Organ chip, lung organ model, construction method and simulation method

Abstract

The invention provides an organ chip, a lung organ model, a construction method and a simulation method, the organ chip comprises a culture membrane and an actuating membrane, the organ chip is further provided with a first culture chamber, a second culture chamber, an actuating chamber and a perfusion channel, the culture membrane separates the first culture chamber and the second culture chamber, the perfusion channel is communicated with the first culture chamber, and the actuating chamber is communicated with the second culture chamber. A communication port of the perfusion channel and the first culture chamber faces the culture membrane, preferably directly faces the culture membrane, the perfusion channel is used for connecting an external air source, the actuating chamber separates the second culture chamber and the actuating chamber, and the actuating chamber can deform in response to a pressure change of the actuating chamber so as to conduct the pressure change between the actuating chamber and the second culture chamber; further, the culture membrane is deformed. The organ chip can more truly simulate the respiratory movement process of pulmonary alveoli and the multi-directional shear stress generated by gas to cells, and is beneficial to meeting the research requirements of respirator-related lung injury.

Description

Technical Field

[0001] This application relates to the field of organ-on-a-chip, in particular to organ-on-a-chip, lung organ model, construction method and simulation method. Background Art

[0002] Related research believes that during the treatment of critically ill patients, the lung injury caused by mechanical ventilation using a ventilator mainly comes from alveolar overexpansion, alveolar epithelial and vascular endothelial damage caused by shear stress, and inflammatory response. However, the dynamic damage and repair mechanism of human alveoli at the cellular level has not been fully clarified. The diagnosis of such ventilator-induced lung injury diseases in clinical practice is usually limited to oxygenation index and imaging features, lacking biomarker-based diagnosis and treatment methods and clinical treatment strategies for effectively reducing ventilator-associated lung injury. Therefore, how to construct a more realistic and effective disease research model in vitro, analyze such complex lung-related diseases, develop biomarker-based diagnosis and treatment methods, and provide a "visible window" for the early diagnosis of ventilator-associated lung injury is an urgent problem to be solved in clinical medicine.

[0003] An organ-on-a-chip is a highly simulated in vitro organ microsystem constructed on a microfluidic chip, which can reconstruct the real dynamic microenvironment of the human body in vitro. Compared with two-dimensional cell culture, organoids and animal models, organ-on-a-chip has great potential advantages in in vitro disease model simulation and construction, drug screening, and precision medicine.

[0004] In 2010, Ingber et al. used an organ-on-a-chip to construct a human alveolar-lung microvascular interface and first proposed a lung organ model that simulates the cyclic respiratory movement of the alveoli. The chip structure contains two parallel microchannels above and below, separated by a porous membrane of polydimethylsiloxane (PDMS) covered with extracellular matrix and permeable to stretching in the middle; human alveolar epithelial cells are inoculated on one side of the membrane, and gas is introduced into the upper channel to simulate air entering the alveoli; human lung microvascular endothelial cells are inoculated on the other side of the membrane, and fluid is introduced into the lower channel to simulate blood flow and introduce circulating immune cells, thereby simulating the alveolar-lung microvascular gas-blood barrier; the chambers on both sides of the channel act by vacuum, causing the bionic alveolar gas-blood barrier membrane in the middle to be stretched and contracted, simulating the physiological cyclic respiratory movement.

[0005] However, there are still certain differences between the two-dimensional single-direction mechanical cyclic stretching in such lung organ models and the expansion and contraction of the air-blood barrier during the in-vivo breathing process, making it difficult to simulate the real three-dimensional breathing movement process of the alveoli. For this reason, in related technologies, a lung organ model based on three-dimensional deformation is further proposed. The chip structure includes two upper and lower culture chambers and a polydimethylsiloxane porous membrane separating the two culture chambers. The three-dimensional expansion or contraction of the polydimethylsiloxane porous membrane is caused by the pressure difference between the two culture chambers.

[0006] In related technologies, lung organ models are usually used to simulate the physiological environment of alveoli during natural breathing in the human body. When introducing culture medium into the organ chip and introducing air into the organ chip to simulate alveolar breathing, the same channel is often used, and the channel does not face the culture membrane, which results in the lung organ model not being able to truly simulate the damage suffered by alveoli in the mechanical ventilation scenario.

[0007] Moreover, during the process of introducing culture medium into the culture chamber, air bubbles are likely to remain on the wall surface of the culture chamber. The air bubbles in contact with the cells in the culture chamber will prevent the cells from obtaining the culture medium, resulting in poor growth of this part of the cells and even potentially damaging the cells, thus affecting the growth consistency of the cells. Summary of the Invention

[0008] This application aims to solve at least one of the technical problems existing in the prior art. For this reason, this application provides an organ chip, a lung organ model, a construction method and a simulation method. The organ chip can more truly simulate the breathing movement process of alveoli and the multi-directional shear stress generated by gas on cells, which is beneficial to meeting the research needs of ventilator-associated lung injury.

[0009] According to the organ chip provided by this application, a first culture chamber, a second culture chamber, an actuator chamber and a perfusion channel are formed. The organ chip includes a culture membrane and an actuator membrane. The culture membrane separates the first culture chamber and the second culture chamber. The perfusion channel is communicated with the first culture chamber, and the communication port of the perfusion channel with the first culture chamber faces the culture membrane, preferably directly facing the culture membrane. The perfusion channel is used to connect to an external gas source. The actuator membrane separates the second culture chamber and the actuator chamber. The actuator membrane 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, thereby causing the culture membrane to deform.

