Construction method and application of children Crohn disease intestinal tract organoid

Abstract

The invention relates to the technical field of cell biology, in particular to a construction method and application of intestinal organs for children with Crohn's disease, the intestinal organs for children with Crohn's disease are successfully constructed by utilizing intestinal tissues from children with Crohn's disease, and the intestinal organs have various characteristic cells of the intestinal tissues; besides, immune cells are extracted from peripheral blood of a child patient homologous with intestinal tissues, a co-culture system is established with the organ-like model, the intestinal immune microenvironment of the child patient suffering from the Crohn's disease can be reproduced, and the system provides a new model for related mechanism exploration and drug screening of the child Crohn's disease.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and particularly relates to a method for constructing and applying an intestinal organoid of pediatric Crohn's disease. Background Art

[0002] An organoid is a 3D organotypic structure formed by the self-assembly of stem cells in vitro, which can differentiate into various characteristic cells in tissues, and can exhibit the interactions and spatial position morphologies between cells and between cells and their surrounding matrix, reproducing some key functions and structures of real human organs in vitro, and having stable phenotypic and genetic characteristics.

[0003] The Chinese utility model patent specification CN116496980A discloses an intestinal crypt isolation solution, isolation method and in vitro 3D organoid culture method for inflammatory bowel disease. This method provides an intestinal crypt isolation solution including potassium chloride, sodium chloride, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, ethylenediaminetetraacetic acid, and ethylene glycol bis(tetraacetic acid), which can quickly isolate intestinal crypts of inflammatory bowel disease and form a crypt organoid model in vitro. However, the organoids constructed using patients with inflammatory bowel disease in this method only contain intestinal epithelial cells, and do not further reconstruct the inflammatory microenvironment, and cannot reflect the role played by immune cells in the real disease development process, and there are defects in the ability to simulate the immune microenvironment of inflammatory bowel disease tissues. Crohn's disease is a chronic, recurrent, non-specific inflammatory bowel disease with unknown etiology, and its incidence in children has gradually increased in recent years. Crohn's disease lacks early diagnostic markers and there are multiple treatment options. In fact, children and adults have different disease characteristics, and currently there is no method specifically for constructing an organoid model for pediatric Crohn's disease. Summary of the Invention

[0004] In order to solve the problem of the lack of an organoid model for pediatric Crohn's disease mentioned in the above background art, the present invention provides a method for constructing and applying an intestinal organoid of pediatric Crohn's disease. Using intestinal tissues from pediatric Crohn's disease patients, an intestinal organoid for pediatric Crohn's disease patients is successfully constructed, and immune cells are extracted from the peripheral blood of the child homologous to the intestinal tissue, and a co-culture system is established with the organoid model to better restore the intestinal immune microenvironment of pediatric Crohn's disease patients, providing a new experimental model for the study of pediatric Crohn's disease.

[0005] The specific technical solution of the present invention is: A method for constructing an intestinal organoid of pediatric Crohn's disease, comprising the following steps: 1) washing the intestinal tissue 3 - 4 times in a PBS solution containing 5 - 7 vol% penicillin-streptomycin double antibody; 2) After the washed intestinal tissue is digested with tissue digestion solution, collect the cell suspension, centrifuge, collect the cell pellet, resuspend it with Matrigel solution, inoculate it on a cell culture plate, and after solidification, add intestinal organoid medium and culture it in a cell incubator at 36.5 - 37°C and 4.5 - 5% CO 2 to obtain primary organoids.

[0006] The present invention uses intestinal tissue from children with Crohn's disease to successfully construct an intestinal organoid model for children with Crohn's disease, which has various characteristic cells of intestinal tissue and better maintains the tissue pathological phenotype of children with Crohn's disease, and can provide a new model for the study of the pathological mechanism of children with Crohn's disease.

[0007] Preferably, the intestinal tissue is freshly obtained intestinal tissue.

[0008] Furthermore, the tissue digestion solution is an Advanced DMEM / F12 solution containing 1 - 1.2 mg / mL of type I collagenase and 0.5 - 0.6 mg / mL of DNase.

