Construction method of children Crohn disease intestinal tract organoid

Abstract

The invention relates to the technical field of cell biology, in particular to a construction method of children Crohn's disease intestinal tract organoid. According to the invention, a natural immune microenvironment is established by using intestinal tissues from children patients suffering from Crohn's disease and immune cells residing in the tissues, so that the experimental limitation of insufficient peripheral blood volume of children is avoided; p-nitro branstatin is innovatively introduced into a culture system, so that the formation of organoid is effectively increased. Compared with the traditional inhibitor blebbistatin of NMII, the p-nitro branstatin has the advantages that the defects of poor solubility and poor light stability are avoided, the composition stability of the culture medium is favorably maintained, and the p-nitro branstatin is more suitable for a system which is a gas-liquid type organ and is longer in liquid change interval and needs to be cultured for a long time. The invention provides a new model for exploration of related mechanisms of children Crohn's disease and evaluation of drug efficacy.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, and in particular to a method for constructing intestinal organoids in children with Crohn's disease. Background Technology

[0002] Organoids are 3D organoid structures formed by the in vitro self-assembly of stem cells. They can differentiate into various characteristic cells found in tissues and exhibit cell-cell interactions and spatial morphology, reproducing key functions and structures of real human organs in vitro, and possessing stable phenotypic and genetic characteristics. To simulate the intestinal immune microenvironment of children with Crohn's disease, and addressing the lack of organoid models for pediatric Crohn's disease, Chinese patent CN120041371A established a drug sensitivity evaluation system by co-culturing intestinal organoids derived from pediatric inflammatory bowel disease patients with immune cells derived from the patients' peripheral blood. However, this system has high requirements for simultaneously obtaining tissue and peripheral blood samples; the amount of blood collected from children is sometimes limited, and the resuscitation viability of frozen immune cells is poor, resulting in insufficient numbers of immune cells for related experiments. Furthermore, the method provided by Chinese patent CN120041371A requires the organoids to form and expand to a sufficient quantity before being mixed with peripheral blood mononuclear cells for co-culture experiments. From obtaining the tissue to passage expansion and completing the co-culture experiment, it is estimated to take about three weeks, which is inefficient. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a method for constructing intestinal organoids for children with Crohn's disease. The method provided by this invention eliminates the need for peripheral blood co-culture, utilizing tissue-resident immune cells and gut-associated cells to construct an intestinal organoid model for children with Crohn's disease. The experimental cycle is approximately 10 days, shortening the experimental time, lowering the entry threshold for experimental samples, and improving experimental efficiency.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for constructing intestinal organoids in children with Crohn's disease, comprising the following steps: Intestinal tissue derived from children with Crohn's disease was seeded onto the upper chamber membrane of a Transwell plate, solidified, and then cultured in the lower chamber with organoid culture medium to obtain intestinal organoids from children with Crohn's disease. The organoid culture medium is based on Advanced DMEM / F12 medium and also includes the following components at the following concentrations: 10-12 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 100-110 U / mL penicillin, 100-110 µg / mL streptomycin, 50-55 µg / mL primordial antibiotic, 2-2.2 mM GlutaMAX supplement, 1×B27 supplement, 1×N2 supplement, 5-6 mM nicotinamide, and 1.25-1.30 mM... N-acetylcysteine, 100-110 ng / mL recombinant human Noggin protein, 50-55 ng / mL recombinant human EGF protein, 500-550 ng / mL recombinant human R-spondin1 protein, 200-300 ng / mL recombinant human WNT3a protein, 50-60 ng / mL recombinant human FGF10 protein, 20-22 ng / mL recombinant human FGF7 protein, 25-28 ng / mL recombinant human HGF protein, 50-75 ng / mL recombinant human IL-2 protein, 0.5-0.6 μM A83-01, 10-12 μM SB202190, 10-12 μM Y-27632 and 10-15 μM p-nitrobrestatin.

