Organ chip, lung gas exchange model, preparation method and three-dimensional deformation method

Abstract

The invention provides an organ chip, a lung gas exchange model, a preparation method and a three-dimensional deformation method, the organ chip comprises a culture membrane and actuating membranes, a culture chamber and at least two actuating chambers are further formed in the organ chip, the culture chamber is divided into a first culture chamber and a second culture chamber by the culture membrane, the second culture chamber and the actuating chambers are separated by the actuating membranes, and the actuating chambers are arranged in the first culture chamber and the second culture chamber. The actuating chambers define a deformable region of an actuating membrane, the at least two actuating chambers are arranged equidistantly spaced circumferentially around the axis of the culture chamber, the actuating membrane located in the actuating chamber being deformable in response to a change in pressure of the actuating chamber to conduct a change in pressure between the actuating chamber and the second culture chamber, the culture membrane is deformable in response to a change in pressure of the second culture chamber. The organ chip can simulate the physiological change of the pulmonary alveoli in the breathing process more truly.

Description

Technical Field

[0001] This application relates to the field of organ-on-a-chip, in particular to an organ-on-a-chip, a lung gas exchange model, a preparation method and a three-dimensional deformation method. Background Art

[0002] An organ-on-a-chip is a bionic cell microculture system that can simulate the physiological processes of organs in the body to a certain extent, predict drug responses, avoid problems such as ethics, high costs, and species differences brought about by animal experiments, and provide strong assistance for the exploration of disease mechanisms, preclinical research, and drug development.

[0003] Among them, the classic lung gas exchange model based on an organ-on-a-chip disclosed by Emulate in 2010 includes two upper and lower main channels and two left and right air chambers. Lung epithelial cells and vascular endothelial cells are cultured on both sides of a polydimethylsiloxane porous membrane between the two upper and lower main channels to construct a pulmonary air-blood barrier; gases and flowing culture media are introduced into the upper and lower main channels respectively to provide gas shear force and liquid shear force, simulating the gas entering the alveoli and the blood flow shear in the pulmonary capillaries during the breathing process respectively; a pressure change is applied to the side chamber to drive the deformation of the side wall between the side chamber and the main channel, and the dynamic stretching of the middle polydimethylsiloxane porous membrane in the two-dimensional plane can be realized, thereby simulating the expansion and retraction of the alveolar wall during the breathing process.

[0004] This type of classic lung gas exchange model has been widely used in the establishment of various disease models and drug screening, and plays an important role in the establishment of infectious disease (such as COVID-19, influenza), lung cancer, obstructive pulmonary disease and asthma disease models and the research and development of vaccines.

[0005] The lung gas exchange model constructs a stretchable and deformable alveolar-pulmonary capillary air-blood exchange barrier structure, which can simulate the in-vivo dynamic breathing microenvironment to a certain extent. However, this type of lung gas exchange model produces stretching and retraction in a single direction on a two-dimensional scale, and it is difficult to truly simulate the three-dimensional expansion and contraction of the alveoli driven by the movement of the diaphragm and pleural cavity in the body, and the simulation effect needs to be further improved.

[0006] Some related technologies have proposed a technical solution of directly applying pressure to the main channel to cause three-dimensional deformation of the polydimethylsiloxane porous membrane. However, in the current solution, there is still a problem that the deformation degrees of each region of the polydimethylsiloxane porous membrane are not uniform, and it is difficult to truly simulate the expansion of the alveolar wall.

[0007] In addition, when constructing a lung gas exchange model, the polydimethylsiloxane porous membrane also has some other defects, which are manifested in the following points:

[0008] (1) In the related art, the thickness of the polydimethylsiloxane porous membrane is generally 10 - 50 μm, while the average thickness of the air-blood barrier in the lungs in vivo is close to 0.1 μm, so the simulation is not accurate enough.

[0009] (2) The polydimethylsiloxane material lacks hydrophilicity and permeability. Therefore, after forming the film, through holes need to be processed to improve the permeability.

[0010] (3) The polydimethylsiloxane material is relatively hard and lacks elasticity, making it difficult to simulate the alveolar deformation process.

[0011] (4) The polydimethylsiloxane material cannot be degraded in a short time and is difficult to form a barrier for cell fusion between the upper and lower layers, such as the air-blood barrier. SUMMARY OF THE INVENTION

[0012] This application aims to solve at least one of the technical problems existing in the prior art. For this purpose, this application provides an organ chip, a lung gas exchange model, a preparation method, and a three-dimensional deformation method. The organ chip can more realistically simulate the physiological changes of the alveoli during the breathing process.

[0013] The organ chip provided by this application includes a culture membrane and an actuator membrane. The organ chip further forms a culture chamber and at least two actuator chambers. The culture membrane divides the culture chamber into a first culture chamber and a second culture chamber. The actuator membrane divides the second culture chamber and each of the actuator chambers. The actuator chambers define the deformable regions of the actuator membrane. At least two of the actuator chambers are arranged at equal intervals along the circumference around the axis of the culture chamber. The actuator membrane located in the actuator chamber can deform in response to the pressure change in the actuator chamber to conduct the pressure change between the actuator chamber and the second culture chamber. The culture membrane can deform in response to the pressure change in the second culture chamber.

[0014] The organ-on-a-chip provided by the present application has at least the following technical effects: By adjusting the pressure in the actuation chamber, the present application causes the actuation membrane between the actuation chamber and the culture chamber to deform, thereby causing a pressure change in the culture chamber and enabling the culture membrane to undergo three-dimensional deformation. Compared with two-dimensional tensile deformation, the three-dimensional deformation is closer to the real physiological changes of the alveoli, which is more conducive to establishing information exchange and gas exchange between alveolar lung epithelial cells and vascular endothelial cells in a three-dimensional dynamic microenvironment, truly simulating the physiological expansion and contraction as well as biological information exchange and effective gas exchange of the alveoli during the breathing process, and providing a reliable technical platform for preclinical research on respiratory diseases and drug development; On this basis, the present application uses multiple actuation chambers for braking. When simulating the reciprocating expansion and contraction of the alveolar wall, the multiple actuation chambers are arranged at equal intervals along the circumference around the axis of the culture chamber (i.e., evenly distributed), and at the same time generate acting forces to traction the culture membrane, so that the acting forces received by the culture membrane are more uniform, reducing or even eliminating the uneven stress phenomenon during the expansion of the culture membrane, and enabling the organ-on-a-chip to more truly simulate the physiological changes of the alveoli during the breathing process.

[0015] According to some embodiments of the present application, the culture membrane is a hydrogel porous membrane.

[0016] According to some embodiments of the present application, the hydrogel porous membrane is degradable.