[0009] Furthermore, in step 2), after adding the tissue digestion solution, a large number of crypts fall off, and an Advanced DMEM / F12 medium containing 3 - 5 vol% fetal bovine serum is added.

[0010] A method for subculture of intestinal organoids of children with Crohn's disease, characterized by comprising the following steps: S1) Add 5 - 6 times the volume of organoid digestion solution to the primary organoids, shake in a shaker at 30 - 37°C for 3 - 5 min, and the rotation speed is 600 - 700 rpm; S2) Add an Advanced DMEM / F12 medium containing 3 - 5 vol% fetal bovine serum with a volume 3 - 5 times that of the organoid digestion solution, centrifuge at high speed for 5 - 6 min, and add an Advanced DMEM / F12 solution for washing; S3) Add a Matrigel mixture, let it stand in a cell incubator at 36.5 - 37°C and 4.5 - 5% CO 2 for 10 - 15 min, add human intestinal organoid medium, and culture it in a cell incubator at 36.5 - 37°C and 4.5 - 5% CO 2 for 72 - 75 h, and replace the human intestinal organoid medium once to obtain subcultured organoids after one passage; S4) Repeat steps S1) - S3) to obtain subcultured organoids after n passages.

[0011] Furthermore, the diameter of the organoids is 100 - 200 μm.

[0012] A method for establishing a co - culture system of intestinal organoids and peripheral blood mononuclear cells (PBMCs) in children with Crohn's disease, comprising the following steps: S1) Culture the thawed peripheral blood mononuclear cells in X - VIVO15 medium containing 10 - 12 vol% fetal bovine serum, 1 - 1.2 vol% penicillin - streptomycin double - antibody solution, and 500 - 510 ng / mL human recombinant IL - 2 protein for 24 - 30 h; S2) After adding matrix gel lysate to the organoids and centrifuging, collect the precipitate, add 500 - 550 μL of pre - cooled Advanced DMEM / F12 solution, break it to obtain a cell mass suspension, take 50 - 60 μL of the cell suspension, add trypsin to obtain single cells and count to obtain the cell density of the organoids; S3) Mix the organoid cell mass and peripheral blood mononuclear cells at a ratio of 1:(2.9 - 3.1) by the number of cells, plate them, add a human intestinal organoid medium containing 5 - 10 vol% matrix gel, and culture for more than 72 h.

[0013] In view of the important role of the inflammatory microenvironment in the development of Crohn's disease, the present invention extracts immune cells from the peripheral blood of children homologous to the intestinal tissue, and establishes a co - culture system with the organoid model, so as to better restore the intestinal immune microenvironment of patients with Crohn's disease, provide a new model for the mechanism research and clinical drug screening of childhood Crohn's disease, and provide a new method for the individualized precision treatment of childhood Crohn's disease.

[0014] Further, in S2), the organoids are the organoids within 3 passages described in S4) of claim 4, and the diameter of the organoids is 100 - 120 μm.

[0015] Further, after adding the human intestinal organoid medium containing 5 - 10 vol% matrix gel in S3), the cell density is 5 - 5.5×10 6 cells / mL.

[0016] Further, the well - plate used in S3) is a low - adhesion cell well - plate.

[0017] Furthermore, the components of the human intestinal organoid culture medium include: Advanced DMEM / F12, 10 - 12 mM HEPES, 100 - 110 U / mL penicillin, 100 - 110 μg / mL streptomycin, 50 - 55 μg / mL Primocin antibiotic, 2 - 2.2 mM GlutaMAX supplement, 1x B27 supplement, 1x N2 supplement, 5 - 6 mM nicotinamide, 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, 0.5 - 0.6 μM A83-01, 10 - 12 μM SB20219, and 10 - 12 μM Y-27632.

[0018] Preferably, the components of the human intestinal organoid culture medium include: Advanced DMEM / F12, 10 mM HEPES, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μg / mL Primocin antibiotic, 2 mM GlutaMAX supplement, 1x B27 supplement, 1x 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, 0.5 μM A83-01, 10 μM SB20219, and 10 μM Y-27632.