[0005] Preferably, the organoid culture medium is based on Advanced DMEM / F12 medium and further comprises the following components at the following concentrations: 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 100 U / mL penicillin, 100 µg / mL streptomycin, 50 µg / mL Primocin antibiotic, 2 mM GlutaMAX supplement, 1×B27 supplement, 1×N2 supplement, 5 mM nicotinamide, 1.25 mM N-acetylcysteine, 100 ng / mL recombinant human Noggin protein, 50 ng / mL recombinant human EGF protein, 500 ng / mL recombinant human R-spondin1 protein, 200 ng / mL recombinant human WNT3a protein, 50 ng / mL recombinant human FGF10 protein, 20 ng / mL recombinant human FGF7 protein, 25 ng / mL recombinant human HGF protein, 75 ng / mL recombinant human IL2 protein, 0.5 μM A83-01, 10 μM SB202190, and 10 µM... Y-27632 and 10μM p-nitrobrestatin.

[0006] Preferably, the culture conditions include a temperature of 36.5~37℃ and a CO2 concentration of 4.5~5%.

[0007] Preferably, the organoid culture medium is replaced every 72-96 hours during the culture process.

[0008] Preferably, the culture time is 3 to 14 days.

[0009] Preferably, the intestinal tissue is pretreated before inoculation; the pretreatment includes washing the intestinal tissue 3-4 times in a PBS solution containing 5-7 vol% penicillin-streptomycin-gentamicin triple antibodies, and then cutting it into 1-3 mm pieces. 3 The organizational block.

[0010] Preferably, the intestinal tissue includes one or more of fresh intestinal tissue biopsy samples and surgically removed samples.

[0011] Preferably, the biopsy samples are ≥6, the fresh intestinal tissue biopsy samples are obtained within 6 hours, and the surgically removed samples are obtained within 24 hours.

[0012] Preferably, the curing time is 10-15 minutes.

[0013] This invention provides the application of the construction method described above to obtain intestinal organoids for Crohn's disease in the study of related mechanisms and / or drug efficacy evaluation of Crohn's disease in children.

[0014] Beneficial effects: This invention utilizes intestinal tissue derived from children with Crohn's disease and the immune cells residing in the tissue to construct a natural immune microenvironment, avoiding experimental limitations due to insufficient peripheral blood volume in children. The innovative introduction of para-nitroblebbistatin into the culture system successfully constructed inflammatory intestinal organoids associated with children with Crohn's disease, effectively increasing organoid formation. Compared to the traditional NMII inhibitor blebbistatin, para-nitroblebbistatin avoids its poor solubility and photostability, facilitating the maintenance of stable culture medium composition and making it more suitable for long-term culture systems like "gas-liquid" organoids, which require long-term culture and long media exchange intervals. This invention provides a new model for exploring the mechanisms of Crohn's disease in children and evaluating drug efficacy. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1-3 Immunofluorescence staining image of intestinal tissue samples from pediatric Crohn's disease patients; Figure 4 Bright-field plots of intestinal organoids constructed for different NHII inhibitors in pediatric Crohn's disease patients; Figure 5 Bright-field growth map of intestinal organoids in pediatric Crohn's disease patients constructed for this invention; Figure 6 Immunofluorescence staining image of intestinal organoids from pediatric Crohn's disease patients constructed for this invention; Figure 7 This invention provides an immunocellular staining pattern for identifying intestinal organoids from pediatric Crohn's disease patients. Detailed Implementation

[0017] This invention provides a method for constructing intestinal organoids in children with Crohn's disease, comprising the following steps: Intestinal tissue derived from children with Crohn's disease was seeded onto the upper chamber membrane of a Transwell plate, solidified, and then cultured in the lower chamber with organoid culture medium to obtain intestinal organoids from children with Crohn's disease. The organoid culture medium is based on Advanced DMEM / F12 medium and also includes the following components at the following concentrations: 10-12 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 100-110 U / mL penicillin, 100-110 µg / mL streptomycin, 50-55 µg / mL primordial antibiotic, 2-2.2 mM GlutaMAX supplement, 1×B27 supplement, 1×N2 supplement, 5-6 mM nicotinamide, and 1.25-1.30 mM... N-acetylcysteine, 100-110 ng / mL recombinant human Noggin protein, 50-55 ng / mL recombinant human EGF protein, 500-550 ng / mL recombinant human R-spondin1 protein, 200-300 ng / mL recombinant human WNT3a protein, 50-60 ng / mL recombinant human FGF10 protein, 20-22 ng / mL recombinant human FGF7 protein, 25-28 ng / mL recombinant human HGF protein, 50-75 ng / mL recombinant human IL-2 protein, 0.5-0.6 μM A83-01, 10-12 μM SB202190, 10-12 μM Y-27632 and 10-15 μM p-nitrobrestatin.

