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

Abstract

The application 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 further comprises 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 a communication opening of the perfusion channel to 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 membrane separates the second culture chamber and the actuating chamber. The actuating membrane can be deformed in response to pressure change of the actuating chamber, so as to conduct pressure change between the actuating chamber and the second culture chamber, and further cause the culture membrane to be deformed. The organ chip can more truly simulate the breathing movement process of alveoli and the multidirectional shear stress generated by gas on cells, and is beneficial to meet the research demand of ventilator-associated lung injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of organ-on-chip, in particular, organ-on-chip, lung organ model, construction method and simulation method. BACKGROUND

[0002] Related studies show that in the treatment of severe patients, the lung injury caused by mechanical ventilation using a ventilator is mainly derived from alveolar overexpansion, alveolar epithelial and vascular endothelial injury caused by shear force and inflammatory response. However, the dynamic injury and repair mechanism of alveoli in the human body at the cellular level has not been fully understood, and such ventilator-induced lung injury diseases are usually limited to oxygenation index and imaging characteristics in clinical disease diagnosis, lacking biomarker-based diagnosis and treatment methods and effective clinical treatment strategies to 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, and develop biomarker-based diagnosis and treatment methods to provide a "visible window" for early diagnosis of ventilator-associated lung injury is a difficult problem to be solved in clinical medicine.

[0003] Organ-on-chip is a highly simulated microsystem of an in-vitro organ 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-chip has great potential advantages in the simulation and construction of in-vitro disease models, drug screening, and precision medicine, etc.

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

[0005] However, the two-dimensional single-direction mechanical cyclic stretching in such a lung organ model still has certain differences from the expansion and contraction of the gas-blood barrier in the respiratory process in vivo, and it is difficult to simulate the real three-dimensional respiratory motion process of the alveoli. Therefore, 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 three-dimensional expansion or contraction of the polydimethylsiloxane porous membrane is caused by the pressure difference between the two culture chambers.

[0006] In the related art, the lung organ model is usually used to simulate the physiological environment of the alveoli during natural human respiration. The same channel is often used when the culture solution is introduced into the organ chip and when the air is introduced into the organ chip to simulate the alveolar respiration. The channel is not directed towards the culture membrane, which causes the lung organ model to not truly simulate the damage to the alveoli in the mechanical ventilation scene.

[0007] In addition, during the process of introducing the culture solution into the culture chamber, bubbles are easily left on the wall surface of the culture chamber. The bubbles in contact with the cells in the culture chamber will hinder the cells from obtaining the culture solution, causing the growth of this part of the cells to be poor, and even possibly damaging the cells, thereby affecting the growth consistency of the cells. SUMMARY

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

[0009] According to the organ chip provided by the present application, a first culture chamber, a second culture chamber, an actuating chamber and a perfusion channel are formed. The organ chip includes a culture membrane and an actuating membrane. The culture membrane 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 towards the culture membrane, preferably directly opposite the culture membrane. The perfusion channel is used to connect an external gas source. The actuating membrane separates the second culture chamber and the actuating chamber. The actuating membrane can deform in response to the pressure change of the actuating chamber to conduct the pressure change between the actuating chamber and the second culture chamber, thereby causing the culture membrane to deform.

[0010] According to the organ chip provided in the application, at least the following technical effects are achieved: the organ chip blows gas towards the culture membrane through the perfusion channel to the first culture chamber, so that the culture membrane generates three-dimensional deformation, thereby simulating the multidirectional shear stress generated by mechanical ventilation of a breathing machine on cells; in addition, the perfusion channel is directed towards the culture membrane, so that the gas introduced through the perfusion channel can impact the culture membrane, more realistically simulating the damage to alveoli during mechanical ventilation, and the organ chip is conducive to meeting the research needs of ventilator-associated lung injury.

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

[0012] According to some embodiments of the application, the surface of the culture membrane is coated with a biological macromolecule for simulating an extracellular matrix.

