Bionic micro-lung chip, bionic micro-lung chip model and construction method and application of bionic micro-lung chip model

Abstract

The invention provides a bionic micro-lung chip, a bionic micro-lung chip model and a construction method and application of the bionic micro-lung chip model. The bionic micro-lung chip provided by the invention is structurally formed by cascading complex multi-layer structures so as to establish pulmonary epithelium and pulmonary interstitial structures, and can simulate pulmonary alveolar respiratory rhythm and hypoxia state by being connected with a micro-pressure device. Various pathological models such as pneumonia, pulmonary fibrosis and interstitial pulmonary diseases can be established by introducing foreign stimulation factors. The lung tissue structure constructed by the bionic micro-lung chip model is closer to the lung distal tissue, and a platform is created for lung tissue disease research and treatment and drug research and development.

Description

Technical Field

[0001] The present invention relates to a bionic micro-lung chip and a construction method and application of a bionic micro-lung chip model, belonging to the technical field of tissue engineering and biomanufacturing. Background Art

[0002] The lung is an important respiratory organ in the human body. From the rare lymphangioleiomyomatosis to the common idiopathic pulmonary fibrosis, there is a lack of effective research models. In particular, the unknown etiology of most interstitial lung diseases and the large differences in pathological characteristics have led to slow research progress.

[0003] In vitro two-dimensional cell culture models and animal models are traditionally used methods for disease mechanism research and preclinical drug screening. However, the accuracy and credibility of these two models are not high, which may cause huge cost losses. Therefore, the establishment of an in vitro bionic model that can simulate the specificity of human tissues / organs has important scientific research significance and application prospects.

[0004] Organ chips are an emerging method used in recent years to simulate human organ functions and study drugs, and are mainly used in disease research and drug screening. However, the lung chip models that have been established at this stage are mostly dedicated to mimicking the blood-gas barrier, that is, the alveolar epithelium-endothelial barrier, mainly simulating a single layer of alveolar epithelium and a single layer of endothelial cells. This type of model is widely used in the study of diseases related to blood-gas barrier function lesions, and is not suitable for lung diseases where the lesions involve the interstitial space of the lung, such as interstitial lung disease. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a bionic micro-lung chip, which has a lung epithelium-pulmonary interstitial structural unit and can be applied to disease mechanism research and drug screening.

[0006] The present invention also aims to provide a bionic micro-lung chip model based on the above bionic micro-lung chip and a construction method thereof.

[0007] To achieve the above object, the present invention first provides a bionic micro-lung chip, which includes an upper culture layer, a porous membrane layer, and a lower culture layer; the porous membrane layer is located between the upper culture layer and the lower culture layer;

[0008] The upper culture layer includes a lung epithelial structure inlet hole, a lung interstitial structure inlet hole, a lung epithelial structure microchannel, a lung epithelial structure outlet hole, a lung interstitial structure outlet hole, a culture medium upper layer inlet hole, and a culture medium upper layer outlet hole;

[0009] The lower culture layer includes a lower layer outlet pool of the pulmonary interstitial structure, a lower layer outlet pool of the culture medium, a microcolumn structure, a culture medium microfluidic channel, a lower layer inlet pool of the culture medium, a lower layer inlet pool of the pulmonary interstitial structure, and a pulmonary interstitial structure microfluidic channel;

[0010] in:

[0011] The lung epithelial structure inlet hole and the lung epithelial structure outlet hole penetrate the upper culture layer and are respectively arranged at two ends of the lung epithelial structure microchannel;

[0012] The entrance hole of the pulmonary interstitial structure and the exit hole of the pulmonary interstitial structure penetrate the upper culture layer and are respectively connected to the entrance pool of the lower layer of the pulmonary interstitial structure and the exit pool of the lower layer of the pulmonary interstitial structure;

[0013] The culture medium upper layer inlet hole and the culture medium upper layer outlet hole penetrate the upper culture layer and are respectively connected to the culture medium lower layer inlet pool and the culture medium lower layer outlet pool;

[0014] The lower layer inlet pool of the pulmonary interstitial structure and the lower layer outlet pool of the pulmonary interstitial structure are respectively arranged at two ends of the microchannel of the pulmonary interstitial structure;

[0015] The culture medium lower layer inlet pool and the culture medium lower layer outlet pool are respectively arranged at two ends of the culture medium microfluidic channel;

[0016] The culture medium microchannel is connected with the lung interstitial structure microchannel, and a plurality of microcolumn structures are arranged at the connection point between the two.

[0017] The lung epithelial structure microchannel is arranged above the lung interstitial structure microchannel, and the two are connected through the through holes in the porous membrane layer.

[0018] In the above-mentioned bionic micro-lung chip, the positional relationship of the upper culture layer, the porous membrane layer and the lower culture layer is arranged from top to bottom in sequence, wherein the cells in the alveolar epithelial structure in the upper culture layer and the cells in the pulmonary interstitial structure in the lower culture layer can interact with each other through the porous micro-membrane.