[0010] The organ-on-a-chip provided by the present application has at least the following technical effects: The organ-on-a-chip blows gas towards the culture membrane into the first culture chamber through the perfusion channel, causing three-dimensional deformation of the culture membrane, so as to simulate the multi-directional shear stress generated by mechanical ventilation of the ventilator on cells; in addition, the perfusion channel faces the culture membrane, so the gas introduced into the perfusion channel can impact the culture membrane, more realistically simulating the damage suffered by the alveoli during mechanical ventilation. The organ-on-a-chip is conducive to meeting the research needs of ventilator-associated lung injury.

[0011] According to some embodiments of the present application, the culture membrane adopts a polydimethylsiloxane porous membrane.

[0012] According to some embodiments of the present application, the surface of the culture membrane is coated with biomacromolecules for simulating the extracellular matrix.

[0013] According to some embodiments of the present application, the biomacromolecules include collagen, fibronectin and laminin, and the collagen includes type I collagen or type IV collagen.

[0014] According to some embodiments of the present application, the culture membrane is located on one side of the first culture chamber for culturing epithelial cells, and the culture membrane is located on one side of the second culture chamber for culturing vascular endothelial cells.

[0015] According to some embodiments of the present application, the perfusion channel faces the center of the deformable region of the culture membrane.

[0016] According to some embodiments of the present application, the first culture chamber defines the deformable region of the culture membrane, and the shape of the culture membrane exposed to the first culture chamber is a circular shape.

[0017] According to some embodiments of the present application, the perfusion channel and the first culture chamber are coaxially arranged.

[0018] According to some embodiments of the present application, in the region close to the culture membrane, the cross-section of the first culture chamber gradually increases from the side close to the culture membrane to the side far from the culture membrane.

[0019] According to some embodiments of the present application, the volume of the second culture chamber is larger than the volume of the first culture chamber, and the second culture chamber is used for storing the culture medium.

[0020] According to some embodiments of the present application, the surface area of the culture membrane exposed to the second culture chamber is larger than the surface area of the culture membrane exposed to the first culture chamber.

[0021] According to some embodiments of the present application, the actuating membrane adopts a polydimethylsiloxane porous membrane.

[0022] According to some embodiments of the present application, the organ chip includes a first culture layer, a second culture layer, and an actuation layer. The first culture layer, the culture membrane, the second culture layer, the actuation membrane, and the actuation layer are stacked in sequence longitudinally. The first culture chamber and the perfusion channel are located in the first culture layer, the second culture chamber is located in the second culture layer, and the actuation chamber is located in the actuation layer.

[0023] According to some embodiments of the present application, the organ chip is formed with a pressure channel, which communicates with the actuation chamber and is used to connect to an external pressure source.

[0024] According to some embodiments of the present application, the pressure channel extends from the actuation layer to the first culture layer along the longitudinal direction, and the pressure channel penetrates through the first culture layer to form an interface connecting to the outside.

[0025] According to some embodiments of the present application, the perfusion channel extends along the longitudinal direction and penetrates through the first culture layer to form an interface connecting to the outside.

[0026] According to some embodiments of the present application, the organ chip includes a first liquid inlet channel and a first liquid outlet channel, and the first liquid inlet channel and the first liquid outlet channel communicate with the first culture chamber respectively.

[0027] According to some embodiments of the present application, the first liquid inlet channel and the first liquid outlet channel are located in the first culture layer, and the first liquid inlet channel and the first liquid outlet channel extend along the longitudinal direction and penetrate through the first culture layer to form an interface connecting to the outside.

[0028] According to some embodiments of the present application, the organ chip includes a second liquid inlet channel and a second liquid outlet channel, and the second liquid inlet channel and the second liquid outlet channel communicate with the second culture chamber respectively.

[0029] According to some embodiments of the present application, the second liquid inlet channel and the second liquid outlet channel extend from the second culture layer to the first culture layer along the longitudinal direction, and the second liquid inlet channel and the second liquid outlet channel penetrate through the first culture layer to form an interface connecting to the outside.

[0030] According to some embodiments of the present application, the first culture layer includes a first sheet, a second sheet, and a third sheet stacked in sequence.

[0031] According to some embodiments of the present application, the shape of the part of the first culture chamber located in the third sheet is frustum-shaped, and the cross-section of the frustum gradually increases from the side close to the culture membrane to the side far from the culture membrane.

[0032] The method for constructing a lung organ model provided by the present application includes the following steps: providing the organ chip of the present application; inoculating and culturing epithelial cells on the surface of the culture membrane located in the first culture chamber; inoculating and culturing vascular endothelial cells on the surface of the culture membrane located in the second culture chamber.

[0033] According to some embodiments of the present application, inoculating and culturing the epithelial cells includes: placing the first culture chamber upward, introducing a solution carrying the epithelial cells into the first culture chamber; standing the organ chip in a cell culture incubator so that the epithelial cells settle and adhere to the surface of the culture membrane; providing a flowing cell culture medium to the first culture chamber.

[0034] According to some embodiments of the present application, inoculating and culturing the vascular endothelial cells includes: placing the second culture chamber upward, introducing a solution carrying the vascular endothelial cells into the second 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 second culture chamber.

[0035] According to some embodiments of the present application, before inoculating the epithelial cells and the vascular endothelial cells, the method for constructing the lung organ model further includes: coating the surface of the culture membrane with a biomacromolecule for simulating the extracellular matrix.

[0036] According to some embodiments of the present application, coating the surface of the culture membrane with the biomacromolecule includes: introducing a composite solution composed of collagen, fibronectin and laminin into the first culture chamber and the second culture chamber, and the collagen is type I collagen or type IV collagen; standing the organ chip and waiting for the solution to react.

