Organ chip and lung organ model

Abstract

The organ chip comprises a culture membrane and an actuating membrane, a first culture chamber, a second culture chamber, an actuating chamber and a perfusion channel are further formed in the organ chip, the culture membrane divides 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] The present application relates to the field of organ chips, in particular to organ chips and lung organ models. Background Art

[0002] Relevant studies have shown that during the treatment of critically ill patients, lung injury caused by mechanical ventilation using a ventilator is mainly caused by excessive expansion of the alveoli, damage to the alveolar epithelium and vascular endothelium caused by shear force, and inflammatory response. However, the dynamic damage and repair mechanism of the alveoli in the human body at the cellular level has not been fully clarified. The clinical diagnosis of lung injury caused by this type of ventilator is usually limited to oxygenation index and imaging features. There is a lack of biomarker-based diagnosis and treatment methods and clinical treatment strategies that effectively reduce 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 "visual window" for the early diagnosis of ventilator-associated lung injury is a difficult problem that needs to be solved in clinical medicine.

[0003] Organ chips are highly simulated microsystems of in vitro organs built with microfluidic chips as carriers, which can reconstruct the real dynamic microenvironment of the human body in vitro. Compared with two-dimensional cell culture, organoids and animal models, organ chips have huge potential advantages in in vitro disease model simulation, drug screening and precision medicine.

[0004] In 2010, Ingber et al. constructed the human alveolar-pulmonary microvascular interface using organ chips, and proposed for the first time a lung organ model that simulates alveolar circulation and breathing movements. The chip structure contains two parallel microchannels, separated by a layer of permeable and stretchable polydimethylsiloxane (PDMS) porous membrane covered with extracellular matrix; 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 pulmonary 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-pulmonary microvascular air-blood barrier; the chambers on both sides of the channel are vacuumed to stretch and contract the bionic alveolar air-blood barrier membrane in the middle, simulating physiological circulation and breathing movements.

[0005] However, the two-dimensional single-direction mechanical cyclic stretching in such lung organ models is still different from the expansion and contraction of the air-blood barrier during in vivo breathing, making it difficult to simulate the real three-dimensional respiratory movement of alveoli. To this end, a lung organ model based on three-dimensional deformation is further proposed in the related art. The chip structure includes two upper and lower culture chambers, and a polydimethylsiloxane porous membrane separating the two culture chambers. The pressure difference between the two culture chambers triggers the three-dimensional expansion or contraction of the polydimethylsiloxane porous membrane.

[0006] In related technologies, lung organ models are usually used to simulate the physiological environment of alveoli during natural breathing of the human body. The same channel is often used when culture fluid is introduced into the organ chip and when air is introduced into the organ chip to simulate alveolar breathing. The channel is not facing the culture membrane, which results in the lung organ model being unable to realistically simulate the damage to the alveoli in a mechanical ventilation scenario.

[0007] Moreover, in the process of introducing culture medium into the culture chamber, bubbles are likely to remain on the wall of the culture chamber. The bubbles in contact with the cells in the culture chamber will hinder the cells from obtaining the culture medium, causing these cells to grow poorly or even damage the cells, thereby 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. To this end, this application proposes an organ chip and a lung organ model, which can more realistically simulate the respiratory movement process of the alveoli and the multi-directional shear stress generated by gas on cells, which is conducive to meeting the research needs of ventilator-related lung injury.

[0009] According to the organ chip provided in the present application, a first culture chamber, a second culture chamber, an actuation chamber and a perfusion channel are formed. The organ chip includes a culture membrane and an actuation membrane. The culture membrane separates the first culture chamber and the second culture chamber. The perfusion channel is connected to the first culture chamber, and the connecting port of the perfusion channel and the first culture chamber faces the culture membrane, preferably facing the culture membrane. The perfusion channel is used to connect to an external gas source. The actuation membrane separates the second culture chamber and the actuation chamber. The actuation membrane can deform in response to pressure changes in the actuation chamber to transmit pressure changes between the actuation chamber and the second culture chamber, thereby causing the culture membrane to deform.

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

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

[0012] According to some embodiments of the present application, the surface of the culture membrane is coated with biological macromolecules 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 is oriented toward the center of the deformable region of the culture membrane.