[0018] In one embodiment, the organoid culture medium is based on Advanced DMEM / F12 medium and further comprises the following components at the following concentrations: 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 100 U / mL penicillin, 100 µg / mL streptomycin, 50 µg / mL Primocin antibiotic, 2 mM GlutaMAX supplement, 1×B27 supplement, 1×N2 supplement, 5 mM nicotinamide, 1.25 mM N-acetylcysteine, 100 ng / mL recombinant human Noggin protein, 50 ng / mL recombinant human EGF protein, 500 ng / mL recombinant human R-spondin1 protein, 200 ng / mL recombinant human WNT3a protein, 50 ng / mL recombinant human FGF10 protein, 20 ng / mL recombinant human FGF7 protein, 25 ng / mL recombinant human HGF protein, 75 ng / mL recombinant human IL2 protein, 0.5 μM A83-01, 10 μM MSB202190, and 10 µM Y-27632 and 10μM p-nitrobrestatin.

[0019] Unlike the embedding method where the matrix gel is immersed in the culture system, in the "air-liquid" method, the upper layer of tissue is in contact with air, while the lower culture medium permeates through the membrane into the base gel and then into contact with the tissue sample. Therefore, to improve the formation of intestinal organoids in the "air-liquid" culture system, this invention innovatively introduces p-nitroblebbistatin into this culture system, effectively increasing organoid formation. Compared to the traditional NMII inhibitor blebbistatin, para-nitroblebbistatin avoids the drawbacks of poor solubility and photostability, which helps maintain the stability of the culture medium composition and is more suitable for systems like "air-liquid" organoid culture where medium change intervals are long and long-term culture is required.

[0020] As one embodiment, the method for preparing the transwell plate includes the following steps: S1) Transwell plates should be made of transparent membranes to facilitate continuous observation during culture. The pore size should be between 0.4 and 3.0 μm to facilitate nutrient penetration. S2) Prepare the base coat by mixing solutions A, B, and C in an 8:1:1 ratio according to the instructions. Use a 1 mL pipette to quickly spread 300-450 μL of the base coat onto the upper membrane of the chamber of a 12-well transwell plate. Gently tap the plate to spread the coat evenly and avoid introducing air bubbles. For 6-well transwell plates, add 1000-1250 μL of base coat and follow the same procedure. S3) Transfer the well plates to a cell culture incubator at 36.5-37℃ and 4.5-5% CO2 for 10-15 min for curing. After curing, place 1 mL and 250 μL of PBS solution containing 5-7 vol% penicillin-streptomycin-gentamicin triple antibodies in the lower chamber and upper layer of the base gel, respectively, in a 12-well plate. Similarly, place 3 mL and 1 mL of PBS solution in the lower chamber and upper layer of the base gel, respectively, in a 6-well plate, and incubate at 36.5-37℃ and 4.5-5% CO2 for 10-15 min to moisten the base gel. S4) Discard the PBS, add an equal volume of organoid culture medium from S2 to the lower layer of the chamber and the upper layer of the base gel, and incubate in a cell culture incubator at 36.5~37℃ and 4.5~5%CO2 to allow the culture medium to soak into the base gel until the tissue block is planted. Then discard the culture medium in the upper layer of the chamber.

[0021] S5) Resuspend the cleaned and shredded tissue blocks with the base coat, and add 100 μL of tissue resuspension to the base coat that has been cured on the upper layer of the 12-well chamber; similarly, add 450 μL of tissue resuspension to the base coat that has been cured on the upper layer of the 6-well chamber, and culture in a cell culture incubator at 36.5~37℃ and 4.5~5% CO2.

[0022] In one implementation, two tissue samples are implanted in each well of the transwell plate. In another implementation, the curing time is 10-15 minutes.

[0023] As one implementation method, 1-1.2 mL of organoid culture medium was added to the lower layer of a 12-well chamber after solidification, and 2.5-3 mL of human organ culture medium was added to the lower layer of a 6-well chamber. The chambers were then incubated at 36.5-37°C in a 4.5-5% CO2 cell culture incubator.

[0024] In one embodiment, the culture conditions include a temperature of 36.5–37°C and a CO2 concentration of 4.5–5%. In another embodiment, the organoid culture medium is replaced every 72–96 hours during the culture process.