[0037] The lung organ model provided by the present application is constructed by using the method for constructing a lung organ model provided by the present application.

[0038] According to the method for simulating lung injury provided by the present application, using the lung organ model provided by the present application and the lung organ chip provided by the present application, the method for simulating lung injury includes introducing air into the first culture chamber through a perfusion channel to simulate mechanical ventilation of the alveoli by a ventilator.

[0039] According to some embodiments of the present application, the method for simulating lung injury further includes periodically changing the pressure in the actuation chamber to simulate lung injury caused by mechanical ventilation in a scenario where the patient has spontaneous breathing; or keeping the pressure in the actuation chamber constant to simulate lung injury caused by mechanical ventilation in a scenario where the patient has no spontaneous breathing.

[0040] The lung organ model provided by the present application includes the organ chip provided by the present application. The method for constructing the lung organ model provided by the present application and the method for simulating lung injury use the organ chip provided by the present application. Therefore, the lung organ model, the method for constructing the lung organ model, and the method for simulating lung injury correspondingly have the beneficial effects provided by the organ chip, which will not be elaborated here. Description of the Drawings

[0041] The above and / or additional aspects and advantages of the present application will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, where: Figure 1 is a schematic diagram of the principle of the organ chip according to an embodiment of the present application; Figure 2 is a schematic diagram of the principle of the lung organ model according to an embodiment of the present application; Figure 3 is a schematic diagram of the deformation of the culture membrane of the organ chip according to an embodiment of the present application obtained under the contour instrument, where part (a) corresponds to the contracted state of the culture membrane, and part (b) corresponds to the expanded state of the culture membrane; Figure 4 is a schematic diagram of the structure of the culture membrane of the lung organ model according to an embodiment of the present application observed under a microscope, where part (a) corresponds to the contracted state of the culture membrane, and it can be seen that cells grow on the culture membrane, and part (b) corresponds to the expanded state; Figure 5 is a top view structural schematic diagram of the organ chip according to an embodiment of the present application; Figure 6 is a cross-sectional structural schematic diagram of the organ chip according to an embodiment of the present application; Figure 7 is a cross-sectional structural schematic diagram of the organ chip according to an embodiment of the present application; Figure 8 is an exploded structural schematic diagram of the organ chip according to an embodiment of the present application; Figure 9 is an exploded structural schematic diagram of the organ chip according to an embodiment of the present application.

[0042] Reference Signs: The first culture chamber 110, the second culture chamber 120, the actuation chamber 130, the perfusion channel 140, the inlet section 141, the connection section 142, the outlet section 143, the pressure channel 150, the first liquid inlet channel 160, the first liquid outlet channel 170, the second liquid inlet channel 180, the second liquid outlet channel 190, The culture membrane 210, the actuation membrane 220, the first culture layer 230, the first sheet 231, the second sheet 232, the third sheet 233, the second culture layer 240, the actuation layer 250. Detailed Embodiments

[0043] Embodiments of the present application will be described in detail below. Examples of the embodiments are shown in the accompanying 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 by referring to the accompanying drawings are exemplary and are only used to explain the present application, and should not be construed as a limitation to the present application.

[0044] In the description of the present application, it should be understood that for the orientation description, such as the orientation or positional relationship indicated by up, down, front, back, left, right, etc. is based on the orientation or positional relationship shown in the accompanying 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 therefore should not be construed as a limitation to the present application.

[0045] In the description of the present application, the meaning of several is one or more, the meaning of multiple is more than two, greater than, less than, exceeding, etc. are understood as not including the present number, and above, below, within, etc. are understood as including the present number. If the first and second are described only for the purpose of distinguishing technical features, they should not 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.

[0046] In the description of the present application, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above terms in the present application in combination with the specific content of the technical solution.

[0047] The dynamic damage and repair mechanism of alveoli in the human body at the cellular level has not been fully clarified. The lung injury diseases caused by mechanical ventilation of ventilators are usually limited to oxygenation index and imaging features in clinical disease diagnosis, lacking a diagnosis and treatment method based on biomarkers and a clinical treatment strategy for effectively reducing ventilator-associated lung injury.

[0048] Some related technologies use a proportional model of scaled-up alveoli to study the damage caused by mechanical ventilation to alveoli. However, the proportional model cannot truly reflect the physiological state of alveoli in the human body, so it is difficult to accurately restore the damage suffered by alveoli.

[0049] In some related technologies, a lung organ model is constructed based on an organ chip. However, the deformation of the cell culture membrane is two-dimensional stretching and contraction, and it is difficult to accurately restore the three-dimensional expansion and contraction of alveoli during breathing.

[0050] In some related technologies, the deformation of the culture membrane is three-dimensional expansion and contraction, thereby improving the authenticity of the lung organ model.

[0051] In the above related technologies, when introducing the culture medium into the organ-on-a-chip and when introducing air into the organ-on-a-chip to simulate the gas environment of alveolar respiration, the same perfusion channel is often used. Since it is generally necessary to avoid the impact of the culture medium on the culture membrane and the damage to cells caused by the bubbles generated during the culture process when introducing the culture medium, the perfusion channel is often designed not to face the culture membrane (specifically, not to face the deformable area of the culture membrane) and is conical.

[0052] When simulating natural human breathing, the operating principle of the organ-on-a-chip is that air passively flows into or out of the perfusion channel through the deformation of the culture membrane. At this time, the orientation of the perfusion channel basically does not affect the authenticity of the simulation; however, when simulating mechanical ventilation, the operating principle of the organ-on-a-chip is to actively inject positive-pressure air through the perfusion channel, thereby causing the deformation of the culture membrane. At this time, if the positive-pressure gas cannot impact the culture membrane, the damage to the alveoli in the mechanical ventilation scenario cannot be realistically simulated.

