Method for constructing an inflammatory bowel disease model, inflammatory bowel disease model and use

Abstract

The application relates to the technical field of bioengineering, and discloses a method for constructing an inflammatory bowel disease model, which comprises the following steps: constructing an inflammatory intestinal barrier by using intestinal cells from an inflammatory bowel disease patient; adding a stimulating solution containing immune cells and / or immune cell secretions into the inflammatory intestinal barrier, and carrying out co-culture to obtain an inflammatory bowel disease model. The inflammatory intestinal barrier constructed by using intestinal cells from an inflammatory bowel disease patient and adding immune components can retain the gene and cell shape characteristic expression characteristics related to IBD diseases, so that the model is closer to the pathological condition in the patient body; the actual in-vivo pathological damage progress is simulated, the model bionics is improved, the damage of the unbalanced immune cells in the IBD pathology to the intestinal tract is reproduced, and the potential drug targets can be discovered and verified. The application further discloses an inflammatory bowel disease model and an application thereof.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, for example to a method for constructing an inflammatory bowel disease model, as well as the inflammatory bowel disease model and its applications. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of nonspecific chronic inflammatory diseases of the gastrointestinal tract with an unclear etiology. In recent years, the incidence of IBD in China has been rising steadily, coinciding with an aging population, particularly among middle-aged and elderly individuals and children. This not only affects the quality of life of individual patients but also poses new challenges to public health. Therefore, research and the development of treatment options for this disease have become increasingly important. Because a key characteristic of this disease is its unclear etiology, commonly used research and drug development methods, such as animal-based enteritis models or cell-based in vitro disease models, often fail to fully simulate the actual pathological conditions in humans, and these models may not carry certain genetic features associated with IBD. Consequently, research on disease mechanisms and drug development based on existing models will face deviations from actual pathological conditions, leading to a higher risk of drug failure in clinical trials.

[0003] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this application, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0004] To provide a basic understanding of some aspects of the disclosed embodiments, a brief summary is given below. This summary is not intended as a general commentary, nor is it intended to identify key / important components or describe the scope of protection of these embodiments, but rather as a prelude to the detailed description that follows.

[0005] This disclosure provides a method for constructing an inflammatory bowel disease (IBD) model, as well as the IBD model and its applications. Intestinal cells derived from IBD patients are used to construct enteritis organoids or inflammatory intestinal barrier models in vitro to achieve optimal pathological biomimicry, thereby improving the accuracy of understanding the disease mechanism and the effectiveness and efficiency of related drug development.

[0006] In some embodiments, the method for constructing the inflammatory bowel disease model includes: constructing an inflammatory intestinal barrier using intestinal cells derived from patients with inflammatory bowel disease; adding a stimulating solution containing immune cells and / or secretions of immune cells to the inflammatory intestinal barrier, and co-culturing to obtain the inflammatory bowel disease model.

[0007] In some embodiments, the inflammatory bowel disease model is obtained by constructing the aforementioned inflammatory bowel disease model construction method.

[0008] In some embodiments, the inflammatory bowel disease model constructed by the aforementioned method or the aforementioned inflammatory bowel disease model is used in the screening of anti-enteritis drugs.

[0009] The method for constructing the inflammatory bowel disease model, the inflammatory bowel disease model and its application provided in this disclosure can achieve the following technical effects:

[0010] The construction method of this disclosure uses intestinal organoids constructed from cells derived from patients with inflammatory bowel disease (IBD). These organoids not only retain the patient's unique gene expression background but also possess the histopathological features associated with the patient's lesions. Furthermore, immune cells or their related components are added during the construction process. For example, patient-derived intestinal immune cells or immune cells from the blood are used for model construction, which more closely simulates the actual in vivo pathological damage progression. This also provides target cells for testing immunomodulatory drugs, expanding the scope of application for in vitro drug efficacy evaluation. This provides an in vitro IBD model constructed from primary intestinal cells of patients for drug development, disease mechanism research, and personalized treatment plans.

[0011] The above general description and the description below are exemplary and illustrative only and are not intended to limit this application. Attached Figure Description

[0012] One or more embodiments are illustrated by way of example with reference to the accompanying drawings. These illustrations and drawings do not constitute a limitation on the embodiments. Elements having the same reference numerals in the drawings are shown as similar elements. The drawings are not to be scaled. And wherein:

[0013] Figure 1 This is a flowchart illustrating a method for constructing an inflammatory bowel disease model according to an embodiment of this disclosure;

[0014] Figure 2 This is a schematic diagram illustrating the construction of an inflammatory bowel disease model provided in an embodiment of this disclosure;

[0015] Figure 3 This is another schematic diagram illustrating the construction of an inflammatory bowel disease model provided in this embodiment of the present disclosure;

[0016] Figure 4 This is a flowchart of another method for constructing an inflammatory bowel disease model provided in this embodiment of the disclosure;

[0017] Figure 5This is a flowchart of the method for constructing an inflammatory bowel disease model according to Embodiment 1 of this disclosure;

[0018] Figure 6(a) is an intestinal tissue image of an IBD patient according to Embodiment 1 of this disclosure;

[0019] Figure 6(b) is an image of the inflammatory intestinal barrier constructed according to Example 1 of this disclosure;

[0020] Figure 7 This is a bright-field image of the inflammatory intestinal barrier constructed according to Embodiment 1 of this disclosure;

[0021] Figure 8 This is a bright-field photograph of the inflammatory bowel disease model I constructed according to Embodiment 1 of this disclosure;

[0022] Figure 9 This is a bright-field photograph of the inflammatory bowel disease model I constructed according to Example 1 of this disclosure, obtained through drug testing of the drug testing model I;

[0023] Figure 10 and Figure 11 The diagram shows the transmembrane resistance test results and the fluorescent yellow apparent permeability coefficient Papp test results for Example 1 of this disclosure, including the inflammatory intestinal barrier, inflammatory bowel disease model I, and drug testing model I.

[0024] Figure 12 and Figure 13 The graphs show the transmembrane resistance test results and the fluorescent yellow apparent permeability coefficient Papp test results for Example 2 of this disclosure, including the inflammatory intestinal barrier, inflammatory bowel disease model II, and drug testing model II.

[0025] Figure 14 and Figure 15 The graphs show the transmembrane resistance test results and the fluorescent yellow apparent permeability coefficient Papp test results for Example 3 of this disclosure, including the inflammatory intestinal barrier, inflammatory bowel disease model III, and drug testing model III.

[0026] Figure 16 and Figure 17 The graphs show the transmembrane resistance test results and the fluorescent yellow apparent permeability coefficient Papp test results for Example 4 of this disclosure, including the inflammatory intestinal barrier, inflammatory bowel disease model IV, and drug testing model IV.

[0027] Figure 18 and Figure 19 The graphs show the transmembrane resistance test results and the fluorescent yellow apparent permeability coefficient Papp test results for Example 5 of this disclosure, including the inflammatory intestinal barrier, inflammatory bowel disease model V, and drug testing model V.

[0028] Figure label:

[0029] 11. Upper culture well; 12. Lower culture well; 13. Diaphragm; 14. Fluid operation channel;

[0030] 21. Inflammatory intestinal barrier; 22. IBD enteritis model. Detailed Implementation

[0031] To provide a more detailed understanding of the features and technical content of the embodiments of this disclosure, the implementation of the embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. The accompanying drawings are for illustrative purposes only and are not intended to limit the embodiments of this disclosure. In the following technical description, for ease of explanation, several details are used to provide a full understanding of the disclosed embodiments. However, one or more embodiments may still be implemented without these details. In other cases, well-known structures and devices may be simplified in their depiction to simplify the drawings.