[0019] In the above-mentioned bionic micro-lung chip, the lung epithelial structure microfluidic channel and the lung interstitial structure microfluidic channel are in the same vertical plane, the two are separated by a porous membrane layer, and interact with each other through the porous membrane layer.

[0020] In the above-mentioned bionic micro-lung chip, the pulmonary interstitial structure microfluidic channel is used to culture pulmonary interstitial structure and cells.

[0021] In the above-mentioned bionic micro-lung chip, the lung epithelial structure microfluidic channel is used to culture the lung epithelial barrier structure of the alveoli. Preferably, the lung epithelial structure microfluidic channel is composed of a first lung epithelial structure microfluidic channel, a second lung epithelial structure microfluidic channel, and a third lung epithelial structure microfluidic channel; the second lung epithelial structure microfluidic channel is arranged above the lung interstitial structure microfluidic channel, and the two are connected through the through hole in the porous membrane layer. The lung epithelial structure microfluidic channel is located on the lower surface of the upper culture layer, which is a microfluidic channel surrounded by the upper culture layer and the porous membrane layer. The opening bottoms of the lung epithelial structure inlet hole and the lung epithelial structure outlet hole are connected to the lung epithelial structure microfluidic channel, and the porous membrane layer and the lower culture layer form a storage pool for the culture medium.

[0022] In the above-mentioned bionic micro-lung chip, the culture medium microchannel stores culture medium to provide growth substances for cells in the pulmonary interstitial structure in the pulmonary interstitial structure microchannel. Preferably, the culture medium microchannel consists of a first culture medium microchannel, a second culture medium microchannel, and a third culture medium microchannel; the second culture medium microchannel is connected to the pulmonary interstitial structure microchannel.

[0023] In the above-mentioned bionic micro-lung chip, preferably, the thickness of the porous membrane layer is 10 microns-50 microns, the diameter of the through hole is 5 microns-15 microns, and the center distance of adjacent through holes is 10-50 microns. The porous membrane layer can be obtained by high-speed PDMS rotation. The micro-column array mold used in the preparation of the porous membrane layer is made by micro-machining dry etching, wherein the micro-column diameter is 5-15 microns and the height is 30-55 microns. During preparation, a mixture of a curing agent and PDMS in a mass ratio of 1:13-1:15 is formed on the above-mentioned mold using a glue spinning machine to form a PDMS porous membrane, which is dried and used. According to the rotation speed and the glue spinning time, a PDMS porous membrane with a thickness range of 10 microns-50 microns can be made.

[0024] In the above-mentioned bionic micro-lung chip, preferably, the cross-sectional dimensions of the first microfluidic channel of the pulmonary epithelial structure, the second microfluidic channel of the pulmonary epithelial structure, and the third microfluidic channel of the pulmonary epithelial structure are the same, and the height of the cross section is 100-300 microns and the width is 250-650 microns. The lengths of the first microfluidic channel of the pulmonary epithelial structure, the second microfluidic channel of the pulmonary epithelial structure, and the third microfluidic channel of the pulmonary epithelial structure can be selected as needed.

[0025] In the above bionic micro-lung chip, preferably, the height of the cross section of the culture medium micro-channel is 100-300 microns, and the width is 200-600 microns. The culture medium micro-channel is located on the upper surface of the lower culture layer, and is a micro-channel surrounded by the lower culture layer and the porous membrane layer.

[0026] In the above bionic micro-lung chip, preferably, the height of the cross section of the pulmonary interstitial structure microchannel is 100-300 microns, and the width is 200-600 microns. The pulmonary interstitial structure microchannel is located on the upper surface of the lower culture layer, and is a microchannel surrounded by the lower culture layer and the porous membrane layer.

[0027] In the above-mentioned bionic micro-lung chip, the lung epithelial structure entrance hole and the lung epithelial structure exit hole are the entrances and exits for planting the lung epithelial structure in the upper culture layer, and are also the storage pools for the culture medium, and their diameters can be set to 2-6 mm.

[0028] In the above-mentioned bionic micro-lung chip, in particular, the inlet hole of the lung epithelial structure is connected to a micro-pressure device, such as: ELVE FLOW's microfluidic pressure and flow controller, Fluigent precision pressure controller, peristaltic pump, etc. The lung epithelial structure microchannel is connected to the output tube of the micro-pressure device through the inlet hole of the lung epithelial structure, which can simulate the alveolar breathing rhythm and pass hypoxic gas to simulate hypoxic conditions; and it can also realize circulatory ventilation and real-time detection of changes in in vitro three-dimensional tissue function, such as barrier integrity, inflammation and cell migration of the lung epithelium.

[0029] In the bionic micro-lung chip, in particular, the thickness of the lower culture layer is 100-150 micrometers. By controlling the thickness of the lower culture layer within the above range, the chip can be easily used for confocal microscopy observation.