[0019] An intestinal organoid pathological model established by co-culturing intestinal organoids of children with Crohn's disease and peripheral blood mononuclear cells can be used to screen and evaluate drugs with therapeutic effects on Crohn's disease, including small molecules, macromolecules, probiotics, metabolites, etc.

[0020] Compared with the prior art, the beneficial effects of the present invention are: 1) The present invention uses intestinal tissues from children with Crohn's disease to construct an intestinal organoid model for children with Crohn's disease, which has various characteristic cells of intestinal tissues and better maintains the tissue pathological phenotype of children with Crohn's disease.

[0021] 2) The present invention extracts immune cells from the peripheral blood of children with the same origin as intestinal tissue and establishes a co-culture system with the organoid model, which can better reproduce the intestinal immune microenvironment of children with Crohn's disease.

[0022] 3) The present invention can provide a new model system for the research of children with Crohn's disease, and can provide new methods for exploring the mechanism of the disease, evaluating clinical individualized precision treatment, etc.

[0023] 4) The present invention can be used to screen and evaluate drugs with therapeutic effects on Crohn's disease, including small molecules, macromolecules, probiotics, metabolites, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Growth diagram for the construction of organoids from samples of children with Crohn's disease.

[0025] Figure 2 Figure A showing the identification results of characteristic cells in tissues and organoids of children with Crohn's disease.

[0026] Figure 3 Figure B showing the identification results of characteristic cells in tissues and organoids of children with Crohn's disease.

[0027] Figure 4 Figure showing the colonization situation after co-culture of PBMC and organoids.

[0028] Figure 5 Figure showing the immunofluorescence staining results of organoids after co-culture of PBMC and organoids.

[0029] Figure 6 Figure showing the HE staining results of organoids after co-culture of PBMC and organoids.

[0030] Figure 7 Figure showing the analysis of changes in the expression levels of various inflammatory factors after co-culture of PBMC and organoids.

[0031] Figure 8 Figure showing the analysis of the improvement of inflammatory factor levels by various monoclonal antibody-based biological agents in the intestinal organoid-PBMC co-culture system.

[0032] Figure 9 Figure showing the analysis of the improvement of inflammatory factor levels by some probiotics and intestinal-related metabolites in the intestinal organoid-PBMC co-culture system. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be further described below in conjunction with the embodiments.

[0034] Example 1 Construction of intestinal organoids from pediatric Crohn's disease patients, establishment of a co - culture system of organoids and PBMCs, and application of testing the efficacy of monoclonal biological agents.

[0035] 1) Construction of intestinal organoids from pediatric Crohn's disease patients After the sample is taken, it is placed on ice and transferred to the laboratory for sample processing within 2 hours. Use sterilized ophthalmic forceps to transfer the tissue to 1x PBS (containing 5% penicillin - streptomycin double - antibody solution) for rinsing to remove blood foam. Then transfer it to a 1.5 mL centrifuge tube, add an appropriate amount of 1x PBS (containing 5% penicillin - streptomycin double - antibody solution), and shake 2 - 3 times for deep cleaning.

[0036] In a 1.5 mL centrifuge tube, use sterilized ophthalmic scissors to cut the tissue into small pieces (about 1 - 3 mm 3 ). Add 1 mL of tissue digestion solution (Advanced DMEM / F12 solution containing 1 mg / mL of type I collagenase and 0.5 mg / mL of DNase) to the tissue. Place the centrifuge tube in a thermostatic shaker bath and digest the sample at 37°C and 700 rpm. Take out the centrifuge tube every 10 minutes and pipette the digested tissue fragments to separate the crypts from the basal layer by the mechanical shear force generated by the tissue passing through the pipette. Place the centrifuge tube under a microscope and stop digestion when a large number of crypts fall off. Transfer the digested cell suspension to a 15 mL centrifuge tube, add an equal volume of Advanced DMEM / F12 medium containing 5% fetal bovine serum to terminate the action of the digestive enzyme. Centrifuge the above cell suspension at 2000 rpm for 5 minutes to collect the crypt precipitate. Add an appropriate amount of fresh basal medium and count the crypts under a microscope. Calculate and add the corresponding volume of organoid culture matrix glue according to the ratio of 50 crypts per well corresponding to 20 μL of matrix glue. Mix well on ice and use a pipette to aspirate the cell and matrix glue mixture and transfer it to a 24 - well plate, 20 μL of glue droplets per well. Place the culture plate in a 37°C, 5% CO 2 cell incubator to solidify. Take it out after 10 minutes and slowly add 700 μL of human intestinal organoid medium along the well wall. Place the 24 - well plate in a 37°C, 5% CO 2 cell incubator for culture, observe and take pictures every day, and change the medium every 3 days.