[0032] The terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this disclosure are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such terms can be used interchangeably where appropriate to understand the embodiments of this disclosure described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion.

[0033] In this disclosure, the terms "upper," "lower," "inner," "middle," "outer," "front," and "rear," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These terms are primarily for better description of the embodiments of this disclosure and their implementations, and are not intended to limit the indicated devices, elements, or components to having a specific orientation, or to require them to be constructed and operated in a specific orientation. Furthermore, some of the aforementioned terms may be used to indicate other meanings besides orientation or positional relationship; for example, the term "upper" may in some cases indicate a dependency or connection relationship. Those skilled in the art can understand the specific meaning of these terms in the embodiments of this disclosure according to the specific circumstances.

[0034] Furthermore, the terms "set up," "connect," and "fix" should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral structure; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be an internal connection between two devices, components, or parts. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of this disclosure according to the specific circumstances.

[0035] Unless otherwise stated, the term "multiple" means two or more.

[0036] In this embodiment of the disclosure, the character " / " indicates that the objects before and after it are in an "or" relationship. For example, A / B means: A or B.

[0037] The term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0038] It should be noted that, unless otherwise specified, the embodiments and features described in the present disclosure can be combined with each other.

[0039] Combination Figure 1 , Figure 2 and Figure 3 As shown in the embodiments of this disclosure, a method for constructing an inflammatory bowel disease model is provided, including the following steps:

[0040] S10. Constructing an inflammatory intestinal barrier using intestinal cells derived from patients with inflammatory bowel disease (e.g.) Figure 2 The densely packed structural layer beneath the diaphragm 13, indicated by the "dashed line" in the middle, is the inflammatory intestinal barrier 21.

[0041] S20. Add a stimulating solution (e.g., in a container such as...) to the inflammatory intestinal barrier. Figure 2 Stimulating solution was added to the upper culture well above the septum 13 (shown by the dotted line) for co-culture to obtain an IBD colitis model (e.g., Figure 3 The damaged structural layer beneath the diaphragm 13, indicated by the dotted line, represents the IBD colitis model 22, i.e., the inflammatory bowel disease model. The stimulating solution contains immune cells and / or their secretions.

[0042] The method for constructing an inflammatory bowel disease (IBD) model provided in this disclosure first utilizes intestinal cells derived from IBD patients to construct an inflammatory intestinal barrier. This addresses the issues of species differences in animal models or gene mutation problems present in traditional cell lines (e.g., the Coco-2 intestinal epithelial cell line). Simultaneously, it preserves the expression characteristics of IBD-related genes and cell morphology, ensuring not only the patient-specific gene expression background but also the patient-specific histopathological features and phenotypic characteristics, making the model more closely resemble the pathological condition in vivo. Then, a stimulating solution containing immune cells and / or immune cell secretions is added to the inflammatory intestinal barrier construction system, generating an immunoinflammatory environment. This causes damage to the intestinal barrier during co-culture, more closely mimicking the actual in vivo pathological damage progression. The stimulation and damage to intestinal cells more closely resemble in vivo immune cell stimulation or immune cell secretion stimulation, better reflecting physiological damage, improving the model's biomimicry, and reproducing the imbalanced immune cell damage to the intestine in IBD pathology. This helps in the discovery and validation of potential drug targets. It also provides target cells for testing immunomodulatory drugs, expanding the scope of application for in vitro drug efficacy evaluation, for example, to evaluate the repair or anti-damage effects of immunomodulatory drugs on in vitro intestinal models of IBD.

[0043] The IBD enteritis model constructed in this embodiment fills the gap in existing enteritis models by lacking the important component of the immune system, enabling better in vitro research on the pathogenesis and development mechanisms of the disease, and providing the possibility of efficacy evaluation for various novel inflammatory enteritis drugs that regulate the state of immune cells.

[0044] Optionally, the IBD colitis model constructed according to the embodiments of this disclosure has one or more of the following characteristics:

[0045] The inflammatory intestinal barrier maintains genetic characteristics and transcriptional levels similar to those of patient tissues.

[0046] The inflammatory intestinal barrier maintains the expression of characteristic markers of various cell subtypes in the human gut, including the absorptive cell marker VIL1, the stem cell marker Ki67, and the secretory cell marker MUC2.

[0047] Before being stimulated, the inflammatory intestinal barrier maintains an appropriate barrier function similar to that of the intestine, manifested in a small molecule permeability (<10e). -6 (cm / s) or cell transmembrane resistance (>200 Ω·cm) 2 Within the approximate physiological range.

[0048] The inflammatory intestinal barrier exhibits significant apoptotic damage after being stimulated by factors related to real pathology and after being cultured in contact with immune cells.

[0049] The inflammatory intestinal barrier exhibits significant impairment of barrier function after being stimulated by factors related to real pathology and after contact culture with immune cells.

[0050] After being stimulated by factors related to real pathology and cultured with immune cells, the inflammatory intestinal barrier exhibits the expression of key biochemical markers of clinical inflammatory bowel disease, such as supernatant interferon-γ, interleukin-8, interleukin-17, and calprotectin.

[0051] In the construction method of this disclosure embodiment, the intestinal cells derived from inflammatory bowel disease patients (IBD patients) used in step S10 to construct the inflammatory bowel barrier are obtained from surgically removed intestinal tissue or intestinal biopsy tissue of IBD patients.

[0052] In some embodiments, the construction method of this disclosure employs a culture device with a porous membrane or a membrane capable of forming a semi-permeable cell barrier, wherein each culture well has a septum to divide the culture well into an upper culture well and a lower culture well. Optionally, an inflammatory intestinal barrier is constructed in the upper culture well, and an stimulating solution is added to the lower culture well for co-culture to obtain an IBD colitis model. Optionally, combined with Figure 2 and Figure 3 As shown, an inflammatory intestinal barrier 21 is constructed in the lower culture well 12, and an irritant solution is added to the upper culture well 11 for co-culture to obtain an IBD colitis model 22. The culture device also includes a fluid manipulation channel 14 for manipulating the lower culture well 12, such as seeding cells and adding culture medium.

[0053] In some embodiments, combined with Figure 2 and Figure 4 As shown, step S10, which involves constructing an inflammatory intestinal barrier using intestinal cells derived from patients with inflammatory bowel disease, includes:

[0054] S11. Digest the intestinal tissue of IBD patients to obtain intestinal extract cells containing intestinal stem cells.

[0055] In step S11, the digestion process includes tissue digestion using biological enzymes. Of course, physical disruption can also be performed before enzyme digestion to facilitate the process. Conventional biological enzymes can be used, such as trypsin, collagenase, and dispersase.

[0056] S12. Intestinal cells extracted from the cells are encapsulated in an extracellular matrix (ECM) gel, and then cultured in vitro with a first culture medium that promotes the proliferation of intestinal stem cells and the formation of intestinal organoids. This promotes the proliferation and spontaneous organoid formation of intestinal stem cells from the extracted cells, resulting in inflammatory intestinal organoids (IBD intestinal organoids). In step S12, the obtained inflammatory intestinal organoids can be expanded and cultured in vitro for a long period and cryopreserved, providing a reserve of patient-derived intestinal cells for subsequent model construction and application.