[0017] According to some embodiments of the present application, the initial thickness of the culture membrane is greater than or equal to 5 μm and less than or equal to 100 μm.

[0018] According to some embodiments of the present application, the entire culture membrane is impregnated with biological macromolecules.

[0019] According to some embodiments of the present application, the biological macromolecules include collagen, fibronectin, and laminin, and the collagen includes type I collagen or type IV collagen.

[0020] According to some embodiments of the present application, the cross-section of the first culture chamber and / or the second culture chamber is circular.

[0021] According to some embodiments of the present application, the cross-section of the first culture chamber and / or the second culture chamber gradually increases from the side close to the culture membrane to the side far from the culture membrane.

[0022] According to some embodiments of the present application, the organ-on-a-chip is formed with pressure holes, the pressure holes are communicated with the actuation chambers, and the pressure holes are used to connect an external pressure end.

[0023] According to some embodiments of the present application, the pressure holes are respectively communicated with each of the actuation chambers through symmetric pressure channels.

[0024] According to some embodiments of the present application, the actuating membrane is a polydimethylsiloxane membrane.

[0025] According to some embodiments of the present application, the first culture chamber is a lung epithelial cell culture chamber, and the second culture chamber is a vascular endothelial cell culture chamber.

[0026] According to some embodiments of the present application, the organ chip includes a first layer and a second layer, the first layer and the second layer are respectively adhered to two sides of the culture membrane, the first culture chamber is located in the first layer, and the second culture chamber is located in the second layer.

[0027] According to some embodiments of the present application, the actuating membrane is attached to the side of the second layer away from the culture membrane, the organ chip includes a third layer, the actuating chamber is located in the third layer, and the third layer is attached to the side of the actuating membrane away from the culture membrane.

[0028] According to some embodiments of the present application, the organ chip includes a fourth layer, which is attached to the side of the first layer facing away from the culture membrane, and the fourth layer is formed with a liquid inlet and a liquid outlet, two groups of the liquid inlet holes are respectively connected to the first culture chamber and the second culture chamber, and two groups of the liquid outlet holes are respectively connected to the first culture chamber and the second culture chamber.

[0029] According to some embodiments of the present application, the first layer is formed with a first flow channel and a second flow channel, the first flow channel connects the liquid inlet and the first culture chamber, and the second flow channel connects the liquid outlet and the first culture chamber; the second layer is formed with a third flow channel and a fourth flow channel, the third flow channel connects the liquid inlet and the second culture chamber, and the fourth flow channel connects the liquid outlet and the second culture chamber.

[0030] According to some embodiments of the present application, the first layer is further formed with a fifth flow channel and a sixth flow channel, the liquid inlet is connected to the third flow channel through the fifth flow channel, and the liquid outlet is connected to the fourth flow channel through the sixth flow channel.

[0031] According to some embodiments of the present application, the first layer is formed with glue overflow grooves, and two groups of the glue overflow grooves respectively surround the fifth flow channel and the sixth flow channel, and the glue overflow grooves are used to accommodate excess glue generated when the first layer is bonded.

[0032] According to some embodiments of the present application, the first flow-through channel and the second flow-through channel penetrate through the first layer in the longitudinal direction, the third flow-through channel and the fourth flow-through channel penetrate through the second layer in the longitudinal direction, and the first flow-through channel, the second flow-through channel, the third flow-through channel, and the fourth flow-through channel are staggered from each other.

[0033] According to some embodiments of the present application, the organ chip includes clamping plates, and the two clamping plates are respectively located at both ends of the organ chip in the longitudinal direction, and the clamping plates clamp the remaining part of the organ chip.

[0034] According to the method for preparing a lung gas exchange model provided by the present application, the method includes the following steps: providing the organ chip of the present application; inoculating and culturing lung epithelial cells on one surface of the culture membrane; inoculating and culturing vascular endothelial cells on the other surface of the culture membrane.

[0035] According to some embodiments of the present application, inoculating and culturing the lung epithelial cells includes: making the surface of the culture membrane to be inoculated face upward, introducing a solution carrying the lung epithelial cells into the lung epithelial cell culture chamber; standing the organ chip in a cell culture incubator so that the lung epithelial cells settle and adhere to the surface of the culture membrane; providing a flowing cell culture medium to the lung epithelial cell culture chamber.

[0036] According to some embodiments of the present application, inoculating and culturing the vascular endothelial cells includes: making the surface of the culture membrane to be inoculated face upward, introducing a solution carrying the vascular endothelial cells into the vascular endothelial cell culture chamber; standing the organ chip in a cell culture incubator so that the vascular endothelial cells settle and adhere to the surface of the culture membrane; providing a flowing cell culture medium to the vascular endothelial cell culture chamber.

[0037] According to some embodiments of the present application, the culture membrane adopts a degradable hydrogel porous membrane, and the lung epithelial cells and the vascular endothelial cells are cultured until the culture membrane degrades to a thickness less than or equal to 100 nm.

[0038] According to some embodiments of the present application, the lung epithelial cells and the vascular endothelial cells are cultured until the culture membrane is completely degraded.

[0039] According to some embodiments of the present application, the method for preparing the lung gas exchange model further includes: detecting and evaluating the degradation status of the culture membrane and the growth status of the lung epithelial cells and the vascular endothelial cells.

[0040] According to some embodiments of the present application, before inoculating the lung epithelial cells and the vascular endothelial cells, the method for preparing the lung gas exchange model further includes: integrally impregnating the culture membrane with biological macromolecules.

[0041] According to some embodiments of the present application, the whole culture membrane is impregnated with biological macromolecules, including: introducing a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide solution with a concentration range of 0.1% to 1% into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber; introducing a composite solution composed of collagen, fibronectin, and laminin into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber, where the collagen is type I collagen or type IV collagen; leaving the organ chip standing and waiting for the solution to diffuse and react; introducing a buffer solution into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber to wash away the molecules that have not impregnated the culture membrane and the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide solution; introducing a cell culture medium into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber to infiltrate the culture membrane.

[0042] The lung gas exchange model provided by the present application is prepared based on the preparation method of the lung gas exchange model provided by the present application.

[0043] According to the three-dimensional deformation method based on the lung gas exchange model provided by the present application, using the lung gas exchange model provided by the present application, the three-dimensional deformation method includes the following steps: adjusting the pressure of the actuator chamber so that the pressure of each actuator chamber changes periodically according to a set curve synchronously, thereby causing the culture membrane to expand and contract periodically.