[0037] Among them, the human intestinal organoid culture medium contains the following components: Advanced DMEM / F12, 10 mM HEPES, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μg / mL Primocin antibiotic, 2 mM GlutaMAX supplement, 1x B27 supplement, 1x 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, 0.5 μM A83-01, 10 μM SB20219, and 10 μM Y-27632.

[0038] The construction and growth diagram of intestinal organoids from children with Crohn's disease is as Figure 1 shown. In Figure 1 , P0 day0 is the diagram of primary crypts extracted; P0 day2 and day5 are the diagrams of the status of organoids growing to the 2nd and 5th days respectively during primary growth; P1 day4 is the diagram of the status of organoids growing to the 4th day after being digested once in the p0 generation; P3 day3 is the diagram of the status of organoids growing to the 3rd day after being digested three times in the p0 generation. The scale bar is 200 μm.

[0039] The constructed intestinal organoids were identified by immunofluorescence based on multiple characteristic cell markers in intestinal tissues. As Figure 2 and Figure 3 shown, the intestinal epithelial cell markers E-cadherin and CDX2, the intestinal columnar epithelial marker KRT8, and the intestinal endocrine cell marker MUC2 in the intestinal organoids constructed by this method all showed positive expression consistent with the tissue.

[0040] 2) Subculture of intestinal organoids from children with Crohn's disease When the organoids are cultured to a size of 100 - 200 μm in diameter, subculture can be performed. Remove the culture medium and add fresh pre-cooled Advanced DMEM / F12 solution. Use a pipette tip to scrape the mixture of Matrigel and organoids from the well plate and transfer it to a 15 mL centrifuge tube. Pipette several times to separate the organoids from Matrigel. Centrifuge at 2000 rpm for 5 min to remove the culture medium and Matrigel.

[0041] Add 5 times the volume of the organoid digestive solution as precipitation, and shake it on a shaker at 37°C for 3 - 5 minutes for digestion. Observe the digestion status of the organoids under the microscope at regular intervals. When more cell clusters are observed, add Advanced DMEM / F12 medium containing 5% fetal bovine serum with a volume 5 times that of the digestive solution to terminate digestion.

[0042] Centrifuge at 2000 rpm for 5 minutes, add Advanced DMEM / F12 solution to wash once to remove the residual digestive solution. Pipette the cell and Matrigel mixture and transfer it to a 24-well plate, with 20 μL of gel droplets in each well. Place the culture plate in a 37°C, 5% CO 2 Cell incubator to solidify for 10 minutes, and slowly add human intestinal organoid medium along the well wall. Place the 24-well plate in a 37°C, 5% CO 2 Cell incubator for culture, observe and take pictures every day, and change the medium every 3 days.

[0043] 3) Establishment of the co-culture system of intestinal organoids and PBMC from children with Crohn's disease. Take out the cryopreserved PBMC cells and quickly warm them up by shaking in a 37°C water bath. Suspend and culture them in X-VIVO15 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin double antibody solution, and 500 ng / mL human recombinant IL-2 protein. The co-culture experiment can be carried out after 24 hours.

[0044] Use organoids that have been passaged within 3 times for the experiment. When the organoids grow to a diameter of 100 μm, they can be co-cultured with PBMC. Use Matrigel lysis solution to separate the organoids from the Matrigel and collect the organoids. Add 500 μL of fresh pre-cooled Advanced DMEM / F12 solution to the organoid precipitate, and continuously pipette with a pipette tip to break the organoids. Take 50 μL of the cell suspension and further digest it into single cells with trypsin, and use trypan blue staining for cell counting to determine the cell density in the organoid suspension.

