Preparation method and application of allergic rhinitis model

Abstract

The invention discloses a micro-fluidic chip for an allergic rhinitis model, and a preparation method and application of the allergic rhinitis model, wherein the allergic rhinitis model has better physiological relevance, high repeatability and short operation period. According to the method, the human primary nasal epithelium organ is induced and differentiated by utilizing gas-liquid interface culture, and the epithelium model with a cilia structure and a barrier function is formed and is closer to a real physiological state. A micro-fluidic chip is adopted to simulate a local microenvironment of a human nasal cavity, the chip structure has a multi-channel separation design, nasal epithelial cells, mast cells and endothelial cells are integrated, and local allergic inflammatory response is accurately reconstructed. Specific allergens can be introduced into the chip to induce sensitization and excitation, a rapid and standardized allergic rhinitis in-vitro model is established, and experiment repeatability and data comparability are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a preparation method of an allergic rhinitis model and application thereof. BACKGROUND

[0002] Allergic rhinitis is a chronic inflammatory disease of nasal cavity caused by the immune system overreaction to common environmental allergens (such as pollen, dust mites, mold, animal dander, etc.), mainly manifested as nasal congestion, rhinorrhea, sneezing, nasal itching, often accompanied by eye discomfort. Its pathological mechanism involves IgE-mediated immune response, inducing mast cells to release histamine and other inflammatory factors, and then triggering local allergic reactions. This disease is often associated with asthma, eczema and other allergic diseases, and can be secondary to chronic sinusitis or otitis media when severe.

[0003] Existing preclinical studies of allergic rhinitis mostly rely on animal models (such as mice), but such models have obvious limitations in terms of construction period, physiological relevance and experimental controllability, mainly including: 1) Long modeling period and low experimental efficiency. Traditional animal models require multiple stages of sensitization and challenge, and the complete modeling period usually takes about 3 weeks, which is a long experimental period with many operation steps, limiting the large-scale drug screening and mechanism research.

[0004] 2) Strong stress response of animals and prominent ethical issues. Such models usually require multiple intraperitoneal injections and nasal drops, which cause significant stress response in experimental animals, not only increasing ethical controversy, but also greatly affecting the experimental results due to the animal state, resulting in poor repeatability and stability of the model.

[0005] 3) Limited simulation effect due to species differences. There are significant differences between animals and humans in terms of nasal anatomy, immune cell composition and inflammatory response mechanism, which leads to the fact that some drugs are effective in animal models but ineffective in human clinical trials, seriously affecting the translation value.

[0006] In summary, existing animal models have been difficult to meet the research needs of simulating human nasal local allergic inflammatory response and efficiently evaluating drug efficacy, and it is urgent to establish an in vitro model system with better physiological relevance, strong repeatability and short operation period. SUMMARY

[0007] The purpose of the present application is to solve the problems in the prior art and provide a microfluidic chip for allergic rhinitis model and a preparation method of an allergic rhinitis model with better physiological relevance, strong repeatability and short operation period and application thereof.

[0008] The technical scheme of the present application is as follows: The present application first provides a microfluidic chip for an allergic rhinitis model, comprising a central channel, a left channel and a right channel; the two ends of the central channel are respectively provided with a central input port and a central output port; the two ends of the left channel are respectively provided with a left input port and a left output port; the two ends of the right channel are respectively provided with a right input port and a right output port; the central channel is isolated from the left channel and the right channel by a porous membrane.

[0009] As an optimization, the width of the central channel is 400-600 μm.

[0010] As an optimization, the width of the left channel and the right channel is 200-300 μm.

[0011] As an optimization, the porous membrane is a PET membrane with a thickness of 10-15 μm and a pore size of 0.4-3 μm.

[0012] As an optimization, the height of the central channel, the left channel and the right channel is 300-600 μm.