[0057] S13. Inflammatory intestinal organoids are processed to obtain inflammatory intestinal cells, including single cells and / or cell clusters, to be inoculated. These cells are then seeded onto one side of a septum within a culture well (e.g., the septum of the upper or lower culture wells of the aforementioned culture device), and a first culture medium is added for cell culture to form an inflammatory intestinal barrier (IBD intestinal barrier). In step S13, the septum is a porous membrane or can form a semi-permeable cell barrier; that is, a culture device with a porous membrane or a septum capable of forming a semi-permeable cell barrier is used in this step, such as Transwell or organ-on-a-chip. After adherent growth, the inflammatory intestinal cells will form a converging and dense IBD intestinal barrier on the surface of the porous membrane of the chip or Transwell.

[0058] In step S11 of this embodiment, the intestinal extract cells containing intestinal stem cells can be all digested products of intestinal tissue after digestion. All digested products include various types of cells that may be contained in the intestinal tissue, such as epithelial cells, immune cells, stem cells, secretory cells, etc. The intestinal extract cells containing intestinal stem cells can also be intestinal extract epithelial cells containing intestinal stem cells obtained after processing. The processing method adopts conventional methods, such as performing flow cytometry sorting on all digested products from intestinal tissue after digestion, and obtaining intestinal stem cells that are positive for stem cell markers (such as Ki67, LGR5).

[0059] In this embodiment, the first culture medium used in steps S12 and S13 has no limited composition, as long as it can promote the proliferation of intestinal stem cells and the formation and growth of intestinal organoids.

[0060] In some embodiments, the first culture medium comprises: 45%–52% DMEM basal medium, 45%–52% F12 basal medium, 0.8%–2% 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), and 0.8%–2% GlutaMAX. TM Supplement, 0.8%~2% B-27 TMThe supplements included 5-20 mM nicotinamide, 50-200 ng / mL epidermal growth factor (EGF), 300-1000 nM TGF-β receptor inhibitor, 50-200 ng / mL BMP pathway inhibitor, 0-500 ng / mL Wnt pathway agonist, and 0-10 μM DAPT ((3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester). The components of the first culture medium in this embodiment synergistically promote the proliferation of intestinal stem cells and the formation and growth of intestinal organoids, resulting in IBD intestinal organoids. Specifically, DMEM, F12, and GlutaMax provide basic nutrients such as sugars, amino acids, and salt ions. This formula provides energy, essential components for material synthesis, and suitable osmotic pressure for basic cellular life activities; B27, as a complex nutritional supplement, contains various cytokines and nutrients to support organoid growth; HEPES stabilizes the pH of the cell culture environment and buffers the effects of acidic waste products during cell growth and metabolism; nicotinamide, a vitamin, participates in various redox reactions, possessing antioxidant and anti-inflammatory effects, and promoting cell self-renewal; EGF, an important epithelial growth factor, stimulates epithelial cell growth and proliferation; and TGFβ, BMP inhibitors, and Wnt activators, as a whole, recreate the core growth factor environment exposed by in vivo intestinal stem cells, inhibiting their differentiation while promoting stem cell proliferation and replication. The percentages of the components are volume percentages. The concentrations of the components are final concentrations, i.e., the concentrations based on the first culture medium obtained after mixing all components.

[0061] Optionally, the concentration of nicotinamide in the first culture medium is 5 to 20 mM, for example, 5 mM, 6 mM, 7 mM, 9 mM, 12 mM, 15 mM, 18 mM, 20 mM, or any value in the range of 5 to 20 mM.

[0062] Optionally, the concentration of epidermal growth factor (EGF) in the first culture medium is 50 to 200 ng / mL, for example, 50 ng / mL, 80 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, or any value within the range of 50 to 200 ng / mL.

[0063] Optionally, the concentration of the TGF-β receptor inhibitor in the first culture medium is 300 to 1000 nM, for example, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1000 nM, or any value within the range of 300 to 1000 nM.

[0064] Optionally, the concentration of the BMP pathway inhibitor in the first culture medium is 50 to 200 ng / mL, for example, 50 ng / mL, 80 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, or any value within the range of 50 to 200 ng / mL.

[0065] Optionally, BMP pathway inhibitors include, but are not limited to, Noggin, LDN-212854, R-spondin, etc.

[0066] Optionally, the concentration of the Wnt pathway agonist in the first culture medium is 0-500 ng / mL, meaning the Wnt pathway agonist may not be added to the first culture medium. For example, in step S12, where in vitro culture is used to obtain inflammatory intestinal organoids, the first culture medium needs to contain a Wnt pathway agonist to promote the self-replication and proliferation of intestinal stem cells. The key to the long-term growth, passage, and reuse of intestinal organoids after cryopreservation and thawing lies in the maintenance of intestinal stem cells. Optionally, the concentration of the Wnt pathway agonist is 50-500 ng / mL, for example, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL, 450 ng / mL, 500 ng / mL, or any value within the range of 50-500 ng / mL.

[0067] For example, in step S13, which involves obtaining the intestinal barrier of IBD, the Wnt pathway agonist may not be added to the first culture medium, depending on the different culture stages.

[0068] Optionally, in step S13, cell culture is performed using a first culture medium to form an inflammatory intestinal barrier; this includes: culturing cells in first culture medium I to allow them to adhere and grow, forming an intestinal barrier layer, and then replacing the culture medium with first culture medium II to induce differentiation and maturation of the intestinal barrier layer, thereby obtaining an inflammatory intestinal barrier. Specifically, first culture medium I contains a Wnt pathway agonist but does not contain DAPT, while first culture medium II does not contain a Wnt pathway agonist but contains DAPT.

[0069] Optionally, Wnt pathway agonists include, but are not limited to, Wnt3A protein and CHIR99021, etc.

[0070] Optionally, the concentration of DAPT in the first culture medium is 0~10 μM, meaning that DAPT may not be added to the first culture medium. For example, in step S12, which involves obtaining inflammatory intestinal organoids through in vitro culture, DAPT is not added to the first culture medium.

[0071] For example, in step S13, which involves obtaining the intestinal barrier for IBD, DAPT is added to the first culture medium to induce intestinal stem cells to differentiate into functional epithelial cells. Therefore, the DAPT factor can be added to the first culture medium when the intestinal barrier model is constructed and differentiation and maturation are required. Optionally, the concentration of DAPT is 1~10 μM, for example, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, or any value within the range of 0~10 μM.

[0072] Optionally, the first culture medium comprises: 48% DMEM basal medium, 48% F12 basal medium, 1% 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), and 1% GlutaMAX. TM Supplement, 2% B-27 TM Supplements include 5–20 mM nicotinamide, 50–200 ng / mL epidermal growth factor (EGF), 300–1000 nM TGF-β receptor inhibitor, 50–200 ng / mL BMP pathway inhibitor (including but not limited to Noggin, LDN-212854, R-spondin, etc.), 50–500 ng / mL Wnt pathway agonist (including but not limited to Wnt3A protein, CHIR99021, etc.), and 1–10 μM DAPT.

[0073] In step S13 of this embodiment, processing the inflammatory intestinal organoids to obtain cells to be inoculated, including single cells and / or cell clusters, includes: mechanically dissociating the inflammatory intestinal organoids and digesting them with enzymes to obtain single cells and / or cell clusters to be inoculated. Mechanical dissociation includes physical methods such as repeated pipetting using a micropipette. Conventional enzymes can be used, such as trypsin or dispersing enzymes.

[0074] In step S13, the seeding amount of inflammatory intestinal cells to be inoculated is adjusted according to the culture chip used. Generally, the seeding amount of the culture chip is usually 1×10⁻⁶. 5 ~5×10 5 Cells per square centimeter.