[0045] Count PBMC cells using trypan blue staining. Mix PBMC and organoids at a cell number ratio of 3:1, add an appropriate amount of human intestinal organoid medium containing 10% Matrigel, and the final cell density is 5x10 6 cells / mL.

[0046] Transfer 100 μL of the cell-gel suspension to a 96-well low-attachment plate for culture and observation. Take pictures of the organoids after 3 days of culture, centrifuge the cell suspension, collect the supernatant for detection of inflammatory factor levels; fix the obtained cell precipitate with 4% paraformaldehyde, embed it in paraffin, and then perform HE and immunofluorescence staining.

[0047] Figure 4 It is the colonization situation diagram after co-culture of PBMC and organoids, asFigure 4 As shown, after co-culturing organoids with PBMCs for 3 days, fluorescently labeled PBMC cells could be observed in the organoids, indicating that PBMCs could colonize in the organoids under this system.

[0048] Immunofluorescence staining was performed using the organoid epithelial cell marker E-cadherin, T lymphocyte marker CD3, monocyte marker CD14, and B lymphocyte marker CD19 respectively. Figure 5 This is the immunofluorescence staining result of the organoids after co-culturing PBMCs with the organoids. As Figure 5 shown, E-cadherin, CD3, CD14, and CD19 were all positively expressed, suggesting that the colonized immune cells included T lymphocytes, B lymphocytes, and monocytes.

[0049] Figure 6 This is the HE staining result of the organoids after co-culturing PBMCs with the organoids. As Figure 6 shown, compared with the organoids cultured alone, the edges and interstitial structures of the intestinal organoid epithelial cells in the co-culture system were no longer clear and orderly, becoming blurred and fragmented; the volume of the cell nuclei became smaller and the number became fewer.

[0050] The levels of inflammatory factors were detected using an Elisa kit. The results were as Figure 7 shown, and the results showed that TNFα, IL-6, IL-12, and IL23 were significantly up-regulated in the co-culture system compared with the single system.

[0051] The above results as a whole indicated that the organoids constructed by this system could reproduce the typical pathology of patients with Crohn's disease.

[0052] 4) Application of the co-culture system of organoids and PBMCs to test the efficacy of monoclonal antibody-based biological agents Monoclonal antibody-based biological agents are a class of drugs prepared using monoclonal antibody technology. They can bind highly specifically to specific immune molecules, reduce the activation and migration of inflammatory cells by inhibiting the activity of pro-inflammatory molecules, thereby alleviating symptoms and intervening in the occurrence and development process of diseases. Currently, there are various monoclonal antibody-based biological agents that can be applied to inflammatory bowel disease. Based on this, in this example, referring to the co-culture experimental procedure in step 3), after co-culturing organoids and PBMCs in a low-adhesion 96-well plate for 3 days, they were diluted with intestinal organoid medium, and 10 ng / mL IFX monoclonal antibody, 10 μg / mL adalimumab, 1 μg / mL ustekinumab, and 1 μg / mL vedolizumab were respectively added, and then continued to be treated for 48 hours. The supernatant was collected for the determination of the levels of inflammatory factors.

[0053] As Figure 8As shown, compared with the co-culture system, the anti-TNFα IFX monoclonal antibody and adalimumab can significantly down-regulate the levels of TNFα, IL-6, IL-12 and IL23; ustekinumab targeting IL-12 / IL-23 can better reverse the levels of IL-12 and IL23, but has little effect on the regulation of TNFα and IL-6 levels; vedolizumab targeting α4β7 integrin only has a good regulatory effect on the levels of IL-12 and TNFα. In summary, different Crohn's disease treatment drugs show different improved drug effects under this co-culture system, indicating that this co-culture system has the potential to predict multiple therapies for Crohn's disease.

[0054] Example 2 Construction of intestinal organoids from pediatric Crohn's disease patients, establishment of a co-culture system of organoids and PBMC, and application of testing drug efficacy.

