Three-dimensional bionic lung chip and preparation method thereof

Abstract

The invention relates to a three-dimensional bionic lung chip and a preparation method thereof. The three-dimensional bionic lung chip comprises an upper chip layer and a lower chip layer, wherein the upper chip layer is provided with an inlet channel, an outlet channel and a first gas channel; the lower chip layer is arranged below the upper chip layer, the upper layer of the lower chip layer is provided with a second gas channel, a plurality of connecting channels and a plurality of culture chambers, one ends of the plurality of connecting channels are connected with the second gas channel at intervals, the other end of each connecting channel is connected with the culture chamber, and the culture chamber penetrates to the bottom of the lower chip layer; a bionic alveolar sac structure prepared from hydrogel microspheres by adopting a hanging drop method is arranged in each culture chamber. The bionic alveolar sac structure is prepared by connecting the channel and the culture chamber by adopting a hanging drop method, so that a bionic hollow alveolar structure is constructed, alveolar epithelial cells can be attached to the surface of the bionic hollow alveolar structure, a curved surface environment is provided for growth of the alveolar epithelial cells, cell growth and functional expression are facilitated, the alveolar epithelial cells are closer to real conditions in vivo, and the accuracy of a research result is ensured.

Description

Technical Field

[0001] The present application relates to the technical field of organ chips, and in particular to a three-dimensional bionic lung chip and a preparation method thereof. Background Art

[0002] Increasingly severe lung diseases, continuously mutating coronaviruses, and new compounds that are constantly being developed all require in-depth research on the pathology or toxicology of the human lung and the development of related new drugs. Constructing an in vitro lung tissue model with a bionic structure can provide a convenient and effective basis for lung pathology, toxicology, and pharmacology.

[0003] The alveoli are the basic structural and functional units of the lungs, with a unique physiological structure, namely hollow microspheres formed by a single layer of alveolar epithelial cells, which grow in a curved pattern on the alveolar surface. Studies have shown that the phenotype of cells growing in three-dimensional curved patterns is different from that of flat cultures. For certain cells, curved growth helps them get closer to the in vivo growth environment and express their functions normally. As the site of gas exchange in the human body, the alveoli have a typical structure of an air-liquid interface. The inside of the alveoli is a gas environment, and the outside is a rich alveolar microvascular network and matrix environment. In addition to being a site for oxygen and carbon dioxide exchange, this structure also acts as a barrier.

[0004] At present, the lung chip with terminal bronchus and curved structure has accurately designed and manufactured the terminal bronchial channel and the alveolar sac structure connected to the channel. However, due to the limitations of traditional microfluidic chip manufacturing technology, it can only provide a two-dimensional structure, that is, all flow channel structures are in the same plane, and cells cannot be introduced for culture, which is very different from the real lung tissue structure. The simulation results can only provide a reference at the fluid flow level, and cannot simulate the real lung tissue structure, which in turn affects the accuracy of the research results. Summary of the invention

[0005] Based on this, it is necessary to provide a three-dimensional bionic lung chip and its preparation method that can enable cells to grow on a curved surface model and simulate the real alveolar structure, in order to address the problem that the flow channels in the lung chip currently manufactured by fluidic chips are in the same plane, resulting in the inability to truly simulate the lung structure.

[0006] A three-dimensional bionic lung chip, comprising:

[0007] an upper chip layer, the upper chip layer having an inlet channel, an outlet channel and a first gas channel, the first gas channel connecting the inlet channel and the outlet channel; and

[0008] The lower chip layer is arranged below the upper chip layer. The upper layer of the lower chip layer has a second gas channel, multiple connecting channels and multiple culture chambers. The second gas channel corresponds to the first gas channel and is connected to form a gas channel. One end of the multiple connecting channels is connected to the second gas channel at intervals, and the other end of each connecting channel is connected to the culture chamber. The culture chamber passes through to the bottom of the lower chip layer. Each culture chamber has a bionic alveolar sac structure prepared by hydrogel microspheres using a hanging drop method.

[0009] In one embodiment of the present application, a cross-sectional dimension of the connecting channel is smaller than a cross-sectional dimension of the culture chamber.

[0010] In one embodiment of the present application, the cross-sectional dimension of the connecting channel is 1 / 10 to 1 / 2 of the cross-sectional dimension of the culture chamber.

[0011] In one embodiment of the present application, the surface of the connection between the culture chamber and the connection channel is hydrophilic treated.

[0012] In one embodiment of the present application, the upper chip layer is made of PDMS, the lower chip layer is made of glass or organic glass, and the upper chip layer is sealed and connected to the lower chip layer.

