition excluding TNFα among the inflammation-inducing factors, i.e. M2, was given priority.

[0142] Experimental Example 3. Transcriptome Analysis in an Intestinal Organoid Inflammation Model

[0143] The results of transcriptome analysis before and after inflammation in the IBD patient-derived intestinal organoid inflammation model manufactured according to the above experimental example 2 are shown in Fig. 8.

[0144] According to Figures 8a and 8b, more than 500 genes showed significant changes before and after inflammation in the intestinal organoid inflammation models of patients 12, 14, and 20.

[0145] According to Fig. 8c, differential gene expression analysis confirmed that the expression of cytokine, immune, and inflammation-related genes significantly increased after inflammation in all patients' intestinal organoids, and that there were differences in the expression of genes and changes in response to inflammation depending on the intestinal organoid of the patient.

[0146] According to Figure 8d, the results of Gene ontology (GO) term analysis showed that cytokine and inflammation-related network genes were upregulated, and cell junction assembly-related genes were downregulated.

[0147] Experimental Example 4. Evaluation of IBD therapeutic drugs in an intestinal organoid inflammation model derived from IBD patients.

[0148] In the IBD patient-derived intestinal organoid inflammation model constructed according to the above Experimental Example 3, the following seven reference drugs for IBD were included and cultured, and the viability and IL-8 secretion amount according to the culture period were analyzed, and the results are shown in Figures 9a and 9b.

[0149] According to Figures 9a and 9b, when the intestinal organoid inflammation model derived from an IBD patient was cultured for 20 days with drugs included, it was confirmed that the survival rate was higher for all therapeutic drugs and the amount of IL-8 secreted was reduced compared to when the model was cultured for 6 days.

[0150] Accordingly, in order to establish a method for evaluating IBD therapeutic drugs, it was found that a maturation model in an IBD patient-derived intestinal organoid inflammation model was important, and a drug evaluation method using an IBD patient-derived intestinal organoid inflammation model was established.

[0151] [Reference drug]

[0152] NC: negative control

[0153] PC: positive control (inflammation w / o drug)

[0154] 5-ASA: 5-Aminosalicylic acid (anti-inflammatory)

[0155] SSZ: Sulfasalazine (anti-inflammatory)

[0156] DEX: Dexamethasone (anti-inflammatory, corticosteroids)

[0157] AZA: Azathioprine (immune suppressor)

[0158] 6-MP: 6-Mercaptopurine (immune suppressor)

[0159] MTZ: Metronidazole (antibiotic)

[0160] INF: Infliximab (anti-TNFa)

[0161] Experimental Example 5. Profiling the Effects of IBD Treatment Drugs in Patient-Specific Inflammation Models

[0162] The results of confirming differences in drug response by patient using the drug evaluation method of the IBD patient-derived intestinal organoid model constructed according to the above experimental example 4 are shown in Figures 10 and 11.

[0163] Figures 10a and 10b show the results of analyzing the viability and IL-8 secretion amount according to the drug for each patient. It was confirmed that the efficacy of three representative IBD treatment drugs (5-ASA, AZA, and INF) differed significantly among patients, and thus the link between the drug efficacy in the patient's intestinal organoid model and the actual clinical profile established in Example 1 was confirmed.

[0164] Specifically, in the case of patient 12, the actual clinical profile showed that the drug response was maintained with 5-ASA and AZA (AZA was administered in cases where 5-ASA did not improve well), and it was confirmed that the drug response using the intestinal organoid of patient 12 was significantly greater for AZA than for 5-ASA.

[0165] In the case of patient 14, according to the actual clinical profile, the condition worsened while being maintained on 5-ASA and there was insufficient response to AZA, so anti-TNFa was administered. However, it was confirmed that the drug response using the intestinal organoid of patient 14 was higher for 5-ASA and INF than for AZA.

[0166] In the case of patient 20, according to the actual clinical profile, there was a clinical record of improvement in symptoms with 5-ASA alone, and in the drug response using the intestinal organoid of the patient, significant alleviation of IL-8 levels was observed not only with 5-ASA but also with other drugs.

[0167] Figure 11 shows the results of analyzing the viability and IL-8 secretion amount according to the drug in each part of the patient. Specifically, the results of the drug evaluation profiling of intestinal organoids according to the normal and lesion parts of the patient showed that the drug responsiveness tended to increase in organoids derived from the lesion or affected part compared to the normal part.

