Organ chip and lung gas exchange model

Abstract

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

Description

Technical Field

[0001] The present application relates to the field of organ chips, and in particular to an organ chip and a lung gas exchange model. Background Art

[0002] Organ chips are a bionic cell microculture system that can simulate the physiological processes of organs in the body to a certain extent, predict drug responses, and avoid the ethical, high-cost, and species differences problems brought about by animal experiments, providing powerful assistance for disease mechanism exploration, preclinical research, and drug development.

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

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

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

[0006] Some related technologies have proposed a technical solution of directly applying pressure to the main channel to cause three-dimensional deformation of the polydimethylsiloxane porous membrane. However, in the current solution, there is still a problem that the deformation degree of each area of ​​the polydimethylsiloxane porous membrane is uneven, making it difficult to truly simulate the expansion of the alveolar wall.

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

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

[0009] (2) Polydimethylsiloxane materials lack hydrophilicity and permeability. Therefore, after film formation, through holes need to be processed to improve permeability.

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

[0011] (4) Polydimethylsiloxane materials cannot be degraded in a short period of time, and it is difficult to form a barrier for the fusion of upper and lower cells, such as the air-blood barrier. Summary of the invention

[0012] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes an organ chip and a lung gas exchange model, which can more realistically simulate the physiological changes of alveoli during breathing.

[0013] The organ chip provided according to the present application includes a culture membrane and an actuation membrane. The organ chip is also formed with a culture chamber and at least two actuation chambers. The culture membrane separates the culture chamber into a first culture chamber and a second culture chamber, the actuation membrane separates the second culture chamber and each of the actuation chambers, the actuation chamber defines a deformable area of ​​the actuation membrane, at least two of the actuation chambers are equidistantly arranged along a circle around the axis of the culture chamber, the actuation membrane located in the actuation chamber can deform in response to pressure changes in the actuation chamber to transmit pressure changes between the actuation chamber and the second culture chamber, and the culture membrane can deform in response to pressure changes in the second culture chamber.

[0014] According to the organ chip provided by the present application, at least the following technical effects are achieved: the present application adjusts the pressure of the actuation chamber to deform the actuation membrane between the actuation chamber and the culture chamber, thereby causing the pressure in the culture chamber to change, causing the culture membrane to deform in three dimensions. Compared with two-dimensional tensile deformation, three-dimensional deformation is closer to the real physiological changes of the alveoli, and is more conducive to establishing information exchange and gas exchange between alveolar lung epithelial cells and vascular endothelial cells in a three-dimensional dynamic microenvironment, and truly simulates the physiological expansion and contraction of the alveoli during breathing, as well as biological information exchange and effective gas exchange, and provides a reliable technical platform for preclinical research and drug development of respiratory diseases; on this basis, the present application uses multiple actuation chambers for braking. When simulating the reciprocating expansion and contraction of the alveolar wall, the multiple actuation chambers are arranged equidistantly around the axis of the culture chamber (that is, evenly distributed) along the circumference, and at the same time generate a force to pull the culture membrane, so that the traction force on the culture membrane is more uniform, reducing or even eliminating the uneven force phenomenon during the expansion of the culture membrane, and the organ chip can more realistically simulate the physiological changes of the alveoli during breathing.

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

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

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

[0018] According to some embodiments of the present application, the culture membrane is entirely impregnated with biomacromolecules.

[0019] 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.

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

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

[0022] According to some embodiments of the present application, the first culture chamber is a lung epithelial cell culture chamber, and the second culture chamber is a vascular endothelial cell culture chamber.

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

[0024] According to some embodiments of the present application, the pressure holes are respectively connected to the actuating chambers through symmetrical pressure channels.

[0025] According to some embodiments of the present application, the actuating membrane is a polydimethylsiloxane membrane.

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

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

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

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

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

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

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

[0033] According to some embodiments of the present application, the organ chip includes a splint, and two of the splints are respectively located at two ends of the organ chip in the longitudinal direction, and the splints clamp the rest of the organ chip.