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

[0014] According to some embodiments of the 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 application, the perfusion channel is directed towards the center of the deformable region of the culture membrane.

[0016] According to some embodiments of the 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 application, the perfusion channel is coaxially arranged with the first culture chamber.

[0018] According to some embodiments of the 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 away from the culture membrane.

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

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

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

[0022] According to some embodiments of the present application, the organ chip comprises 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 sequentially stacked in a 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, the pressure channel is in communication with the actuation chamber, and the pressure channel is used to connect 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 out of 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 out of the first culture layer along the longitudinal direction 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 in communication with 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 out of the first culture layer along the longitudinal direction 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 in communication with 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 penetrate out of 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 comprises a first sheet, a second sheet, and a third sheet which are sequentially stacked.

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

[0032] According to the lung organ model construction method provided in the present application, the following steps are included: providing the organ chip of the present application; seeding and culturing epithelial cells on the surface of the culture membrane located in the first culture chamber; seeding 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, seeding and culturing the epithelial cells includes: placing the first culture chamber upward, introducing a solution carrying the epithelial cells into the first culture chamber; placing the organ chip in a cell culture box to allow the epithelial cells to settle and adhere to the surface of the culture membrane; and providing a flowing cell culture medium to the first culture chamber.

[0034] According to some embodiments of the present application, seeding 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; placing the organ chip in a cell culture box to allow the vascular endothelial cells to settle and adhere to the surface of the culture membrane; and providing a flowing cell culture medium to the second culture chamber.

[0035] According to some embodiments of the present application, before seeding the epithelial cells and the vascular endothelial cells, the lung organ model construction method further includes: coating a biological macromolecule for simulating extracellular matrix on the surface of the culture membrane.

[0036] According to some embodiments of the present application, coating the biological macromolecule on the surface of the culture membrane includes: introducing a complex solution composed of collagen, fibronectin and laminin into the first culture chamber and the second culture chamber, the collagen being type I collagen or type IV collagen; and placing the organ chip to allow the solution to react.

[0037] The lung organ model provided in the present application is obtained by using the lung organ model construction method provided in the present application.

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

[0039] According to some embodiments of the present application, the lung injury simulation method further includes periodically changing the pressure of the actuation chamber to simulate lung injury caused by mechanical ventilation in the presence of spontaneous breathing of a patient; or keeping the pressure of the actuation chamber constant to simulate lung injury caused by mechanical ventilation in the absence of spontaneous breathing of a patient.

[0040] The lung organ model provided in the present application comprises the organ chip provided in the present application, and the lung organ model construction method and the lung injury simulation method provided in the present application use the organ chip provided in the present application. Therefore, the lung organ model, the lung organ model construction method and the lung injury simulation method correspondingly have the beneficial effects provided by the organ chip, which will not be described herein again. BRIEF DESCRIPTION OF DRAWINGS

[0041] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the references to the figures, in which:

[0042] Figure 1 is a schematic diagram of the principle of the organ chip of the embodiment of the present application;

[0043] Figure 2 is a schematic diagram of the principle of the lung organ model of the embodiment of the present application;

[0044] Figure 3 is a schematic diagram of the deformation of the culture membrane of the organ chip of the embodiment of the present application under the characterization of a profilometer, wherein part (a) corresponds to the contraction state of the culture membrane, and part (b) corresponds to the expansion state of the culture membrane;

[0045] Figure 4 is a 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 contraction state of the culture membrane, and cells are observed to grow on the culture membrane, and part (b) corresponds to the expansion state of the culture membrane;

[0046] Figure 5 is a schematic diagram of the top view structure of the organ chip of the embodiment of the present application;

[0047] Figure 6 is a schematic diagram of the cross-sectional structure of the organ chip of the embodiment of the present application;

[0048] Figure 7 is a schematic diagram of the cross-sectional structure of the organ chip of the embodiment of the present application;

[0049] Figure 8 is a schematic diagram of the exploded structure of the organ chip of the embodiment of the present application;

[0050] Figure 9 is a schematic diagram of the exploded structure of the organ chip of the embodiment of the present application.