[0044] The lung gas exchange model provided by the present application includes the organ chip provided by the present application. The preparation method of the lung gas exchange model provided by the present application is used to make the lung gas exchange model provided by the present application. The three-dimensional deformation method provided by the present application uses the lung gas exchange model provided by the present application. Therefore, the lung gas exchange model, the preparation method of the lung gas exchange model, and the three-dimensional deformation method correspondingly have the beneficial effects provided by the organ chip, which will not be elaborated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, where:

[0046] Figure 1 is a schematic diagram of the principle of the organ chip according to an embodiment of the present application;

[0047] Figure 2 is a schematic diagram of the results of the control experiment of the organ chip according to an embodiment of the present application;

[0048] Figure 3 is an exploded structural diagram of the organ chip according to an embodiment of the present application;

[0049] Figure 4 is a schematic cross-sectional structure diagram of the organ chip according to an embodiment of the present application;

[0050] Figure 5 is an exploded structure diagram of the organ chip according to an embodiment of the present application;

[0051] Figure 6 is a schematic cross-sectional structure diagram of the organ chip according to an embodiment of the present application;

[0052] Figure 7 is a schematic structure diagram of the organ chip according to an embodiment of the present application.

[0053] Reference numerals:

[0054] culture membrane 100,

[0055] actuation membrane 200,

[0056] first layer 300, first culture chamber 310, first flow-through channel 320, second flow-through channel 330, fifth flow-through channel 340, sixth flow-through channel 350, overflow glue groove 360,

[0057] second layer 400, second culture chamber 410, third flow-through channel 420, fourth flow-through channel 430,

[0058] third layer 500, actuation chamber 510, pressure hole 520, pressure flow channel 530,

[0059] fourth layer 600, liquid inlet hole 610, liquid outlet hole 620,

[0060] clamping plate 700,

[0061] lung epithelial cell layer 910, vascular endothelial cell layer 920. Detailed implementation manners

[0062] The embodiments of the present application will be described in detail below. The examples of the embodiments are shown in the drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the drawings are exemplary and are only used to explain the present application, and should not be construed as a limitation to the present application.

[0063] In the description of the present application, it should be understood that the orientation or positional relationship indicated by terms such as up, down, front, back, left, right, etc., is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation to the present application.

[0064] In the description of the present application, the meaning of "several" is one or more, the meaning of "multiple" is more than two, and understandings such as "greater than", "less than", "exceeding", etc. do not include the base number, and understandings such as "above", "below", "within", etc. include the base number. If there is a description of "first" and "second", it is only for the purpose of distinguishing technical features and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features or implicitly indicating the sequence relationship of the indicated technical features.

[0065] In the description of the present application, unless otherwise clearly defined, words such as "set", "installed", "connected", etc. should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meaning of the above words in the present application in combination with the specific content of the technical solution.

[0066] The alveolus is the terminal part of the bronchial tree and is the place where gas exchange occurs between the human body and the outside world. The structure through which gas molecules are exchanged between the gas in the alveolus and the gas in the blood is called the air-blood barrier, which includes the alveolar surface liquid layer, type I alveolar cells and basement membrane, thin connective tissue, capillary basement membrane and endothelium. The air-blood barrier is very thin, with a total thickness of 0.2 μm to 0.5 μm, which is conducive to rapid gas exchange.

[0067] Pulmonary respiration refers to the action of gas exchange during the contraction and expansion of the lungs. During the breathing process, the pressure in the thoracic cavity changes, resulting in a pressure difference between the pressure in the lungs and the outside pressure, which causes gas flow, and the deformation of normal alveoli is uniform deformation.

[0068] The lung gas exchange model is a system for studying the physiological process of alveoli. The lung gas exchange model includes an organ chip. Lung epithelial cells and vascular endothelial cells are respectively cultured on both surfaces of the culture membrane of the organ chip to simulate the alveolar wall. By applying pressure to the culture membrane, the culture membrane deforms in response to the change of pressure to simulate the expansion and contraction of the alveolar wall during breathing.

[0069] However, in the related art, the pressure distribution on the surface of the culture membrane is not uniform enough, resulting in inconsistent deformation degrees in different regions of the culture membrane, and further resulting in differences in the states of cell tissues in different regions, affecting the simulation effect.

[0070] The following introduces the organ chip provided by the present application.

[0071] Refer to Figure 1, according to the embodiments of the first aspect provided by the present application, the organ-on-a-chip includes a culture membrane 100 and an actuating membrane 200. The organ-on-a-chip further forms a culture chamber and at least two actuating chambers 510. The culture membrane 100 divides the culture chamber into a first culture chamber 310 and a second culture chamber 410. The actuating membrane 200 separates the second culture chamber 410 and each actuating chamber 510. The actuating chamber 510 defines a deformable region of the actuating membrane 200. At least two actuating chambers 510 are arranged at equal intervals along the circumference around the axis of the culture chamber. The actuating membrane 200 located in the actuating chamber 510 can deform in response to the pressure change in the actuating chamber 510 to conduct the pressure change between the actuating chamber 510 and the second culture chamber 410. The culture membrane 100 can deform in response to the pressure change in the second culture chamber 410.

[0072] The present application also provides a lung gas exchange model including the organ-on-a-chip of the present application. When constructing the lung gas exchange model, lung epithelial cells and vascular endothelial cells are respectively introduced into the first culture chamber 310 and the second culture chamber 410, so that the cells grow on the two surfaces of the culture membrane 100, and a lung epithelial cell layer 910 and a vascular endothelial cell layer 920 are respectively generated, thereby simulating the air-blood barrier of the alveoli. At this time, the first culture chamber 310 and the second culture chamber 410 are respectively a lung epithelial cell culture chamber and a vascular endothelial cell culture chamber.

[0073] By adjusting the pressure in the actuating chamber 510, the organ-on-a-chip provided by the present application causes the actuating membrane 200 between the actuating chamber 510 and the culture chamber to deform, thereby causing a pressure change in the culture chamber and causing the culture membrane 100 to undergo three-dimensional deformation. Compared with two-dimensional tensile deformation, three-dimensional deformation is closer to the real physiological changes of the alveoli, which is more conducive to establishing information exchange and gas exchange between alveolar lung epithelial cells and vascular endothelial cells in a three-dimensional dynamic microenvironment, truly simulating the physiological expansion and contraction and bioinformation exchange and effective gas exchange of the alveoli during the breathing process, and providing a reliable technical platform for preclinical research of respiratory diseases and drug development.

[0074] Furthermore, the present application uses multiple actuating chambers 510 for actuation. When simulating the reciprocating expansion and contraction of the alveolar wall, multiple actuating chambers 510 are arranged at equal intervals along the circumference around the axis of the culture chamber (that is, evenly distributed). The acting forces generated by the multiple actuating chambers 510 can balance each other, so that the traction force received by the culture membrane 100 is more uniform, reducing or even eliminating the uneven stress phenomenon during the expansion of the culture membrane 100, and the organ-on-a-chip can more truly simulate the physiological changes of the alveoli during the breathing process.