[0034] The pulmonary gas exchange model provided in the present application includes the organ chip provided in the present application.

[0035] The pulmonary gas exchange model provided in the present application includes the organ chip provided in the present application, so the pulmonary gas exchange model correspondingly possesses the beneficial effects provided by the organ chip, which will not be elaborated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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:

[0037] Figure 1 is a schematic diagram of the principle of the organ chip of the embodiment of the present application;

[0038] Figure 2 is a schematic diagram of the results of the control experiment of the organ chip of the embodiment of the present application;

[0039] Figure 3 is a schematic diagram of the exploded structure of the organ chip according to an embodiment of the present application;

[0040] Figure 4 is a schematic cross-sectional structural diagram of an organ chip according to an embodiment of the present application;

[0041] Figure 5 is a schematic diagram of the exploded structure of the organ chip according to an embodiment of the present application;

[0042] Figure 6 is a schematic cross-sectional structural diagram of an organ chip according to an embodiment of the present application;

[0043] Figure 7 It is a schematic diagram of the structure of the organ chip according to an embodiment of the present application.

[0044] Reference numerals:

[0045] Culture membrane 100,

[0046] Actuating membrane 200,

[0047] The first layer 300, the first culture chamber 310, the first flow channel 320, the second flow channel 330, the fifth flow channel 340, the sixth flow channel 350, the overflow groove 360,

[0048] The second layer 400, the second culture chamber 410, the third flow channel 420, the fourth flow channel 430,

[0049] The third layer 500, the actuation chamber 510, the pressure hole 520, the pressure channel 530,

[0050] The fourth layer 600, the liquid inlet hole 610, the liquid outlet hole 620,

[0051] Splint 700,

[0052] Lung epithelial cell layer 910 and vascular endothelial cell layer 920 . DETAILED DESCRIPTION

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] The alveoli are the terminal part of the bronchial tree and are the place where the human body exchanges gases with the outside world. The structure through which the gas molecules in the alveoli exchange with the gas molecules in the blood is called the gas-blood barrier, which includes the alveolar surface liquid layer, type I alveolar cells and basement membrane, thin layer of connective tissue, capillary basement membrane and endothelium. The gas-blood barrier is very thin, with a total thickness of 0.2μm to 0.5μm, which is conducive to rapid gas exchange.

[0058] Pulmonary breathing refers to the action of exchanging gases during the process of lung contraction and expansion. During breathing, the pressure in the chest cavity changes, resulting in a difference between the pressure in the lungs and the external pressure, which causes gas flow and changes the deformation of normal alveoli into uniform deformation.

[0059] The pulmonary gas exchange model is a system used to study the physiological processes of the alveoli. The pulmonary gas exchange model includes an organ chip. Lung epithelial cells and vascular endothelial cells are cultured on both sides of the culture membrane of the organ chip to simulate the alveolar wall. By applying pressure to the culture membrane, the culture membrane deforms in response to the change in pressure to simulate the expansion and contraction of the alveolar wall during breathing.

[0060] However, the pressure distribution on the surface of the culture membrane in the related technology is not uniform enough, resulting in inconsistent deformation degrees in different areas of the culture membrane, which in turn leads to differences in the state of cell tissues in different areas, affecting the simulation effect.

[0061] The organ chip provided by this application is introduced below.

[0062] Reference Figure 1 According to an embodiment of the first aspect provided by the present application, the organ chip includes a culture membrane 100 and an actuating membrane 200, and the organ chip is also formed with a culture chamber and at least two actuating chambers 510, the culture membrane 100 separates the culture chamber into a first culture chamber 310 and a second culture chamber 410, the actuating membrane 200 separates the second culture chamber 410 and each actuating chamber 510, the actuating chamber 510 defines a deformable area of ​​the actuating membrane 200, at least two actuating chambers 510 are arranged equidistantly along a circle around the axis of the culture chamber, the actuating membrane 200 located in the actuating chamber 510 can deform in response to pressure changes in the actuating chamber 510 to transmit pressure changes between the actuating chamber 510 and the second culture chamber 410, and the culture membrane 100 can deform in response to pressure changes in the second culture chamber 410.