[0025] In one implementation method, the culture time is 3 to 14 days.

[0026] As one implementation method, the intestinal tissue is pretreated before inoculation; the pretreatment includes: washing the intestinal tissue 3-4 times in a PBS solution containing 5-7 vol% penicillin-streptomycin-gentamicin triple antibodies, and then cutting it into 1-3 mm pieces. 3 The organizational block.

[0027] In one embodiment, the intestinal tissue includes one or more of fresh intestinal tissue biopsy samples and surgically removed samples.

[0028] In one implementation, the fresh intestinal tissue biopsy samples are ≥6 pieces, meaning the fresh intestinal tissue biopsy samples are those obtained within 6 hours; the surgically removed samples are those obtained within 24 hours. This invention selects biopsy or surgically removed samples within specific timeframes, ensuring higher cell viability and improving the success rate of model construction.

[0029] The method provided by this invention requires only the patient's intestinal tissue without the addition of peripheral blood. It utilizes the tissue's own resident immune cells and gut-associated cells to construct an intestinal organoid model for childhood Crohn's disease, solving the problem of model construction being impossible when peripheral blood is difficult to obtain or when peripheral blood cells are in poor condition, thus improving model construction efficiency. Secondly, Chinese patent CN120041371A requires organoid formation and expansion to a sufficient quantity before co-culturing with peripheral blood mononuclear cells, which takes approximately three weeks from obtaining the tissue to passage expansion and co-culturing completion. The method provided by this invention achieves P0 generation organoid formation in approximately 10 days, shortening the experimental time and further improving model construction efficiency. This invention provides technical support for exploring the mechanisms of childhood Crohn's disease and evaluating personalized and precise clinical treatment.

[0030] This invention utilizes intestinal tissue derived from children with Crohn's disease to successfully construct an intestinal organoid model for childhood Crohn's disease. This model retains the functional cells of intestinal tissue while preserving the immune cells residing in the tissue, providing a model system for the study of the pathological mechanisms of childhood Crohn's disease, clinical personalized precision treatment, and drug efficacy evaluation.

[0031] Based on the above advantages, the present invention provides the application of the construction method described in the above technical solution to obtain intestinal organoids for Crohn's disease in the study of related mechanisms and / or drug efficacy evaluation of Crohn's disease in children.

[0032] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a method for constructing intestinal organoids for Crohn's disease in children, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Preparation Example The culture media used in the following examples are as follows: The organoid culture medium contains the following components: Advanced DMEM / F12, 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 100 U / mL penicillin, 100 µg / mL streptomycin, 50 μg / mL Primocin antibiotic, 2 mM GlutaMAX supplement, 1×B27 supplement, 1×N2 supplement, 5 mM nicotinamide, 1.25 mM N-acetylcysteine, 100 ng / mL recombinant human Noggin protein, 50 ng / mL recombinant human EGF protein, 500 ng / mL recombinant human R-spondin1 protein, 200 ng / mL recombinant human WNT3a protein, 50 ng / mL recombinant human FGF10 protein, 20 ng / mL recombinant human FGF7 protein, 25 ng / mL recombinant human HGF protein, 75 ng / mL recombinant human IL2 protein, and 0.5 μM A83-01, 10 μM MSB202190, 10 μM Y-27632 and 10 μM p-nitroblebbistatin.

[0034] The recombinant proteins were sourced as follows: recombinant human Noggin protein (10267-HNAH), recombinant human EGF protein (10605-HNAE), recombinant human R-spondin1 protein (11083-HNAS), recombinant human FGF10 protein (10573-HNAE), recombinant human FGF7 protein (10210-H07E), recombinant human HGF protein (10463-HNAS), and recombinant human IL-2 protein (GMP-11848-HNAE) were all purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (China); recombinant human WNT3a protein (C22R) was purchased from Suzhou Nearshore Protein Technology Co., Ltd.