[0013] A method for preparing a three-dimensional bionic lung chip, used to prepare a three-dimensional bionic lung chip as described in any of the above technical features, the preparation method comprising the following steps:

[0014] The upper chip layer is prepared by soft lithography, and the lower chip layer is prepared by film building method;

[0015] The hydrogel microspheres were prepared by microfluidics;

[0016] extruding a liquid hydrogel containing hydrogel microspheres as hanging drops into a culture chamber, wherein the hydrogel microspheres degrade to form alveolar sac structures;

[0017] Cells are implanted into the alveolar sac structure to simulate the real alveolar structure.

[0018] In one embodiment of the present application, the method of preparing the upper chip layer by soft lithography and preparing the lower chip layer by film building method comprises the following steps:

[0019] The upper chip layer is prepared by PDMS soft lithography, and a first gas channel, an inlet channel and an outlet channel are prepared on the upper chip layer;

[0020] Using a membrane-building method to prepare a second gas channel, a connecting channel and the culture chamber on the lower chip layer;

[0021] The surface where the culture chamber is connected to the connecting channel is subjected to a hydrophilic treatment.

[0022] In one embodiment of the present application, the step of extruding a liquid hydrogel containing hydrogel microspheres into a culture chamber as a hanging drop, wherein the hydrogel microspheres degrade to form an alveolar sac structure, comprises the following steps:

[0023] Controlling the printer to extrude the hydrogel microspheres into the connecting channel;

[0024] The hydrogel microspheres flow toward the culture chamber under the action of gravity to form the hanging drops;

[0025] The hydrogel microspheres are cross-linked with the degradable material and degraded.

[0026] The hydrogel microspheres are sacrificed and connected to form the alveolar sac structure.

[0027] In one embodiment of the present application, the hydrogel microspheres are made of gelatin, sodium alginate or F127;

[0028] The degradable material is different from the material selected for the hydrogel microspheres.

[0029] In one embodiment of the present application, the step of implanting cells into the alveolar sac structure to simulate a real alveolar structure comprises the following steps:

[0030] Encapsulating vascular endothelial cells, fibroblasts and alveolar epithelial cells in the hydrogel microspheres; or encapsulating vascular endothelial cells and fibroblasts in the hydrogel microspheres, and injecting alveolar epithelial cells into the alveolar sac structure;

[0031] The vascular endothelial cells, the fibroblasts and the alveolar epithelial cells grow on the surface of the alveolar sac structure to construct a bionic alveolar model.

[0032] After adopting the technical solutions of the above embodiments, the present application has at least the following technical effects:

[0033] The three-dimensional bionic lung chip of the present application and its preparation method, in which the lower chip layer is arranged below the upper chip layer, the first gas channel in the upper chip layer connects the inlet channel and the outlet channel, the top of the lower chip layer has a second gas channel, multiple connecting channels and multiple culture chambers, the second gas channel is connected to the first gas channel and forms a gas channel, one end of the connecting channel is connected to the second gas channel, the other end of the connecting channel is connected to the culture chamber, and the culture chamber runs through to the bottom of the lower chip layer. Hydrogel microspheres are formed in the culture chamber as hanging drops, and after degradation to form hydrogel microspheres, multiple hydrogel microspheres are sacrificed and connected to form a bionic alveolar sac structure, and the hydrogel microspheres are used as cell carriers, which can make cells grow on the surface of the hydrogel microspheres to simulate the real alveolar structure.

[0034] This three-dimensional bionic lung chip uses the hanging drop method to prepare a bionic alveolar sac structure by connecting the channel and the culture chamber to construct a bionic hollow alveolar structure, and alveolar epithelial cells can be attached to its surface to provide a curved surface environment for their growth, which is conducive to cell growth and functional expression, closer to the real situation in the body, and ensures the accuracy of the research results. In addition, the alveolar sac structure provides an air-liquid culture environment interface for the model, simulating the real air-liquid exchange environment of alveolar epithelial cells when working. Based on this air-liquid functional interface, the exchange simulation of inhaled substances in the alveoli and the evaluation of their toxicity, particulate matter, pollutant toxicity or drug effectiveness can also be carried out. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG1 is a schematic diagram of the structure of a three-dimensional bionic lung chip according to an embodiment of the present application.

[0036] FIG. 2 is a side partial cross-sectional view of the three-dimensional bionic lung chip shown in FIG. 1 .

[0037] Figure 3 Schematic diagram of the formation of hanging drops in the lower chip layer of the three-dimensional bionic lung chip shown in FIG2 .

[0038] Figure 4 Figure 3 Schematic diagram of the lower chip layer forming a hanging drop in a culture dish.

[0039] FIG. 5 is a flow chart of the preparation of the three-dimensional bionic lung chip shown in FIG. 1 .

[0040] Among them: 100, three-dimensional bionic lung chip; 110, upper chip layer; 111, inlet channel; 112, outlet channel; 113, first gas channel; 120, lower chip layer; 121, second gas channel; 122, connecting channel; 123, culture chamber 200, hanging drop; 300, culture dish. DETAILED DESCRIPTION

[0041] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0042] In the description of the present application, it should be understood that if the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. appear, the orientation or position relationship indicated by these terms is based on the orientation or position relationship shown in the accompanying drawings, which is only for the convenience of describing the present application and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present application.