[0063] The present application also provides a lung gas exchange model including the organ chip of the present application. When constructing the lung gas exchange model, lung epithelial cells and vascular endothelial cells are introduced into the first culture chamber 310 and the second culture chamber 410 respectively, so that the cells grow on the two surfaces of the culture membrane 100, and generate a lung epithelial cell layer 910 and a vascular endothelial cell layer 920 respectively, thereby simulating the alveolar gas-blood barrier. At this time, the first culture chamber 310 and the second culture chamber 410 are the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber respectively.

[0064] The organ chip provided in the present application adjusts the pressure of the actuation chamber 510 to deform the actuation membrane 200 between the actuation chamber 510 and the culture chamber, thereby causing the pressure in the culture chamber to change, causing the culture membrane 100 to undergo three-dimensional deformation. Compared with the two-dimensional tensile deformation, the three-dimensional deformation is closer to the real physiological changes of the alveoli, and is more conducive to establishing information exchange and gas exchange between alveolar pulmonary epithelial cells and vascular endothelial cells in a three-dimensional dynamic microenvironment, truly simulating the physiological expansion and contraction of the alveoli during breathing, as well as the biological information exchange and effective gas exchange, providing a reliable technical platform for preclinical research and drug development of respiratory diseases.

[0065] Furthermore, the present application adopts multiple actuation chambers 510 for braking. When simulating the reciprocating expansion and contraction of the alveolar wall, the multiple actuation chambers 510 are arranged at equal intervals along the circumference around the axis of the culture chamber (that is, evenly distributed). The forces generated by the multiple actuation chambers 510 can balance each other, so that the traction force on the culture membrane 100 is more uniform, reducing or even eliminating the uneven force phenomenon when the culture membrane 100 expands. The organ chip can more realistically simulate the physiological changes of the alveoli during the breathing process.

[0066] It is understandable that the alveoli are always cycling between expansion and contraction. In order to more realistically simulate the dynamic environment of cell and tissue growth, the pressure of the actuation chamber 510 also needs to change back and forth according to the set conditions. The reciprocating pressure change will affect the flow field in the culture chamber, but this factor is often ignored in the relevant technology, and the number and layout of the actuation chamber 510 are not limited. If there is only one actuation chamber 510 that is not coaxial with the culture membrane 100, when the culture membrane 100 expands, it is easy to cause uneven force in different areas under the influence of the flow field.

[0067] The actuation chamber 510 design adopted in the present application can overcome this defect. Figure 2 The results of the control experiments based on the organ chip embodiments of the present application are shown. Figure 2 Part A in the middle shows the expansion and contraction of the culture membrane 100 when all the actuation chambers 510 are used simultaneously. Figure 2 Part B shows the expansion and contraction states of the culture membrane 100 when only one actuation chamber 510 that is not coaxial with the culture chamber is used and the remaining actuation chambers 510 are closed. By comparing them, it can be seen that when there is only one actuation chamber 510, there is obvious uneven deformation when the culture membrane 100 expands, and the use of multiple actuation chambers 510 can improve this defect.

[0068] The organ chip may have one or more culture chambers, and correspondingly, the number of the actuation chambers 510 may be one group or multiple groups.

[0069] For convenience, in the embodiment of the first aspect, each actuation chamber 510 has the same shape and size, and the distance from the culture chamber to each actuation chamber 510 along the axial direction is equal, so as to reduce the variables affecting the force, and only need to control the pressure of each actuation chamber 510 to keep synchronization, so as to achieve the balance of the forces generated by the multiple actuation chambers 510. However, it is not excluded that in some embodiments, each actuation chamber 510 has different shapes and sizes, the distance from the culture chamber to each actuation chamber 510 along the axial direction is not equal, and the pressure change curves of each actuation chamber 510 are different, but the variables are balanced, and the forces generated by the multiple actuation chambers 510 are still balanced.