[0030] In the present invention, the preparation of the upper culture layer and the lower culture layer can be carried out with reference to the method commonly used for preparing microfluidic chips. For example, the lower culture layer can be formed by using a glue spreader to slowly throw a mixture of a curing agent and PDMS in a mass ratio of 1:10 on a mold of the lower culture layer, vacuuming to remove bubbles, and heating for 1 hour to solidify. The thickness of the upper culture layer is 2-3 mm. The mixture of a curing agent and PDMS in a mass ratio of 1:10 is poured on a mold of the upper culture layer, vacuuming to remove bubbles, and heating for 1 hour to solidify. The membrane molds of the upper culture layer and the lower culture layer can be prepared according to the required shape using soft lithography technology, and the molds prepared by this method can be reused.

[0031] In the above bionic micro-lung chip, in particular, the culture medium upper layer inlet hole and the culture medium lower layer inlet pool are connected to a micro-pressure device. The culture medium micro-channel is connected to the micro-pressure device through the culture medium upper layer inlet hole and the culture medium lower layer inlet pool, which can provide dynamic culture conditions and realize a dynamic culture system.

[0032] The above-mentioned bionic micro-lung chip provided by the present invention can be assembled by plasma treatment-hot pressing-uncovering-plasma treatment-hot pressing, which can specifically include the following steps: firstly, the microchannel side of the upper culture layer and the PDMS porous membrane are plasma treated, and the treated two sides are gently bonded together, and after hot pressing on a heating plate at 95°C for 1 hour, the upper culture layer is gently uncovered from the microporous membrane structure, and the PDMS microporous membrane is bonded to the upper culture layer; then the above-mentioned structure and the microfluidic side of the lower culture layer are plasma treated again, and then aligned and bonded together at 95°C for 1 hour to complete the assembly of the bionic micro-lung chip. After high-pressure sterilization and drying, the bionic micro-lung chip is placed in a carbon dioxide incubator for standby use.

[0033] The present invention also provides a method for constructing a bionic micro-lung chip model, which is based on the above-mentioned bionic micro-lung chip and comprises the following steps:

[0034] Step 1: Mix the pulmonary interstitial cells with the biohydrogel material to obtain a pulmonary interstitial cell mixture, and inject the mixture into the pulmonary interstitial structure microchannel through the pulmonary interstitial structure inlet hole and the pulmonary interstitial structure lower layer inlet pool, and fill the pulmonary interstitial structure microchannel, the pulmonary interstitial structure lower layer outlet pool, and the pulmonary interstitial structure outlet hole;

[0035] Solidifying the biohydrogel material by heating or secondary cross-linking;

[0036] Step 2: injecting culture medium into the culture medium microfluidic channel through the culture medium upper layer inlet hole and the culture medium lower layer inlet pool, so that the culture medium fills the culture medium microfluidic channel, the culture medium lower layer outlet pool and the culture medium upper layer outlet hole; adding the matrix glue solution through the lung epithelial structure inlet hole to fill the lung epithelial structure microfluidic channel, and flowing into the lung epithelial structure outlet hole; placing in a carbon dioxide incubator and letting it stand;

[0037] Step 3: removing the matrix glue solution in the inlet hole and the outlet hole of the lung epithelial structure; adding the lung epithelial cell suspension to the inlet hole of the lung epithelial structure, and the lung epithelial cell suspension flows into the microchannel of the lung epithelial structure and the outlet hole of the lung epithelial structure; placing in a carbon dioxide incubator and letting it stand;

[0038] Step 4: Remove the waste liquid from the lung epithelial structure inlet hole, the lung epithelial structure outlet hole, the culture medium upper layer inlet hole, and the culture medium upper layer outlet hole, and add fresh culture medium to the lung epithelial structure inlet hole and the culture medium upper layer inlet hole respectively to complete the construction of the bionic micro-lung chip model.

[0039] The above-mentioned bionic micro-lung chip model provided by the present invention has a higher degree of simulation and is more similar to the distal lung tissue structure. In the construction method of the above-mentioned bionic micro-lung chip model, preferably, the lung interstitial cells include lung fibroblasts and vascular endothelial cells; more preferably, the lung interstitial cells also include one or a combination of more than two of lung myofibroblasts and lung interstitial immune cells. Among them, lung fibroblasts and vascular endothelial cells are necessary cells, and others can be added as appropriate according to the research objectives.

[0040] In the above-mentioned method for constructing the bionic micro-lung chip model, preferably, the biohydrogel material includes one or more mixtures of Gelma, matrigel, fibrinogen, and adhesion protein, or a combination of two or more thereof.

[0041] In the above-mentioned method for constructing the bionic micro-lung chip model, preferably, the hydrogel can be solidified by temperature in the following manner: after mixing lung fibroblasts, vascular endothelial cells, etc. with matrix glue, inject them into the microchannel through the inlet hole, and stand in a carbon dioxide incubator at 37°C for 35 minutes, the matrix glue is completely solidified.