[0013] The present application also provides a method for constructing an allergic rhinitis model using the microfluidic chip, comprising the following steps: 1) obtaining human nasal mucosa epithelial primary organoids, inoculating in the central channel and inducing differentiation of ciliated epithelial structures simulating human nasal mucosa epithelium; 2) resuspending human vascular endothelial cells in endothelial cell culture medium, injecting into the left channel and the right channel of the microfluidic chip at a certain concentration, standing for 4-6 hours to promote adhesion, and then performing perfusion culture for 48-72 hours to make the human vascular endothelial cells grow along the inner surface of the left channel and the right channel and form microvessel structures, simulating the vascular network of the submucosal layer of the nasal cavity; 3) resuspending human mast cells in mixed culture medium, slowly injecting into the left channel and the right channel of the microfluidic chip, standing for 30-60 minutes, and then performing perfusion culture for 48-72 hours using the mixed culture medium; an allergic rhinitis model with a simulated human nasal cavity "epithelial-vascular-immune cell" ternary microenvironment is constructed.

[0014] As an optimization, in step 2), the concentration of the human vascular endothelial cells resuspended in the endothelial cell culture medium is 1×10 5 -1×10 6 cells / mL.

[0015] As an optimization, in step 3), the concentration of the human mast cells resuspended in the mixed culture medium is 1×10 4 -5×10 4 cells / mL.

[0016] As optimization, step 1) is specifically: obtaining human nasal mucosa epithelial primary organoids, preparing a single cell suspension and inoculating into the central channel, introducing nasal epithelial expansion medium into the central channel, left channel and right channel, and placing in a culture box to form a dense monolayer of cells; then removing the culture medium in the central channel to establish an air-liquid interface, replacing the culture medium in the left channel and the right channel with respiratory epithelial ALI differentiation medium to induce a ciliated epithelial structure simulating human nasal mucosa epithelium under the condition of air-liquid interface.

[0017] As optimization, the mixed culture medium is composed of respiratory epithelial differentiation medium, endothelial cell culture medium and immune cell culture medium mixed at a volume ratio of 1:1:1.

[0018] The application provides a use of the allergic rhinitis model for evaluating the effect of an allergic rhinitis drug.

[0019] Compared with the traditional preclinical model of allergic rhinitis, the model of the application is significantly optimized in terms of model construction method, simulation accuracy and experimental efficiency, and has the following technical effects: Traditional animal model construction method: using mice as experimental animals, the experimental period is long, and the sensitization and challenge processes need to be completed in stages. For example, using OVA, the whole process lasts about 3 weeks, the animals are easily affected by stress, and the experimental operation is complicated, with poor repeatability and controllability. Simulation effect: the nasal cavity structure is quite different from that of humans, and some drugs are effective in animal models but ineffective in humans; the local immune mechanism of the nasal cavity is different from that of humans, and false positive or false negative drug screening results are easily generated; the simulated allergic reaction is mainly systemic immune activation, which cannot accurately reflect the local mucosal immune inflammatory process.

[0020] The model of the application uses human nasal mucosa epithelial primary cells, which are expanded and then induced to differentiate under an air-liquid interface, and are assembled into a complete system simulating the nasal cavity microenvironment by using a microfluidic chip. The whole process can be completed in 2 weeks, the time cost is significantly reduced, and the chip platform operation is highly standardized and easy to repeat. Simulation effect: the ciliated epithelium with functionality is constructed in the chip, combined with mast cells and endothelial cells, to form a complete local microvascular-immune microenvironment; after exposure to allergens, the expression of inflammatory factors and mast cell secreted factors is significantly up-regulated, the epithelial barrier function is decreased (TEER value is decreased), and the mucus secretion is increased, which are consistent with the characteristics of allergic reactions in clinical practice; multiple parameters can be quantified in real time to provide high sensitivity for drug efficacy evaluation.