[0043] In addition, if the terms "first" or "second" appear, these terms are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, if the term "plurality" appears, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0044] In this application, unless otherwise clearly specified and limited, if the terms "installed", "connected", "connected", "fixed" and the like appear, these terms should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integrated connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to the specific circumstances.

[0045] In the present application, unless otherwise clearly specified and limited, if there is a description that a first feature is "above" or "below" a second feature, etc., or similar descriptions appear, it may mean that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature being "above", "above" and "above" the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. The first feature being "below", "below" and "below" the second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0046] It should be noted that if an element is referred to as being "fixed to" or "disposed on" another element, it may be directly on the other element or there may be a central element. If an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be a central element at the same time. If any, the terms "vertical", "horizontal", "upper", "lower", "left", "right" and similar expressions used in this application are for illustrative purposes only and do not represent the only implementation method.

[0047] Referring to Figure 1, the present application provides a three-dimensional bionic lung chip 100. The three-dimensional bionic lung chip 100 can construct a bionic alveolar model with a hollow spherical three-dimensional cell structure to simulate the real alveolar structure. In this way, the three-dimensional bionic lung chip 100 can perform culture medium perfusion culture or gas flow exchange, providing an effective in vitro three-dimensional model for research on the lungs, and is applied to the research of lung tissue physiology, pathology and toxicology, etc., providing a convenient and effective basis for research. Figure 1 is a structural schematic diagram of a three-dimensional bionic lung chip 100 of an embodiment of the present application.

[0048] It is understandable that the current lung chip with terminal bronchus and curved structure has precisely designed and manufactured the terminal bronchial channel and the alveolar sac structure connected to the channel. However, due to the limitations of traditional microfluidic chip manufacturing technology, it can only provide a two-dimensional structure, that is, all flow channel structures are in the same plane, and cells cannot be introduced for culture, which is very different from the real lung tissue structure. The simulation results can only provide a reference at the fluid flow level, and cannot simulate the real lung tissue structure, which in turn affects the accuracy of the research results.

[0049] To this end, the present application provides a novel three-dimensional bionic lung chip 100, which constructs a bionic hollow alveolar structure and can attach alveolar epithelial cells on its surface, providing a curved surface environment for their growth, which is conducive to cell growth and functional expression, closer to the real situation in the body, and ensures the accuracy of the research results. The specific structure of the three-dimensional bionic lung chip 100 of an embodiment is introduced below.

[0050] Referring to FIGS. 1 to 4 , in one embodiment, a three-dimensional bionic lung chip 100 includes an upper chip layer 110 and a lower chip layer 120. The upper chip layer 110 has an inlet channel 111, an outlet channel 112, and a first gas channel 113, wherein the first gas channel 113 connects the inlet channel 111 and the outlet channel 112. The lower chip layer 120 is disposed below the upper chip layer 110, and the upper layer of the lower chip layer 120 has a second gas channel 121, a plurality of connecting channels 122, and a plurality of culture chambers 123, wherein the second gas channel 121 corresponds to the first gas channel 113 and is connected to form a gas channel, wherein one end of the plurality of connecting channels 122 is connected to the second gas channel 121 at intervals, and the other end of each connecting channel 122 is connected to the culture chamber 123, wherein the culture chamber 123 passes through the bottom of the lower chip layer 120, and each culture chamber 123 has a bionic alveolar sac structure prepared by a hanging drop method with hydrogel microspheres. FIG. 2 is a side partial cross-sectional view of the three-dimensional bionic lung chip 100 shown in FIG. 1 . Figure 3 FIG. 4 is a schematic diagram of forming a hanging drop 200 in the lower chip layer 120 of the three-dimensional bionic lung chip 100 shown in FIG. 2 , and FIG. Figure 3 The schematic diagram shown is that the lower chip layer 120 forms a hanging drop 200 in a culture dish 300 .

[0051] Based on the direction shown in FIG. 1 , the upper chip layer 110 is located at the upper layer, and the lower chip layer 120 is located at the lower layer. The upper chip layer 110 and the lower chip layer 120 are stacked and sealed to prevent gas leakage. The upper chip layer 110 and the lower chip layer 120 constitute the main structure of the three-dimensional bionic lung chip 100, and the hydrogel microspheres are supported by the upper chip layer 110 and the lower chip layer 120 to simulate the real alveolar structure.

[0052] The upper chip layer 110 is provided with a channel to simulate the bronchial structure of the lungs. Specifically, the upper chip layer 110 has an inlet channel 111, an outlet channel 112 and a first gas channel 113. The first gas channel 113 is connected to the inlet channel 111 and the outlet channel 112 respectively. The inlet channel 111 and the outlet channel 112 simulate the proximal bronchial structure of the lungs. In the direction shown in FIG. 1 , the bottom is the bottom of the upper chip layer 110, and the first gas channel 113 is located in the upper chip layer 110 and passes through to the bottom of the upper chip layer 110.