[0042] In the above-mentioned method for constructing a bionic micro-lung chip model, preferably, the cells in the cell suspension include one or a combination of two or more of lung epithelial cells, alveolar macrophages, and neutrophils. Among them, alveolar epithelial cells are necessary cells, and immune cells such as alveolar macrophages can be added as appropriate according to the research purpose.

[0043] In the above-mentioned method for constructing the bionic micro-lung chip model, preferably, the construction method also includes: when the growth density of lung epithelial cells is 90-95%, replacing the culture medium in the entrance hole of the lung epithelial structure with a culture medium containing 1 micromole of dexamethasone, and continuing to culture for at least 2 days to enhance the tight connection between the lung epithelial cells so that they form a dense single layer of lung epithelium.

[0044] In the above-mentioned method for constructing the bionic micro-lung chip model, preferably, the method further comprises:

[0045] adding immune cells to the entrance hole of the lung epithelial structure, allowing the immune cells to flow into the microchannel of the lung epithelial structure, thereby obtaining a bionic micro-lung chip model with immune function;

[0046] Alternatively, a target agent is added into the lung epithelial structure microchannel through the lung epithelial structure inlet hole, and the lung epithelial structure microchannel is dynamically perfused and cultured to obtain a bionic micro-lung chip model with disease analysis function.

[0047] The present invention also provides a bionic micro-lung chip model, which is constructed by the above construction method.

[0048] The present invention also provides the application of the above-mentioned bionic micro-lung chip model in the detection of causes of disease, toxins and drugs, which includes:

[0049] Introducing the substance to be tested into the lung epithelial structure microfluidic channel through the lung epithelial structure inlet hole, and / or introducing the substance to be tested into the culture medium microfluidic channel through the culture medium upper layer inlet hole;

[0050] The regulation result of the test substance on the alveolar epithelial structure and / or the pulmonary interstitial structure is obtained.

[0051] The bionic micro-lung chip model provided by the present invention is a micro-lung chip model comprising alveolar epithelium-pulmonary interstitial space units, and is particularly suitable for mechanism research and drug screening of interstitial lung diseases.

[0052] The technical solution of the present invention has the following beneficial effects:

[0053] 1) The bionic micro-lung chip model provided by the present invention is formed by a cascade of complex multi-layered structures. The model highly simulates the structure and function of the smallest lung tissue and is closer to the distal lung tissue;

[0054] 2) The bionic micro-lung chip model provided by the present invention has lung epithelial and interstitial structures, and can simulate alveolar respiratory rhythm and hypoxia by connecting a micro-pressure device. Therefore, various pathological models such as pneumonia, pulmonary fibrosis, and interstitial lung disease can be established by introducing exogenous stimuli (such as drugs, viruses, and poisons);

[0055] 3) The bionic micro-lung chip model provided by the present invention can introduce resident immune cells (such as alveolar macrophages and monocytes) to achieve inflammatory cascade reactions and their research in vitro;

[0056] 4) The present invention provides an in vitro model construction method of a monolayer cell tissue-three-dimensional tissue coupling unit. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 is a three-dimensional schematic diagram of the bionic micro-lung chip of Example 1;

[0058] Figure 2 Schematic diagram of the structure of the upper culture layer of the bionic micro-lung chip of Example 1;

[0059] Figure 3 Schematic diagram of the structure of the lower culture layer of the bionic micro-lung chip of Example 1;

[0060] Figure 4 This is a schematic diagram of the structure of the lung epithelial structure microchannel of the bionic micro-lung chip of Example 1;

[0061] Figure 5 This is a schematic diagram of the structure of the culture medium microfluidic channel of the bionic micro-lung chip of Example 1;

[0062] Figure 6 Schematic diagram of the micro-column structure position of the bionic micro-lung chip of Example 1;

[0063] Figure 7 is a partial cross-sectional view of the bionic micro-lung chip of Example 1;

[0064] Figure 8 The flowchart of the preparation of the PDMS porous membrane in Example 1;

[0065] Fig. 9 The assembly process, physical diagram and cross-sectional diagram of the bionic micro-lung chip model of Example 1;

[0066] Fig.10 The implantation and cultivation process of the bionic micro-lung chip model of Example 1;

[0067] Fig.11 This is a diagram of the status of lung epithelial cells on the 5th day of culture;

[0068] Fig.12For biological characterization of the microlung chip model;

[0069] Fig.13 It is a paraquat-induced pulmonary fibrosis pathological model based on the microlung chip model.