[0070] The pulmonary gas exchange model includes an organ chip, and thus has the corresponding beneficial effects of the organ chip of the present application, which will not be described in detail here.

[0071] In the human body, the deformation of alveoli comes from the pressure change in the chest cavity. When the chest cavity expands, the chest cavity pressure is less than the atmospheric pressure, causing the alveoli to expand and inhale air. When the chest cavity shrinks, the chest cavity pressure is greater than the atmospheric pressure, causing the alveoli to contract and expel air. Figure 1 When used to construct a pulmonary gas exchange model that simulates a real lung environment, some embodiments represented by the embodiments of the first aspect tend to also use negative pressure to cause the culture membrane 100 to deform, that is, the actuating chamber 510 and the second culture chamber 410 are on the same side of the culture membrane 100, and the actuating membrane 200 separates the vascular endothelial cell culture chamber (that is, the second culture chamber 410) and the actuating chamber 510, and the pressure in the vascular endothelial cell culture chamber changes through the actuating membrane 200, thereby causing the culture membrane 100 to deform.

[0072] Correspondingly, when the pressure in the actuating chamber 510 decreases, the actuating membrane 200 expands and deforms toward one side of the actuating chamber 510, simulating the expansion of the chest cavity causing the human body to inhale. The actuating chamber 510 serves to limit the deformable area of ​​the actuating membrane 200. When the pressure in the actuating chamber 510 is restored, the actuating membrane 200 returns to a state of approximate planarity, simulating the contraction of the chest cavity causing the human body to exhale.

[0073] Of course, it is not excluded that the organ chip adopts a different design in other simulation scenarios. For example, in the scenario of simulating active ventilation of a ventilator, the first culture chamber 310 can be used as a vascular endothelial cell culture chamber, and the second culture chamber 410 can be used as a lung epithelial cell culture chamber. The actuating membrane 200 separates the lung epithelial cell culture chamber and the actuating chamber 510. The actuating membrane 200 can expand and deform toward one side of the lung epithelial cell culture chamber, thereby causing the pressure in the lung epithelial cell culture chamber to increase, simulating the ventilator to actively introduce air into the lungs. Alternatively, two sets of actuating chambers 510 can be set on both sides of the culture membrane 100.

[0074] In addition to the lung gas exchange model, the organ chip of the present application can also be used to construct other organ models, depending on the cell types grown on the two layers of the culture membrane 100 of the organ chip, such as fibroblasts, dendritic cells and other cells. In addition, when constructing the lung gas exchange model, the vascular endothelial cells can use human lung microvascular endothelial cells or human umbilical vein endothelial cells.

[0075] Back to the organ chip of this application. In the organ chip, on the one hand, the culture membrane 100 needs to provide elasticity to meet the needs of expansion and deformation, and on the other hand, the culture membrane 100 also serves as a substrate for cell growth and plays a role in simulating the interstitial matrix between cells. In the related art, the culture membrane 100 usually adopts a porous membrane based on polydimethylsiloxane material as the culture membrane 100.

[0076] However, the porous membrane based on polydimethylsiloxane material has defects. On the one hand, the deformability of polydimethylsiloxane material is poor, which makes it difficult for the culture membrane 100 to expand and deform significantly. Therefore, when applied to the lung gas exchange model, the simulation effect of alveolar expansion is not good; on the other hand, the porous membrane based on polydimethylsiloxane material is specially processed with holes after the film is formed, and the cells on both sides exchange substances through the holes in the membrane, which is different from the real physiological environment in which cells exchange substances through the interstitium, and will also have a negative impact on the authenticity of the simulation.