[0053] The lower chip layer 120 also has channels. In the direction shown in FIG. 1 , the upper side is the top of the lower chip layer 120, and the lower side is the bottom of the lower chip layer 120. The lower chip layer 120 has a second gas channel 121, a connecting channel 122, and a culture chamber 123. The second gas channel 121 is arranged in the lower chip layer 120 and passes through to the bottom of the upper chip layer 110. When the upper chip layer 110 and the lower chip layer 120 are stacked, the first gas channel 113 and the second gas channel 121 are arranged correspondingly and communicated to form a gas channel to simulate the distal bronchial structure of the lung.

[0054] It is worth noting that the layout of the first gas channel 113 and the second gas channel 121 is not restricted in principle, as long as the first gas channel 113 and the second gas channel 121 can provide gas flow. Optionally, the first gas channel 113 and the second gas channel 121 are arranged in a regular shape or an irregular shape. In this embodiment, the gas channel includes a plurality of branch channels and two switching channels, and the plurality of branch channels are arranged in parallel, and the two ends of the plurality of branch channels are respectively connected to the two switching channels, and the two switching channels are respectively connected to the inlet channel 111 and the outlet channel 112. Of course, in other embodiments of the present application, the gas channel may also be in other structural forms.

[0055] Arranged in the up-down direction, a plurality of connecting channels 122 are connected to the second gas channel 121 at intervals, and one end of the connecting channel 122 is connected to the second gas channel 121, and the other end of the connecting channel 122 is connected to the culture chamber 123. The second gas channel 121, the connecting channel 122 and the culture chamber 123 are connected in the up-down direction, the connecting channel 122 connects the second gas channel 121 and the culture chamber 123, and the culture chamber 123 passes through to the bottom of the lower chip layer 120.

[0056] The culture chamber 123 can use the hanging drop method to prepare the hydrogel microspheres into a bionic alveolar sac structure, and the bionic alveolar sac structure can simulate the real alveolar structure. It can be understood that the hydrogel microspheres are placed in the culture chamber 123 as hanging drops 200, and the hydrogel microspheres are degraded by the culture chamber 123. The degradation of the hydrogel microspheres produces a bionic alveolar sac structure formed by sacrificial connection of the microspheres, thereby constructing a bionic alveolar model to simulate the real lung structure. Moreover, the three-dimensional bionic lung chip 100 is prepared in a hanging drop 200 in a culture dish 300, such as Figure 3 As shown in FIG. 4 , the lower chip layer 120 is placed in the culture dish 300 , and the upper chip layer 110 is disposed above the lower chip layer 120 .

[0057] After the air enters the gas channel formed by the first gas channel 113 and the second gas channel 121 through the inlet channel 111, it enters the culture chamber 123 through the connecting channel 122 and contacts the bionic alveolar sac structure to form a hanging drop 200. The bionic alveolar sac structure provides a gas-liquid culture environment interface for the model to simulate the real gas-liquid exchange environment of the lung structure when it is working. In this way, after the gas is exchanged with the bionic alveolar sac structure, it is discharged through the outlet channel 112.

[0058] The three-dimensional bionic lung chip 100 structure of the above embodiment adopts the hanging drop method to prepare a bionic alveolar sac structure by connecting the channel 122 and the culture chamber 123 to construct a bionic hollow alveolar structure, and alveolar epithelial cells can be attached to its surface to provide a curved surface environment for its growth, which is conducive to cell growth and functional expression, closer to the real situation in the body, and ensures the accuracy of the research results. In addition, the alveolar sac structure provides an air-liquid culture environment interface for the model, simulating the real air-liquid exchange environment of alveolar epithelial cells when working. Based on this air-liquid functional interface, the exchange simulation of inhaled substances in the alveoli and the evaluation of their poisons, particulate matter, pollutant toxicity or effective effects of drugs can also be carried out.

[0059] When constructing the bionic alveolar sac structure in the culture chamber 123, the hollow bionic alveolar sac structure is constructed by the hanging drop method. A hydrogel solution (liquid hydrogel) containing sacrificial hydrogel microspheres is extruded in the connecting channel 122 based on quantitative micro-extrusion. The hydrogel microspheres enter the culture chamber 123 along the connecting channel 122 under the action of gravity to form a hanging drop 200. The hanging drop 200 is cross-linked with the degradation solution to degrade the hydrogel microspheres in the hanging drop 200, thereby generating a bionic alveolar sac structure formed by sacrificial connection of the microspheres.