[0070] Description of main figures:

[0071] Upper culture layer 1, lung epithelial structure inlet hole 2, lung interstitial structure inlet hole 3, lung epithelial structure microfluidic channel 4, lung epithelial structure first microfluidic channel 401, lung epithelial structure second microfluidic channel 402, lung epithelial structure third microfluidic channel 403, lung epithelial structure outlet hole 5, lung interstitial structure outlet hole 6, culture medium upper layer inlet hole 7, culture medium upper layer outlet hole 8, lung interstitial structure lower layer outlet pool 9, culture medium lower layer outlet pool 10, microcolumn structure 11, culture medium microfluidic channel 12, culture medium first microfluidic channel 1201, culture medium second microfluidic channel 1202, culture medium third microfluidic channel 1203, culture medium lower layer inlet pool 13, lung interstitial structure lower layer inlet pool 14, lung interstitial structure microfluidic channel 15, lower culture layer 16, porous membrane layer 17, through hole 1701. DETAILED DESCRIPTION

[0072] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0073] Example 1

[0074] This embodiment provides a bionic micro-lung chip, and its three-dimensional schematic diagram is as follows: Figure 1 The bionic micro-lung chip comprises an upper culture layer 1, a porous membrane layer 17, and a lower culture layer 16; the porous membrane layer 17 is located between the upper culture layer 1 and the lower culture layer 16;

[0075] like Figure 2 As shown, the upper culture layer 1 includes a lung epithelial structure inlet hole 2, a lung interstitial structure inlet hole 3, a lung epithelial structure microchannel 4, a lung epithelial structure outlet hole 5, a lung interstitial structure outlet hole 6, a culture medium upper layer inlet hole 7, and a culture medium upper layer outlet hole 8;

[0076] like Figure 3 As shown, the lower culture layer 16 includes a lung interstitial structure lower layer outlet pool 9, a culture medium lower layer outlet pool 10, a micro-column structure 11, a culture medium micro-channel 12, a culture medium lower layer inlet pool 13, a lung interstitial structure lower layer inlet pool 14, and a lung interstitial structure micro-channel 15;

[0077] The thickness of the porous membrane layer 17 is 15 microns, the diameter of the through hole 1701 is 15 microns, and the distance between the centers of adjacent through holes 1701 is 45 microns;

[0078] The lengths of the first lung epithelial structure microchannel 401, the second lung epithelial structure microchannel 402, and the third lung epithelial structure microchannel 403 are 8 mm, 5 mm, and 8 mm, respectively, and the cross-sectional dimensions of the three are the same, with a height of 200 μm and a width of 520 μm.

[0079] The lengths of the first culture medium microfluidic channel 1201, the second culture medium microfluidic channel 1202, and the third culture medium microfluidic channel 1203 of the culture medium microfluidic channel 12 are 8 mm, 5 mm, and 8 mm, respectively, and the cross-sectional dimensions of the three are the same, with a height of 200 μm and a width of 500 μm;

[0080] The length of the lung interstitial structure microchannel 15 is 13 mm, the height of the cross section is 200 μm, and the width is 400 μm;

[0081] The diameter of the lung epithelial structure inlet hole 2 is 3 mm, and the diameter of the lung epithelial structure outlet hole 5 is 3 mm.

[0082] in:

[0083] The lung epithelial structure inlet hole 2 and the lung epithelial structure outlet hole 5 penetrate the upper culture layer 1 and are respectively arranged at two ends of the lung epithelial structure microchannel 4;

[0084] The pulmonary interstitial structure inlet hole 3 and the pulmonary interstitial structure outlet hole 6 penetrate the upper culture layer 1 and are respectively connected to the pulmonary interstitial structure lower layer inlet pool 14 and the pulmonary interstitial structure lower layer outlet pool 9;

[0085] The upper culture medium layer inlet hole 7 and the upper culture medium layer outlet hole 8 penetrate the upper culture layer 1 and are connected to the lower culture medium layer inlet pool 13 and the lower culture medium layer outlet pool 10 respectively;

[0086] The lower layer inlet pool 14 of the pulmonary interstitial structure and the lower layer outlet pool 9 of the pulmonary interstitial structure are respectively arranged at the two ends of the pulmonary interstitial structure microchannel 15;

[0087] The culture medium lower layer inlet pool 13 and the culture medium lower layer outlet pool 10 are respectively arranged at two ends of the culture medium microfluidic channel 12;

[0088] like Figure 4 As shown, the lung epithelial structure microchannel 4 is composed of a first lung epithelial structure microchannel 401, a second lung epithelial structure microchannel 402, and a third lung epithelial structure microchannel 403;

[0089] like Figure 5 As shown, the culture medium micro-channel 12 is composed of a first culture medium micro-channel 1201, a second culture medium micro-channel 1202, and a third culture medium micro-channel 1203;

[0090] like Figure 6As shown, the second microchannel 1202 of the culture medium is connected to the pulmonary interstitial structure microchannel 15, and a plurality of trapezoidal micro-column structures 11 are provided at the connection point between the two, with the short sides of the trapezoid facing the pulmonary interstitial microchannel 15, and its function is to effectively prevent the hydrogel material from leaking to the side channel when it is perfused;

[0091] like Figure 7 As shown, the second microchannel 402 of the lung epithelial structure is arranged above the microchannel 15 of the lung interstitial structure, and the two are connected through the through hole 1701 in the porous membrane layer 17.