[0051] Reference Signs:

[0052] The first culture chamber 110, the second culture chamber 120, the actuating chamber 130, the perfusion channel 140, the inlet section 141, the connecting 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,

[0053] The culture membrane 210, the actuating 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 actuating layer 250. DETAILED DESCRIPTION

[0054] Embodiments of the present application are described below in detail, examples of which are shown in the drawings, wherein the same or similar reference signs represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the drawings are exemplary, only for explaining the present application, and cannot be understood as a limitation on the present application.

[0055] In the description of the present application, it is understood that the orientation description, such as the orientation or position relationship indicated by up, down, front, back, left, right, etc. is based on the orientation or position relationship shown in the drawings, only for the purpose of describing the present application and simplifying the description, and therefore cannot be understood as a limitation on the present application.

[0056] 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, more than, etc. are understood as not including the number, above, below, etc. are understood as including the number. If it is described as first, second, only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features or implicitly indicating the sequence of indicated technical features.

[0057] In the description of the present application, unless otherwise explicitly limited, the words such as setting, installing, connecting, etc. should be broadly understood, and the person skilled in the art can reasonably determine the specific meaning of the above words in the present application in combination with the specific content of the technical solution.

[0058] The dynamic injury and repair mechanism of alveoli in the human body at the cellular level is not fully clear, and the lung injury disease caused by mechanical ventilation of a ventilator is usually limited to oxygenation index and imaging characteristics in clinical disease diagnosis, lacking a biomarker-based diagnosis and treatment method and an effective clinical treatment strategy for reducing ventilator-associated lung injury.

[0059] Some related technologies use a scaled-up alveolus proportion model to study the damage to alveolus caused by mechanical ventilation, but the proportion model cannot truly reflect the physiological state of alveolus in the human body, and thus it is difficult to accurately restore the damage to alveolus.

[0060] Some related technologies construct a lung organ model based on an organ chip, but the deformation of the culture membrane of cells is two-dimensional stretching and contraction, and it is difficult to accurately restore the three-dimensional expansion and contraction of alveolus when breathing.

[0061] 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.

[0062] In the above related technologies, the same perfusion channel is often used when the culture solution is introduced into the organ chip and when air is introduced into the organ chip to simulate the gas environment of alveolus breathing. Since the culture solution is generally avoided from impacting the culture membrane when the culture solution is introduced, and the damage to cells caused by the bubbles generated during the culture process is avoided, the perfusion channel is often designed to be not directed to the deformable region of the culture membrane (specifically, not directed to the deformable region of the culture membrane) and to be conical.

[0063] When simulating natural human respiration, the operating principle of the organ chip is that air is passively introduced or discharged from 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 that the perfusion channel actively injects positive pressure air, thereby causing the culture membrane to deform, at this time, if the positive pressure gas cannot impact the culture membrane, the damage to the alveolus in the mechanical ventilation scene cannot be truly simulated.

[0064] With reference to Figure 1 , the organ chip provided by the embodiment of the first aspect of the present application comprises a culture membrane 210 and an actuating membrane 220. The organ chip further forms 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 in communication with the first culture chamber 110, and the communication port of the perfusion channel 140 with the first culture chamber 110 is directed to the culture membrane 210, in particular, directly opposite the culture membrane 210. The perfusion channel 140 is used to connect 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, so as to conduct the pressure change between the actuating chamber 130 and the second culture chamber 120, and further cause the culture membrane 210 to deform.

[0065] With reference to Figure 2This application also provides a lung organ model based on the organ-on-a-chip of this application. The lung organ model is constructed by the 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 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.