[0060] It can be understood that the hydrogel microspheres are degradable parts, and the hydrogel material used for the hanging drop 200 that wraps the hydrogel microspheres is a non-degradable material, which is used for: 1. Carrying the hydrogel microspheres to form the hanging drop 200, 2. After the hydrogel microspheres are degraded, the pattern is formed in the material, and 3. A support for subsequent cell culture. Moreover, the extrusion of the hanging drop 200 is achieved by printing with a printer, and the print head of the printer extends into the connecting channel 122 to extrude the hydrogel microspheres as the hanging drop 200.

[0061] After constructing the bionic alveolar sac structure, cells are inoculated in the bionic alveolar sac to construct a bionic alveolar model, thereby simulating a real alveolar structure. Hydrogel microspheres are used as carriers to encapsulate vascular endothelial cells and fibroblasts through the hydrogel microspheres, so that when the hanging drop 200 is extruded, the vascular endothelial cells and fibroblasts are extruded at the same time. When the hydrogel microspheres are degraded and sacrificed, the vascular endothelial cells and fibroblasts can simulate the composition of the extra-alveolar vascular network and matrix environment in the subsequent model culture process. Moreover, alveolar epithelial cells can also be encapsulated in the hydrogel microspheres, or the alveolar epithelial cell suspension can be perfused in the bionic alveolar sac structure, so that the alveolar epithelial cells can grow on the surface of the bionic alveolar sac structure to simulate the real alveolar structure.

[0062] 1 to 4 , in one embodiment, the cross-sectional dimension of the connecting channel 122 is smaller than the cross-sectional dimension of the culture chamber 123. That is, in the horizontal plane direction, the dimension of the connecting channel 122 is smaller than the dimension of the culture chamber 123. In this way, the hydrogel microspheres can enter the culture chamber 123 along the connecting channel 122 to form a hanging drop 200.

[0063] Referring to FIGS. 1 to 4 , in one embodiment, the cross-sectional dimension of the connecting channel 122 is 1 / 10 to 1 / 2 of the cross-sectional dimension of the culture chamber 123. That is, in the horizontal plane direction, the dimension of the connecting channel 122 is smaller than the dimension of the culture chamber 123. When the cross-sectional dimension of the connecting channel 122 and the cross-sectional dimension of the culture chamber 123 are within the above range, it is possible to form the hanging drop 200, thereby facilitating the preparation of the bionic alveolar sac structure.

[0064] Referring to FIGS. 1 to 4 , in one embodiment, the connecting channel 122 and the culture chamber 123 are both cylindrical. That is, the connecting channel 122 is cylindrical, and the culture chamber 123 is also cylindrical. This facilitates the flow of the hydrogel microspheres to form a hanging drop 200, avoiding the occurrence of edges and corners that affect the flow. Of course, in other embodiments of the present application, the cross-sections of the connecting channel 122 and the culture chamber 123 may also be elliptical or polygonal, etc.

[0065] In one embodiment, the surface of the connection between the culture chamber 123 and the connection channel 122 is treated hydrophilically. That is, the top surface of the culture chamber 123 is treated hydrophilically to improve the suspension ability of the culture chamber 123 for droplets, thereby facilitating the droplets to form hanging drops 200 on the top surface of the culture chamber 123. That is, after the top surface of the culture chamber 123 is treated hydrophilically, it is possible to facilitate the formation of hanging drops 200 on the top surface of the culture chamber 123 by the hydrophilic treatment of the top surface of the culture chamber 123.

[0066] Optionally, the top surface of the culture chamber 123 is treated with oxygen plasma surface hydrophilicity. In this way, the suspension ability of the top surface of the culture chamber 123 for droplets is guaranteed. Of course, in other embodiments of the present application, other methods can also be used to hydrophilize the top surface of the culture chamber 123, as long as the suspension ability of the top surface of the culture chamber 123 for droplets can be improved.

[0067] In one embodiment, the upper chip layer 110 is made of PDMS, the lower chip layer 120 is made of glass or organic glass, and the upper chip layer 110 is sealed and connected to the lower chip layer 120. It can be understood that the upper chip layer 110 is thinner, so it is made of PDMS material, and the lower chip layer 120 is thicker, so it is made of glass or organic glass.

[0068] Moreover, the upper chip 110 is prepared by PDMS soft lithography, and the first gas channel 113, the inlet channel 111 and the outlet channel 112 are prepared by soft lithography technology on the upper chip layer 110, and the first gas channel 113 connects the inlet channel 111 and the outlet channel 112, and the inlet channel 111 and the outlet channel 112 simulate the proximal bronchial structure of the lung. The lower chip layer 120 is prepared by the membrane building method, and the second gas channel 121, the connecting channel 122 and the culture chamber 123 are prepared by the membrane building method on the lower chip layer 120. After the first gas channel 113 and the second gas channel 121 form a gas channel, the distal bronchial structure of the lung is simulated, and the connecting channel 122 simulates the terminal bronchial structure of the lung.