[0053] Refer to Figure 1 , the organ-on-a-chip provided by the embodiment of the first aspect of the present application includes a culture membrane 210 and an actuator membrane 220. The organ-on-a-chip further forms a first culture chamber 110, a second culture chamber 120, an actuator chamber 130, and a perfusion channel 140. The culture membrane 210 separates the first culture chamber 110 and the second culture chamber 120. The perfusion channel 140 communicates with the first culture chamber 110, and the communication port of the perfusion channel 140 with the first culture chamber 110 faces the culture membrane 210, especially directly facing the culture membrane 210. The perfusion channel 140 is used to connect to an external gas source. The actuator membrane 220 separates the second culture chamber 120 and the actuator chamber 130. The actuator membrane 220 can deform in response to the pressure change in the actuator chamber 130 to conduct the pressure change between the actuator chamber 130 and the second culture chamber 120, thereby causing the deformation of the culture membrane 210.

[0054] Refer to Figure 2 , the present application also provides a lung organ model based on the organ-on-a-chip of the present application. The lung organ model is obtained by a lung organ model construction method. In the lung organ model, the culture membrane 210 is located on one side of the first culture chamber 110 for culturing epithelial cells, and the culture membrane 210 is located on one side of the second culture chamber 120 for culturing vascular endothelial cells. In other words, the lung organ model further includes an epithelial cell layer and a vascular endothelial cell layer. The epithelial cell layer grows on the side of the culture membrane 210 located in the first culture chamber 110, and the vascular endothelial cell layer grows on the side of the culture membrane 210 located in the second culture chamber 120.

[0055] The organ-on-a-chip can construct a controllable minimum functional unit that mimics the physiological state of the human body at the micron scale, thus more truly and accurately simulating cell activities. Generally speaking, the average diameter of the alveoli is about 0.2 mm. Therefore, the diameter of the culture membrane 210 exposed in the first culture chamber 110 can also be set at about 0.2 mm to be closer to the actual alveolar size. Combining Figure 1 and Figure 2 , it is defined that the culture membrane 210 longitudinally separates the first culture chamber 110 and the second culture chamber 120. When the perfusion channel 140 is not ventilated and the actuation chamber 130 is at normal pressure, the culture membrane 210 is hardly affected by the acting force longitudinally and is in a contracted state; while when the perfusion channel 140 ventilates the first culture chamber 110 and / or the actuation chamber 130 is under negative pressure, the culture membrane 210 is affected by the acting force longitudinally and expands toward the second culture chamber 120 side, being in an expanded state.

[0056] Exemplarily, through a profilometer, Figure 3 the deformation of the culture membrane 210 in a certain embodiment of the organ-on-a-chip was detected and characterized. Figure 3 Part (a) of Figure 3 reflects the profile of the culture membrane 210 in the contracted state. It can be observed from the figure that the undulation degree of the culture membrane 210 longitudinally can be ignored.

[0057] Exemplarily, Figure 4 shows the observation results of the culture membrane 210 under a microscope in a certain embodiment of the lung organ model. Figure 4 Part (a) of Figure 4 reflects the profile of the culture membrane 210 in the contracted state. It can be seen that cells grow on the surface of the culture membrane 210;

[0058] When used to simulate the alveoli, the contracted state of the organ-on-a-chip can simulate the contracted state of the alveoli during exhalation, and the expanded state of the organ-on-a-chip can simulate the expanded state of the alveoli during inhalation. The organ-on-a-chip causes three-dimensional expansion and contraction of the culture membrane 210 through the periodic pressure change in the actuation chamber 130, thus being able to more truly simulate the respiratory movement process of the alveoli during human active breathing; and, the organ-on-a-chip blows gas towards the culture membrane 210 into the first culture chamber 110 through the perfusion channel 140, causing three-dimensional deformation of the culture membrane 210, thus being able to simulate the multi-directional shear stress generated by mechanical ventilation of the ventilator on cells.

[0059] In addition, referring to Figure 2, by making the communication port of the perfusion channel 140 with the first culture chamber 110 face the culture membrane 210, the gas introduced into the perfusion channel 140 can impact the culture membrane 210, more realistically simulating the damage to the alveoli during mechanical ventilation. In particular, referring to Figure 1 , when the communication port of the perfusion channel 140 with the first culture chamber 110 is directly opposite the culture membrane 210, the gas can generate an impact force perpendicular to the surface of the culture membrane 210 in the static state, and simulate the mechanical ventilation effect when the culture membrane 210 undergoes three-dimensional deformation during the respiratory movement, making the impact effect more ideal.

[0060] Therefore, the organ chip is conducive to meeting the research needs of ventilator-associated lung injury.

[0061] The lung organ model based on the organ chip correspondingly also has the beneficial effects brought by the organ chip, which will not be elaborated here.

[0062] It can be understood that according to whether the actuation chamber 130 generates negative pressure, the lung organ model can simulate different ventilator-associated lung injuries. When the pressure of the actuation chamber 130 is periodically changed, the lung organ model simulates the lung injury caused by mechanical ventilation in the scenario where the patient has spontaneous breathing; when the pressure of the actuation chamber 130 is constant, the lung organ model simulates the lung injury caused by mechanical ventilation in the scenario where the patient has no spontaneous breathing.