[0066] Organ-on-a-chip technology can construct controllable, minimal functional units at the micrometer scale to simulate human physiological states, thereby more realistically and accurately simulating cellular activity. Typically, the average diameter of alveoli is around 0.2 mm; therefore, the diameter of the culture membrane 210 exposed in the first culture chamber 110 can also be set to around 0.2 mm to more closely approximate the size of real alveoli. Figure 1 and Figure 2 The culture membrane 210 is defined to longitudinally separate 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 under normal pressure, the culture membrane 210 is almost not subjected to any force in the longitudinal direction and is in a contracted state. However, 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 subjected to a force in the longitudinal direction and expands towards the second culture chamber 120 and is in an expanded state.

[0067] For example, using a profilometer, Figure 3 Deformation of culture membrane 210 in one embodiment of organ-on-a-chip was detected and characterized. Figure 3 Part (a) shows the outline of the culture membrane 210 in the contracted state. It can be observed from the figure that the longitudinal undulation of the culture membrane 210 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 exhibits obvious expansion deformation in the longitudinal direction, thereby simulating the three-dimensional expansion of alveoli during inhalation.

[0068] For example, Figure 4 The results of microscopic observation of the culture membrane 210 in one embodiment of the lung organ model are shown. Figure 4 Part (a) shows the outline of the culture membrane 210 in the contracted state, and cells can be seen growing on the surface of the culture membrane 210; Figure 4 Part (b) reflects the outline of the culture membrane 210 in the expanded state, showing that the culture membrane 210 expands uniformly.

[0069] When used to simulate alveoli, the contracted state of the organ chip can simulate the contracted state of alveoli during exhalation, and the expanded state of the organ chip can simulate the expanded state of alveoli during inhalation. The organ chip can simulate the breathing movement process of alveoli in the human body during active breathing by causing the three-dimensional expansion and contraction of the culture membrane 210 through the periodic pressure change of the actuation chamber 130, and can simulate the multi-directional shear stress on cells generated by mechanical ventilation of a breathing machine by blowing gas towards the culture membrane 210 through the perfusion channel 140 into the first culture chamber 110 to cause the culture membrane 210 to produce three-dimensional deformation.

[0070] In addition, by directing the communication opening of the perfusion channel 140 and the first culture chamber 110 towards the culture membrane 210, the gas introduced through the perfusion channel 140 can impact the culture membrane 210, more realistically simulating the damage to alveoli during mechanical ventilation, especially when the communication opening of the perfusion channel 140 and 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 a static state, and simulate the mechanical ventilation effect when the culture membrane 210 breathing movement occurs three-dimensional deformation, so that the impact effect is more ideal. Figure 2 Figure 1

[0071] Therefore, the organ chip is beneficial to meet the research needs of ventilator-associated lung injury.

[0072] The lung organ model based on the organ chip also has the beneficial effects brought by the organ chip, which will not be described here.

[0073] 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 presence of spontaneous breathing of a patient; when the pressure of the actuation chamber 130 is constant, the lung organ model simulates the lung injury caused by mechanical ventilation in the absence of spontaneous breathing of a patient.

[0074] 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, so as to study the effect of the environment on alveoli, such as controlling parameters such as the oxygen content, humidity, carbon dioxide concentration of the gas, introducing gas containing irritating gas components (such as cigarette smoke, chemical irritating gas, toxic gas, etc.), introducing gas containing pseudo-virus particles, etc., so as to simulate different test environments.

[0075] In addition to gas, the perfusion channel 140 can also be used to introduce liquid, such as introducing alveolar lavage fluid derived from a patient, to simulate a corresponding disease model.​​

[0076] 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 fibrocytes, dendritic cells, and other cells, etc. In other use scenarios, the cells grown on the two sides of the culture membrane 210 can be the same or different.

[0077] In the present application, the culture membrane 210 can adopt 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 is filled with gas, thereby improving the reliability of the organ chip. The polydimethylsiloxane porous membrane allows the cells on the two sides to exchange gas through the pores of the culture membrane 210. The size of the pores is generally in the micron level.

[0078] The actuating membrane 220 can also adopt a polydimethylsiloxane porous membrane or a polydimethylsiloxane non-porous membrane.