[0069] Referring to Figures 1 to 4, the three-dimensional bionic lung chip 100 of the present application extrude hydrogel microspheres into the connecting channel 122, and allow the hydrogel microspheres to flow to the top surface of the culture chamber 123 under the action of gravity to form a hanging drop 200. When the hydrogel microspheres are sacrificed, multiple hydrogel microspheres are connected to form a bionic alveolar sac structure. Since the outer surface of the hydrogel microspheres is curved, after the bionic alveolar sac structure is prepared using hydrogel microspheres, the bionic alveolar sac can have a curved structure to simulate the real alveolar structure. In this way, after the cells are implanted in the bionic alveolar sac structure, a curved growth environment can be provided for the cells, that is, on the basis of having a bionic three-dimensional cell growth environment, the effect of curvature on cell growth behavior is introduced, which helps cell growth and functional expression to be closer to the real situation in vivo to simulate the real alveolar structure.

[0070] Moreover, the three-dimensional bionic lung chip 100 uses hydrogel materials to construct a three-dimensional structure of a bionic alveolar sac structure. In a cell matrix environment dominated by a hanging drop solution with good biocompatibility, vascular endothelial cells and fibroblasts are introduced to simulate the extra-alveolar vascular network and matrix environment composition, respectively, to construct a more complex and bionic alveolar microenvironment, as well as real cell-to-cell interactions. Specifically, vascular endothelial cells, fibroblasts and other important microenvironmental components are introduced by hydrogel wrapping to reproduce the real alveolar microenvironment in vivo and the interaction process between multiple cells.

[0071] After the bionic alveolar sac structure is prepared in the three-dimensional bionic lung chip 100, a functional alveolar air-blood barrier can be constructed. Based on the constructed hollow bionic alveolar sac structure, microfluidic flow channel and hanging drop culture technology principle, the controllable gas input and output of the microfluidic flow channel can be realized, the gel micro-hanging droplets provide a liquid culture environment, and because the hydrogel has a certain elasticity, the contraction and relaxation during alveolar breathing can be simulated. Moreover, the bionic alveolar sac structure provides an air-liquid culture environment interface for the model, simulating the real air-liquid exchange environment of alveolar epithelial cells at work. Based on this gas-liquid functional interface, the exchange simulation of inhaled substances in the alveoli and the evaluation of their poisons, particulate matter, pollutant toxicity or effective drug effects can also be carried out.

[0072] Referring to Figures 1 to 5, Figure 5 is a flow chart of the preparation of the three-dimensional bionic lung chip 100 shown in Figure 1. The present application also provides a method for preparing a three-dimensional bionic lung chip 100, which is used to prepare the three-dimensional bionic lung chip 100 as described in any of the above embodiments, and the preparation method comprises the following steps:

[0073] The upper chip layer 110 is prepared by soft lithography, and the lower chip layer 120 is prepared by film building method;

[0074] The hydrogel microspheres were prepared by microfluidics;

[0075] Extruding a liquid hydrogel containing hydrogel microspheres into the culture chamber 123 as a hanging drop 200, wherein the hydrogel microspheres degrade to form an alveolar sac structure;

[0076] Cells are implanted into the alveolar sac structure to simulate the real alveolar structure.

[0077] When preparing the three-dimensional bionic lung chip 100 of the present application, the upper chip layer 110 is first prepared by soft lithography technology, and the lower chip layer 120 is prepared by film building method to form the main structure of the three-dimensional bionic lung chip 100. Subsequently, hydrogel microspheres are prepared by microfluidics, and the hydrogel microspheres are extruded into the culture chamber 123 by a printer to form a hanging drop 200. After the hydrogel microspheres are degraded, a bionic alveolar sac structure formed by sacrificial connection of the microspheres is generated. Cells are inoculated in the bionic alveolar sac to construct a bionic alveolar model to simulate the real alveolar structure.

[0078] In one embodiment, the method of preparing the upper chip layer 110 by soft lithography and preparing the lower chip layer 120 by film building method comprises the following steps:

[0079] The upper chip layer 110 is prepared by PDMS soft lithography, and a first gas channel 113, an inlet channel 111 and an outlet channel 112 are prepared in the upper chip layer 110;

[0080] The second gas channel 121, the connecting channel 122 and the culture chamber 123 are prepared in the lower chip layer 120 by using a membrane-building method;

[0081] The surface where the culture chamber 123 is connected to the connection channel 122 is subjected to a hydrophilic treatment.