[0075] In the construction method of this disclosure embodiment, the stimulating solution containing immune cells and / or immune cell secretions in step S20 specifically includes three types: one is a stimulating solution containing immune cells (referred to as the first stimulating solution), one is a stimulating solution containing immune cell secretions (referred to as the second stimulating solution), and one is a stimulating solution containing immune cells and immune cell secretions (referred to as the third stimulating solution).

[0076] In some embodiments, the first stimulating solution, comprising immune cells, includes an immune cell resuspension obtained by resuspending immune cells in a second culture medium; wherein the immune cells include commercially available immune cells, intestinal immune cells derived from IBD patients, or immune cells from the blood of IBD patients. During co-culture, damage to the intestinal barrier occurs through the self-contact cell growth of activated immune cells and biological mediators such as cytokines and proteases secreted by the immune cells. In this embodiment, the concentration of immune cells in the immune cell resuspension is not limited, and can be determined based on the inoculation ratio of immune cells to intestinal cells used to construct the inflammatory intestinal barrier (e.g., the inflammatory intestinal cells inoculated in step S13) and the inoculation volume of the stimulating solution.

[0077] Optionally, the ratio (e.g., the quantity ratio, defined as the first dosage ratio) of immune cells to intestinal cells used to construct the inflammatory intestinal barrier (inflammatory intestinal cells seeded in step S13) in the first stimulating solution is 1:5 to 1:50. Optionally, the first dosage ratio is 1:5, 1:8, 1:10, 1:25, 1:30 or 1:50, or any ratio within the range of 1:(5 to 50).

[0078] For example, the cell density of immune cells in the first type of stimulation solution is 50,000 to 200,000 cells / mL.

[0079] Optionally, commercially available immune cells include one or more of peripheral blood lymphocytes (PBMCs), T cells, and monocytes.

[0080] Optionally, intestinal immune cells derived from IBD patients are obtained through the following steps: physically disrupting and enzymatically digesting the intestinal tissue of IBD patients to obtain intestinal extract cells containing intestinal stem cells; centrifuging the obtained intestinal extract cells using a cell separation medium to separate intestinal immune cells. In this embodiment, the cell separation medium includes Percoll cell separation medium. Conventional enzymes can be used in the enzymatic digestion process, such as trypsin, collagenase, and dispersant enzymes.

[0081] Optionally, immune cells in the blood of IBD patients are obtained through the following steps: mixing the blood of IBD patients (using peripheral lymphoid blood) with a cell separation medium and centrifuging to separate the immune cells from the blood of IBD patients. In this embodiment, the cell separation medium includes Percoll cell separation medium.

[0082] Of course, alternatively, intestinal immune cells derived from IBD patients or immune cells from the blood of IBD patients (e.g., peripheral blood lymphocytes) can be cultured in a second culture medium (using conventional immune cell culture methods, such as suspension cell culture) for further expansion or purification. Optionally, relevant immune cell activators (e.g., IL-2, Anti-CD3, PMA, etc.) can be added to the culture medium during the culture process to expand specific types of immune cells, or specific cell types can be purified using cell sorting methods (e.g., flow cytometry cell sorting). Some of the expanded or purified immune cells can be directly used for subsequent model applications (e.g., resuspended in a second culture medium to obtain the first stimulation solution), or cryopreserved for cell storage, with subsequent cell resuscitation and utilization as needed.

[0083] In some embodiments, the first stimulating solution, including the stimulating solution containing immune cells, further includes an immune cell stimulating reagent and / or a first culture medium. That is, the stimulating solution containing immune cells is based on an immune cell resuspension, with the addition of an immune cell stimulating reagent, or the addition of a first culture medium, or the addition of both an immune cell stimulating reagent and a first culture medium. In this embodiment, the immune cell stimulating reagent can stimulate the growth of immune cells during co-culture. Depending on the actual situation, relevant immune cell activators can be added to amplify specific types of immune cells. The addition of a first culture medium is used to adjust the components and content of the stimulating solution, promote the growth of immune cells, and adapt to different application scenarios of the constructed IBD colitis model. The volume of the first culture medium added is not limited and can be determined according to the actual situation.

[0084] Optionally, the immune cell stimulating agent includes IL-2, Anti-CD3, PMA, etc. Optionally, the concentration of the immune cell stimulating agent in the stimulating solution is 0.1–20 μg / mL.

[0085] Optionally, when the stimulation solution containing immune cells includes a first culture medium, the volume ratio of the second culture medium to the first culture medium is 1:1 to 1:20. For example, 1:1, 1:5, 1:10, 1:15, 1:20, or any ratio within the range.

[0086] In one example, the stimulation solution containing immune cells includes a resuspension obtained by resuspending commercially available immune cells in a second culture medium and an immune cell stimulation reagent. In this embodiment, the first stimulation solution uses commercially available immune cells with low activity; the addition of the immune cell stimulation reagent promotes the co-culture effect during the co-culture process.

[0087] In another example, the stimulating solution containing immune cells includes a resuspension obtained by resuspending intestinal immune cells derived from IBD patients in a second culture medium and a first culture medium, with a volume ratio of the second culture medium to the first culture medium of 1:1. In this embodiment, the first stimulating solution uses intestinal immune cells derived from IBD patients, which are highly active. Co-culturing with a mixture of the first and second culture media can simultaneously promote the culture of both immune cells and the inflammatory intestinal barrier.

[0088] In some embodiments, the second culture medium includes 29-32% DMEM basal medium, 29-32% F12 basal medium, 29-32% RPMI-1640 basal medium, 1%-5% fetal bovine serum, and 1%-2% GlutaMAX. TM Supplements include 1-2% N2 supplement, 10-100 ng / mL epidermal growth factor (EGF), and 0-20 μg / mL immune cell activator. In this embodiment, the immune cell activator is determined according to actual needs and includes, but is not limited to, IL-2, Anti-CD3, and PMA (phorbol ester). In this embodiment, the second culture medium uses a composite of three culture media, supplemented with fetal bovine serum and GlutaMAX. TM Supplements, N2 supplements, EGF, and immune cell activators can effectively adapt to the co-culture process. Among them, multiple basal culture media can comprehensively provide the basic nutrients required by two different cell types, as well as provide relevant salt ions to maintain a suitable ionic environment and osmotic pressure for both cell types, maintain epidermal growth factor, and maintain the basic cell growth capacity of epithelial cells. Fetal bovine serum and immune cell activators can promote the growth, activity, and function of immune cells, and construct a more biomimetic IBD colitis model.

[0089] Optionally, the second culture medium includes 29–32% DMEM basal medium, 29–32% F12 basal medium, 29–32% RPMI-1640 basal medium, 1%–5% fetal bovine serum, and 1%–2% GlutaMAX. TM Supplements: 1-2% N2 supplement, 10-100 ng / mL epidermal growth factor (EGF), 0-20 μg / mL immune cell activator.

[0090] Optionally, the second culture medium includes 30% DMEM basal medium, 30% F12 basal medium, 30% RPMI-1640 basal medium, 3% fetal bovine serum, and 2% GlutaMAX. TM The supplement contains 2% N2 supplement, 5 μg / mL immune cell activator, and 10–100 ng / mL epidermal growth factor (EGF).

[0091] In some embodiments, the second stimulating solution I, comprising the stimulating solution containing immune cell secretions, includes: the culture supernatant from the immune cell culture process. In this embodiment, during the immune cell culture process, the corresponding secretions produced during immune cell growth enter the culture medium, and the supernatant containing the corresponding secretions is directly used as the stimulating solution. This homology better balances the culture of immune cells and the inflammatory intestinal barrier, thereby constructing a more realistic IBD colitis model. In this embodiment, the cultured immune cells include commercially available immune cells, intestinal immune cells derived from IBD patients, or immune cells from the blood of IBD patients. Commercially available immune cells include one or more of peripheral blood lymphocytes (PBMCs), T cells, and monocytes. The methods for obtaining intestinal immune cells derived from IBD patients and immune cells from the blood of IBD patients can adopt the aforementioned methods.