[0079] In the human body, cells are filled with extracellular matrix, so the surface of the culture membrane 210 can be further coated with a biomacromolecule for simulating the extracellular matrix. This makes the growth environment of the cells closer to the physiological environment of the human body, which helps the organ chip to more realistically simulate the alveoli or other human tissues and organs, and also helps the lung organ model to more accurately simulate the damage to the cells during mechanical ventilation.

[0080] Exemplarily, the biomacromolecule can include collagen, fibronectin, and laminin, wherein the collagen includes type I collagen or type IV collagen. Of course, in other embodiments, the biomacromolecule can also adopt different designs, such as adding, reducing, or replacing certain components.

[0081] In order to improve the consistency of different regions on the surface of the culture membrane 210 during expansion deformation, in the present application, the perfusion channel 140 can be designed to be directed towards the center of the deformable region of the culture membrane 210. At this time, referring to Figure 2 The gas from the perfusion channel 140 acts on the center of the deformable region, so as to prevent the culture membrane 210 from being eccentric during expansion deformation when the gas is filled.

[0082] The deformable region refers to a region of the culture membrane 210 that is not constrained and has a degree of freedom of deformation. In order to further improve the consistency of different regions on the surface of the culture membrane 210 during expansion deformation, the shape of the deformable region can be designed to be circular.

[0083] In the organ chip, the culture membrane 210 is usually bonded with other parts of the organ chip, so in the longitudinal direction, the deformable region of the culture membrane 210 is not bonded with other parts of the organ chip on both sides. In other words, in this 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.

[0084] For example, referring to Figure 1 In the embodiment of the first aspect of the application, the area of the culture membrane 210 exposed to the first culture chamber 110 is smaller than 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, so the first culture chamber 110 defines the deformable region of the culture membrane 210.

[0085] It is self-evident that 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 arranged coaxially with the first culture chamber 110, so as to realize the center of the deformable region of the culture membrane 210.

[0086] In this application, in order to improve the consistency of the growth environment of different regions on the surface of 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 the region close to 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 (such as bubbles generated during the manufacture of the organ chip or bubbles generated by cells during the culture process) to remain near the side wall, avoiding damage to cells located at the edge of the deformable region, on the other hand, it also improves the flowability of the region near the side wall, thereby ensuring the consistency of the supply of culture medium in different regions, and further improving the consistency of the growth environment of different regions on the surface of the culture membrane 210.

[0087] For example, referring to Figure 1 In the embodiment of the first aspect of the application, since the deformable region of the culture membrane 210 is defined by the first culture chamber 110, the side wall of the second culture chamber 120 is away from the edge of the deformable region of the culture membrane 210, so the side wall of the second culture chamber 120 theoretically will not 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 away from the culture membrane 210.

[0088] And, referring to Figure 1 In embodiments of the first aspect of the application, the organ-on-chip is further designed such that the volume of the second culture chamber 120 is greater than the volume of the first culture chamber 110, so that the second culture chamber 120 is used to store culture medium.

[0089] It can be understood that, when simulating the breathing of alveoli or performing other experiments, the second culture chamber 120 is preferably closed, 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, energy can only be provided to the cells by the culture medium stored in the second culture chamber 120, and therefore the design of the second culture chamber 120 with a large volume can prevent the death of cells caused by the drying of the stored culture medium.

[0090] The organ-on-chip is manufactured by microfabrication processes such as photolithography. In the related art, the organ-on-chip is generally divided into multiple parts, and corresponding cavities and channels are processed on each part, and then combined to form the organ-on-chip. Depending on the division method, the organ-on-chip can have different configurations.

[0091] Exemplarily, referring to Figure 5 , Figure 6 , Figure 7 , Figure 8 and Figure 9 According to embodiments of the second aspect of the application, the organ-on-chip comprises 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 use of a stacked design can facilitate the processing and assembly of the organ-on-chip.