[0082] The upper chip layer 110 is thinner and therefore made of PDMS material, and the lower chip layer 120 is thicker and therefore made of glass or organic glass. Moreover, the upper chip is prepared by PDMS soft lithography, and the first gas channel 113, the inlet channel 111 and the outlet channel 112 are prepared by soft lithography technology on the upper chip layer 110, and the first gas channel 113 connects the inlet channel 111 and the outlet channel 112, and the inlet channel 111 and the outlet channel 112 simulate the proximal bronchial structure of the lung. The lower chip layer 120 is prepared by a membrane-building method, and the second gas channel 121, the connecting channel 122 and the culture chamber 123 are prepared by a membrane-building method on the lower chip layer 120. After the first gas channel 113 and the second gas channel 121 form a gas channel, the distal bronchial structure of the lung is simulated, and the connecting channel 122 simulates the terminal bronchial structure of the lung. Subsequently, the top of the culture chamber 123 is hydrophilic to improve the suspension ability of the culture chamber 123 to droplets.

[0083] Optionally, the top surface of the culture chamber 123 is treated with oxygen plasma surface hydrophilicity. In this way, the suspension ability of the top surface of the culture chamber 123 for droplets is guaranteed. Of course, in other embodiments of the present application, other methods can also be used to hydrophilize the top surface of the culture chamber 123, as long as the suspension ability of the top surface of the culture chamber 123 for droplets can be improved.

[0084] In one embodiment, the preparation of hydrogel microspheres by microfluidics comprises the following steps:

[0085] Selecting liquid hydrogel with reversible gel properties as the preparation material of hydrogel microspheres;

[0086] Passing the hydrogel into the water phase inlet channel of the microfluidic chip layer;

[0087] Passing mineral oil into the oil phase inlet channel of the microfluidic chip layer;

[0088] The hydrogel microspheres with uniform size are generated by the shearing action of the oil phase on the water phase.

[0089] The hydrogel microspheres are prepared based on the microfluidic method. Hydrogel with reversible gel properties is selected as the sacrificial microsphere preparation material, which is passed into the water phase inlet channel of the microfluidic chip, and mineral oil + 2% SPAN is passed into the oil phase inlet channel. Through the rapid shearing of the oil phase on the water phase, hydrogel microspheres with uniform size are produced.

[0090] In one embodiment, the step of extruding a liquid hydrogel containing hydrogel microspheres into the culture chamber 123 as a hanging drop 200, wherein the hydrogel microspheres degrade to form an alveolar sac structure, comprises the following steps:

[0091] Controlling the printer to extrude the hydrogel microspheres toward the connecting channel 122;

[0092] The hydrogel microspheres flow toward the culture chamber 123 under the action of gravity to form the hanging drop 200;

[0093] The hydrogel microspheres are cross-linked with the degradable material and degraded.

[0094] The hydrogel microspheres are sacrificed and connected to form the alveolar sac structure.

[0095] When constructing the bionic alveolar sac structure in the culture chamber 123, the hollow bionic alveolar sac structure is constructed by the hanging drop method. A hydrogel solution containing sacrificial hydrogel microspheres is extruded in the connecting channel 122 based on quantitative micro-extrusion, and the hydrogel microspheres enter the culture chamber 123 along the connecting channel 122 under the action of gravity to form a hanging drop 200. The hanging drop 200 is cross-linked with the degradation solution to degrade the hydrogel microspheres in the hanging drop 200, thereby generating a bionic alveolar sac structure formed by sacrificial connection of the microspheres. Moreover, the extrusion of the hanging drop 200 is achieved by printing with a printer, and the print head of the printer extends into the connecting channel 122 to extrude the hydrogel microspheres as the hanging drop 200.

[0096] It is understandable that the hydrogel solution contains a plurality of hydrogel microspheres. After the printer extrude the hydrogel solution containing the hydrogel microspheres, the hydrogel microspheres enter the culture chamber 123 along the connecting channel 122 under the action of gravity to form a hanging drop 200. The hydrogel solution can be selected from GelMA with photocuring properties or sodium alginate with ion crosslinking properties.

[0097] In one embodiment, the hydrogel microspheres are made of materials such as gelatin, sodium alginate or F127. The hanging drop hydrogel material (the hydrogel material used for the hanging drop 200 that wraps the hydrogel microspheres is a non-degradable material, which is used for: 1. Carrying the hydrogel microspheres to form a hanging drop 200, 2. After the hydrogel microspheres are degraded, the pattern is formed in the material, and 3. The support for subsequent cell culture) is different from the material selected for the hydrogel microspheres. That is to say, when the hydrogel microspheres are made of gelatin, the hydrogel microsphere material is made of sodium alginate. In this way, after the degradable material contacts the hydrogel microspheres, the hydrogel microspheres in the hanging drop 200 can be degraded.

[0098] In one embodiment, the step of implanting cells into the alveolar sac structure to simulate a real alveolar structure comprises the following steps:

[0099] Encapsulating vascular endothelial cells, fibroblasts and alveolar epithelial cells in the hydrogel microspheres; or encapsulating vascular endothelial cells and fibroblasts in the hydrogel microspheres, and injecting alveolar epithelial cells into the alveolar sac structure;

[0100] The vascular endothelial cells, the fibroblasts and the alveolar epithelial cells grow on the surface of the alveolar sac structure to construct a bionic alveolar model.