[0075] It is understandable that the alveoli are constantly cycling between expansion and contraction. In order to more realistically simulate the dynamic environment of cell and tissue growth, the pressure in the actuator chamber 510 also needs to change reciprocally according to set conditions. The reciprocating pressure change will affect the flow field in the culture chamber, and this factor is often ignored in related technologies, and there are no restrictions on the number and layout of the actuator chambers 510. If there is only one actuator chamber 510 that is not coaxial with the culture membrane 100, the culture membrane 100 is prone to uneven force in different regions under the influence of the flow field when it expands.

[0076] The design of the actuator chamber 510 adopted in this application can overcome this defect. Figure 2 Shows the results of a control experiment conducted on an organ chip embodiment based on this application. Figure 2 Part A in it shows the state of expansion and contraction of the culture membrane 100 when all the actuator chambers 510 are used simultaneously. Figure 2 Part B in it shows the state of expansion and contraction of the culture membrane 100 when only one actuator chamber 510 that is not coaxial with the culture chamber is used and the other actuator chambers 510 are closed. By comparing them, it can be seen that when there is only one actuator chamber 510, there is an obvious uneven deformation phenomenon when the culture membrane 100 expands, and using multiple actuator chambers 510 can improve this defect.

[0077] The organ chip can have one or more culture chambers. Correspondingly, the number of actuator chambers 510 can be one group or multiple groups.

[0078] For convenience, in the embodiments of the first aspect, the shapes and sizes of the respective actuator chambers 510 are the same, and the distances from the culture chamber to the respective actuator chambers 510 along the axis are equal, thereby reducing the variables affecting the acting force. It only needs to control the pressures of the respective actuator chambers 510 to be synchronized to achieve the balance of the acting forces generated by the multiple actuator chambers 510. However, it is not excluded that in some embodiments, the shapes and sizes of the respective actuator chambers 510 are different, the distances from the culture chamber to the respective actuator chambers 510 along the axis are not equal, and the pressure change curves of the respective actuator chambers 510 are different, but the variables are balanced with each other, and still achieve the balance of the acting forces generated by the multiple actuator chambers 510.

[0079] The lung gas exchange model includes the organ chip and thus correspondingly has the beneficial effects of the organ chip of this application, which will not be elaborated here.

[0080] In the human body environment, the deformation of the alveoli comes from the pressure change in the thoracic cavity. When the thoracic cavity expands, the thoracic cavity pressure is less than the atmospheric pressure, causing the alveoli to expand and inhale air. When the thoracic cavity contracts, the thoracic cavity pressure is greater than the atmospheric pressure, causing the alveoli to contract and exhale air. Therefore, referring to Figure 1, when used to construct a lung gas exchange model that simulates the real lung environment, some embodiments represented by the embodiments of the first aspect tend to also use a negative pressure method to cause the culture membrane 100 to deform, that is, to make the actuation chamber 510 and the second culture chamber 410 on the same side of the culture membrane 100. The actuation membrane 200 separates the vascular endothelial cell culture chamber (that is, the second culture chamber 410) and the actuation chamber 510, and causes a pressure change in the vascular endothelial cell culture chamber through the actuation membrane 200, thereby causing the culture membrane 100 to deform.

[0081] Correspondingly, when the pressure in the actuation chamber 510 decreases, the actuation membrane 200 expands and deforms toward the actuation chamber 510 side, simulating the expansion of the thoracic cavity to cause inhalation in the human body. The actuation chamber 510 plays a role in defining the deformable area of the actuation membrane 200. When the pressure in the actuation chamber 510 is restored, the actuation membrane 200 returns to an approximately planar state, simulating the contraction of the thoracic cavity to cause exhalation in the human body.

[0082] Of course, it does not exclude that in other simulation scenarios, the organ chip adopts different designs. Exemplarily, in the scenario of simulating the active ventilation of a ventilator, the first culture chamber 310 can be used as the vascular endothelial cell culture chamber, and the second culture chamber 410 can be used as the lung epithelial cell culture chamber. The actuation membrane 200 separates the lung epithelial cell culture chamber and the actuation chamber 510, and the actuation membrane 200 can expand and deform toward the lung epithelial cell culture chamber side, thereby causing an increase in the pressure in the lung epithelial cell culture chamber, simulating the active introduction of air into the lung by the ventilator. Or, two groups of actuation chambers 510 can be respectively arranged on both sides of the culture membrane 100.

[0083] In addition to the lung gas exchange model, the organ chip of the present application can also be used for the construction of other organ models, which depends on the cell types grown on the two layers of the culture membrane 100 of the organ chip, such as fibroblasts, dendritic cells and other cells. In addition, when constructing the lung gas exchange model, human lung microvascular endothelial cells or human umbilical vein endothelial cells can be used as the vascular endothelial cells.

[0084] Returning to the organ chip of the present application. In the organ chip, on the one hand, the culture membrane 100 needs to provide elasticity to meet the requirements of expansion and deformation. On the other hand, the culture membrane 100 simultaneously serves as the substrate for cell growth and plays a role in simulating the interstitial substance between cells. In the related art, the culture membrane 100 usually uses a porous membrane based on polydimethylsiloxane material as the culture membrane 100.

[0085] However, the porous membrane based on polydimethylsiloxane material has defects. On the one hand, the deformability of the polydimethylsiloxane material is poor, which makes it difficult for the culture membrane 100 to expand and deform significantly. Therefore, when applied to the lung gas exchange model, the simulation effect of alveolar inflation is not good. On the other hand, the porous membrane based on polydimethylsiloxane material is specially processed to form holes after film formation, and the cells on both sides exchange substances through the holes on the membrane, which is different from the substance exchange of cells through the interstitial tissue in the real physiological environment, and will also have a negative impact on the authenticity of the simulation.

[0086] In view of this, in the present application, a hydrogel porous membrane can be used as the culture membrane 100. The hydrogel porous membrane has a microscopic pore structure, so there is no need to additionally process holes on the culture membrane 100. The elasticity of the hydrogel material is better than that of the polydimethylsiloxane material. Therefore, under the same pressure regulation, the hydrogel porous membrane has a greater degree of expansion and can better simulate the expansion of alveoli. The hydrogel also has the characteristic of good biocompatibility, which is convenient for the growth of cells on both sides. However, the material of the culture membrane 100 itself and the thickness of the culture membrane 100 will also affect the substance exchange of cells on both sides. Therefore, further, the culture membrane 100 can adopt a degradable hydrogel porous membrane. After the cells on both sides have completed growth, the culture membrane 100 will partially or even completely degrade, thereby further reducing the impact of the material of the culture membrane 100 on the authenticity of the simulation of the cell interstitial tissue.