[0021] The application utilizes gas-liquid interface culture to induce differentiation of human primary nasal epithelial organoids, forms an epithelial model with cilia structure and barrier function, and is closer to the real physiological state. A microfluidic chip is used to simulate the local microenvironment of human nasal cavity, the chip structure has a multi-channel separation design, integrates nasal epithelial cells, mast cells and endothelial cells, and precisely reconstructs local allergic inflammatory response. Specific allergens can be introduced into the chip to induce allergy and trigger, establish a rapid and standardized allergic rhinitis in vitro model, and significantly improve the repeatability of experiments and data comparability. The application can realize multi-parameter comprehensive evaluation of drug effect and inflammation degree by detecting multiple indexes such as cytokines, mucus secretion and epithelial barrier function, improve research efficiency and clinical transformation value. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a three-dimensional schematic diagram of the microfluidic chip in Example 1.

[0023] Figure 2 It is a three-dimensional cross-sectional schematic diagram of the microfluidic chip in Example 1.

[0024] Figure 3 It is a cross-sectional structure schematic diagram of the microfluidic chip in Example 1.

[0025] Figure 4 It is an immunofluorescence staining phenotype identification diagram of the nasal cavity organoid in Example 2.

[0026] Figure 5 It is an immunofluorescence staining diagram of the nasal cavity organoid in Example 2.

[0027] Figure 6 It is a schematic diagram of cell morphology in the three-channel cross-section of the microfluidic chip in Example 2.

[0028] In the figure: 1, central channel; 11, central input port; 12, central output port; 2, left side channel; 21, left side input port; 22, left side output port; 3, right side channel; 31, right side input port; 32, right side output port; 4, multi-hole membrane. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all the embodiments. Example 1

[0030] As Figures 1-3As shown, the embodiment provides a microfluidic chip for allergic rhinitis model, which comprises a central channel 1, a left channel 2 and a right channel 3; the two ends of the central channel 1 are respectively provided with a central input port 11 and a central output port 12; the two ends of the left channel 2 are respectively provided with a left input port 21 and a left output port 22; the two ends of the right channel 3 are respectively provided with a right input port 31 and a right output port 32; the central channel 1 is isolated from the left channel 2 and the right channel 3 by a porous membrane 4. The width of the central channel 1 is 500 μm. The width of the left channel 2 and the right channel 3 is 200 μm. The porous membrane 4 is a PET film with a pore size of 3 μm. The height of the central channel 1, the left channel 2 and the right channel 3 is 400 μm. Example 2

[0031] The method for constructing an allergic rhinitis model using the microfluidic chip of Example 1 comprises the following steps:

[0032] The excess nasal mucosa tissue in the middle turbinate region was obtained from patients undergoing nasal polyp surgery after ethical approval, and immediately placed in a 4°C pre-cooled tissue preservation solution after collection, and transported to the laboratory within 40 minutes after the operation. The tissue was cut into about 1 mm 3 pieces and added to a TrypLE Express digestion solution containing 10 mM Y27632, and digested at 37°C water bath for 40 minutes, while intermittently blowing and dispersing the tissue. After filtering through a 70 μm cell filter, the cell mass was collected and resuspended in 30 μL Matrigel to form a round dot-shaped gel in a 24-well plate. The 24-well plate was incubated in a 37°C, 5% CO2 cell incubator for 30 minutes until the gel was completely solidified, then 600 μL of nasal epithelial expansion medium (Biozellen CM Human Nasal Mucosa Organoid Culture Medium, Catalog No.: B-MN-00001-500, Brand: Biozellen) was added, and the medium was replaced every two days, and the culture was continued for 7 days. The formed vesicular nasal cavity organoids were uniform in size and complete in structure. They were used for freezing or subsequent chip experiments.

[0033] The nasal cavity organoids were digested with trypsin (0.25% Trypsin-EDTA) for 5 minutes to obtain a single cell suspension, and then inoculated into the central channel 1 of the microfluidic chip coated with Matrigel at a density of 5×10 4 cells / 10 μL, and then nasal epithelial expansion medium (Biozellen CM Human Nasal Mucosa Organoid Culture Medium, Catalog No.: B-MN-00001-500, Brand: Biozellen) was introduced into the central channel 1, left channel 2 and right channel 3, and then placed in the incubator for continuous culture for 48 hours to form a dense monolayer of cells.