[0092] The bionic micro-lung chip of this embodiment can be prepared by the following method:

[0093] 1. Prepare PDMS porous membrane, the production flow chart is as follows Figure 8 As shown, specifically including:

[0094] Prepare a silicon wafer with a thickness of 500 μm, spin-coat photoresist according to size requirements, perform photolithography, deep silicon etching (30 μm), desizing, and cleaning to obtain a micropillar array mold;

[0095] A mixture of curing agent and PDMS in a mass ratio of 1:13 was thrown at high speed on the mold using a spinning machine to form a PDMS porous membrane, which was dried and set aside for use.

[0096] 2. Assembly process of bionic micro-lung chip Fig. 9 As shown, specifically including:

[0097] The microchannel side and the PDMS porous membrane of the upper culture layer were plasma treated, and the treated two sides were gently bonded together. After hot pressing on a hot plate at 65°C for 1 hour, the upper culture layer was gently peeled off from the microporous membrane structure, and the PDMS microporous membrane was now bonded to the upper culture layer;

[0098] Then the above structure and the microfluidic side of the lower culture layer were plasma treated again, aligned, and hot-pressed for 1 hour to complete the assembly of the bionic micro-lung chip;

[0099] After high-pressure sterilization and drying, the bionic micro-lung chip is placed in a carbon dioxide incubator for use.

[0100] Example 2

[0101] This embodiment provides a method for constructing a bionic micro-lung chip model, which is based on the above-mentioned bionic micro-lung chip. The exemplary process is as follows: Fig.10 As shown, the following steps are included:

[0102] Step 1: mixing pulmonary interstitial cells (pulmonary epithelial cells, macrophages and pulmonary fibroblasts are added separately to construct their respective bionic micro-lung chip models) with matrix glue to obtain a pulmonary interstitial cell mixture, injecting the mixture into the pulmonary interstitial structure microchannel 15 through the pulmonary interstitial structure entrance hole 3 and the pulmonary interstitial structure lower layer entrance pool 14, and filling the pulmonary interstitial structure microchannel 15; solidifying the matrix glue by heating or secondary cross-linking;

[0103] Step 2: injecting culture medium into the culture medium microfluidic channel 12 through the culture medium upper layer inlet hole 7 and the culture medium lower layer inlet pool 13, so that the culture medium fills the culture medium microfluidic channel 12, the culture medium lower layer outlet pool 10 and the culture medium upper layer outlet hole 8; adding the matrix glue solution through the lung epithelial structure inlet hole 2 and filling the lung epithelial structure microfluidic channel 4, and flowing into the lung epithelial structure outlet hole 5, and placing it in a carbon dioxide incubator to stand;

[0104] Step 3: remove the matrix glue solution in the lung epithelial structure inlet hole 2 and the lung epithelial structure outlet hole 5; add the cell suspension to the lung epithelial structure inlet hole 2, and the cell suspension flows into the lung epithelial structure microchannel 4 and the lung epithelial structure outlet hole 5; place it in a carbon dioxide incubator and let it stand;

[0105] Step 4: remove the waste liquid in the lung epithelial structure inlet hole 2, the lung epithelial structure outlet hole 5, the culture medium upper layer inlet hole 7, and the culture medium upper layer outlet hole 8, and add fresh culture medium to the lung epithelial structure inlet hole 2 and the culture medium upper layer inlet hole 7, respectively, to complete the construction of the bionic micro-lung chip model;

[0106] Step 5: Replace the culture medium in the entrance hole 2 of the lung epithelial structure with a culture medium containing 1 micromolar dexamethasone, and continue culturing for 2 days to enhance the tight connection between lung epithelial cells and form a dense single layer of lung epithelium.

[0107] The micro-lung chip model constructed in Example 2 was cultured for 5 days and then subjected to immunofluorescence analysis, and the processing steps were as follows:

[0108] Step 1: Use a pipette to remove the culture medium in the microlung chip model and wash the microlung chip three times with phosphate buffer solution, soaking for 5 minutes each time.

[0109] Step 2: Remove the phosphate buffer solution with a pipette, add a 4% paraformaldehyde solution, treat the micro-lung chip model for 30 minutes, then remove it, and wash the micro-lung chip with phosphate buffer solution 3 times, soaking for 5 minutes each time.

[0110] Step 3: Remove the phosphate buffer solution with a pipette, add 0.1% Triton X-100 to treat the micro-lung chip model for 60 minutes and then remove it.

[0111] Step 4: Continue to treat the microlung chip model with 3% BSA solution for 60 minutes, remove it and add primary antibody dilution solution, and place it at 4°C overnight.