[0087] The preparation materials of the hydrogel porous membrane include natural proteins (such as gelatin, sericin, etc.), natural polysaccharides (such as dextran, alginate, hyaluronic acid, dextran, etc.) and synthetic materials (such as polyester, polyamide, polyvinyl alcohol, etc.). For example, a double-network hydrogel composed of sodium alginate - polyacrylamide can be selected to prepare a hydrogel porous membrane with good hydrophilicity, high porosity and high transparency. Generally speaking, the prepared hydrogel porous membrane needs to be washed with pure water, Hank's balanced salt solution, phosphate buffered saline, etc. to remove unreacted monomers before it can be used for the assembly of the organ chip.

[0088] The organ chip with a degradable hydrogel porous membrane can be applied to the lung gas exchange model or other organ models with similar requirements. Ideally, in the lung gas exchange model, the culture membrane 100 can degrade after the growth of the lung epithelial cell layer 910 and the vascular endothelial cell layer 920 is completed.

[0089] In a real physiological environment, the thickness of the extracellular matrix is about 100 nm. Therefore, under ideal conditions, the culture membrane 100 should be set to be able to degrade to a thickness less than or equal to 100 nm. To reduce the processing difficulty and facilitate the initial growth of cells, the initial thickness of the culture membrane 100 can be set to be greater than or equal to 5 μm and less than or equal to 100 μm. It can be understood that the initial thickness refers to the thickness of the culture membrane 100 when the organ chip has not been put into use and the degradation of the culture membrane 100 has not started.

[0090] To form an environment suitable for cell growth, more realistically simulate the extracellular matrix, and improve the growth effect of cells on the surface of the culture membrane 100, the culture membrane 100 can also be impregnated with biomacromolecules, that is, the entire culture membrane 100 is impregnated with biomacromolecules.

[0091] Specifically, the biomacromolecules can include collagen, fibronectin, and laminin, and the collagen can include type I collagen or type IV collagen.

[0092] In addition, since the shapes of the first culture chamber 310 and the second culture chamber 410 define the deformable region (which is also the cell growth region) of the culture membrane 100, to make the deformation of the culture membrane 100 more uniform, the cross-section of the first culture chamber 310 and / or the second culture chamber 410 can be designed to be circular.

[0093] Cells grow on the surface of the culture membrane 100 exposed to the first culture chamber 310 and the second culture chamber 410. When cells grow, it is necessary to continuously introduce cell culture medium into the culture chamber to provide energy for the cells. In some dead-end positions, such as the outer edge of the exposed culture membrane 100, that is, the junction of the first culture chamber 310 and the culture membrane 100 and the junction of the second culture chamber 410 and the culture membrane 100, the fluidity of the cell culture medium is poor, and the cells are prone to poor growth.

[0094] To address this problem, the first culture chamber 310 and / or the second culture chamber 410 can be designed such that the cross-section gradually increases from the side close to the culture membrane 100 to the side far from the culture membrane 100, thereby improving the fluidity of the cell culture medium at the outer edge position. Exemplarily, referring to Figure 3 、 Figure 4 In the embodiment of the second aspect of the present application, the first culture chamber 310 is designed such that the cross-section gradually increases from the side close to the culture membrane 100 to the side far from the culture membrane 100.

[0095] The actuating membrane 200 needs to have a certain elasticity and needs to separate the culture chamber and the actuating chamber 510 to avoid mass exchange. Therefore, the actuating membrane 200 often uses a non-porous membrane made of polydimethylsiloxane material. Generally speaking, the pressure in the actuating chamber 510 can be adjusted by changing the fluid volume in the actuating chamber 510. For example, the organ chip can include a pressure hole 520, which is connected to the actuating chamber 510, and the pressure hole 520 is used to connect to an external pressure end. The pressure end can provide a gaseous or liquid fluid, thereby causing a pressure change in the actuating chamber 510.

[0096] Furthermore, in order to improve the consistency of the deformation of the actuating membrane 200 in each actuating chamber 510, it can be designed such that the pressure hole 520 is respectively connected to each actuating chamber 510 through a symmetric pressure flow channel 530.

[0097] Changing the pressure by inputting and outputting fluids helps to simplify the structure of the organ chip, thereby facilitating the miniaturization of the organ chip and better simulating structures with a relatively small unit size such as alveoli. In some scenarios, it is also possible to consider designing a movable micro-mechanical structure in the organ chip, such as a micro piston structure, etc. By changing the volume of the actuating chamber 510 through the movement of the structure, the pressure can be adjusted. For specific design solutions, reference can be made to the related technologies in this field and related technical fields (such as the MEMS field), which will not be elaborated here.

[0098] In this application, the organ chip can form spatial structures such as a culture chamber and an actuating chamber 510 in different ways. Exemplarily, it can be selected to form structures such as a culture chamber and an actuating chamber 510 by stacking layered structures. Figure 3 and Figure 4 shows an embodiment of the second aspect based on this concept. Figure 5 and Figure 6 shows an embodiment of the third aspect based on this concept. It can be understood that Figure 3 and Figure 4 、 Figure 5 and Figure 6 are some examples of forming structures such as a culture chamber and an actuating chamber 510 by stacking layered structures. Figure 5 and Figure 6 The first culture chamber 310 shown is funnel-shaped (that is, the cross-section gradually increases from the side close to the culture membrane 100 to the side far from the culture membrane 100), which can avoid dead corners of liquid flow and increase the volume. Based on the concept of stacking layered structures, the organ chip can also have other different stacking formation methods.

[0099] Refer to Figure 3 and Figure 4 、 Figure 5 and Figure 6, Exemplarily, the organ-on-a-chip may include a first layer 300 and a second layer 400. The first layer 300 and the second layer 400 are respectively attached to both sides of the culture membrane 100. The first culture chamber 310 is located in the first layer 300, and the second culture chamber 410 is located in the second layer 400. The first layer 300 and the second layer 400 sandwich the culture membrane 100 by attachment, and expose the area of the culture membrane 100 for culturing cells to the culture chambers.

[0100] Continue to refer to Figure 3 and Figure 4 、 Figure 5 and Figure 6 , Since the organ-on-a-chip in the embodiment is used to simulate human inspiration caused by thoracic cavity expansion, the actuating membrane 200 is attached to the side of the second layer 400 facing away from the culture membrane 100. The organ-on-a-chip may further include a third layer 500. The actuating chamber 510 is located in the third layer 500. The third layer 500 is attached to the side of the actuating membrane 200 facing away from the culture membrane 100. The second layer 400 and the third layer 500 sandwich the actuating membrane 200.