[0168] At this time, odd numbers indicate normal areas, and even numbers indicate lesion areas. For example, #1 and #2 indicate normal and lesion areas in the same patient. (#1 and #2, #3 and #4, #5 and #6, #7 and #8, #9 and #10; #11 and #12; #13 and #14; #15 and #16; #19 and #20; #21 and #22; #23 and #24; and #25 and #26; a total of 12 patients)

[0169] Experimental Example 6. Analysis of changes in intestinal cell wall markers after IBD drug treatment in a patient-specific inflammation model.

[0170] According to the drug evaluation method of the IBD patient-derived intestinal organoid model constructed according to the above experimental example 4, the results of comparative analysis of organoid cell junction markers after drug-induced inflammation relief in the patient-derived intestinal organoid inflammation model are shown in Fig. 12.

[0171] According to Figures 12a and 12b, the expression of E-cadherin protein, which was reduced after inflammation occurred, was found to increase after drug treatment, confirming that the damaged junction protein was restored by the therapeutic drug.

[0172] The results according to the above examples and experimental examples demonstrate that inflammatory bowel disease can be successfully modeled using a patient-derived intestinal organoid model with inflammatory bowel disease.

[0173] In addition, by referring to the responses of candidate substances or therapeutic drugs shown in patient-derived intestinal organoid models, it is possible to select agents for the prevention or treatment of inflammatory bowel disease in actual patients, as well as provide information for selecting patient-tailored treatment methods.

[0174] As the specific parts of the present invention have been described in detail above, it will be understood by those skilled in the art that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention, and it is clear that the scope of the present invention is not limited thereto.

[0175] Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims

1. 1) Step of culturing patient-derived intestinal organoids; 2) A step of culturing the cultured intestinal organoids of the above 1) to induce differentiation in a medium containing Wnt3a, R-spondin, and Noggin (WRN-conditioned medium, WRN-CM) and maturation; and 3) A method for producing an inflammatory bowel disease model, comprising: a step of inducing inflammation in an intestinal organoid matured according to 2); 2. In paragraph 1, A method for producing an inflammatory bowel disease model, wherein the patient in step 1) is an inflammatory bowel disease (IBD) patient with at least one selected from the group consisting of intestinal lesions accompanying Behcet's disease, ulcerative colitis, Crohn's disease, hemorrhagic rectal ulcer, and ileal pouchitis.

3. In paragraph 1, A method for producing an inflammatory bowel disease model, wherein the patient-derived intestinal organoid of step 1) is cultured from stem cells derived from a normal part or a lesion part of the patient's colon.

4. In paragraph 3, A method for producing an inflammatory bowel disease model, wherein the above stem cells are human adult stem cells or human induced pluripotent stem cells.

5. In paragraph 1, A method for producing an inflammatory bowel disease model, wherein the maturation culture of step 2) above is performed in an intestinal organoid maintenance medium containing WRN-CM at a concentration of 20 to 40% (v / v).

6. In paragraph 1, A method for producing an inflammatory bowel disease model, wherein the maturation culture of step 2) above is performed for 15 to 22 days.

7. In paragraph 1, A method for producing an inflammatory bowel disease model, wherein the step of inducing inflammation in step 3) above is induced for 30 to 60 hours with at least one inflammatory inducing factor selected from the group consisting of TNF-α (Tumor necrosis factor-α), IL-1b (Interleukin 1 beta), and Flagellin.

8. An inflammatory bowel disease model manufactured by the method for manufacturing an inflammatory bowel disease model according to any one of claims 1 to 7.

9. A step of treating a candidate substance in the inflammatory bowel disease model of Article 8; and A method for screening for an agent for preventing or treating inflammatory bowel disease, comprising a step of comparing the level of inflammatory factor expression or cell viability by the candidate substance in an inflammatory bowel disease model treated with the candidate substance with a positive control model.

10. In paragraph 9, A method for screening an agent for preventing or treating inflammatory bowel disease, wherein the inflammatory bowel disease model treated with the above candidate substance has an increased expression level of E-cadherin protein.

11. In paragraph 9, A method for screening for an agent for preventing or treating inflammatory bowel disease, wherein the candidate substance is determined to be an agent for preventing or treating inflammatory bowel disease in the inflammatory bowel disease model when the level of expression of the inflammatory factor is reduced or the level of cell viability is increased compared to the positive control model.

12. A step of treating a candidate substance in the inflammatory bowel disease model of Article 8; and Including a step of selecting the candidate substance as a preventive or therapeutic agent for inflammatory bowel disease in a patient by comparing the level of inflammatory factor expression or cell viability level by the candidate substance in an inflammatory bowel disease model treated with the candidate substance with a positive control model. A method for providing information for selecting a patient-tailored method of preventing or treating inflammatory bowel disease.

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