[0034] As shown in Figure 4

[0035] Then the nasal epithelial expansion medium in the central channel 1 was removed using a pipette to establish an air-liquid interface (ALI). The medium in the left channel 2 and the right channel 3 was replaced with a respiratory epithelial ALI-specific differentiation medium from STEMCELL (STEMCELL, item number: #05001), which was replaced every two days. The induction culture was continued under ALI conditions for 10 days.

[0036] On the 10th day, the chip was taken for immunofluorescence staining. The results showed that the cells in the central channel 1 expressed β-tubulin positive cilia structure, arranged uniformly, and some areas showed Muc5ac positive goblet cells, indicating that multiple types of functional respiratory epithelial structures had been successfully induced under the air-liquid interface, as shown in Figure 5 It can be seen that the nasal epithelial gland structure (green: Phalloidine) was formed by induction and differentiation, and the ciliated epithelial structure (red: βIV-Tubulin) was formed at the edge. Similarly, DAPI (blue) was used to locate the position of the cell nucleus, and after merging, the ciliated epithelial structure could be clearly seen covering the periphery of the nasal epithelial gland, with distinct structures.

[0037] In the central channel 1 of the microfluidic chip that has completed the air-liquid interface induction and differentiation, the nasal epithelial cells have formed a continuous and functional monolayer. To simulate the local blood vessels and immune microenvironment of the nasal cavity, human umbilical vein endothelial cells (HUVEC) were inoculated in the left channel 2 and the right channel 3 of the microfluidic chip, with a concentration of 1×10 6 cells / mL, and 20 μL of cell suspension was injected into each channel. After 6 hours of standing, perfusion culture was started, with a flow rate of 0.5 μL / min, using EGM-2 medium for continuous culture for 72 hours to form an endothelial wrapping structure.

[0038] Then human mast cells (LUVA, item number: CTCC-001-0351, source: Kerafast Cell Bank, USA) were introduced, with a concentration of 2×10 4 ​cells / mL, 20 μL of mast cell suspension was injected into each of left channel 2 and right channel 3, and after standing for 30 minutes, the mixed medium was continuously used to perfuse and culture for 48 hours. The mixed medium was composed of the respiratory epithelial ALI special differentiation medium, the endothelial cell culture medium (ECM endothelial cell culture medium, model number: CTCC-002-031, brand: Meisen CTCC) and the immune cell culture medium (StemProTM-34 SFM (1X) medium, model number: 1063901, brand: Gibco) mixed at a ratio of 1:1:1, so as to ensure the stable function state of the three types of cells.

[0039] Figure 6 The microfluidic chip three-channel section is a schematic diagram of cell morphology, and the blood vessel endothelial cells in the left and right channels form a tubular structure, and the mixed medium containing mast cells can flow between them; the central channel is differentiated to form a respiratory epithelial structure with cup cell function and cilia structure, and the medium containing allergen can be reperfused between them.

[0040] After the microfluidic chip model is constructed, it is processed in three groups: a blank control group: the central channel 1 is not loaded with an antigen; a model group: the central channel 1 is loaded with 100 μg / mL ovalbumin (OVA) allergen; and a drug group: OVA is combined with loratadine (10 μM) and montelukast sodium (5 μM).

[0041] Each group is processed by the microfluidic perfusion system (chip perfusion and ring control module Avt-Perfusion / IN0401 / IN0301) of Avatarget, and the perfusion rate is set to 0.5 μL / min, and the stimulation is continued for 72 hours. During the stimulation, the changes in the morphology of the epithelial cells on the chip are observed, and the phenomena such as increased mucus secretion and cell edge contraction are recorded. After the perfusion is completed, the effluent is collected, and the histamine level is detected by ELISA (histamine ELISA kit, model number: BGT-KET-336, brand: Biogradetech). The results show that the histamine concentration of the model group is significantly increased, and the histamine concentration of the drug group is decreased to be much lower than that of the model group but higher than that of the blank group, indicating that the drug treatment can effectively alleviate the inflammatory reaction of mast cells.