[0063] In addition to conducting research on ventilator-associated lung injury, the organ chip can also be used for other research work. The perfusion channel 140 can be connected to a gas control system to control the composition of the gas introduced through the perfusion channel 140, so as to study the impact of the environment on the alveoli, such as controlling parameters such as the oxygen content, humidity, carbon dioxide concentration of the gas, introducing gases containing irritating gas components (such as cigarette smoke, chemical irritant gases, poisonous gases, etc.), introducing gases containing pseudovirus particles, etc., so as to simulate different test environments.

[0064] In addition to gases, the perfusion channel 140 can also be used to introduce liquids, such as introducing bronchoalveolar lavage fluid from patients to simulate corresponding disease models.

[0065] In addition to being applied to the lung organ model, the organ chip can also be used to construct other types of organ models. In other words, the two side surfaces of the culture membrane 210 can also be used to culture other types of cells, such as fibroblasts, dendritic cells and other cells. In other usage scenarios, the cells growing on both sides of the culture membrane 210 can be the same or different.

[0066] In the present application, the culture membrane 210 can be a polydimethylsiloxane porous membrane, that is, a PDMS membrane. On the one hand, the PDMS material has good biocompatibility, which facilitates the growth of cells on the surface of the culture membrane 210. On the other hand, the PDMS membrane has high strength, which helps the culture membrane 210 withstand the impact generated when the perfusion channel 140 intakes air, thereby improving the reliability of the organ chip. The polydimethylsiloxane porous membrane allows cells on both sides to exchange gases through the pores of the culture membrane 210, and the scale of the pores is generally in the micron level.

[0067] The actuating membrane 220 can also be a polydimethylsiloxane porous membrane or a polydimethylsiloxane non-porous membrane.

[0068] In the human body, the space between cells is filled with the extracellular matrix. Therefore, it is possible to choose to further coat the surface of the culture membrane 210 with biomacromolecules for simulating the extracellular matrix. This makes the cell growth environment closer to the physiological environment of the human body, helps the organ chip more realistically simulate the alveoli or other human tissues and organs, and also helps the lung organ model more accurately simulate the damage to cells during mechanical ventilation.

[0069] Exemplarily, the biomacromolecules can include collagen, fibronectin, and laminin, where the collagen includes type I collagen or type IV collagen. Of course, in other embodiments, the biomacromolecules can also adopt different designs, such as adding, reducing, or replacing certain components.

[0070] To improve the consistency of different regions on the surface of the culture membrane 210 during expansion and deformation, in the present application, the perfusion channel 140 can be designed to be oriented towards the center of the deformable region of the culture membrane 210. At this time, referring to Figure 2 , the gas introduced from the perfusion channel 140 acts on the center of the deformable region, so as to prevent the intake culture membrane 210 from being eccentric during expansion and deformation.

[0071] The deformable region refers to the region of the culture membrane 210 that is not restricted and has the freedom of deformation. To further improve the consistency of different regions on the surface of the culture membrane 210 during expansion and deformation, the shape of the deformable region can be designed as a circle.

[0072] In the organ chip, the culture membrane 210 is usually bonded to other parts of the organ chip. Therefore, longitudinally, neither side of the deformable region of the culture membrane 210 is bonded to other parts of the organ chip. In other words, in the present application, the overlapping region of the culture membrane 210 exposed to the first culture chamber 110 and the culture membrane 210 exposed to the second culture chamber 120 is the deformable region of the culture membrane 210, and the range of the deformable region is defined by the first culture chamber 110 and / or the second culture chamber 120.

[0073] Exemplarily, referring to Figure 1 , in the embodiment of the first aspect of the present application, the area of the culture membrane 210 exposed to the first culture chamber 110 is smaller than that of the culture membrane 210 exposed to the second culture chamber 120, and the projection of the culture membrane 210 exposed to the first culture chamber 110 in the longitudinal direction falls within the range of the culture membrane 210 exposed to the second culture chamber 120. Therefore, the first culture chamber 110 defines a deformable region of the culture membrane 210.

[0074] It goes without saying that at this time, the shape of the culture membrane 210 exposed to the first culture chamber 110 can be designed as a circular shape. In addition, at this time, the perfusion channel 140 can be coaxially arranged with the first culture chamber 110, so as to achieve that the perfusion channel 140 faces the center of the deformable region of the culture membrane 210.

[0075] In the present application, in order to improve the consistency of the growth environment of different regions on the surface of the culture membrane 210, in the region close to the culture membrane 210, the cross-section of the first culture chamber 110 can be designed to gradually increase from the side close to the culture membrane 210 to the side far from the culture membrane 210. In other words, this makes the angle α between the side wall of the first culture chamber 110 and the culture membrane 210 less than 90°. On the one hand, this makes it not easy for air bubbles to remain near the side wall (such as air bubbles generated during the manufacture of the organ chip or air bubbles generated by cells during the culture process), avoiding air bubble damage to the cells located at the edge of the deformable region. On the other hand, it also improves the fluidity of the region near the side wall, thus ensuring the consistency of the medium supply in different regions, and further improving the consistency of the growth environment of different regions on the surface of the culture membrane 210.

[0076] Exemplarily, referring to Figure 1 , in the embodiment of the first aspect of the present application, since the deformable region of the culture membrane 210 is defined by the first culture chamber 110, and the side wall of the second culture chamber 120 is far from the edge of the deformable region of the culture membrane 210, therefore, the side wall of the second culture chamber 120 will not theoretically affect the cells located at the edge of the deformable region. The organ chip can be designed such that in the region close to the culture membrane 210, the cross-section of the first culture chamber 110 gradually increases from the side close to the culture membrane 210 to the side far from the culture membrane 210.

[0077] And, referring to Figure 1 , in the embodiment of the first aspect of the present application, the organ chip is also designed such that the volume of the second culture chamber 120 is larger than that of the first culture chamber 110, so that the second culture chamber 120 is used to store the culture medium.