[0092] Optionally, the culture supernatant obtained during the immune cell culture process includes: adding a second culture medium to the immune cells for culturing (using conventional immune cell culture methods, such as suspension cell culture). After a preset culturing time, the culture supernatant is obtained. Optionally, during the culturing process, relevant immune cell activators (such as IL-2, Anti-CD3, PHA, etc.) can be added to the culture medium to amplify specific types of immune cells, thereby increasing the concentration of secretions from that specific type of immune cell in the culture supernatant.

[0093] In some embodiments, the second stimulation solution I, which contains secretions from immune cells, also includes a first culture medium. That is, the culture supernatant from the immune cell culture process is mixed with the first culture medium. In this embodiment, the first culture medium is added to adjust the components and concentrations in the stimulation solution to suit different application scenarios of the constructed IBD colitis model. The volume of the first culture medium added to the second stimulation solution I is not limited and can be determined according to actual conditions.

[0094] Optionally, when the stimulating solution containing immune cell secretions includes a first culture medium, the volume ratio of the culture supernatant to the first culture medium is 1:1 to 1:20. For example, 1:1, 1:5, 1:10, 1:15, 1:20, or any ratio within that range.

[0095] In other embodiments, the second stimulating solution II, comprising the secretions of immune cells, includes a recombinant protein solution of recombinant inflammatory cytokine proteins. In this embodiment, the recombinant inflammatory cytokine proteins may be designed based on drug development or based on inflammatory cytokines found in the intestines of IBD patients. The concentration of each recombinant inflammatory cytokine protein is not limited; optionally, the concentration of each recombinant inflammatory cytokine protein (defined as the second concentration) in the recombinant protein solution is 0.1 ng / mL to 500 ng / mL. Optionally, the second concentration is 0.1 ng / mL to 300 ng / mL. Optionally, the second concentration is 0.1 ng / mL to 250 ng / mL.

[0096] Optionally, the recombinant inflammatory factor proteins include one or more of the interleukin (IL) family proteins, tumor necrosis factor (TNF) family proteins, and interferon (IFN) family proteins.

[0097] Optionally, the interleukin family proteins include, but are not limited to, one or more of the following: interleukin 1β (IL1β), interleukin 4 (IL4), interleukin 5 (IL5), interleukin 6 (IL6), interleukin 8 (IL8), interleukin 10 (IL10), interleukin 12 (IL12), and interleukin 23 (IL23).

[0098] Optionally, the concentration of each interleukin family protein is 0.1–25 ng / mL.

[0099] Optionally, tumor necrosis factor family proteins include, but are not limited to, one or more of tumor necrosis factor α, apoptosis-associated factor ligand FasL, etc.

[0100] Optionally, the concentration of each tumor necrosis factor family protein is 25–200 ng / mL.

[0101] Optionally, interferon family proteins include, but are not limited to, one or more of interferon β, interferon γ, etc.

[0102] Optionally, the concentration of each interferon family protein is 25–200 ng / mL.

[0103] Optionally, the recombinant protein solution includes recombinant inflammatory cytokine proteins and a culture medium, wherein the culture medium is not limited, as long as it can be used for culturing the intestinal barrier. Optionally, the culture medium includes a second culture medium or a mixture of a second culture medium and a first culture medium. When the culture medium includes a mixture of a second culture medium and a first culture medium, the mixing ratio is not limited. Optionally, when the culture medium includes a mixture of a second culture medium and a first culture medium, the volume ratio of the second culture medium to the first culture medium is 1:1 to 1:20. For example, 1:1, 1:5, 1:10, 1:15, 1:20, or any ratio within this range.

[0104] In one example, the recombinant protein solution includes interleukin family proteins, tumor necrosis factor family proteins, and a second culture medium. Optionally, the interleukin family proteins include IL12, IL23, IL6, and IL10; the tumor necrosis factor family proteins include tumor necrosis factor α. Optionally, the concentration of each inflammatory cytokine recombinant protein is 0.1 ng / mL to 500 ng / mL. Optionally, the concentration of each inflammatory cytokine recombinant protein is as follows: IL12 concentration is 0.01–2 ng / mL, IL23 concentration is 0.1–5 ng / mL, IL6 concentration is 0.1–10 ng / mL, IL10 concentration is 0.01–1 ng / mL, and tumor necrosis factor α concentration is 50–100 ng / mL.

[0105] In some embodiments, the third stimulating solution I, comprising immune cells and their secretions, includes the supernatant from an in vitro culture of intestinal tissue from an IBD patient. In this embodiment, the acquisition of the supernatant from the in vitro culture of intestinal tissue from an IBD patient is not limited. Optionally, the supernatant from the in vitro culture of intestinal tissue from an IBD patient is obtained through the following steps: physically disrupting and enzymatically digesting the intestinal tissue of an IBD patient to obtain intestinal cells; encapsulating the intestinal cells in an extracellular matrix gel, then adding a culture medium (e.g., a second culture medium) for in vitro culture (the culture time is determined according to actual needs, e.g., 3–7 days); and collecting the culture supernatant to obtain the stimulating solution containing immune cells and their secretions. See steps S11 and S12 above for details. Conventional intestinal cell culture medium can be used as the culture medium; optionally, the aforementioned first culture medium can be used.

[0106] In other embodiments, the third stimulating solution II, comprising immune cells and their secretions, includes a first mixture of in vitro culture supernatant from IBD patient intestinal tissue and immune cells. In this embodiment, the in vitro culture supernatant from IBD patient intestinal tissue is as described above. Optionally, the immune cells include commercially available immune cells, intestinal immune cells derived from IBD patients, or immune cells from the blood of IBD patients. The selection and acquisition of each immune cell are detailed in the aforementioned relevant content and will not be repeated here.

[0107] Optionally, when the third stimulating solution II is a first mixture comprising the supernatant of in vitro cultured intestinal tissue from IBD patients and immune cells, the immune cells are obtained from the cultured tissue obtained from the in vitro culture of intestinal tissue from IBD patients. That is, the immune cells of the cultured tissue and the culture supernatant are obtained in the same culture process and are homologous, in order to obtain a more biomimetic IBD colitis model. Specifically, the method for obtaining the first mixture in this embodiment includes: physically disrupting and enzymatically digesting the intestinal tissue of IBD patients to obtain intestinal extract cells; encapsulating the intestinal extract cells in an extracellular matrix gel, then adding a culture medium (e.g., a second culture medium) for in vitro culture and collecting the culture supernatant; dissociating the cultured tissue to separate the immune cells; and mixing the separated intestinal immune cells with the collected culture supernatant to obtain the first mixture.

[0108] In other embodiments, the third stimulating solution III, comprising immune cells and their secretions, includes a second mixture of recombinant inflammatory factor proteins and immune cells. In this embodiment, the recombinant inflammatory factor proteins may be designed based on drug development or based on inflammatory factors found in the intestines of IBD patients. Immune cells include commercially available immune cells, intestinal immune cells derived from IBD patients, or immune cells from the blood of IBD patients. The selection and acquisition of each immune cell are detailed in the foregoing and will not be repeated here. The types of recombinant inflammatory factor proteins are the same as described in the foregoing and will not be repeated here. Compared to the recombinant protein solution of the second stimulating solution, the concentration of each recombinant inflammatory factor protein in the third stimulating solution III is appropriately reduced. Optionally, the third concentration of each recombinant inflammatory factor protein in the third stimulating solution III is 1 / 10 to 1 / 5 of the second concentration of each recombinant inflammatory factor protein in the recombinant protein solution of the second stimulating solution. Optionally, the third concentration of each recombinant inflammatory factor protein in the third stimulating solution III is 0.1 ng / mL to 100 ng / mL.