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

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

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

[0019] According to some embodiments of the present application, the volume of the second culture chamber is greater than that of the first culture chamber, and the second culture chamber is used to store culture medium.

[0020] According to some embodiments of the present application, a surface area of ​​the culturing membrane exposed to the second culturing chamber is greater than a surface area of ​​the culturing membrane exposed to the first culturing chamber.

[0021] According to some embodiments of the present application, the actuating membrane is 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 sequentially in the longitudinal direction, 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 is communicated with the actuation chamber, and the pressure channel 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 passes through the first culture layer to form an interface connected to the outside.

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

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

[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 pass through the first culture layer to form an interface connected to the outside.

[0028] According to some embodiments of the present application, the organ chip comprises 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 connected to the second culture chamber.

[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 pass through the first culture layer to form an interface connected to the outside.

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

[0031] According to some embodiments of the present application, the shape of the portion of the first culture chamber located at the third sheet is a truncated cone, and the cross-section of the truncated cone gradually increases from a side close to the culture membrane to a side away from the culture membrane.

[0032] The lung organ model provided in the present application includes the organ chip provided in the present application.

[0033] The lung organ model provided in the present application includes the organ chip provided in the present application, so the lung organ model correspondingly possesses the beneficial effects provided by the organ chip, which will not be repeated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0035] Figure 1 is a schematic diagram of the principle of the organ chip of the embodiment of the present application;

[0036] Figure 2 is a schematic diagram of the principle of the lung organ model of an embodiment of the present application;

[0037] Figure 3 Schematic diagram of the deformation of the culture membrane of the organ chip of the embodiment of the present application under the profilometer characterization, wherein part (a) corresponds to the contracted state of the culture membrane, and part (b) corresponds to the expanded state of the culture membrane;

[0038] Figure 4 Schematic diagram of the structure of the culture membrane of the lung organ model of the embodiment of the present application observed under a microscope, wherein part (a) corresponds to the contracted state of the culture membrane, and cells can be seen growing on the culture membrane, and part (b) corresponds to the expanded state of the culture membrane;

[0039] Figure 5 is a schematic diagram of the top view of the organ chip according to an embodiment of the present application;

[0040] Figure 6 is a schematic cross-sectional structural diagram of an organ chip according to an embodiment of the present application;

[0041] Figure 7 is a schematic cross-sectional structural diagram of an organ chip according to an embodiment of the present application;

[0042] Figure 8 is a schematic diagram of the exploded structure of the organ chip according to an embodiment of the present application;

[0043] Fig. 9 It is a schematic diagram of the exploded structure of the organ chip according to an embodiment of the present application.

[0044] Reference numerals:

[0045] 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,

[0046] 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 , and the actuation layer 250 . DETAILED DESCRIPTION

[0047] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0048] In the description of the present application, it should be understood that descriptions involving orientation, such as up, down, front, back, left, right, etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0049] In the description of this application, "several" means one or more, "more" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0050] In the description of this application, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in this application based on the specific content of the technical solution.

[0051] The dynamic injury and repair mechanism of alveoli in the human body at the cellular level has not yet been fully understood. Lung injury caused by mechanical ventilation is usually limited to oxygenation index and imaging features in clinical diagnosis. There is a lack of biomarker-based diagnostic and treatment methods and clinical treatment strategies that effectively reduce ventilator-associated lung injury.

[0052] Some related technologies use scale models of enlarged alveoli to study the damage caused by mechanical ventilation to the alveoli, but the scale model cannot truly reflect the physiological state of the alveoli in the human body, so it is difficult to accurately restore the damage to the alveoli.

[0053] Some related technologies build lung organ models based on organ chips, but the deformation of the cell culture membrane is two-dimensional stretching and contraction, which makes it difficult to accurately restore the three-dimensional expansion and contraction of the alveoli during breathing.

[0054] In some related technologies, the deformation of the culture membrane is three-dimensional expansion and contraction, thereby improving the realism of the lung organ model.