[0078] It is understandable that when simulating the breathing of alveoli or conducting other experiments, it is preferable to seal the second culture chamber 120 so that the second culture chamber 120 can accurately respond to the pressure change in the actuation chamber 130, thereby causing the contraction and expansion of the culture membrane 210. At this time, only the culture medium stored in the second culture chamber 120 can provide energy for the cells. Therefore, designing a relatively large second culture chamber 120 can prevent the stored culture medium from drying up and causing cell death.

[0079] The organ-on-a-chip is fabricated through microfabrication processes such as photolithography. In related technologies, generally, the organ-on-a-chip is disassembled into multiple parts, and corresponding cavities and channels are respectively fabricated on each part, and then combined to form the organ-on-a-chip. According to different disassembly methods, the organ-on-a-chip can have different constitutions.

[0080] Exemplarily, referring to Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 and Figure 9 According to the organ-on-a-chip provided by the embodiment of the second aspect of the present application, the organ-on-a-chip includes a first culture layer 230, a second culture layer 240, and an actuation layer 250. The first culture layer 230, the culture membrane 210, the second culture layer 240, the actuation membrane 220, and the actuation layer 250 are stacked in sequence in the longitudinal direction. The first culture chamber 110 and the perfusion channel 140 are located in the first culture layer 230, the second culture chamber 120 is located in the second culture layer 240, and the actuation chamber 130 is located in the actuation layer 250. The stacked design can facilitate the processing and assembly of the organ-on-a-chip.

[0081] Before using the organ-on-a-chip, it is necessary to first culture cells on the surface of the culture membrane 210, and then continuously supply the culture medium to the first culture chamber 110 and the second culture chamber 120 to provide the nutrients required for cell growth. Therefore, referring to Figure 5 and Figure 6 The organ-on-a-chip also needs to include a first liquid inlet channel 160 and a first liquid outlet channel 170, and the first liquid inlet channel 160 and the first liquid outlet channel 170 are respectively communicated with the first culture chamber 110. The organ-on-a-chip also needs to include a second liquid inlet channel 180 and a second liquid outlet channel 190, and the second liquid inlet channel 180 and the second liquid outlet channel 190 are respectively communicated with the second culture chamber 120.

[0082] During use, solutions containing cells, culture medium, and other required materials (such as biomacromolecules simulating the extracellular matrix) are respectively introduced into both sides of the culture membrane 210 through the first liquid inlet channel 160 and the second liquid inlet channel 180, so that the cells grow on the surface of the culture membrane 210 and form good tight junctions between the cells, thereby simulating the state of cells in the body. The waste liquid is discharged through the first liquid outlet channel 170 and the second liquid outlet channel 190.

[0083] The pressure in the actuation chamber 130 can be changed by supplying a pressure medium to the actuation chamber 130 or by changing the volume of the actuation chamber 130. Exemplarily, referring to Figure 6 、 Figure 7 、 Figure 8 and Figure 9 , in an embodiment of the second aspect, the organ-on-a-chip is formed with a pressure channel 150 that communicates with the actuation chamber 130, and the pressure channel 150 is used to connect to an external pressure source to supply a pressure medium to the actuation chamber 130.

[0084] Referring to Figure 6 and Figure 7 , in an embodiment of the second aspect, longitudinally corresponds to the up-and-down direction. For the convenience of placing and connecting the organ-on-a-chip, the perfusion channel 140, the pressure channel 150, the first liquid inlet channel 160, the second liquid inlet channel 180, the second liquid inlet channel 180, and the second liquid outlet channel 190, etc. can be designed to penetrate out from the top surface of the organ-on-a-chip, so that the side surface of the organ-on-a-chip can be conveniently grasped, while the bottom surface can be stably placed on the workbench.

[0085] Specifically, referring to Figure 6 、 Figure 7 、 Figure 8 and Figure 9 , the pressure channel 150 extends longitudinally from the actuation layer 250 to the first culture layer 230, and the pressure channel 150 penetrates out of the first culture layer 230 to form an interface connecting to the outside.

[0086] The perfusion channel 140 also extends longitudinally and penetrates out of the first culture layer 230 to form an interface connecting to the outside. Exemplarily, referring to Figure 6 , the perfusion channel 140 can be further divided into an inlet section 141, a connection section 142, and an outlet section 143. The inlet section 141 penetrates out of the first culture layer 230 longitudinally to form an interface connecting to the outside; the outlet section 143 communicates with the first culture chamber 110 and also extends longitudinally to face the culture membrane; the connection section 142 extends in a direction perpendicular to the longitudinal direction to connect the inlet section 141 and the outlet section 143 located at different positions, so as to flexibly adjust the outlet position of the perfusion channel 140 and avoid interference. The connection section 142 can not only extend in a straight line as shown in Figure 6 , but also be designed to extend along a curve or other forms of routes according to the need for avoidance, which will not be elaborated here.

[0087] The first liquid inlet channel 160 and the first liquid outlet channel 170 are located in the first culture layer 230. The first liquid inlet channel 160 and the first liquid outlet channel 170 extend longitudinally and penetrate through the first culture layer 230 to form interfaces connecting to the outside. The second liquid inlet channel 180 and the second liquid outlet channel 190 extend longitudinally from the second culture layer 240 towards the first culture layer 230, and the second liquid inlet channel 180 and the second liquid outlet channel 190 penetrate through the first culture layer 230 to form interfaces connecting to the outside.