[0035] Example: Construction of inflammatory bowel organoids from children with Crohn's disease using the "gas-liquid" method After obtaining intestinal tissue biopsy samples from children with inflammatory bowel disease, place them in 1.5 mL sterile centrifuge tubes and keep them on ice. First, rinse with PBS solution containing 1% penicillin-streptomycin, then transfer to tissue preservation solution. Transfer to the laboratory for sample processing within 2 hours. Using sterile ophthalmic forceps, transfer the tissue to 1×PBS (containing 5% penicillin-streptomycin-gentamicin triple antibody solution) and rinse. Then transfer to 1.5 mL centrifuge tubes, add an appropriate amount of 1×PBS (containing 5% penicillin-streptomycin-gentamicin triple antibody solution), shake 2-3 times for deep cleaning, and keep on ice until needed.

[0036] Following the instructions of the collagen reagent kit (WAKO Collagen Gel Culturing Kit, 638-00781), solutions A, B, and C were mixed sequentially in an 8:1:1 ratio to prepare the base gel. Using a 1 mL pipette, 300 μL of the base gel was quickly spread onto the upper membrane of a 12-well transwell plate. The plate was gently tapped to ensure even distribution of the gel, avoiding the introduction of air bubbles. The plate was then transferred to a 37°C, 5% CO2 cell culture incubator for curing for 10 min. After curing, 1 mL and 250 μL of PBS solution containing 5% penicillin-streptomycin-gentamicin triple antibodies were placed in the lower chamber and upper layer of the base gel, respectively. The plates were incubated at 37°C, 5% CO2 for 15 min to moisten the base gel. The PBS was then discarded and replaced with 1 mL and 250 μL of organoid culture medium, and the plates were incubated at 37°C, 5% CO2.

[0037] Remove the cleaned intestinal tissue, place it in a 35 mm petri dish, place it on an ice pack, and cut it into pieces approximately 1 mm thick. 3 One-third of the tissue blocks of various sizes were randomly selected, fixed in 4% paraformaldehyde, and embedded in paraffin. Immunofluorescence staining was performed on the tissue blocks using KRT8 (intestinal columnar epithelial marker), CD3 (T lymphocyte marker), CD14 (monocyte marker), and CD19 (B lymphocyte marker). Normal intestinal tissue was used as a control group. The staining results are as follows: Figures 1-3 As shown in the figure. The results showed that the patient's intestinal tissue exhibited positive expression of immune cell markers CD3 (T lymphocyte marker), CD14 (monocyte marker), and CD19 (B lymphocyte marker), and the number of these markers was significantly increased compared to the control tissue. The biopsy sampling and delivery conditions of this invention effectively maintained the immune cells in the patient's tissue samples, demonstrating the potential to provide a natural immune microenvironment.

[0038] The remaining tissue blocks were washed once with PBS solution containing 5 vol% penicillin-streptomycin-gentamicin triple antibodies, resuspended in base gel, and seeded onto coated chamber membranes. After solidification in a 37°C, 5% CO2 cell culture incubator, organoid culture medium was added to the lower layer of the chamber for culture and observation. The medium was changed every 3 days (denoted as the para-nitroblebbistatin group). Control group 1 (without NMII inhibitor) and control group 2 (with blebbistatin) were also set up. The differences between control group 1 and control group 2 and the para-nitroblebbistatin group are as follows: Control group 1: The organoid culture medium was similar to that of the para-nitroblebbistatin group, except that it did not contain para-nitroblebbistatin; Control group 2: The organoid culture medium was similar to that of the para-nitroblebbistatin group, except that para-nitroblebbistatin was replaced with blebbistatin.

[0039] Results after 3 days of cultivation Figure 4 As shown, compared to tissues without NMII inhibitors, tissue samples with para-nitroblebbistatin showed some fusion, and organoids formed by day 3. While some organoids formed in the blebbistatin group, they were loosely structured and of poor condition. The organoids from tissues with para-nitroblebbistatin were more translucent and structurally compact than those from the blebbistatin group. These results indicate that the addition of NMII inhibitors is beneficial to organoid formation in the gas-liquid culture system, and the organoids formed in the para-nitroblebbistatin group are more stable. Further continuous culture observation of the organoids in the para-nitroblebbistatin group revealed that the organoid volume gradually increased with prolonged culture time. Figure 5 The organoids grew well.

[0040] Immunofluorescence identification of intestinal organoids cultured for 3 days was performed using intestinal epithelial cell markers CDX2 and KRT8, immune cell markers CD3 (T lymphocytes), CD14 (monocytes), and CD19 (B lymphocytes). The results are as follows: Figure 6 and Figure 7 As shown in the figure. The results show that the intestinal organoids constructed in this invention significantly express the intestinal epithelial cell marker CDX2 and the intestinal columnar epithelial marker KRT8; at the same time, the immune cell-related markers CD3, CD14 and CD19 also show positive expression in the organoids, indicating that the intestinal organoids constructed in this invention have immune cells.