[0101] It can be understood that the attachment can be by applying a force to press or bond together, or by using methods such as glue or tape to paste together. By decomposing the organ-on-a-chip into multiple different layers, the processing of structures such as the culture chamber and the actuating chamber 510 can be facilitated.

[0102] Furthermore, the organ-on-a-chip may further include a fourth layer 600. The fourth layer 600 is formed with a liquid inlet hole 610 and a liquid outlet hole 620. The liquid inlet hole 610 and the liquid outlet hole 620 communicate with the culture chamber to supply materials such as cells and cell culture medium to the culture chamber.

[0103] Generally speaking, the cells cultured on both sides of the culture membrane 100 are different, and there are differences in the required culture environments. Therefore, the liquid inlet hole 610 and the liquid outlet hole 620 are divided into two groups to supply materials such as cells and cell culture medium to the first culture chamber 310 and the second culture chamber 410 respectively. The organ-on-a-chip may include only one fourth layer 600. The fourth layer 600 has two groups of liquid inlet holes 610 and two groups of liquid outlet holes 620. One fourth layer 600 is responsible for supplying materials to both the first culture chamber 310 and the second culture chamber 410 at the same time, or the organ-on-a-chip may include two fourth layers 600. Each fourth layer 600 has a group of liquid inlet holes 610 and a group of liquid outlet holes 620. The two fourth layers 600 are respectively responsible for supplying materials to the first culture chamber 310 and the second culture chamber 410.

[0104] Correspondingly, the organ chip is formed with a first flow channel 320 and a second flow channel 330, wherein the first flow channel 320 connects the liquid inlet 610 and the first culture chamber 310, and the second flow channel 330 connects the liquid outlet 620 and the first culture chamber 310. The organ chip is also formed with a third flow channel 420 and a fourth flow channel 430, wherein the third flow channel 420 connects the liquid inlet 610 and the second culture chamber 410, and the fourth flow channel 430 connects the liquid outlet 620 and the second culture chamber 410.

[0105] For example, refer to Figure 3 In an embodiment of the second aspect, there is only one fourth layer 600, and the fourth layer 600 is attached to the side of the first layer 300 away from the culture membrane 100, and the fourth layer 600 has two groups of liquid inlet holes 610 and two groups of liquid outlet holes 620, the two groups of liquid inlet holes 610 are respectively connected to the first culture chamber 310 and the second culture chamber 410, and the two groups of liquid outlet holes 620 are respectively connected to the first culture chamber 310 and the second culture chamber 410. At the same time, the first layer 300 forms a first flow channel 320 and a second flow channel 330, and the second layer 400 forms a third flow channel 420 and a fourth flow channel 430.

[0106] In this case, the first layer 300 also needs to be formed with a fifth flow channel 340 and a sixth flow channel 350, and the liquid inlet 610 and the liquid outlet 620 are respectively connected to the second culture chamber 410 located in the second layer 400 through the fifth flow channel 340 and the sixth flow channel 350. It goes without saying that the fifth flow channel 340 and the sixth flow channel 350 are not connected to the first culture chamber 310 located in the first layer 300 to prevent cross contamination of cells.

[0107] For ease of processing, the first flow channel 320 and the second flow channel 330 can pass through the first layer 300 in the stacking direction (i.e., longitudinal direction) of the organ chip, and the third flow channel 420 and the fourth flow channel 430 can pass through the second layer 400 in the stacking direction of the organ chip. At this time, the first flow channel 320 and the second flow channel 330 located in the first layer 300 and the third flow channel 420 and the fourth flow channel 430 located in the second layer 400 need to be staggered to avoid mutual connection between different flow channels.

[0108] In order to ensure the light transmittance of the organ chip, the materials of the first layer 300 , the second layer 400 , the third layer 500 , and the fourth layer 600 can all be polymethyl methacrylate.

[0109] Exemplarily, the actuating membrane 200 made of polydimethylsiloxane material can be bonded to the second layer 400 and the third layer 500 by means of a plasma cleaning process. The specific assembly process is as follows: Spin-coat a thin layer of polydimethylsiloxane on the second layer 400 and the third layer 500. After curing at 60 °C, bond the spin-coated polydimethylsiloxane surface of the second layer 400 to the actuating membrane 200 after plasma surface treatment, and bond the spin-coated polydimethylsiloxane surface of the actuating membrane 200 to the third layer 500 in the same way. The assembly order of the second layer 400 and the third layer 500 can be swapped.

[0110] Exemplarily, the culture membrane 100 made of hydrogel material can be bonded to the first layer 300 and the second layer 400 by means of tape. The culture membrane 100 is flattened on the first layer 300 with tape covering the surface; alternatively, glue (such as AB glue, UV-curable glue, and polydimethylsiloxane prepolymer, etc.) can also be coated on the surface of the first layer 300 and then bonded. After the culture membrane 100 is bonded to the first layer 300, the culture membrane 100 is bonded to the second layer 400 in the same way. It goes without saying that the bonding order of the first layer 300 and the second layer 400 can be swapped.

[0111] Exemplarily, the first layer 300 and the fourth layer 600 can be bonded by means of hot pressing, or can also be bonded by coating a strong glue on the surface.

[0112] Bonding the organ chip with glue can achieve a good sealing effect, but when bonding, it is necessary to first coat glue on the surface of the first layer 300 to be glued, and then press the first layer 300 and the fourth layer 600 together. The uncured glue may flow into the fifth flow channel 340 and the sixth flow channel 350 during extrusion, resulting in poor fluidity or even blockage of the fifth flow channel 340 and the sixth flow channel 350. Therefore, the first layer 300 can further be formed with glue overflow grooves 360. Two groups of glue overflow grooves 360 respectively surround the fifth flow channel 340 and the sixth flow channel 350. The glue overflow grooves 360 are used to accommodate the excess glue generated during the bonding of the first layer 300.

[0113] After the organ chip is fabricated, the deformation effect of the culture membrane 100 can be evaluated first to ensure that the organ chip meets the design requirements. For example, the culture membrane 100 can be observed through a microscope under applied pressure, and the deformation effect can be evaluated based on the observation results; or, fluorescent microspheres can be incorporated into the culture membrane 100 in advance. After the organ chip is assembled, the displacement process of the fluorescent microspheres is observed under excitation light, and the deformation effect is evaluated based on the observation results.