[0055] In the above-mentioned related technologies, the same perfusion channel is often used when introducing culture fluid into the organ chip and when introducing air into the organ chip to simulate the gas environment of alveolar breathing. When introducing culture fluid, it is generally necessary to avoid the culture fluid from impacting the culture membrane and to avoid damage to the cells caused by bubbles generated during the culture process. Therefore, the perfusion channel is often designed to be not facing the culture membrane (specifically, not facing the deformable area of ​​the culture membrane) and to be conical.

[0056] When simulating natural human breathing, the operating principle of the organ chip is to allow air to passively flow in or out of the perfusion channel through the deformation of the culture membrane. At this time, the direction of the perfusion channel basically does not affect the authenticity of the simulation; but when simulating mechanical ventilation, the operating principle of the organ chip is to actively inject positive pressure air through the perfusion channel, thereby causing the culture membrane to deform. 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.

[0057] Reference Figure 1 According to the embodiment of the first aspect of the present application, the organ chip provided includes a culture membrane 210 and an actuating membrane 220. The organ chip is also formed with a first culture chamber 110, a second culture chamber 120, an actuating 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 is connected to the first culture chamber 110, and the connecting port of the perfusion channel 140 and the first culture chamber 110 faces the culture membrane 210, especially facing the culture membrane 210. The perfusion channel 140 is used to connect to an external gas source. The actuating membrane 220 separates the second culture chamber 120 and the actuating chamber 130. The actuating membrane 220 can deform in response to the pressure change of the actuating chamber 130 to transmit the pressure change between the actuating chamber 130 and the second culture chamber 120, thereby causing the culture membrane 210 to deform.

[0058] Reference Figure 2 The present application also provides a lung organ model based on the organ chip of the present application, and the lung organ model is constructed 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 also 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 on the first culture chamber 110, and the vascular endothelial cell layer grows on the side of the culture membrane 210 located on the second culture chamber 120.

[0059] Organ chips can construct controllable minimum functional units that simulate the physiological state of the human body at the micrometer scale, thereby simulating cell activities more realistically and accurately. Generally speaking, the average diameter of alveoli is about 0.2 mm, so the diameter of the culture membrane 210 exposed in the first culture chamber 110 can also be set to about 0.2 mm to be closer to the actual alveolar size. Figure 1 and Figure 2, it is defined that the culture membrane 210 separates the first culture chamber 110 and the second culture chamber 120 in the longitudinal direction. When the perfusion channel 140 is not ventilated and the actuation chamber 130 is at normal pressure, the culture membrane 210 is almost not affected by any force in the longitudinal direction and is in a contracted state; when the perfusion channel 140 is ventilated to the first culture chamber 110 and / or the actuation chamber 130 is at negative pressure, the culture membrane 210 is affected by a force in the longitudinal direction and expands toward the second culture chamber 120, and is in an expanded state.

[0060] For example, by a profilometer, Figure 3 The deformation of the culture membrane 210 in one embodiment of the Organ Chip was detected and characterized. Figure 3 Part (a) reflects the outline of the culture membrane 210 in the contracted state. It can be observed from the figure that the undulation of the culture membrane 210 in the longitudinal direction is negligible. Figure 3 Part (b) reflects the outline of the culture membrane 210 in the expanded state. It can be observed from the figure that the culture membrane 210 has obvious expansion deformation in the longitudinal direction, thereby simulating the three-dimensional expansion of the alveoli during inhalation.

[0061] For example, Figure 4 The results of observing the culture membrane 210 under a microscope in a certain embodiment of the lung organ model are shown. Figure 4 Part (a) reflects the outline of the culture membrane 210 in the contracted state, and it can be seen that cells grow on the surface of the culture membrane 210; Figure 4 Part (b) reflects the outline of the culture membrane 210 in the expanded state, and it can be seen that the culture membrane 210 is uniformly expanded.

[0062] When used to simulate alveoli, the contraction state of the organ chip can simulate the contraction state of the alveoli during exhalation, and the expansion state of the organ chip can simulate the expansion state of the alveoli during inhalation. The organ chip causes the three-dimensional expansion and contraction of the culture membrane 210 through the periodic pressure changes of the actuation chamber 130, so as to more realistically simulate the respiratory movement process of the alveoli during active breathing of the human body; and the organ chip blows gas toward the culture membrane 210 into the first culture chamber 110 through the perfusion channel 140, so that the culture membrane 210 produces a three-dimensional deformation, thereby simulating the multi-directional shear stress generated by mechanical ventilation of the ventilator on the cells.