[0092] Before using the organ-on-chip, cells need to be cultured on the surface of the culture membrane 210, and then culture medium needs to be continuously supplied to the first culture chamber 110 and the second culture chamber 120 to provide the cells with the nutrients required for growth. Therefore, referring to Figure 5 and Figure 6 The organ-on-chip also needs to include a first liquid inlet channel 160 and a first liquid outlet channel 170, which are respectively in communication with the first culture chamber 110. The organ-on-chip also needs to include a second liquid inlet channel 180 and a second liquid outlet channel 190, which are respectively in communication with the second culture chamber 120.

[0093] In use, the solution containing cells, culture medium and other required materials (e.g. biomacromolecules simulating extracellular matrix) are introduced into the two 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 the cells in vivo. The waste liquid is discharged through the first liquid outlet channel 170 and the second liquid outlet channel 190.

[0094] The pressure of the actuation chamber 130 can be changed by providing a pressure medium to the actuation chamber 130 or changing the volume of the actuation chamber 130. Exemplarily, with reference to Figure 6 、 Figure 7 、 Figure 8 and Figure 9 In an embodiment of the second aspect, the organ chip is formed with a pressure channel 150, the pressure channel 150 being in communication with the actuation chamber 130, and the pressure channel 150 being configured to connect to an external pressure source to provide a pressure medium to the actuation chamber 130.

[0095] With reference to Figure 6 and Figure 7 In an 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 outlet channel 190, etc. can be designed to pass through the top surface of the organ chip, so that the side surface of the organ chip can be conveniently gripped, and the bottom surface can be stably placed on the workbench.

[0096] Specifically, with reference to Figure 6 、 Figure 7 、 Figure 8 and Figure 9 The pressure channel 150 extends along the longitudinal direction 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.

[0097] The perfusion channel 140 also extends along the longitudinal direction to pass through the first culture layer 230 to form an interface connected to the outside. Exemplarily, with reference 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 passes through the first culture layer 230 along the longitudinal direction to form an interface connected to the outside; the outlet section 143 is in communication with the first culture chamber 110 and also extends along the longitudinal direction to be opposite to the culture membrane; the connection section 142 extends in a direction perpendicular to the longitudinal direction to communicate 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 to avoid interference. The connection section 142 can be designed as a straight line as shown in Figure 6The first liquid inlet channel 160 and the first liquid outlet channel 170 are located in the first culture layer 230, and extend longitudinally and pass through the first culture layer 230 to form interfaces 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 pass through the first culture layer 230 to form interfaces connected to the outside.

[0098] The first liquid inlet channel 160 and the first liquid outlet channel 170 are located in the first culture layer 230, and extend longitudinally and pass through the first culture layer 230 to form interfaces 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 pass through the first culture layer 230 to form interfaces connected to the outside.

[0099] In an embodiment of the second aspect, the first culture chamber 110 is designed to gradually increase in cross-section from the side close to the culture membrane 210 to the side away from the culture membrane 210 in the area close to the culture membrane 210, so as to improve the culture effect of the cells. In order to facilitate the processing of the first culture chamber 110, the first culture chamber 110 is designed to gradually increase in cross-section from the side close to the culture membrane 210 to the side away from the culture membrane 210 in the area close to the culture membrane 210. Figure 8 In an embodiment of the second aspect, the first culture layer 230 can 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 in the third sheet 233 is a circular truncated cone shape, and the cross-section of the circular truncated cone gradually increases from the side close to the culture membrane 210 to the side away from the culture membrane 210.

[0100] The lung organ model is obtained by a lung organ model construction method. The lung organ model construction method includes providing an organ chip; seeding and culturing epithelial cells on the surface of the culture membrane 210 located in the first culture chamber 110, and seeding and culturing vascular endothelial cells on the surface of the culture membrane 210 located in the second culture chamber 120.