[0114] Refer to Figure 7 , the organ chip of the present application can further include clamping plates 700. The two clamping plates 700 are longitudinally located at both ends of the organ chip respectively. The clamping plates 700 clamp the rest of the organ chip (in Figure 7Specifically, they are the culture membrane 100, the actuating membrane 200, the first layer 300, the second layer 400, the third layer 500, and the fourth layer 600. Adding the clamping plate 700 can facilitate the carrying of the organ chip. Different positioning structures can also be designed on the clamping plate 700 according to needs, which is convenient for positioning and installing the organ chip on other devices. In the embodiment of the organ chip using a layered structure, the clamping plate 700 can also provide a clamping force to prevent slippage and misalignment of the layers of the organ chip.

[0115] This application also provides a method for preparing a lung gas exchange model. The method for preparing the lung gas exchange model includes the following steps:

[0116] Providing the organ chip of this application;

[0117] Inoculating and culturing lung epithelial cells on one surface of the culture membrane 100;

[0118] Inoculating and culturing vascular endothelial cells on the other surface of the culture membrane 100.

[0119] Exemplarily, inoculating and culturing lung epithelial cells includes:

[0120] Turning the surface of the culture membrane 100 to be inoculated upwards, and introducing a solution carrying lung epithelial cells into the lung epithelial cell culture chamber;

[0121] Placing the organ chip in a cell culture incubator to allow the lung epithelial cells to settle and adhere to the surface of the culture membrane 100;

[0122] Providing a flowing cell culture medium to the lung epithelial cell culture chamber.

[0123] Inoculating and culturing vascular endothelial cells includes:

[0124] Turning the surface of the culture membrane 100 to be inoculated upwards, and introducing a solution carrying vascular endothelial cells into the vascular endothelial cell culture chamber;

[0125] Placing the organ chip in a cell culture incubator to allow the vascular endothelial cells to settle and adhere to the surface of the culture membrane 100;

[0126] Providing a flowing cell culture medium to the vascular endothelial cell culture chamber.

[0127] The standing time can be set to 0.5 h to 24 h to ensure that sufficient cells settle on the surface of the culture membrane 100.

[0128] For the lung gas exchange model using a degradable hydrogel porous membrane as the culture membrane 100, the preparation method of the lung gas exchange model can be designed to culture lung epithelial cells and vascular endothelial cells until the culture membrane 100 degrades to a thickness less than or equal to 100 nm. Further, it can be designed to culture lung epithelial cells and vascular endothelial cells until the culture membrane 100 is completely degraded.

[0129] Correspondingly, since the degradation of the culture membrane 100 takes a long time, in the preparation method of the lung gas exchange model, it is also necessary to detect and evaluate the degradation status of the culture membrane 100 and the growth status of lung epithelial cells and vascular endothelial cells.

[0130] Exemplarily, the growth status of lung epithelial cells and vascular endothelial cells can be evaluated by fluorescence staining observation. The specific implementation of fluorescence staining observation can refer to related technologies, such as staining and observing dead cells and live cells separately, staining and observing under pressure, etc., which will not be elaborated here.

[0131] Exemplarily, the culture membrane 100 can be fluorescently labeled, so as to monitor the integrity and thickness of the culture membrane 100 according to the fluorescence signal, thereby evaluating the degradation status of the culture membrane 100.

[0132] In order to form an environment suitable for cell growth and more realistically simulate the extracellular matrix, before inoculating lung epithelial cells and vascular endothelial cells, the preparation method of the lung gas exchange model further includes: immersing the entire culture membrane 100 in biomacromolecules.

[0133] Exemplarily, immersing the entire culture membrane 100 in biomacromolecules includes:

[0134] Introducing a solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide with a concentration range of 0.1% to 1% into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber;

[0135] Introducing a composite solution composed of collagen, fibronectin and laminin into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber, and the collagen is type I collagen or type IV collagen;

[0136] Let the organ chip stand still and wait for the solution to react;

[0137] Introducing a buffer solution into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber to wash away the molecules that have not infiltrated the culture membrane 100 and the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide solution;

[0138] Introducing a cell culture medium into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber to infiltrate the culture membrane 100.

[0139] The present application also provides a three-dimensional deformation method based on a lung gas exchange model, and the three-dimensional deformation method uses the lung gas exchange model of the present application. The three-dimensional deformation method includes the following steps: adjusting the pressure of the actuating chamber 510 so that the pressures of the respective actuating chambers 510 change periodically according to a set curve synchronously, thereby causing the culture membrane 100 to expand and contract periodically.

[0140] The periodic expansion and contraction of the culture membrane 100 can truly simulate the physiological activities of the alveoli, so that cells can grow and be observed in a more real environment, which helps to improve the accuracy of the experiment.

[0141] In the description of this specification, the descriptions with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0142] In some alternative embodiments, the functions / operations mentioned in the block diagrams may not occur in the order mentioned in the operation diagrams. For example, depending on the functions / operations involved, two consecutively shown blocks can actually be executed substantially simultaneously or the blocks can sometimes be executed in the reverse order. In addition, the embodiments presented and described in the flowcharts of the present application are provided by way of example for the purpose of providing a more comprehensive understanding of the technology. The disclosed method is not limited to the operations and logical flows presented herein. Alternative embodiments are foreseeable, in which the order of various operations is changed and the sub-operations described as part of a larger operation are executed independently.

[0143] Although the embodiments of the present application have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. An organ-on-a-chip, characterized in that, The organ chip is formed with a culture chamber and at least two actuation chambers, and the organ chip comprises: a culture membrane, wherein the culture membrane separates the culture chamber into a first culture chamber and a second culture chamber; an actuating membrane, the actuating membrane separates the second culture chamber and each of the actuating chambers, the actuating chambers define a deformable region of the actuating membrane, at least two of the actuating chambers are equidistantly arranged around the axis of the culture chamber along a circle, the actuating membrane located in the actuating chamber is deformable in response to a pressure change in the actuating chamber to transmit the pressure change between the actuating chamber and the second culture chamber, and the culture membrane is deformable in response to a pressure change in the second culture chamber.

2. The organ-on-a-chip according to claim 1, characterized in that, The culture membrane is a hydrogel porous membrane.

3. The organ-on-a-chip according to claim 2, characterized in that, The hydrogel porous membrane is degradable.

4. The organ-on-a-chip according to claim 3, characterized in that, The initial thickness of the culture membrane is greater than or equal to 5 μm and less than or equal to 100 μm.

5. The organ-on-a-chip according to claim 1, characterized in that, The culture membrane is entirely impregnated with biomacromolecules.

6. The organ-on-a-chip according to claim 5, characterized in that, The biomacromolecules include collagen, fibronectin and laminin, and the collagen includes type I collagen or type IV collagen.

7. The organ-on-a-chip according to claim 1, characterized in that, The cross section of the first culture chamber and / or the second culture chamber is circular.