[0063] In addition, refer to Figure 2 By making the communication port between the perfusion channel 140 and 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, especially referring to Figure 1When the connecting port of the perfusion channel 140 and the first culture chamber 110 is facing the culture membrane 210, the gas can generate an impact force perpendicular to the surface of the culture membrane 210 in a static state, as well as a mechanical ventilation effect when simulating the three-dimensional deformation of the culture membrane 210 during respiratory movement, making the impact effect more ideal.

[0064] Therefore, organ chips are conducive to meeting the research needs of ventilator-associated lung injury.

[0065] The lung organ model based on the organ chip also has the beneficial effects brought by the organ chip, which will not be elaborated here.

[0066] It can be understood that, depending on whether the actuation chamber 130 generates negative pressure, the lung organ model can simulate different ventilator-related lung injuries. When the pressure of the actuation chamber 130 is periodically changed, the lung organ model simulates the lung damage caused by mechanical ventilation in a patient's spontaneous breathing scenario; when the pressure of the actuation chamber 130 is constant, the lung organ model simulates the lung damage caused by mechanical ventilation in a patient's non-spontaneous breathing scenario.

[0067] In addition to studying 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 to study the impact of the environment on the alveoli, such as controlling the oxygen content, humidity, carbon dioxide concentration and other parameters of the gas, introducing gas containing irritating gas components (such as cigarette smoke, chemical irritant gas, poison gas, etc.), introducing gas containing pseudovirus particles, etc., so as to simulate different test environments.

[0068] In addition to gas, the perfusion channel 140 can also be used to introduce liquid, such as alveolar lavage fluid from a patient, to simulate a corresponding disease model.

[0069] In addition to being used in lung organ models, organ chips can also be used to construct other types of organ models. In other words, the surfaces on both sides 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 grown on both sides of the culture membrane 210 can be the same or different.

[0070] 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 and can facilitate 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 to withstand the impact generated when the perfusion channel 140 is inflated, 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 size of the pores is generally in the micrometer level.

[0071] The actuating membrane 220 may also be a polydimethylsiloxane porous membrane or a polydimethylsiloxane non-porous membrane.

[0072] In the human body, cells are filled with extracellular matrix, so the surface of the culture membrane 210 can be further coated with biomacromolecules used to simulate the extracellular matrix. This makes the cell growth environment closer to the physiological environment of the human body, helps the organ chip to simulate alveoli or other human tissues and organs more realistically, and also helps the lung organ model to more accurately simulate the damage to cells during mechanical ventilation.

[0073] Exemplarily, the biomacromolecule may include collagen, fibronectin and laminin, wherein the collagen includes type I collagen or type IV collagen. Of course, in other embodiments, the biomacromolecule may also be designed differently, such as by adding, reducing or replacing certain components.

[0074] In order to improve the consistency of different areas 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 toward the center of the deformable area of ​​the culture membrane 210. Figure 2 The gas introduced from the perfusion channel 140 acts on the center of the deformable area to prevent the air-intake culture membrane 210 from being eccentric when expanding and deforming.

[0075] The deformable region refers to an area in the culture membrane 210 that is not constrained and has the freedom to deform. In order to further improve the consistency of different areas on the surface of the culture membrane 210 during expansion and deformation, the shape of the deformable region can be designed to be circular.

[0076] In the organ chip, the culture membrane 210 is usually bonded to other parts of the organ chip, so in the longitudinal direction, the deformable region of the culture membrane 210 is not bonded to other parts of the organ chip on both sides. 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.

[0077] For example, refer to Figure 1 In an 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 the area of ​​the culture membrane 210 exposed to the second culture chamber 120, and the longitudinal projection of the culture membrane 210 exposed to the first culture chamber 110 falls within the range of the culture membrane 210 exposed to the second culture chamber 120, so the first culture chamber 110 limits the deformable area of ​​the culture membrane 210.