[0041] In summary, when it is difficult to obtain peripheral blood from children or the peripheral blood cells are in poor condition, this invention directly utilizes intestinal tissue and tissue-resident immune cells derived from children with Crohn's disease to construct an intestinal organoid model with an immune microenvironment, providing a new model system.

[0042] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing intestinal organoids in children with Crohn's disease, characterized in that, Includes the following steps: Intestinal tissue derived from children with Crohn's disease was seeded onto the upper chamber membrane of a Transwell plate, solidified, and then cultured in the lower chamber with organoid culture medium to obtain intestinal organoids from children with Crohn's disease. The organoid culture medium is based on Advanced DMEM / F12 medium and also includes the following components at the following concentrations: 10-12 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 100-110 U / mL penicillin, 100-110 µg / mL streptomycin, 50-55 µg / mL Primocin antibiotic, 2-2.2 mM GlutaMAX supplement, 1×B27 supplement, 1×N2 supplement, 5-6 mM nicotinamide, and 1.25-1.30 mM... N-acetylcysteine, 100-110 ng / mL recombinant human Noggin protein, 50-55 ng / mL recombinant human EGF protein, 500-550 ng / mL recombinant human R-spondin1 protein, 200-300 ng / mL recombinant human WNT3a protein, 50-60 ng / mL recombinant human FGF10 protein, 20-22 ng / mL recombinant human FGF7 protein, 25-28 ng / mL recombinant human HGF protein, 50-75 ng / mL recombinant human IL-2 protein, 0.5-0.6 μM A83-01, 10-12 μM SB202190, 10-12 μM Y-27632 and 10-15 μM p-nitrobrestatin.

2. The construction method according to claim 1, characterized in that, The organoid culture medium is based on Advanced DMEM / F12 medium and also includes the following components at the following concentrations: 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 100 U / mL penicillin, 100 µg / mL streptomycin, 50 µg / mL Primocin antibiotic, 2 mM GlutaMAX supplement, 1×B27 supplement, 1×N2 supplement, 5 mM nicotinamide, 1.25 mM N-acetylcysteine, 100 ng / mL recombinant human Noggin protein, 50 ng / mL recombinant human EGF protein, 500 ng / mL recombinant human R-spondin1 protein, 200 ng / mL recombinant human WNT3a protein, 50 ng / mL recombinant human FGF10 protein, 20 ng / mL recombinant human FGF7 protein, 25 ng / mL recombinant human HGF protein, 75 ng / mL recombinant human IL2 protein, 0.5 μM A83-01, 10 μM SB202190, and 10 µM... Y-27632 and 10μM p-nitrobrestatin.

3. The construction method according to claim 1, characterized in that, The cultivation conditions include a temperature of 36.5~37℃ and a CO2 concentration of 4.5~5%.

4. The construction method according to claim 1 or 3, characterized in that, The organoid culture medium should be replaced every 72-96 hours during the culture process.

5. The construction method according to claim 1 or 3, characterized in that, The culture time is 3 to 14 days.

6. The construction method according to claim 1, characterized in that, The intestinal tissue was pretreated before inoculation; the pretreatment included washing the intestinal tissue 3-4 times in PBS solution containing 5-7 vol% penicillin-streptomycin-gentamicin triple antibodies, and then cutting it into 1-3 mm pieces. 3 The organizational blocks.

7. The construction method according to claim 1 or 6, characterized in that, The intestinal tissue includes one or more of the following: fresh intestinal tissue biopsy samples and surgically removed samples.

8. The construction method according to claim 7, characterized in that, The biopsy samples are ≥6, and the fresh intestinal tissue biopsy samples are those obtained within 6 hours; the surgically removed samples are those obtained within 24 hours.

9. The construction method according to claim 1, characterized in that, The curing time is 10-15 minutes.

10. The application of the construction method described in any one of claims 1 to 9 to obtain intestinal organoids for Crohn's disease in the study of related mechanisms of Crohn's disease and / or the evaluation of drug efficacy.

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