[0109] Optionally, the second mixture includes recombinant inflammatory cytokine proteins, immune cells, and a second culture medium. The recombinant inflammatory cytokine proteins include interleukin family proteins and tumor necrosis factor family proteins; the immune cells include helper T cells, monocytes, and NK cells. Optionally, the interleukin family proteins include IL12, IL23, IL1, IL6, IL8, and IL10; the tumor necrosis factor family proteins include tumor necrosis factor α. Optionally, the helper T cells include one or more of Th1 cells and Th17 cells. This is a mixture of immune cells and recombinant inflammatory cytokine proteins mimicking Crohn's disease. Optionally, the concentration of each recombinant inflammatory cytokine protein is 0.1 ng / mL to 100 ng / mL. Optionally, the concentrations of the recombinant proteins of each inflammatory factor are as follows: IL12 concentration is 0.01–0.4 ng / mL, IL23 concentration is 0.1–1 ng / mL, IL6 concentration is 0.1–2 ng / mL, IL10 concentration is 0.01–0.2 ng / mL, and tumor necrosis factor α concentration is 10–20 ng / mL.

[0110] The method for obtaining the second mixture in this embodiment includes: culturing immune cells to obtain an immune cell culture suspension; mixing the immune cell culture suspension with a second culture medium, and then adding recombinant inflammatory factor protein to obtain a third stimulating solution III.

[0111] In this embodiment of the disclosure, the number of immune cells in the third stimulation solution II and the third stimulation solution III can be appropriately reduced compared to the first stimulation solution. Optionally, the third amount (e.g., the number) of immune cells in the third stimulation solution II and the third stimulation solution III is 1 / 2 to 1 / 5 of the first amount of immune cells in the first stimulation solution. That is, when the stimulation solution is a stimulation solution containing immune cells and immune cell secretions, the ratio of immune cells to intestinal cells used to construct the inflammatory intestinal barrier (defined as the second ratio) is 1:10 to 1:200. Optionally, the second ratio is 1:10, 1:20, 1:30, 1:40, 1:50, 1:80, 1:100, 1:150 or 1:200, or any ratio within the range of 1:(10 to 200).

[0112] In step S20 of this embodiment, a stimulating solution is added to the inflammatory intestinal barrier. The amount of stimulating solution added is not limited and is determined based on the composition of the stimulating solution and the preset required degree of IBD colitis damage. For example, if the stimulating solution includes immune cells (e.g., the first or second stimulating solution), the amount is determined by combining the required degree of IBD colitis damage with the ratio of immune cells to intestinal cells used to construct the inflammatory intestinal barrier (e.g., the inflammatory intestinal cells inoculated in step S13).

[0113] In step S20 of the construction method of this embodiment, a stimulating solution is added to the inflammatory intestinal barrier. It is understood that the culture medium used in the construction of the inflammatory intestinal barrier is removed before adding the stimulating solution, and then the stimulating solution is added to the inflammatory intestinal barrier. Based on the structure of the culture device used, the inflammatory intestinal barrier is located on a diaphragm. The location of the stimulating solution addition is determined according to the actual situation. It can be added to the upper culture well on the upper side of the inflammatory intestinal barrier (in which case conventional culture medium, such as the second culture medium, is added to the lower side), or it can be added to both the upper and lower sides of the inflammatory intestinal barrier, i.e., the upper and lower culture wells.

[0114] Optionally, when the stimulating solution is a first stimulating solution or a third stimulating solution, in step S20, the first stimulating solution or the third stimulating solution is added to the upper side of the inflammatory intestinal barrier, that is, into the upper culture well of the culture device.

[0115] Optionally, when the stimulating solution is a second type of stimulating solution, in step S20, the second type of stimulating solution is simultaneously added to the upper and lower sides of the inflammatory intestinal barrier, that is, to the upper culture well and the lower culture well of the culture device.

[0116] This disclosure also provides an inflammatory bowel disease model, which is constructed using the inflammatory bowel disease model construction method of any of the foregoing embodiments.

[0117] The inflammatory bowel disease model of this disclosure is constructed using the aforementioned construction method. Therefore, the constructed inflammatory bowel disease model has all the beneficial effects of the construction method, which will not be repeated here.

[0118] This disclosure also provides the application of inflammatory bowel disease models in the screening of anti-enteritis drugs.

[0119] The inflammatory bowel disease model in this embodiment is constructed using the construction method of any of the foregoing embodiments.

[0120] The following specific embodiments illustrate the construction method of the inflammatory bowel disease model, the inflammatory bowel disease model, and its application according to the present disclosure.

[0121] Example 1

[0122] like Figure 2 , Figure 3 and Figure 5 As shown, a method for constructing an inflammatory bowel disease model includes the following steps:

[0123] S101. The intestinal tissue of IBD patients is physically broken down and bioenzymatically digested to obtain intestinal extract cells containing intestinal stem cells. The intestinal extract cells containing intestinal stem cells can be any digested material from the intestinal tissue after digestion.

[0124] S102. Intestinal cells were extracted and encapsulated in an extracellular matrix (ECM) gel, then the first culture medium was added for in vitro culture to obtain IBD intestinal organoids. The first culture medium included 48% DMEM basal medium, 48% F12 basal medium, 1% 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 1% GlutaMAX™ supplement, 2% B-27™ supplement, 5-20 mM nicotinamide, 50-200 ng / mL epidermal growth factor (EGF), 300-1000 nM TGF-β receptor inhibitor, 50-200 ng / mL BMP pathway inhibitor (including but not limited to Noggin, LDN-212854, R-spondin, etc.), and 50-500 ng / mL Wnt pathway agonist (including but not limited to Wnt3A protein, CHIR99021, etc.).

[0125] S103. After physical disruption and enzymatic digestion of IBD intestinal organoids, inflammatory intestinal cells, including single cells and / or cell clusters, are obtained for inoculation. The obtained inflammatory intestinal cells are then inoculated (1×10⁻⁶ cells / cells). 5 ~5×10 5 Cells / square centimeter) on one side of the septum in a culture well with a septum (e.g., Figure 2 and Figure 3 As shown, cells to be inoculated are seeded into the lower culture wells 12 of the culture device through the fluid operation channel 14, and the device is inverted so that the inoculated cells adhere to the septum 13. Then, the first culture medium I is added for cell culture, allowing the cells to adhere and grow to form an intestinal barrier layer. Then, the first culture medium II is replaced to induce the differentiation and maturation of the intestinal barrier layer, forming an inflammatory bowel barrier (IBD intestinal barrier). Figure 2 The cell structure layer on the lower surface of the septum 13 of the lower culture well 12 of the culture device shown is densely packed. The first culture medium I is the first culture medium of step S102; the first culture medium II is obtained by removing the Wnt pathway agonist and adding 1~10μM DAPT to the first culture medium I.