[0042] Therefore, the model of the component of the application has good anti-allergic drug reaction recognition ability, and is suitable for in vitro evaluation applications such as efficacy screening of candidate drugs, mechanism research and optimization of drug administration strategy.

[0043] The above is only an example of the characteristic implementation of the application, and does not constitute any limitation on the protection scope of the application. Any technical solution formed by equivalent exchange or equivalent replacement falls within the scope of the protection of the application.

Claims

1. A microfluidic chip for an allergic rhinitis model, characterized in that: The invention comprises a central channel (1), a left channel (2) and a right channel (3); the central channel (1) is provided with a central input port (11) and a central output port (12) at both ends; the left channel (2) is provided with a left input port (21) and a left output port (22) at both ends; the right channel (3) is provided with a right input port (31) and a right output port (32) at both ends; the central channel (1) is isolated from the left channel (2) and the right channel (3) by a porous film (4).

2. The microfluidic chip for an allergic rhinitis model according to claim 1, wherein: The width of the central channel (1) is 400-600 μm.

3. The microfluidic chip for the allergic rhinitis model according to claim 2, characterized in that: The width of the left channel (2) and the right channel (3) is 200-300 μm.

4. The microfluidic chip for the allergic rhinitis model according to claim 3, characterized in that: The porous film (4) is a PET film with a thickness of 10-15 μm and a pore size of 0.4-3 μm.

5. The microfluidic chip for the allergic rhinitis model according to claim 4, characterized in that: The heights of the central channel (1), the left channel (2) and the right channel (3) are 300-600 μm.

6. A method for constructing an allergic rhinitis model using the microfluidic chip according to any one of claims 1 to 5, characterized in that: The following steps are involved: 1) Obtain primary human nasal epithelial organoids, seed them in the central channel (1) and induce differentiation into ciliated epithelial structures that mimic human nasal epithelial epithelium; 2) Resuspend human vascular endothelial cells in endothelial cell culture medium and inject them into the left channel (2) and right channel (3) of the microfluidic chip. Let them stand for 4–6 hours to promote their adhesion to the wall. Then, perform perfusion culture for 48–72 hours to allow the human vascular endothelial cells to grow along the inner surface of the left channel (2) and right channel (3) and form microvascular structures, simulating the vascular network of the nasal submucosal layer. 3) Resuspend the human mast cells in a mixed culture medium and slowly inject them into the left channel (2) and the right channel (3) of the microfluidic chip. After standing for 30-60 minutes, perfuse the culture medium with the mixed culture medium for 48-72 hours. An allergic rhinitis model with a ternary microenvironment simulating the human nasal cavity "epithelium-blood vessels-immune cells" is constructed.

7. The method for constructing an allergic rhinitis model according to claim 6, wherein: In step 2), the concentration of human vascular endothelial cells after resuspending in endothelial cell culture medium is 1×10 5 –1×10 6 cells / mL; in step 3), the concentration of human mast cells after resuspending in mixed culture medium is 1×10 4 –5×10 4 cells / mL.

8. The method for constructing an allergic rhinitis model according to claim 7, characterized in that: Step 1) is specifically as follows: obtaining primary organoids of human nasal mucosal epithelium, preparing a single cell suspension and inoculating it into the central channel (1), introducing nasal epithelial expansion culture medium into the central channel (1), the left channel (2) and the right channel (3), and culturing them in an incubator to form a dense monolayer of cells; then removing the culture medium in the central channel (1) to establish an air-liquid interface, replacing the culture medium in the left channel (2) and the right channel (3) with respiratory epithelial ALI differentiation culture medium to induce and culture a ciliated epithelial structure that simulates the human nasal mucosal epithelium under air-liquid interface conditions.

9. The method for constructing an allergic rhinitis model according to claim 8, characterized in that: In step 3), the mixed culture medium is composed of respiratory epithelial differentiation medium, endothelial cell culture medium and immune cell culture medium in a volume ratio of 1:1:

1.

10. Use of an allergic rhinitis model constructed by the method according to any one of claims 6 to 9, characterized in that: Used to evaluate the efficacy of drugs for allergic rhinitis.

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