[0078] It goes without saying that the shape of the culture membrane 210 exposed to the first culture chamber 110 can be designed to be circular. In addition, the perfusion channel 140 can be coaxially arranged with the first culture chamber 110 so that the perfusion channel 140 is oriented toward the center of the deformable area of ​​the culture membrane 210.

[0079] In the present application, in order to improve the consistency of the growth environment in different areas on the surface of the culture membrane 210, in the area 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 away 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 difficult for bubbles to remain near the side wall (such as bubbles generated when manufacturing organ chips or bubbles generated by cells during the culture process), avoiding bubbles from damaging cells at the edge of the deformable area. On the other hand, it also improves the fluidity of the area near the side wall, thereby ensuring the consistency of the culture medium supply in different areas, thereby improving the consistency of the growth environment in different areas on the surface of the culture membrane 210.

[0080] For example, refer 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 away from the edge of the deformable region of the culture membrane 210, the side wall of the second culture chamber 120 will not affect the cells at the edge of the deformable region in theory. 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 away from the culture membrane 210.

[0081] And, refer to Figure 1 In an embodiment of the first aspect of the present application, the organ chip is also designed so that the volume of the second culture chamber 120 is larger than the volume of the first culture chamber 110, so that the second culture chamber 120 is used to store culture medium.

[0082] It is understandable that when simulating alveolar breathing or conducting other experiments, it is preferred to close the second culture chamber 120 so that the second culture chamber 120 can accurately respond to the pressure change of the actuation chamber 130, thereby causing the contraction and expansion of the culture membrane 210. At this time, the cells can only be provided with energy by the culture medium stored in the second culture chamber 120, so the second culture chamber 120 with a larger volume can prevent the stored culture medium from drying up and causing cell death.

[0083] Organ chips are made through micro-machining processes such as photolithography. In related technologies, organ chips are generally split into multiple parts, and corresponding cavities and channels are processed on each part, and then combined to form an organ chip. Depending on the way the parts are split, organ chips can have different structures.

[0084] For example, refer to Figure 5 , Figure 6 , Figure 7 and Figure 8 According to the organ chip provided by the embodiment of the second aspect of the present application, the organ 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 chip.

[0085] Before using the organ chip, cells need to be cultured on the surface of the culture membrane 210, and then the culture medium is continuously delivered to the first culture chamber 110 and the second culture chamber 120 to provide the cells with the nutrients required for growth. Figure 5 and Figure 6 The organ chip also needs to include a first liquid inlet channel 160 and a first liquid outlet channel 170, which are respectively connected to the first culture chamber 110. The organ chip also needs to include a second liquid inlet channel 180 and a second liquid outlet channel 190, which are respectively connected to the second culture chamber 120.

[0086] When in use, a solution containing cells, a culture medium, and other required materials (such as biological macromolecules simulating extracellular matrix) are introduced into both sides of the culture membrane 210 through the first liquid inlet channel 160 and the second liquid inlet channel 180, respectively, so that the cells grow on the surface of the culture membrane 210 and form good tight connections between the cells, thereby simulating the state of the cells in the body. Waste liquid is discharged through the first liquid outlet channel 170 and the second liquid outlet channel 190.

[0087] The pressure of the actuation chamber 130 may be changed by providing a pressure medium to the actuation chamber 130 or by changing the volume of the actuation chamber 130. Figure 6 , Figure 7 and Figure 8 In an embodiment of the second aspect, the organ chip is formed with a pressure channel 150 , which is connected to the actuation chamber 130 . The pressure channel 150 is used to connect to an external pressure source to provide pressure medium to the actuation chamber 130 .

[0088] Reference Figure 6 and Figure 7In the embodiment of the second aspect, the longitudinal direction corresponds to the up-down direction. In order to facilitate the placement and connection of the organ 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, the second liquid outlet channel 190, etc. can be designed to pass through the top surface of the organ chip, so that the side of the organ chip can be easily grasped, and the bottom surface can be stably placed on the workbench.

[0089] Specifically, refer to Figure 6 , Figure 7 and Figure 8 The pressure channel 150 extends longitudinally from the actuation layer 250 to the first culture layer 230 , and the pressure channel 150 passes through the first culture layer 230 to form an interface connected to the outside.