[0101] Seeding and culturing epithelial cells means making the epithelial cells grow on the surface of the culture membrane 210 and form a tightly connected structure (i.e., an epithelial cell layer). Seeding and culturing epithelial cells specifically include the following steps:

[0102] The first culture chamber 110 is placed upward, and a solution carrying epithelial cells is introduced into the first culture chamber 110;

[0103] The organ chip is placed in the cell culture box to make the epithelial cells settle and adhere to the surface of the culture membrane 210;

[0104] The first culture chamber 110 is provided with a flowing cell culture medium.

[0105] Seeding and culturing vascular endothelial cells means making the vascular endothelial cells grow on the surface of the culture membrane 210 and form a tightly connected structure (i.e., a vascular endothelial cell layer). Seeding and culturing vascular endothelial cells specifically include the following steps:

[0106] The second culture chamber 120 is placed upward, and a solution carrying vascular endothelial cells is introduced into the second culture chamber 120;

[0107] The organ chip is left in the cell culture box to allow the vascular endothelial cells to settle and adhere to the surface of the culture membrane 210;

[0108] The second culture chamber 120 is provided with a flowing cell culture medium.

[0109] Further, the lung organ model construction method further comprises coating biological macromolecules for simulating extracellular matrix on the surface of the culture membrane 210. The coating of the biological macromolecules occurs before the inoculation of the epithelial cells and the vascular endothelial cells, and the coating of the biological macromolecules enables the organ chip to more realistically simulate the physiological environment of the human body, and also helps the growth of the cells on the surface of the culture membrane 210.

[0110] The coating of the biological macromolecules on the surface of the culture membrane 210 specifically comprises the following steps:

[0111] The first culture chamber 110 and the second culture chamber 120 are introduced with a composite solution composed of collagen, fibronectin and laminin, and the collagen is type I collagen or type IV collagen;

[0112] The organ chip is left to wait for the reaction of the solution.

[0113] The application also provides a lung injury simulation method using the lung organ model and the lung organ chip of the application, and the lung injury simulation method comprises introducing air into the first culture chamber 110 through the perfusion channel 140 to simulate the mechanical ventilation of the alveoli by the ventilator.

[0114] On this basis, the lung injury simulation method can further comprise periodically changing the pressure of the actuation chamber 130 to simulate the lung injury caused by mechanical ventilation in the presence of spontaneous breathing of the patient, or keeping the pressure of the actuation chamber 130 constant to simulate the lung injury caused by mechanical ventilation in the absence of spontaneous breathing of the patient.

[0115] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example", 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 the present application. In the present specification, the exemplary description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0116] In some alternative embodiments, the functions / operations referred to in the block diagrams can occur in a different order than the order referred to in the operational illustrations. For example, two blocks shown in succession can in fact be executed substantially concurrently or the blocks sometimes be executed in reverse order, depending on the functionality / operations involved. Also, the embodiments presented and described in the flowcharts of the present application are only examples of implementations. The processes presented and described as part of the flowcharts can be implemented in a variety of other ways. For example, the various operations shown as part of the flowcharts can be executed in a different order, or in parallel, or in reverse order, depending on the functionality / operations involved. Alternative embodiments are possible where sub-operations of described operations are executed independently, and where the order of the sub-operations is changed.

[0117] While embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, combinations, and alterations can be made to the embodiments without departing from the principles and spirit of the application. The scope of the application is defined by the claims and their equivalents.