[0055] 1) Construction of intestinal organoids from pediatric Crohn's disease patients After the samples were taken, they were placed on ice and transferred to the laboratory for sample processing within 2 h. The tissue was transferred to 1x PBS (containing 6% penicillin-streptomycin double antibody solution) using sterilized ophthalmic forceps and rinsed to remove blood foam. Then it was transferred to a 1.5 mL centrifuge tube, and an appropriate amount of 1x PBS (containing 6% penicillin-streptomycin double antibody solution) was added, and shaken 2-3 times for deep cleaning.

[0056] In a 1.5 mL centrifuge tube, the tissue was minced into small pieces (about 3 mm 3 ) using sterilized ophthalmic scissors. 1 mL of tissue digestion solution (Advanced DMEM / F12 solution containing 1 mg / mL type I collagenase and 0.5 mg / mL DNase) was added to the tissue. The centrifuge tube was placed in a thermostatic shaker bath and the samples were digested at 37 °C and 700 rpm. The centrifuge tube was taken out every 10 minutes, and the digested tissue fragments were pipetted to separate the crypts from the basal layer by the mechanical shear force generated by the tissue passing through the pipette. The centrifuge tube was placed under a microscope and digestion was stopped when a large number of crypts fell off. The digested cell suspension was transferred to a 15 mL centrifuge tube, and an equal volume of Advanced DMEM / F12 medium containing 5% fetal bovine serum was added to terminate the action of the digestive enzyme. The above cell suspension was centrifuged at 2000 rpm for 5 min to collect the crypt precipitate. An appropriate amount of fresh basal medium was added, and the crypts were counted under a microscope. According to the ratio of 50 crypts per well corresponding to 20 μL of Matrigel, the corresponding volume of organoid culture Matrigel was calculated and added. After mixing on ice, the cell and Matrigel mixture was aspirated with a pipette and transferred to a 24-well plate, 20 μL of gel drops per well. The culture plate was placed at 37 °C and 5% CO 2The cells were solidified in the cell culture incubator and taken out after 10 minutes. 700 μL of human intestinal organoid culture medium was slowly added along the well wall. The 24-well plate was placed in a 37°C, 5% CO 2 The cells were cultured in a cell culture incubator, observed and photographed every day, and the medium was changed every 3 days.

[0057] Among them, the human intestinal organoid culture medium contains the following components: Advanced DMEM / F12, 10mM HEPES, 100U / mL penicillin, 100μg / mL streptomycin, 50μg / mL Primocin antibiotics, 2mM GlutaMAX supplement, 1x B27 supplement, 1x N2 supplement, 5mM Nicotinamide, 1.25mM N-acetylcysteine, 100ng / mL recombinant human Noggin protein, 50ng / mL recombinant human EGF protein, 500ng / mL recombinant human R-spondin1 protein, 200ng / mL recombinant human WNT3a protein, 50ng / mL recombinant human FGF10 protein, 20ng / mL recombinant human FGF7 protein, 25ng / mL recombinant human HGF protein, 0.5μM A83-01, 10μM SB20219 and 10μM Y-27632.

[0058] 2) Subculture of intestinal organoids from children with Crohn's disease When the organoids are cultured to a diameter of 100 μm, they can be subcultured. Remove the culture medium and add fresh pre-cooled Advanced DMEM / F12 solution. Use a pipette to scrape the mixture of matrix gel and organoids from the well plate and transfer it to a 15 mL centrifuge tube. Pipette several times to separate the organoids and matrix gel. Centrifuge at 2000 rpm for 5 minutes to remove the culture medium and matrix gel.

[0059] Add 5 times the volume of the organoid digestion solution to the pellet and shake at 37°C for 3 minutes for digestion. When more cell clusters are observed, add 5 times the volume of the digestion solution in Advanced DMEM / F12 medium containing 5% fetal bovine serum to terminate the digestion.