[0126] S104. Preparation of a stimulation solution containing immune cells: The intestinal cells extracted in step S101 are separated by gradient centrifugation using an immune cell separation solution (such as Percoll cell separation solution) to obtain intestinal immune cells. These cells are then cultured in a second culture medium. During the culture process, an immune cell activation reagent (such as anti-CD3 antibody) is added to the intestinal immune cell culture system to stimulate the immune cells. After culture, the stimulated intestinal immune cells are obtained by centrifugation. The stimulated intestinal immune cells are then resuspended in the second culture medium to obtain an immune cell resuspension, which is used as stimulation solution I. The second culture medium includes 30% DMEM basal medium, 30% F12 basal medium, 30% RPMI-1640 basal medium, 3% fetal bovine serum, and 2% GlutaMAX. TM The supplement contains 2% N2 supplement, 3% immune cell activator and 10-100 ng / mL epidermal growth factor (EGF).

[0127] S105. Remove the first culture medium II from step S103, and add the stimulating solution I obtained in step S104 to the inflammatory intestinal barrier of step S103. For example, add the stimulating solution I to... Figure 2 and Figure 3 The upper culture well and the lower culture well of the culture device shown are added with a second culture medium for co-culture. The ratio of immune cells in the added stimulation solution I to the inflammatory bowel cells inoculated in step S103 is controlled to be 1:5 to 1:50 to obtain inflammatory bowel disease model I.

[0128] In the inflammatory bowel disease model I obtained in Example 1, intestinal immune cells were used as the stimulus.

[0129] Figure 6(a) shows an image of the intestinal tissue of an IBD patient in step S101, and Figure 6(b) shows an image of the IBD organoid constructed in step S102. It can be seen that the intestinal lesions appear in the patient's intestinal tissue, such as the hyperplastic nodular structure indicated by the red arrow in Figure 6(a) and the atrophic intestinal crypt structure indicated by the blue arrow; the corresponding hyperplastic morphology can be seen in the IBD intestinal organoid (as shown by the green arrow in Figure 6(b)).

[0130] Bright-field photograph of the IBD intestinal barrier constructed in step S103 of Example 1 is shown below. Figure 7 As shown, and the bright-field photograph of the inflammatory bowel disease model I constructed in step S105, as shown. Figure 8 As shown. Comparison Figure 7 and Figure 8 As shown, in the inflammatory bowel disease model, the original intestinal villi structure is lost, cells die, and the cell barrier is damaged.

[0131] Example 2

[0132] The difference between Example 2 and Example 1 is that in step S104, when preparing the stimulation solution containing immune cells, the immune cells used are immune cells from the blood of IBD patients. Specifically, in step S104, the intestinal extracted cells used in step S104 are replaced with peripheral lymphoid blood from IBD patients. Cell separation is performed using a gradient centrifugation method with a cell separation medium (such as Percoll cell separation medium) to separate the immune cells from the blood of IBD patients. Then, the immune cells in the blood are cultured using a second culture medium. During the culture process, an immune cell activation reagent (such as anti-CD3 T cell activation antibody) is added to the immune cell culture system to stimulate the immune cells. After the culture is completed, the stimulated immune cells in the blood are obtained by centrifugation. The stimulated immune cells in the blood are then resuspended in a second culture medium to obtain an immune cell resuspension, which serves as stimulation solution II. In step S105, stimulating solution II is added to the inflammatory intestinal barrier from step S103 for co-culture. The ratio of immune cells in the added stimulating solution II to the inflammatory intestinal cells inoculated in step S103 is controlled to be 1:5 to 1:50, thus obtaining inflammatory bowel disease model II. The remaining steps and parameters are the same as in Example 1.

[0133] In the inflammatory bowel disease model II obtained in this embodiment 2, immune cells in the blood are used as the stimulus source.

[0134] Example 3

[0135] The difference between Example 3 and Example 1 is that in step S104, a stimulating solution containing secretions from immune cells is prepared. Specifically, this is the culture supernatant from the immune cell culture process, with a 1:1 ratio of culture supernatant to the first culture medium, mixed to form stimulating solution III. The culture supernatant used in step S104 of Example 1 for culturing intestinal immune cells is employed. In step S105, stimulating solution III is added to the inflammatory intestinal barrier from step S103. Stimulating solution III is added to both the upper (upper culture well) and lower (lower culture well) sides of the inflammatory intestinal barrier for co-culturing to obtain inflammatory bowel disease model III. The remaining steps and parameters are the same as in Example 1.

[0136] In the inflammatory bowel disease model III obtained in this embodiment 3, immune cell secretions were used as the stimulant.

[0137] Example 4

[0138] The difference between Example 4 and Example 1 is that in step S104, the stimulating solution containing immune cells and their secretions is prepared. Specifically, this is a first mixture of the culture supernatant from the in vitro culture of IBD patient intestinal tissue and immune cells extracted from the intestinal tract of IBD patients, which serves as stimulating solution IV. In step S105, stimulating solution IV is added to the inflammatory intestinal barrier obtained in step S103. Specifically, stimulating solution IV is added to the upper side (upper culture well) of the inflammatory intestinal barrier, and a second culture medium is added to the lower side (lower culture well) for co-culture to obtain inflammatory bowel disease model IV. The remaining steps and parameters are the same as in Example 1.

[0139] In the inflammatory bowel disease model IV obtained in Example 3, immune cells and their secretions were used as stimuli.

[0140] Example 5

[0141] The difference between Example 5 and Example 1 is that in step S104, a stimulation solution containing immune cells and their secretions is prepared, specifically a second mixture containing recombinant inflammatory cytokine proteins and immune cells. Specifically, the second mixture includes recombinant inflammatory cytokine proteins, immune cells, and a second culture medium. The recombinant inflammatory cytokine proteins include interleukin family proteins (IL12, IL23, IL6, and IL10) and tumor necrosis factor family proteins (tumor necrosis factor α); the immune cells include helper T cells (Th1 cells and Th17 cells). The concentrations of each recombinant inflammatory cytokine protein are: IL12 concentration of 0.01–0.4 ng / mL, IL23 concentration of 0.1–1 ng / mL, and IL6 concentration of 0.1–2 ng / mL; the amount of T cells used is 1 / 10 to 1 / 5 of the amount of immune cells used in Example 1 (e.g., 3000–10000 T cells / well). The second mixture serves as stimulation solution V. In step S105, the stimulating solution V is added to the inflammatory intestinal barrier from step S103 and co-cultured to obtain the inflammatory bowel disease model V.

[0142] In the inflammatory bowel disease model V obtained in Example 5, stimulating factors that simulate immune cells and their secretions are used as the stimuli.

[0143] In this embodiment, pharmacodynamic evaluation tests were performed on inflammatory bowel disease (IBD) models I (Example 1) to IBD model V (Example 5). The specific testing method was as follows: After obtaining the corresponding IBD models through co-culturing (e.g., 3-5 days) in each embodiment, anti-enteritis drugs (Anti-JAK small molecule drugs or Anti-TNF monoclonal antibody drugs) were added to both the intestinal barrier side and the immune side, respectively, and treated for 2-7 days to obtain the corresponding drug testing models. Specifically, the barrier density of the inflammatory intestinal barrier was tested at the initial addition of the stimulating solution, after co-culturing, and after 7 days of drug testing. The apparent permeability coefficient P of the anti-enteritis drugs was also measured. app .

[0144] Figure 9 The image shown is a bright-field photograph of the inflammatory bowel disease model I from Example 1 after drug testing. (Comparison) Figure 8 and Figure 9 It can be seen that under drug treatment, the intestinal barrier remains intact, while the original intestinal villi structure is maintained.