[0090] The perfusion channel 140 also extends longitudinally out of the first culture layer 230 to form an interface connected to the outside. Figure 6 The perfusion channel 140 can be further divided into an inlet section 141, a connecting section 142 and an outlet section 143. The inlet section 141 passes through the first culture layer 230 in the longitudinal direction to form an interface connected to the outside; the outlet section 143 is connected to the first culture chamber 110 and also extends in the longitudinal direction so as to face the culture membrane; the connecting section 142 extends in a direction perpendicular to the longitudinal direction, thereby connecting the inlet section 141 and the outlet section 143 at different positions, so as to flexibly adjust the outlet position of the perfusion channel 140 to avoid interference. The connecting section 142 can not only be as Figure 6 It is shown extending along a straight line, and can also be designed to extend along a curve or other forms of routes according to the need for avoidance, which will not be described in detail here.

[0091] The first liquid inlet channel 160 and the first liquid outlet channel 170 are located in the first culture layer 230, and the first liquid inlet channel 160 and the first liquid outlet channel 170 extend longitudinally and pass through the first culture layer 230 to form an interface connected to the outside. The second liquid inlet channel 180 and the second liquid outlet channel 190 extend longitudinally from the second culture layer 240 to the first culture layer 230, and the second liquid inlet channel 180 and the second liquid outlet channel 190 pass through the first culture layer 230 to form an interface connected to the outside.

[0092] In the embodiment of the second aspect, in the area near the culture membrane 210, the first culture chamber 110 adopts a design in which the cross section gradually increases from the side near the culture membrane 210 to the side away from the culture membrane 210, so as to improve the cell culture effect. Figure 8In 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 portion of the first culture chamber 110 located at the third sheet 233 is a truncated cone, and the cross-section of the truncated cone gradually increases from the side close to the culture membrane 210 to the side away from the culture membrane 210.

[0093] The lung organ model is constructed by a lung organ model construction method, which 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.

[0094] Inoculating and culturing epithelial cells refers to making the epithelial cells grow on the surface of the culture membrane 210 and form a tightly connected structure (that is, an epithelial cell layer). Inoculating and culturing epithelial cells specifically includes the following steps:

[0095] The first culture chamber 110 is placed upward, and a solution carrying epithelial cells is introduced into the first culture chamber 110;

[0096] The organ chip is placed in a cell culture incubator to allow the epithelial cells to settle and adhere to the surface of the culture membrane 210;

[0097] The first culture chamber 110 is provided with flowing cell culture medium.

[0098] Inoculating and culturing vascular endothelial cells refers to making the vascular endothelial cells grow on the surface of the culture membrane 210 and form a tightly connected structure (that is, a vascular endothelial cell layer). Inoculating and culturing vascular endothelial cells specifically includes the following steps:

[0099] The second culture chamber 120 is placed upward, and a solution carrying vascular endothelial cells is introduced into the second culture chamber 120;

[0100] The organ chip is placed in a cell culture incubator to allow the vascular endothelial cells to settle and adhere to the surface of the culture membrane 210;

[0101] The second culture chamber 120 is provided with flowing cell culture medium.

[0102] Furthermore, the lung organ model construction method also includes coating the surface of the culture membrane 210 with biomacromolecules used to simulate the extracellular matrix. The coating of biomacromolecules occurs before the inoculation of epithelial cells and endothelial cells. The coating of biomacromolecules enables the organ chip to more realistically simulate the physiological environment of the human body and also facilitates the growth of cells on the surface of the culture membrane 210.

[0103] The biomacromolecules are coated on the surface of the culture membrane 210, specifically comprising the following steps:

[0104] A composite solution consisting of collagen, fibronectin and laminin is introduced into the first culture chamber 110 and the second culture chamber 120, wherein the collagen is type I collagen or type IV collagen;

[0105] The organ chip was left to stand and the solution was allowed to react.

[0106] The present application also provides a lung injury simulation method using the lung organ model and lung organ chip of the present application, the lung injury simulation method comprising introducing air into the first culture chamber 110 through the perfusion channel 140 to simulate the mechanical ventilation of the alveoli by a ventilator.