[0060] Centrifuge at 2000 rpm for 5 min, add Advanced DMEM / F12 solution to wash once, and remove the residual digestion solution. Use a pipette to transfer the cell and matrix gel mixture to a 24-well plate, with 20 μL of gel droplet per well. Place the culture plate in a 37°C, 5% CO 2 The cells were solidified in a cell culture incubator for 10 min, and human intestinal organoid culture medium was slowly added along the well wall. The 24-well plate was placed in a 37°C, 5% CO 2 The cells were cultured in a cell culture incubator, observed and photographed every day, and the medium was changed every 3 days.

[0061] 3) Establishment of a co - culture system of intestinal organoids and PBMC from children with Crohn's disease. After taking out the cryopreserved PBMC cells, quickly shake and warm them in a 37 °C water bath, and suspend and culture them in X - VIVO15 medium containing 10% fetal bovine serum, 1% penicillin - streptomycin double - antibody solution, and 500 ng / mL human recombinant IL - 2 protein. The co - culture experiment can be carried out after 24 hours.

[0062] Use organoids passaged within 3 times for the experiment. When the organoids grow to a diameter of 100 μm, they can be co - cultured with PBMC. Use matrix gel lysis solution to separate the organoids from the matrix gel and collect the organoids. Add 500 μL of fresh pre - cooled Advanced DMEM / F12 solution to the organoid precipitate, and continuously pipette with a pipette tip to break the organoids. Take 50 μL of the cell suspension and further digest it into single cells with trypsin, and use trypan blue staining for cell counting to determine the cell density in the organoid suspension.

[0063] Count the PBMC cells using trypan blue staining. Mix PBMC and organoid cells at a ratio of 3:1, add an appropriate amount of human intestinal organoid medium containing 10% matrix gel, and the final cell density is 5.2x10 6 cells / mL.

[0064] Transfer 100 μL of the cell - gel suspension to a 96 - well low - adhesion plate for culture and observation. Take pictures of the organoids after 3 days of culture, aspirate the cell suspension for centrifugation, and collect the supernatant for detection of inflammatory factor levels.

[0065] 4) Application of the co - culture system of organoids and PBMC in testing drug efficacy In addition to the marketed monoclonal biological agents, some intestinal - related in - vivo metabolites, novel intestinal probiotics, etc. have also been reported to have a certain improvement effect on inflammatory bowel disease. Based on this, in this example, referring to the co - culture experimental procedure in step 3), after co - culturing the organoids and PBMC in a low - adhesion 96 - well plate for 3 days, dilute them with intestinal organoid medium, and respectively add 1x10 7 CFU / mL Lactobacillus acidophilus, 1x10 7 CFU / mL Akkermansia muciniphila, 100 μM cholic acid, and 100 μM butyric acid. Place them on the upper layer of a transwell chamber, continue to process for 48 hours, then discard the chamber, and collect the supernatant to measure the levels of inflammatory factors TNFα, IL - 6, IL - 12, and IL23.

[0066] Such as Figure 9As shown, compared with the co-culture system, the addition of the probiotic Akkermansia muciniphila reduced the levels of TNFα and IL-6 to a certain extent; cholic acid had a certain down-regulating effect on IL-6 and IL-12; while the probiotic Lactobacillus acidophilus and the short-chain fatty acid butyric acid did not show significant regulatory effects on the inflammatory factors TNFα, IL-6, IL-12 and IL23 related to Crohn's disease. This result suggests that the constructed intestinal organoid-PBMC co-culture system can be used as a screening model for effective substances to improve Crohn's disease, and is expected to contribute to the discovery and mechanism research of disease-related active substances.

[0067] In the present invention, the raw materials and equipment used, unless otherwise specified, are common raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0068] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes and equivalent transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for constructing intestinal organoids for children with Crohn's disease, characterized in that: The following steps are involved: 1) Wash the intestinal tissue 3-4 times in a PBS solution containing 5-7 vol% penicillin-streptomycin dual antibody; 2) After the cleaned intestinal tissue is digested with tissue digestion solution, the cell suspension is collected and centrifuged, the cell precipitate is collected, the matrix gel solution is added to resuspend, and the cell culture plate is inoculated. After solidification, intestinal organoid culture medium is added and cultured in a cell culture incubator at 36.5-37°C and 4.5-5% CO2 to obtain primary organoids.