[0088] In an embodiment of the second aspect, in the region close to the culture membrane 210, the first culture chamber 110 is designed with a cross-section gradually increasing from the side close to the culture membrane 210 to the side far from the culture membrane 210 to improve the cell culture effect. For the convenience of processing the first culture chamber 110, referring to Figure 8 , in an embodiment of the second aspect, the first culture layer 230 may include a first sheet 231, a second sheet 232, and a third sheet 233 stacked in sequence; the shape of the part of the first culture chamber 110 located in the third sheet 233 is frustum-shaped, and the cross-section of the frustum gradually increases from the side close to the culture membrane 210 to the side far from the culture membrane 210.

[0089] The lung organ model is obtained by constructing through a lung organ model construction method. The lung organ model construction method includes providing an organ chip; inoculating and culturing epithelial cells on the surface of the culture membrane 210 located in the first culture chamber 110, and inoculating and culturing vascular endothelial cells on the surface of the culture membrane 210 located in the second culture chamber 120.

[0090] Inoculating and culturing epithelial cells means allowing epithelial cells to grow on the surface of the culture membrane 210 and form a tightly connected structure (i.e., an epithelial cell layer). Inoculating and culturing epithelial cells specifically includes the following steps: Place the first culture chamber 110 upward, and introduce a solution carrying epithelial cells into the first culture chamber 110; Let the organ chip stand in the cell culture incubator so that the epithelial cells settle and adhere to the surface of the culture membrane 210; Provide a flowing cell culture medium to the first culture chamber 110.

[0091] Inoculating and culturing vascular endothelial cells means allowing vascular endothelial cells to grow on the surface of the culture membrane 210 and form a tightly connected structure (i.e., a vascular endothelial cell layer). Inoculating and culturing vascular endothelial cells specifically includes the following steps: Place the second culture chamber 120 upward, and introduce a solution carrying vascular endothelial cells into the second culture chamber 120; Let the organ chip stand in the cell culture incubator so that the vascular endothelial cells settle and adhere to the surface of the culture membrane 210; Provide a flowing cell culture medium to the second culture chamber 120.

[0092] Furthermore, the method for constructing a lung organ model further includes coating the surface of the culture membrane 210 with biomacromolecules for simulating the extracellular matrix. The coating of biomacromolecules occurs before inoculating epithelial cells and vascular endothelial cells. Coating the biomacromolecules enables the organ chip to more realistically simulate the physiological environment of the human body, and also helps the growth of cells on the surface of the culture membrane 210.

[0093] Coating the surface of the culture membrane 210 with biomacromolecules specifically includes the following steps: Introduce a composite solution composed of collagen, fibronectin, and laminin into the first culture chamber 110 and the second culture chamber 120. The collagen is type I collagen or type IV collagen; Let the organ chip stand still and wait for the solution to react.

[0094] This application also provides a method for simulating lung injury using the lung organ model and the lung organ chip of this application. The method for simulating lung injury includes introducing air into the first culture chamber 110 through the perfusion channel 140 to simulate the mechanical ventilation of the alveoli by a ventilator.

[0095] On this basis, the method for simulating lung injury may further include periodically changing the pressure in the actuator chamber 130 to simulate the lung injury caused by mechanical ventilation in the scenario where the patient has spontaneous breathing; or keeping the pressure in the actuator chamber 130 constant to simulate the lung injury caused by mechanical ventilation in the scenario where the patient has no spontaneous breathing.

[0096] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples" means 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 this application. In this specification, the schematic expressions 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 a suitable manner in any one or more embodiments or examples.

[0097] In some alternative embodiments, the functions / operations recited in the block diagrams may not occur in the order presented in the operational illustrations. For example, depending on the functions / operations involved, two blocks shown in succession may actually be executed substantially simultaneously or the blocks may sometimes be executed in the reverse order. Further, the embodiments presented and described in the flowcharts of the present application are provided by way of example in order to provide a more thorough understanding of the technology. The disclosed methods are not limited to the operations and logical flows presented herein. Alternative embodiments are contemplated in which the order of various operations is altered and in which sub-operations described as part of a larger operation are executed independently.

[0098] Although the embodiments of the present application have been shown and described, those of ordinary skill in the art will understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. An organ chip, characterized in that, The organ-on-a-chip is formed with a first culture chamber, a second culture chamber, an actuation chamber, and a perfusion channel. The organ-on-a-chip includes: A culture membrane that separates the first culture chamber and the second culture chamber. The perfusion channel communicates with the first culture chamber, and the communication port of the perfusion channel with the first culture chamber faces the culture membrane, preferably directly facing the culture membrane. The perfusion channel is used to connect to an external gas source. An actuation membrane that separates the second culture chamber and the actuation chamber. The actuation membrane can deform in response to a pressure change in the actuation chamber to conduct the pressure change between the actuation chamber and the second culture chamber, thereby causing the culture membrane to deform.

2. The organ chip according to claim 1, characterized in that, The culture membrane is made of a polydimethylsiloxane porous membrane.

3. The organ chip according to claim 1, characterized in that, The surface of the culture membrane is coated with biomacromolecules for simulating the extracellular matrix.

4. The organ chip according to claim 3, characterized in that, The biomacromolecules include collagen, fibronectin, and laminin, and the collagen includes type I collagen or type IV collagen.

5. The organ chip according to any one of claims 1 to 4, characterized in that, The culture membrane is located on one side of the first culture chamber for culturing epithelial cells, and the culture membrane is located on one side of the second culture chamber for culturing vascular endothelial cells.

6. The organ chip according to claim 1, characterized in that, The perfusion channel faces the center of the deformable region of the culture membrane.

7. The organ chip according to claim 6, characterized in that, The first culture chamber defines the deformable region of the culture membrane, and the shape of the culture membrane exposed to the first culture chamber is a circular shape.