[0101] After constructing the bionic alveolar sac structure, cells are inoculated in the bionic alveolar sac to construct a bionic alveolar model, thereby simulating a real alveolar structure. Hydrogel microspheres are used as carriers to encapsulate vascular endothelial cells and fibroblasts through the hydrogel microspheres, so that when the hanging drop 200 is extruded, the vascular endothelial cells and fibroblasts are extruded at the same time. When the hydrogel microspheres are degraded and sacrificed, the vascular endothelial cells and fibroblasts can simulate the composition of the extra-alveolar vascular network and matrix environment in the subsequent model culture process. Moreover, alveolar epithelial cells can also be encapsulated in the hydrogel microspheres, or the alveolar epithelial cell suspension can be perfused in the bionic alveolar sac structure, so that the alveolar epithelial cells can grow on the surface of the bionic alveolar sac structure to simulate the real alveolar structure.

[0102] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0103] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims

1. A three-dimensional bionic lung chip, characterized in that: include: An upper chip layer, the upper chip layer having an inlet channel, an outlet channel and a first gas channel, the first gas channel connecting the inlet channel and the outlet channel; as well as The lower chip layer is arranged below the upper chip layer. The upper layer of the lower chip layer has a second gas channel, multiple connecting channels and multiple culture chambers. The second gas channel corresponds to the first gas channel and is connected to form a gas channel. One end of the multiple connecting channels is connected to the second gas channel at intervals, and the other end of each connecting channel is connected to the culture chamber. The culture chamber passes through to the bottom of the lower chip layer. Each culture chamber has a bionic alveolar sac structure prepared by hydrogel microspheres using a hanging drop method.

2. The three-dimensional bionic lung chip according to claim 1, characterized in that: The cross-sectional dimension of the connecting channel is smaller than the cross-sectional dimension of the culture chamber.

3. The three-dimensional bionic lung chip according to claim 1, characterized in that: The cross-sectional dimension of the connecting channel is 1 / 10 to 1 / 2 of the cross-sectional dimension of the culture chamber.

4. The three-dimensional bionic lung chip according to claim 1, characterized in that: The surface of the connection between the culture chamber and the connection channel is treated with hydrophilicity.

5. The three-dimensional bionic lung chip according to any one of claims 1 to 4, characterized in that: The upper chip layer is made of PDMS, the lower chip layer is made of glass or organic glass, and the upper chip layer is sealed and connected with the lower chip layer.

6. A method for preparing a three-dimensional bionic lung chip, characterized in that: For preparing the three-dimensional bionic lung chip according to any one of claims 1 to 5, the preparation method comprises the following steps: The upper chip layer is prepared by soft lithography, and the lower chip layer is prepared by film building method; The hydrogel microspheres were prepared by microfluidics; extruding a liquid hydrogel containing hydrogel microspheres as hanging drops into a culture chamber, wherein the hydrogel microspheres degrade to form alveolar sac structures; Cells are implanted into the alveolar sac structure to simulate the real alveolar structure.

7. The preparation method according to claim 6, characterized in that: The method of preparing the upper chip layer by soft lithography and preparing the lower chip layer by film building method comprises the following steps: The upper chip layer is prepared by PDMS soft lithography, and a first gas channel, an inlet channel and an outlet channel are prepared on the upper chip layer; Using a membrane-building method to prepare a second gas channel, a connecting channel and the culture chamber on the lower chip layer; The surface where the culture chamber is connected to the connecting channel is subjected to a hydrophilic treatment.

8. The preparation method according to claim 6 or 7, characterized in that: The step of extruding a liquid hydrogel containing hydrogel microspheres into a culture chamber as a hanging drop, wherein the hydrogel microspheres degrade to form an alveolar sac structure, comprises the following steps: Controlling the printer to extrude the hydrogel microspheres into the connecting channel; The hydrogel microspheres flow toward the culture chamber under the action of gravity to form the hanging drops; The hydrogel microspheres are cross-linked with the degradable material and degraded. The hydrogel microspheres are sacrificed and connected to form the alveolar sac structure.

9. The preparation method according to claim 8, characterized in that: The hydrogel microspheres are made of gelatin, sodium alginate or F127; The degradable material is different from the material selected for the hydrogel microspheres.

10. The preparation method according to claim 8, characterized in that: The step of implanting cells into the alveolar sac structure to simulate a real alveolar structure comprises the following steps: Encapsulating vascular endothelial cells, fibroblasts and alveolar epithelial cells in the hydrogel microspheres; or encapsulating vascular endothelial cells and fibroblasts in the hydrogel microspheres, and injecting alveolar epithelial cells into the alveolar sac structure; The vascular endothelial cells, the fibroblasts and the alveolar epithelial cells grow on the surface of the alveolar sac structure to construct a bionic alveolar model.

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