[0112] Step 5: Remove the primary antibody dilution from the microlung chip model and wash the microlung chip three times with phosphate buffer solution, soaking for 15 minutes each time.

[0113] Step 6: Remove the phosphate buffer solution with a pipette, add the secondary antibody diluent, treat at room temperature for 60 minutes, then remove it, and wash the micro-lung chip with phosphate buffer solution 3 times, soaking for 15 minutes each time.

[0114] Step 7: Remove the phosphate buffer solution with a pipette, add DAPI solution, treat for 15 minutes, and then directly photograph with a confocal microscope.

[0115] The micro-lung chip model constructed in Example 2 was cultured for 5 days, and then cultured for 5 days using paraquat, and then immunofluorescence analysis was performed. The process of immunofluorescence analysis was the same as the above process.

[0116] Fig.11 This is the state diagram of lung epithelial cells on the 5th day of culture. Fig.11 It can be seen that the lung epithelial cells form a tight connection.

[0117] Fig.12 For biological characterization of the microlung chip model, Fig.12 It can be seen that: by adopting the bionic micro-lung chip model provided by the present invention, a tight connection is formed between the cultured lung epithelial cells, and the lung epithelial cells, macrophages and lung fibroblasts can normally express their characteristic proteins;

[0118] Fig.13 A paraquat-induced pulmonary fibrosis model based on a microlung chip model. Fig.13 It can be seen that during the paraquat-induced pulmonary fibrosis pathological experiment using the bionic micro-lung chip model provided in Example 2, the lung epithelial cells underwent epithelial-mesenchymal transition and were activated. This indicates that the bionic micro-lung chip model of the present invention can well establish the pathological characteristics of paraquat-induced pulmonary fibrosis and can be used to study the disease mechanism of pulmonary fibrosis and its treatment methods.

Claims

1. A bionic micro-lung chip, comprising an upper culture layer (1), a porous membrane layer (17), and a lower culture layer (16); the porous membrane layer (17) is located between the upper culture layer (1) and the lower culture layer (16); The upper culture layer (1) includes a lung epithelial structure inlet hole (2), a lung interstitial structure inlet hole (3), a lung epithelial structure microchannel (4), a lung epithelial structure outlet hole (5), a lung interstitial structure outlet hole (6), a culture medium upper layer inlet hole (7), and a culture medium upper layer outlet hole (8); The lower culture layer (16) includes a lung interstitial structure lower layer outlet pool (9), a culture medium lower layer outlet pool (10), a microcolumn structure (11), a culture medium microfluidic channel (12), a culture medium lower layer inlet pool (13), a lung interstitial structure lower layer inlet pool (14), and a lung interstitial structure microfluidic channel (15); in: The lung epithelial structure inlet hole (2) and the lung epithelial structure outlet hole (5) are respectively arranged at two ends of the lung epithelial structure microchannel (4); The pulmonary interstitial structure inlet hole (3) and the pulmonary interstitial structure outlet hole (6) penetrate the upper culture layer (1) and are respectively connected to the pulmonary interstitial structure lower layer inlet pool (14) and the pulmonary interstitial structure lower layer outlet pool (9); The culture medium upper layer inlet hole (7) and the culture medium upper layer outlet hole (8) penetrate the upper culture layer (1) and are respectively connected to the culture medium lower layer inlet pool (13) and the culture medium lower layer outlet pool (10); The lower layer entrance pool (14) of the pulmonary interstitial structure and the lower layer exit pool (9) of the pulmonary interstitial structure are respectively arranged at two ends of the pulmonary interstitial structure microchannel (15); The culture medium lower layer inlet pool (13) and the culture medium lower layer outlet pool (10) are respectively arranged at two ends of the culture medium microfluidic channel (12); The culture medium microchannel (12) is connected to the lung interstitial structure microchannel (15), and a plurality of microcolumn structures (11) are provided at the connection point between the two. The lung epithelial structure microchannel (4) is arranged above the lung interstitial structure microchannel (15), and the two are connected through the through hole (1701) in the porous membrane layer (17).

2. The bionic micro-lung chip according to claim 1, wherein: The lung epithelial structure microchannel (4) is composed of a first lung epithelial structure microchannel (401), a second lung epithelial structure microchannel (402), and a third lung epithelial structure microchannel (403); The second microchannel (402) of the lung epithelial structure is arranged above the microchannel (15) of the lung interstitial structure, and the two are connected through the through hole (1701) in the porous membrane layer (17).

3. The bionic micro-lung chip according to claim 1, wherein: The culture medium microfluidic channel (12) is composed of a first culture medium microfluidic channel (1201), a second culture medium microfluidic channel (1202), and a third culture medium microfluidic channel (1203); the second culture medium microfluidic channel (1202) is connected to the pulmonary interstitial structure microfluidic channel (15).