8. The organ-on-a-chip according to claim 7, characterized in that, The cross section of the first culture chamber and / or the second culture chamber gradually increases from a side close to the culture membrane to a side away from the culture membrane.

9. The organ-on-a-chip according to claim 1, characterized in that, The organ chip is formed with a pressure hole, which is communicated with the actuation chamber and is used to connect to an external pressure end.

10. The organ-on-a-chip according to claim 9, characterized in that, The pressure holes are connected to the actuating chambers respectively through symmetrical pressure channels.

11. The organ-on-a-chip according to claim 1, characterized in that, The actuating film is a polydimethylsiloxane film.

12. The organ-on-a-chip according to any one of claims 1 to 11, characterized in that, The first culture chamber is a lung epithelial cell culture chamber, and the second culture chamber is a vascular endothelial cell culture chamber.

13. The organ-on-a-chip according to any one of claims 1 to 11, characterized in that, The organ chip comprises a first layer and a second layer, wherein the first layer and the second layer are respectively attached to two sides of the culture membrane in the longitudinal direction, the first culture chamber is located in the first layer, and the second culture chamber is located in the second layer.

14. The organ-on-a-chip according to claim 13, characterized in that, The actuating membrane is attached to a side of the second layer away from the culture membrane. The organ chip comprises a third layer. The actuating chamber is located in the third layer. The third layer is attached to a side of the actuating membrane away from the culture membrane.

15. The organ-on-a-chip according to claim 14, characterized in that, The organ chip includes a fourth layer, which is attached to the side of the first layer away from the culture membrane, and the fourth layer is formed with a liquid inlet and a liquid outlet, two groups of the liquid inlet are respectively connected to the first culture chamber and the second culture chamber, and two groups of the liquid outlet are respectively connected to the first culture chamber and the second culture chamber.

16. The organ-on-a-chip according to claim 15, characterized in that, The first layer is formed with at least one first flow channel and at least one second flow channel, the first flow channel is connected with the liquid inlet and the first culture chamber, and the second flow channel is connected with the liquid outlet and the first culture chamber; The second layer is formed with at least one third flow channel and at least one fourth flow channel, the third flow channel connects the liquid inlet and the second culture chamber, and the fourth flow channel connects the liquid outlet and the second culture chamber.

17. The organ-on-a-chip according to claim 16, characterized in that, At least one fifth flow channel and at least one sixth flow channel are also formed in the first layer. The liquid inlet hole communicates with the third flow channel through the fifth flow channel, and the liquid outlet hole communicates with the fourth flow channel through the sixth flow channel.

18. The organ-on-a-chip according to claim 17, characterized in that, A glue overflow groove is formed in the first layer. Two groups of the glue overflow grooves respectively surround the fifth flow channel and the sixth flow channel. The glue overflow groove is used to accommodate the excess glue generated during the bonding of the first layer.

19. The organ-on-a-chip according to claim 16, characterized in that,The first flow channel and the second flow channel penetrate through the first layer in the longitudinal direction. The third flow channel and the fourth flow channel penetrate through the second layer in the longitudinal direction. The first flow channel, the second flow channel located in the first layer and the third flow channel, the fourth flow channel located in the second layer are staggered from each other.

20. The organ-on-a-chip according to claim 15, wherein, The organ chip includes clamping plates. The two clamping plates are respectively located at both ends of the organ chip in the longitudinal direction. The clamping plates clamp the rest of the organ chip.

21. A method for preparing a lung gas exchange model, wherein, The method for preparing the lung gas exchange model includes the following steps: Providing the organ chip according to any one of claims 1 to 20; Inoculating and culturing lung epithelial cells on one surface of the culture membrane; Inoculating and culturing vascular endothelial cells on the other surface of the culture membrane.

22. The method for preparing a lung gas exchange model according to claim 21, wherein, Inoculating and culturing the lung epithelial cells includes: Making the surface of the culture membrane to be inoculated face upward, and introducing a solution carrying the lung epithelial cells into the lung epithelial cell culture chamber; Placing the organ chip in a cell culture incubator to allow the lung epithelial cells to settle and adhere to the surface of the culture membrane; Providing a flowing cell culture medium to the lung epithelial cell culture chamber.

23. The method for preparing a lung gas exchange model according to claim 21, wherein, Inoculating and culturing the vascular endothelial cells includes: Making the surface of the culture membrane to be inoculated face upward, and introducing a solution carrying the vascular endothelial cells into the vascular endothelial cell culture chamber; Placing the organ chip in a cell culture incubator to allow the vascular endothelial cells to settle and adhere to the surface of the culture membrane; Providing a flowing cell culture medium to the vascular endothelial cell culture chamber.

24. The method for preparing a lung gas exchange model according to claim 21, wherein, The culture membrane uses a degradable hydrogel porous membrane. The lung epithelial cells and the vascular endothelial cells are cultured until the culture membrane degrades to a thickness less than or equal to 100 nm.

25. The method for preparing a lung gas exchange model according to claim 24, wherein, The lung epithelial cells and the vascular endothelial cells are cultured until the culture membrane is completely degraded.

26. The method for preparing a lung gas exchange model according to claim 24 or 25, wherein, The method for preparing the lung gas exchange model further includes: detecting and evaluating the degradation status of the culture membrane and the growth status of the lung epithelial cells and the vascular endothelial cells.

27. The method for preparing a lung gas exchange model according to claim 21, wherein, Before inoculating the lung epithelial cells and the vascular endothelial cells, the method for preparing the lung gas exchange model further includes: overall impregnating the culture membrane with biological macromolecules.

28. The method for preparing a lung gas exchange model according to claim 27, wherein, Overall impregnating the culture membrane with biological macromolecules includes: Introducing a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide solution with a concentration range of 0.1% to 1% into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber; A composite solution composed of collagen, fibronectin, and laminin is introduced into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber, and the collagen is type I collagen or type IV collagen; The organ chip is left standing to allow the solution to diffuse and react; A buffer solution is introduced into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber to wash away the molecules that have not infiltrated the culture membrane and the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide solution; A cell culture medium is introduced into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber to infiltrate the culture membrane.

29. A lung gas exchange model, wherein, The lung gas exchange model is obtained by preparing according to the preparation method of the lung gas exchange model according to any one of claims 21 to 28.

30. A three-dimensional deformation method based on a lung gas exchange model, wherein, The three-dimensional deformation method uses the lung gas exchange model according to claim 29, and the three-dimensional deformation method includes the following steps: The pressure of the actuator chamber is adjusted so that the pressure of each actuator chamber changes periodically according to a set curve synchronously, thereby causing the culture membrane to expand and contract periodically.

Images