8. The organ chip according to claim 7, characterized in that, The perfusion channel is coaxially arranged with the first culture chamber.

9. The organ chip according to claim 1 or 7, characterized in that, In the region near the culture membrane, the cross-section of the first culture chamber gradually increases from the side close to the culture membrane to the side far from the culture membrane.

10. The organ chip according to claim 1, characterized in that, The volume of the second culture chamber is larger than the volume of the first culture chamber, and the second culture chamber is used to store the culture medium.

11. The organ chip according to claim 10, characterized in that, The surface area of the culture membrane exposed to the second culture chamber is larger than the surface area of the culture membrane exposed to the first culture chamber.

12. The organ chip according to claim 1, characterized in that, The actuation membrane is made of a polydimethylsiloxane porous membrane.

13. The organ chip according to claim 1, characterized in that, The organ-on-a-chip includes a first culture layer, a second culture layer, and an actuation layer. The first culture layer, the culture membrane, the second culture layer, the actuation membrane, and the actuation layer are stacked in sequence longitudinally. The first culture chamber and the perfusion channel are located in the first culture layer, the second culture chamber is located in the second culture layer, and the actuation chamber is located in the actuation layer.

14. The organ chip according to claim 13, characterized in that, The organ-on-a-chip is formed with a pressure channel that communicates with the actuation chamber, and the pressure channel is used to connect to an external pressure source.

15. The organ chip according to claim 14, characterized in that, The pressure channel extends longitudinally from the actuation layer to the first culture layer, and the pressure channel penetrates through the first culture layer to form an interface connecting to the outside.

16. The organ chip according to claim 13, characterized in that, The perfusion channel extends longitudinally and penetrates through the first culture layer to form an interface connecting to the outside.

17. The organ chip according to claim 13, characterized in that, The organ-on-a-chip includes a first liquid inlet channel and a first liquid outlet channel, and the first liquid inlet channel and the first liquid outlet channel communicate with the first culture chamber respectively.

18. The organ chip according to claim 17, characterized in that, The first liquid inlet channel and the first liquid outlet channel are located in the first culture layer, and the first liquid inlet channel and the first liquid outlet channel extend longitudinally and penetrate through the first culture layer to form interfaces connecting to the outside.

19. The organ chip according to claim 13, characterized in that, The organ-on-a-chip includes a second liquid inlet channel and a second liquid outlet channel, and the second liquid inlet channel and the second liquid outlet channel are respectively communicated with the second culture chamber.

20. The organ-on-a-chip according to claim 19, wherein, The second liquid inlet channel and the second liquid outlet channel extend from the second culture layer to the first culture layer along the longitudinal direction, and the second liquid inlet channel and the second liquid outlet channel penetrate through the first culture layer to form an interface connecting to the outside.

21. The organ-on-a-chip according to claim 13, wherein, The first culture layer includes a first sheet, a second sheet, and a third sheet stacked in sequence.

22. The organ-on-a-chip according to claim 21, wherein, The shape of the part of the first culture chamber located in the third sheet is frustum-shaped, and the cross-section of the frustum gradually increases from the side close to the culture membrane to the side far from the culture membrane.

23. A method for constructing a lung organ model, wherein, The method for constructing the lung organ model includes the following steps: Providing the organ-on-a-chip according to any one of claims 1 to 22; Inoculating and culturing epithelial cells on the surface of the culture membrane located in the first culture chamber; Inoculating and culturing vascular endothelial cells on the surface of the culture membrane located in the second culture chamber.

24. The method for constructing a lung organ model according to claim 23, wherein, Inoculating and culturing the epithelial cells includes: Placing the first culture chamber upward, and introducing a solution carrying the epithelial cells into the first culture chamber; Leaving the organ-on-a-chip stationary in a cell culture incubator so that the epithelial cells settle and adhere to the surface of the culture membrane; Providing a flowing cell culture medium to the first culture chamber.

25. The method for constructing a lung organ model according to claim 23, wherein, Inoculating and culturing the vascular endothelial cells includes: Placing the second culture chamber upward, and introducing a solution carrying the vascular endothelial cells into the second culture chamber; Leaving the organ-on-a-chip stationary 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 second culture chamber.

26. The method for constructing a lung organ model according to claim 23, wherein, Before inoculating the epithelial cells and the vascular endothelial cells, the method for constructing the lung organ model further includes: coating a biomacromolecule for simulating the extracellular matrix on the surface of the culture membrane.

27. The method for constructing a lung organ model according to claim 26, wherein, Coating the biomacromolecule on the surface of the culture membrane includes: Introducing a composite solution composed of collagen, fibronectin, and laminin into the first culture chamber and the second culture chamber, and the collagen is type I collagen or type IV collagen; Leaving the organ-on-a-chip stationary and waiting for the solution to react.

28. A lung organ model, wherein, The lung organ model is constructed by using the method for constructing the lung organ model according to any one of claims 23 to 27.

29. A method for simulating lung injury, wherein, The method for simulating lung injury uses the lung organ model according to claim 28 and the organ-on-a-chip according to any one of claims 1 to 22. The method for simulating lung injury includes introducing air into the first culture chamber through a perfusion channel to simulate mechanical ventilation of the alveoli by a ventilator.

30. The method for simulating lung injury according to claim 29, wherein, The method for simulating lung injury further includes periodically changing the pressure of the actuation chamber to simulate lung injury caused by mechanical ventilation in a scenario where the patient has spontaneous breathing; or keeping the pressure of the actuation chamber constant to simulate lung injury caused by mechanical ventilation in a scenario where the patient has no spontaneous breathing.

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