4. The bionic micro-lung chip according to claim 1, wherein: The thickness of the porous membrane layer (17) is 10 micrometers to 50 micrometers, the diameter of the through hole (1701) is 5 micrometers to 15 micrometers, and the distance between the centers of adjacent through holes (1701) is 10-50 micrometers.

5. The bionic micro-lung chip according to claim 2, wherein: The cross-sectional dimensions of the first lung epithelial structure microchannel (401), the second lung epithelial structure microchannel (402), and the third lung epithelial structure microchannel (403) are the same, with a height of 100-300 microns and a width of 250-650 microns. and / or, the height of the cross section of the culture medium microfluidic channel (12) is 100-300 microns and the width is 200-600 microns; And / or, the cross-section of the pulmonary interstitial structure microchannel (15) has a height of 100-300 microns and a width of 200-600 microns.

6. The bionic micro-lung chip according to claim 1, wherein: The lung epithelial structure inlet hole (2) is connected to a micro-pressure device; And / or, the culture medium upper layer inlet hole (7) and the culture medium lower layer inlet pool (13) are connected to a micro-pressure device.

7. A method for constructing a bionic micro-lung chip model, which is based on the bionic micro-lung chip according to any one of claims 1 to 6, comprising the following steps: Step 1: mixing the pulmonary interstitial cells with the biohydrogel material to obtain a pulmonary interstitial cell mixture, and injecting the mixture into the pulmonary interstitial structure microchannel (15) through the pulmonary interstitial structure inlet hole (3) and the pulmonary interstitial structure lower layer inlet pool (14), and filling the pulmonary interstitial structure microchannel (15); Solidifying the biohydrogel material by heating or secondary cross-linking; Step 2: injecting culture medium into the culture medium microfluidic channel (12) through the culture medium upper layer inlet hole (7) and the culture medium lower layer inlet pool (13), so that the culture medium fills the culture medium microfluidic channel (12), the culture medium lower layer outlet pool (10) and the culture medium upper layer outlet hole (8); adding the matrix glue solution through the lung epithelial structure inlet hole (2) to fill the lung epithelial structure microfluidic channel (4), and flowing into the lung epithelial structure outlet hole (5); placing in a carbon dioxide incubator and letting it stand; Step 3: removing the matrix glue solution in the lung epithelial structure inlet hole (2) and the lung epithelial structure outlet hole (5); adding the cell suspension to the lung epithelial structure inlet hole (2), and the cell suspension flows into the lung epithelial structure microchannel (4) and the lung epithelial structure outlet hole (5); placing in a carbon dioxide incubator and letting it stand; Step 4: Remove the waste liquid from the lung epithelial structure inlet hole (2), the lung epithelial structure outlet hole (5), the culture medium upper layer inlet hole (7), and the culture medium upper layer outlet hole (8), and add fresh culture medium to the lung epithelial structure inlet hole (2) and the culture medium upper layer inlet hole (7) respectively to complete the construction of the bionic micro-lung chip model.

8. The method for constructing a bionic micro-lung chip model according to claim 7, wherein: The pulmonary interstitial cells include pulmonary fibroblasts and vascular endothelial cells; preferably, the pulmonary interstitial cells also include one or a combination of two or more of pulmonary myofibroblasts and pulmonary interstitial immune cells; The cells in the cell suspension include one or a combination of two or more of lung epithelial cells, alveolar macrophages, and neutrophils.

9. The method for constructing a bionic micro-lung chip model according to claim 7, wherein: The construction method also includes: When the growth density of lung epithelial cells reached 90-95%, the culture medium in the entrance hole (2) of the lung epithelial structure was replaced with a culture medium containing 1 micromolar dexamethasone and culture was continued for 2 days to enhance the tight connection between lung epithelial cells and form a dense single layer of lung epithelium.

10. The method for constructing a bionic micro-lung chip model according to claim 7, wherein: The construction method also includes: Adding immune cells to the lung epithelial structure inlet hole (2) to allow the immune cells to flow into the lung epithelial structure microchannel (4) to obtain a bionic micro-lung chip model with immune function; Alternatively, a target agent is added into the lung epithelial structure microchannel (4) through the lung epithelial structure inlet hole (2), and the lung epithelial structure microchannel (4) is dynamically perfused and cultured to obtain a bionic micro-lung chip model with disease analysis function.

11. A bionic micro-lung chip model constructed by the construction method described in any one of claims 7 to 10.

12. The use of the bionic micro-lung chip model according to claim 11 in the detection of causes of disease, toxins and drugs, comprising: Introducing a substance to be tested into the lung epithelial structure microfluidic channel (4) through the lung epithelial structure inlet hole (2), and / or introducing a substance to be tested into the culture medium microfluidic channel (12) through the culture medium upper layer inlet hole (7); The regulation result of the test substance on the alveolar epithelial structure and / or the pulmonary interstitial structure is obtained.

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