2. A method for constructing intestinal organoids for children with Crohn's disease according to claim 1, characterized in that: The tissue digestion solution is an Advanced DMEM / F12 solution containing 1-1.2 mg / mL type I collagenase and 0.5-0.6 mg / mL DNase.

3. A method for constructing intestinal organoids for children with Crohn's disease according to claim 1 or 2, characterized in that: In step 2), after adding tissue digestion solution, a large number of crypts fell off, and Advanced DMEM / F12 culture medium containing 3-5 vol% fetal bovine serum was added.

4. A method for subculturing intestinal organoids of children with Crohn's disease obtained according to any one of the construction methods of claims 1 to 3, characterized in that: The following steps are involved: S1) adding 5-6 times the volume of organoid digestion solution to the primary organoid, and shaking in a shaker at 30-37°C for 3-5 minutes; S2) adding 3-5 times the volume of organoid digestion solution to Advanced DMEM / F12 medium containing 3-5 vol% fetal bovine serum, centrifuging at high speed for 5-6 minutes, and adding Advanced DMEM / F12 solution for washing; S3) adding the matrix gel mixture, placing the mixture in a cell culture incubator at 36.5-37° C. and 4.5-5% CO2 for 10-15 min, adding human intestinal organoid culture medium, placing the mixture in a cell culture incubator at 36.5-37° C. and 4.5-5% CO2, and replacing the human intestinal organoid culture medium every 72-75 hours to obtain an organoid that has been passaged once; S4) Repeat steps S1) to S3) to obtain organoids that have been passaged n times.

5. The subculture method according to claim 4, characterized in that: The diameter of organoids in S1) is 100-200 μm.

6. A method for establishing a co-culture system of intestinal organoids from children with Crohn's disease and peripheral blood mononuclear cells obtained by the subculture method according to claim 4 or 5, characterized in that: The following steps are involved: s1) culturing the rewarmed peripheral blood mononuclear cells in X-VIVO15 medium containing 10-12 vol% fetal bovine serum, 1-1.2 vol% penicillin-streptomycin double antibody solution, and 500-510 ng / mL human recombinant IL-2 protein for 24-30 h; s2) Add Matrigel lysis buffer to the organoids and centrifuge to collect the precipitate, add 500-550 μL pre-cooled Advanced DMEM / F12 solution, break the cell suspension to obtain a cell mass, take 50-60 μL of the cell suspension and add trypsin to obtain single cells and count them to obtain the cell density of the organoids; s3) The organoid cell clusters were mixed with peripheral blood mononuclear cells at a ratio of 1:(2.9-3.1) and plated, and human intestinal organoid culture medium containing 5-10 vol% matrix gel was added and cultured for more than 72 h.

7. The establishment method according to claim 6, characterized in that: In s2), the organoids were passaged less than 3 times, and the diameter of the organoids was 100 to 120 μm.

8. The establishment method according to claim 6, characterized in that: s3) After adding human intestinal organoid culture medium containing 5-10 vol% Matrigel, the cell density was 5-5.5×10 6 The cells were 90% / mL and the well plate used was a low-adhesion cell well plate.

9. The establishment method according to claim 6, characterized in that: The components of human intestinal organoid culture medium include: Advanced DMEM / F12, 10-12mM HEPES, 100-110U / mL penicillin, 100-110μg / mL streptomycin, 50-55μg / mL Primocin antibiotics, 2-2.2mM GlutaMAX supplement, 1xB27 supplement, 1xN2 supplement, 5-6mM Nicotinamide, 1.25-1.30mM N-acetylcysteine, 100-110ng / mL recombinant human Noggin protein, 50-55ng / mL recombinant human EGF protein, 500-550ng / mL recombinant human R-spondin1 protein, 200-300ng / mL recombinant human WNT3a protein, 50-60ng / mL recombinant human FGF10 protein, 20-22ng / mL recombinant human FGF7 protein, 25-28ng / mL recombinant human HGF protein, 0.5-0.6μM A83-01, 10-12μM SB20219 and 10-12μM Y-27632.

10. An intestinal organoid pathological model established according to the establishment method according to any one of claims 6 to 10.

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