[0107] On this basis, the lung injury simulation method may also include periodically changing the pressure of the actuation chamber 130 to simulate the lung injury caused by mechanical ventilation in a patient's spontaneous breathing scenario; or keeping the pressure of the actuation chamber 130 constant to simulate the lung injury caused by mechanical ventilation in a patient's non-spontaneous breathing scenario.

[0108] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0109] In some selectable embodiments, the function / operation mentioned in the block diagram may not occur in the order mentioned in the operation diagram. For example, depending on the function / operation involved, the two boxes shown in succession can actually be executed substantially simultaneously or the boxes can sometimes be executed in reverse order. In addition, the embodiment presented and described in the flow chart of the application is provided by way of example, for the purpose of providing a more comprehensive understanding of technology. The disclosed method is not limited to the operation and logic flow presented herein. Selectable embodiments are expected, wherein the order of various operations is changed and the sub-operation of a part described as a larger operation is performed independently.

[0110] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the claims and their equivalents.

Claims

1. An organ chip, characterized in that: The organ chip is formed with a first culture chamber, a second culture chamber, an actuation chamber and a perfusion channel, and the organ chip includes: a culture membrane, wherein the culture membrane separates the first culture chamber and the second culture chamber, the perfusion channel is connected to the first culture chamber, and a communication port between the perfusion channel and the first culture chamber faces the culture membrane, and the perfusion channel is used to connect to an external gas source; An actuating membrane separates the second culture chamber from the actuating chamber, and the actuating membrane 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, thereby causing the culture membrane to deform.

2. The organ chip according to claim 1, characterized in that: The communication port between the perfusion channel and the first culture chamber faces the culture membrane.

3. The organ chip according to claim 1, characterized in that: The culture membrane is a polydimethylsiloxane porous membrane.

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

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

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

7. The organ chip according to claim 1, characterized in that: The perfusion channel is oriented toward the center of the deformable region of the culturing membrane.

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

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

10. The organ chip according to claim 1 or 8, characterized in that: In the area close to the culture membrane, the cross section of the first culture chamber gradually increases from a side close to the culture membrane to a side away from the culture membrane.

11. The organ chip according to claim 1, characterized in that: The volume of the second culture chamber is greater than that of the first culture chamber, and the second culture chamber is used for storing culture medium.

12. The organ chip according to claim 11, 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.

13. The organ chip according to claim 1, characterized in that: The actuating membrane is a polydimethylsiloxane porous membrane.

14. The organ chip according to claim 1, characterized in that: The organ chip comprises a first culture layer, a second culture layer and an actuation layer, wherein the first culture layer, the culture membrane, the second culture layer, the actuation membrane and the actuation layer are stacked sequentially in the longitudinal direction, 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.

15. The organ chip according to claim 14, characterized in that: The organ chip is formed with a pressure channel, which is communicated with the actuation chamber and is used to connect to an external pressure source.

16. The organ chip according to claim 15, characterized in that: The pressure channel extends from the actuation layer to the first culture layer along the longitudinal direction, and the pressure channel passes through the first culture layer to form an interface connected to the outside.

17. The organ chip according to claim 14, characterized in that: The perfusion channel extends along the longitudinal direction and passes through the first culture layer to form an interface connected to the outside.

18. The organ chip according to claim 14, characterized in that: The organ chip comprises a first liquid inlet channel and a first liquid outlet channel, and the first liquid inlet channel and the first liquid outlet channel are respectively connected to the first culture chamber.

19. The organ chip according to claim 18, 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 along the longitudinal direction and pass through the first culture layer to form an interface connected to the outside.

20. The organ chip according to claim 14, characterized in that: The organ chip comprises 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 connected to the second culture chamber.

21. The organ chip according to claim 20, characterized in that: 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 pass through the first culture layer to form an interface connected to the outside.

22. The organ chip according to claim 14, characterized in that: The first culture layer includes a first sheet body, a second sheet body and a third sheet body which are stacked in sequence.

23. The organ chip according to claim 22, characterized in that: The shape of the portion of the first culture chamber located at the third sheet is a truncated cone, and the cross section of the truncated cone gradually increases from a side close to the culture membrane to a side away from the culture membrane.

24. A lung organ model, characterized in that: The lung organ model comprises the organ chip according to any one of claims 1 to 23.

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