[0077] In this regard, in the present application, a hydrogel porous membrane can be used as the culture membrane 100. The hydrogel porous membrane has a microscopic pore structure, so there is no need to process additional holes on the culture membrane 100. The elasticity of the hydrogel material is better than that of the polydimethylsiloxane material. Therefore, under the same pressure regulation, the hydrogel porous membrane has a greater expansion degree and can better simulate the expansion of the alveoli. The hydrogel also has the characteristics of good bioaffinity, which can facilitate the growth of cells on both sides. However, the material of the culture membrane 100 itself and the thickness of the culture membrane 100 will also affect the material exchange of cells on both sides. For this reason, further, the culture membrane 100 can use a degradable hydrogel porous membrane. After the cell growth on both sides is completed, the culture membrane 100 is partially or even completely degraded, thereby further reducing the influence of the material of the culture membrane 100 on the authenticity of the intercellular matrix simulation.

[0078] The materials for preparing hydrogel porous membranes include natural proteins (gelatin, sericin, etc.), natural polysaccharides (glucan, alginate, hyaluronic acid, dextran, etc.) and synthetic materials (polyester, polyamide, polyvinyl alcohol, etc.). For example, a double network hydrogel composed of sodium alginate-polyacrylamide can be selected to prepare hydrogel porous membranes with good hydrophilicity, high porosity and high transparency. Generally speaking, the prepared hydrogel porous membrane needs to be washed with pure water, Hank's balanced salt solution, phosphate buffered saline solution, etc. to remove unreacted monomers before it can be used for the assembly of organ chips.

[0079] The organ chip with a degradable hydrogel porous membrane can be applied to the lung gas exchange model or other organ models with similar requirements. Ideally, in the lung gas exchange model, the culture membrane 100 can be degraded after the growth of the lung epithelial cell layer 910 and the vascular endothelial cell layer 920 is completed.

[0080] In a real physiological environment, the thickness of the intercellular matrix is ​​about 100nm. Therefore, under ideal conditions, the culture membrane 100 should be set to be degradable to a thickness less than or equal to 100nm. In order to reduce the difficulty of processing and facilitate the initial growth of cells, the initial thickness of the culture membrane 100 can be set to be greater than or equal to 5μm and less than or equal to 100μm. It can be understood that the initial thickness refers to the thickness of the culture membrane 100 before the organ chip is put into use and before it begins to degrade.

[0081] In order to form an environment suitable for cell growth, simulate the intercellular matrix more realistically, and improve the growth effect of cells on the surface of the culture membrane 100, the culture membrane 100 can also be impregnated with biomacromolecules, that is, the entire culture membrane 100 is impregnated with biomacromolecules.

[0082] Specifically, the biomacromolecules may include collagen, fibronectin and laminin, wherein the collagen may include type I collagen or type IV collagen.

[0083] In addition, since the shapes of the first culture chamber 310 and the second culture chamber 410 define the deformable area of ​​the culture membrane 100 (which is also the cell growth area), in order to make the deformation of the culture membrane 100 more uniform, the cross-section of the first culture chamber 310 and / or the second culture chamber 410 can be designed to be circular.

[0084] Cells grow on the surface of the culture membrane 100 exposed to the first culture chamber 310 and the second culture chamber 410. When the cells grow, it is necessary to continuously introduce cell culture medium into the culture chamber to provide energy for the cells. In some dead corners, such as the outer edge of the exposed culture membrane 100, that is, the junction of the first culture chamber 310 and the culture membrane 100 and the junction of the second culture chamber 410 and the culture membrane 100, the fluidity of the cell culture medium is poor, and the cells are prone to poor growth.

[0085] To address this problem, the first culture chamber 310 and / or the second culture chamber 410 may be designed to have a cross section that gradually increases from the side close to the culture membrane 100 to the side away from the culture membrane 100, thereby improving the fluidity of the cell culture medium at the outer edge. Figure 3 , Figure 4 In an embodiment of the second aspect of the present application, the first culture chamber 310 is designed to have a cross section that gradually increases from a side close to the culture membrane 100 to a side away from the culture membrane 100 .

[0086] The actuation membrane 200 needs to have a certain elasticity, and needs to separate the culture chamber and the actuation chamber 510 to avoid material exchange, so the actuation membrane 200 often uses a non-porous membrane of polydimethylsiloxane material. Generally speaking, the pressure of the actuation chamber 510 can be adjusted by changing the fluid volume of the actuation chamber 510. For example, the organ chip can include a pressure hole 520, which is connected to the actuation chamber 510, and the pressure hole 520 is used to connect to the external pressure end. The pressure end can provide gaseous or liquid fluid, thereby causing the pressure change of the actuation chamber 510.