Claims

1. An organ-on-a-chip, characterized in that, The organ chip comprises a first culture chamber, a second culture chamber, an actuation chamber and a perfusion channel, and comprises: a culture membrane separating the first culture chamber and the second culture chamber, the perfusion channel being in communication with the first culture chamber, and a communication port of the perfusion channel facing the culture membrane, the perfusion channel being used for connecting an external air source; an actuation membrane separating the second culture chamber and the actuation chamber, the actuation membrane being capable of deforming in response to pressure change of the actuation chamber to conduct pressure change between the actuation chamber and the second culture chamber, thereby causing the culture membrane to deform; The organ chip comprises 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 sequentially stacked 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; the organ chip is formed with a pressure channel, the pressure channel is in communication with the actuation chamber, and the pressure channel is used for connecting an external pressure source; 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 connected to the outside; the perfusion channel extends through the first culture layer along the longitudinal direction to form an interface connected to the outside; the organ chip comprises a first liquid inlet channel and a first liquid outlet channel, the first liquid inlet channel and the first liquid outlet channel are respectively in communication with the first culture chamber; the first liquid inlet channel and the first liquid outlet channel are located in the first culture layer, and extend through the first culture layer along the longitudinal direction to form an interface connected to the outside; the organ chip comprises a second liquid inlet channel and a second liquid outlet channel, the second liquid inlet channel and the second liquid outlet channel are respectively in communication with the second culture chamber; 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 penetrate through the first culture layer to form an interface connected to the outside; the first culture layer comprises a first sheet, a second sheet and a third sheet which are sequentially stacked; the first culture chamber is located in a portion of the third sheet, and the shape of the portion is a circular truncated cone shape, and the cross section of the circular truncated cone gradually increases from the side close to the culture membrane to the side away from the culture membrane.

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

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

4. The organ-chip of claim 3, wherein The biological macromolecule comprises collagen, fibronectin and laminin, and the collagen comprises 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-on-chip of claim 1, wherein, The perfusion channel is opposite to the culture membrane, and the perfusion channel faces the center of the deformable region of the culture membrane.

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

8. The organ-on-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 area 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 away from the culture membrane.

10. The organ-chip of claim 1, wherein, 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.

11. The organ-chip of claim 10, wherein, The surface area of the culture membrane exposed to the second culture chamber is greater than that of the culture membrane exposed to the first culture chamber.

12. The organ-chip of claim 1, wherein, The actuating membrane adopts a polydimethylsiloxane porous membrane.

13. A method of constructing a lung organ model, characterized by, The lung organ model construction method comprises the following steps: providing the organ chip of any one of claims 1 to 12; seeding and culturing epithelial cells on the surface of the culture membrane in the first culture chamber; seeding and culturing vascular endothelial cells on the surface of the culture membrane in the second culture chamber.

14. The lung organ model construction method according to claim 13, wherein, Seeding and culturing the epithelial cells, comprising: placing the first culture chamber upward, and introducing a solution carrying the epithelial cells into the first culture chamber; resting the organ chip in a cell culture box to allow the epithelial cells to settle and adhere to the surface of the culture membrane; providing a flowing cell culture medium to the first culture chamber.

15. The lung organ model construction method according to claim 13, wherein, Seeding and culturing the vascular endothelial cells, comprising: placing the second culture chamber upward, and introducing a solution carrying the vascular endothelial cells into the second culture chamber; resting the organ chip in a cell culture box to allow the vascular endothelial cells to settle and adhere to the surface of the culture membrane; providing a flowing cell culture medium to the second culture chamber.

16. The lung organ model construction method according to claim 13, wherein, Before seeding the epithelial cells and the vascular endothelial cells, the lung organ model construction method further comprises coating a biological macromolecule for simulating extracellular matrix on the surface of the culture membrane.

17. The lung organ model construction method according to claim 16, wherein Coating the biological macromolecule on the surface of the culture membrane, comprising: introducing a complex 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; resting the organ chip to allow the solution to react.

18. A lung organ model, characterized in that, The lung organ model is constructed using the lung organ model construction method of any one of claims 13 to 17.

19. A method of simulating lung injury, comprising, The lung injury simulation method uses the lung organ model of claim 18 and the organ chip of any one of claims 1 to 12, and the lung injury simulation method comprises introducing air into the first culture chamber through the perfusion channel to simulate mechanical ventilation of the alveoli by a ventilator.

20. The lung injury simulation method of claim 19, wherein, The lung injury simulation method further comprises periodically changing the pressure of the actuating chamber to simulate lung injury caused by mechanical ventilation in the presence of spontaneous breathing of a patient; or keeping the pressure of the actuating chamber constant to simulate lung injury caused by mechanical ventilation in the absence of spontaneous breathing of a patient.

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