[0145] The transmembrane resistance test results of the IBD intestinal barrier (corresponding to the single-barrier group in the figure), inflammatory bowel disease model I (corresponding to the co-culture group in the figure), and drug testing model I (corresponding to the positive drug group in the figure) in Example 1 are as follows: Figure 10 As shown, and the test results of the apparent permeability coefficient Papp of fluorescent yellow are as follows. Figure 11 As shown in the figure, the analysis reveals that the transmembrane resistance of the intestinal barrier in the inflammatory bowel disease (IBD) model is significantly lower than that in the IBD model, indicating damage to the intestinal barrier and a decreased ability to block ions in solution, resulting in higher conductivity and lower transmembrane resistance. Similarly, the apparent permeability of fluorescent yellow in the IBD model is significantly increased, indicating damage to the intestinal barrier in this model and a significantly reduced ability to block molecules in solution, allowing a larger number of molecules to escape and penetrate the cell barrier, thus increasing permeability. In contrast, the drug-treated model inhibited barrier damage and maintained barrier function. Therefore, the transmembrane resistance of the drug-treated model was significantly higher than that of the IBD model group, and the apparent permeability of fluorescent yellow was lower than that of the IBD model group, both approaching the state of an undamaged intestinal barrier in IBD.

[0146] The transmembrane resistance test results of the inflammatory intestinal barrier (corresponding to the intestinal barrier in the figure), inflammatory bowel disease model II (corresponding to the co-culture group in the figure), and drug testing model II (corresponding to the positive drug in the co-culture figure) in Example 2 are as follows: Figure 12 As shown, and the test results of the apparent permeability coefficient Papp of fluorescent yellow are as follows. Figure 13As shown in the figure, the analysis reveals that the transmembrane resistance of the intestinal barrier in the inflammatory bowel disease (IBD) model is significantly lower than that in the IBD model, indicating damage to the intestinal barrier and a decreased ability to block ions in solution, resulting in higher conductivity and lower transmembrane resistance. Similarly, the apparent permeability of fluorescent yellow in the IBD model is significantly increased, indicating damage to the intestinal barrier in this model and a significantly reduced ability to block molecules in solution, allowing a larger number of molecules to escape and penetrate the cell barrier, thus increasing permeability. In contrast, the drug-treated model inhibited barrier damage and maintained barrier function. Therefore, the transmembrane resistance of the drug-treated model was significantly higher than that of the IBD model group, and the apparent permeability of fluorescent yellow was lower than that of the IBD model group, both approaching the state of an undamaged intestinal barrier in IBD.

[0147] The transmembrane resistance test results of the inflammatory intestinal barrier, inflammatory bowel disease model III, and drug testing model III in Example 3 are as follows: Figure 14 As shown, and the test results of the apparent permeability coefficient Papp of fluorescent yellow are as follows. Figure 15 As shown.

[0148] The transmembrane resistance test results of the inflammatory intestinal barrier, inflammatory bowel disease model IV, and drug testing model IV in Example 4 are as follows: Figure 16 As shown, and the test results of the apparent permeability coefficient Papp of fluorescent yellow are as follows. Figure 17 As shown.

[0149] The transmembrane resistance test results of the inflammatory intestinal barrier, inflammatory bowel disease model V, and drug testing model V in Example 5 are as follows: Figure 18 As shown, and the test results of the apparent permeability coefficient Papp of fluorescent yellow are as follows. Figure 19 As shown.

[0150] As can be seen, Examples 3, 4 and 5 all successfully constructed the desired inflammatory intestinal barrier damaged by irritant fluid, and all demonstrated the ability to evaluate related drugs.

[0151] The foregoing description and accompanying drawings fully illustrate embodiments of the present disclosure to enable those skilled in the art to practice them. Other embodiments may include structural and other changes. The embodiments represent only possible variations. Individual components and functions are optional unless explicitly required, and the order of operation may vary. Parts and features of some embodiments may be included or substituted for parts and features of other embodiments. Embodiments of the present disclosure are not limited to the structures described above and shown in the accompanying drawings, and various modifications and changes may be made without departing from its scope. The scope of the present disclosure is limited only by the appended claims.

Claims

1. A method for constructing an inflammatory bowel disease model, characterized in that, include: The intestinal tissue of IBD patients was digested and processed to obtain intestinal extract cells containing intestinal stem cells; Intestinal cells were extracted and encapsulated in an extracellular matrix gel, then added to the first culture medium and cultured in vitro to obtain inflammatory intestinal organoids. Inflammatory intestinal organoids are processed to obtain inflammatory intestinal cells, including single cells and / or cell clusters, which are then seeded onto one side of the septum in a culture well with a septum. The first culture medium I is added to the culture to allow the cells to adhere and grow to form an intestinal barrier layer. Then, the culture is replaced with the first culture medium II to induce the differentiation and maturation of the intestinal barrier layer, thus obtaining an inflammatory intestinal barrier. An irritant was added to the inflamed intestinal barrier, and the mixture was co-cultured to obtain an IBD enteritis barrier model. The irritant in the irritant solution is immune cells and / or immune cell secretions; The first culture medium includes: 45%–52% DMEM basal medium, 45%–52% F12 basal medium, 0.8%–2% 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, and 0.8%–2% GlutaMAX. TM Supplement, 0.8%~2% B-27 TM Supplements: 5-20 mM nicotinamide, 50-200 ng / mL epidermal growth factor, 300-1000 nM TGF-β receptor inhibitor, 50-200 ng / mL BMP pathway inhibitor, 0-500 ng / mL Wnt pathway agonist, 0-10 μM DAPT; The first culture medium I is a first culture medium with Wnt pathway agonist added but no DAPT added, and the first culture medium II is a first culture medium without Wnt pathway agonist added but with DAPT added. The ratio of immune cells to inflammatory intestinal cells used to construct the inflammatory intestinal barrier is 1:5 to 1:

50.

2. The construction method according to claim 1, characterized in that, When the stimulus is the secretion of immune cells, the culture supernatant from the immune cell culture process is mixed with the first culture medium as the stimulus solution.

3. The construction method according to claim 2, characterized in that, The volume ratio of culture supernatant to the first culture medium is 1:1 to 1:

20.

4. The construction method according to claim 1, characterized in that, When the stimuli are immune cells and their secretions, the supernatant of in vitro culture of intestinal tissue from IBD patients and immune cells extracted from the intestines of IBD patients are mixed as the stimulant solution.

5. The construction method according to claim 4, characterized in that, The stimulating solution also includes: immune cell stimulating reagents.

6. The construction method according to claim 1, characterized in that, When the stimulus is immune cells, the immune cell resuspension obtained by resuspending the immune cells in the second culture medium is used as the stimulus solution. The second culture medium includes 29-32% DMEM basal medium, 29-32% F12 basal medium, 29-32% RPMI-1640 basal medium, 1%-5% fetal bovine serum, and 1%-2% GlutaMAX. TM Supplements: 1-2% N2 supplement, 10-100 ng / mL epidermal growth factor, 0-20 μg / mL immune cell activator.

7. The construction method according to claim 6, characterized in that, The stimulating solution also includes: immune cell stimulating reagents.

8. The construction method according to claim 5 or 7, characterized in that, The concentration of the immune cell stimulation reagent is 0.1–20 μg / mL.

9. The construction method according to any one of claims 1 to 7, characterized in that, Immune cells include commercially available immune cells, intestinal immune cells derived from IBD patients, or immune cells from the blood of IBD patients.

10. An inflammatory bowel disease model, characterized in that, The model was constructed using the method described in any one of claims 1 to 9 for constructing an inflammatory bowel disease model.

11. The inflammatory bowel disease model constructed by the method of any one of claims 1 to 9, or the inflammatory bowel disease model as described in claim 10, is used in the screening of anti-enteritis drugs.

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