[0087] Furthermore, in order to improve the consistency of deformation of the actuating membrane 200 in each actuating chamber 510 , the pressure hole 520 may be designed to be connected to each actuating chamber 510 through a symmetrical pressure channel 530 .

[0088] Changing the pressure by inputting and outputting fluids helps to simplify the structure of the organ chip, thereby facilitating the miniaturization of the organ chip and better simulating structures with smaller unit sizes such as alveoli. In some scenarios, it is also possible to consider designing a movable micromechanical structure in the organ chip, such as a micro piston structure, etc., to change the volume of the actuation chamber 510 through the movement of the structure, thereby adjusting the pressure. The specific design scheme can refer to the relevant technologies in this field and related technical fields (such as the MEMS field), which will not be repeated here.

[0089] In the present application, the organ chip can form spatial structures such as the culture chamber and the actuation chamber 510 in different ways. For example, the culture chamber and the actuation chamber 510 can be formed by stacking layer structures. Figure 3 and Figure 4 An embodiment of the second aspect based on this concept is shown. Figure 5 and Figure 6 An embodiment of the third aspect based on this concept is shown. It can be understood that Figure 3 and Figure 4 , Figure 5 and Figure 6 These are some examples of structures such as culture chambers and actuation chambers 510 formed by stacking layer structures. Figure 5 and Figure 6 The first culture chamber 310 is funnel-shaped (i.e., the cross-section gradually increases from the side close to the culture membrane 100 to the side away from the culture membrane 100), which can avoid dead corners for liquid flow and increase the volume. Based on the concept of stacking layered structures, the organ chip can also have other different stacking formation methods.

[0090] Reference Figure 3 and Figure 4 , Figure 5 and Figure 6 For example, the organ chip may include a first layer 300 and a second layer 400, the first layer 300 and the second layer 400 are respectively attached to both sides of the culture membrane 100, the first culture chamber 310 is located in the first layer 300, and the second culture chamber 410 is located in the second layer 400. The first layer 300 and the second layer 400 sandwich the culture membrane 100 by attaching, and the area of ​​the culture membrane 100 for culturing cells is exposed to the culture chamber.

[0091] Continue to refer to Figure 3 and Figure 4 , Figure 5 and Figure 6 Since the organ chip in the embodiment is used to simulate chest expansion to cause human inhalation, the actuating membrane 200 is attached to the side of the second layer 400 away from the culture membrane 100, and the organ chip may further include a third layer 500, the actuating chamber 510 is located in the third layer 500, and the third layer 500 is attached to the side of the actuating membrane 200 away from the culture membrane 100. The second layer 400 and the third layer 500 sandwich the actuating membrane 200.

[0092] It is understandable that the bonding may be by applying a force to press or bond together, or by sticking together by glue, tape, etc. By decomposing the organ chip into multiple different layers, the processing of structures such as the culture chamber and the actuation chamber 510 can be facilitated.

[0093] Furthermore, the organ chip may also include a fourth layer 600, which is formed with a liquid inlet 610 and a liquid outlet 620, and the liquid inlet 610 and the liquid outlet 620 are connected to the culture chamber to supply cells, cell culture medium and other materials to the culture chamber.

[0094] Generally speaking, the cells cultured on both sides of the culture membrane 100 are different, and the required culture environments are different, so the liquid inlet holes 610 and the liquid outlet holes 620 are divided into two groups to supply cells, cell culture medium and other materials to the first culture chamber 310 and the second culture chamber 410 respectively. The organ chip can include only one fourth layer 600, the fourth layer 600 has two groups of liquid inlet holes 610 and two groups of liquid outlet holes 620, and one fourth layer 600 is responsible for supplying materials to the first culture chamber 310 and the second culture chamber 410 at the same time, or the organ chip can include two fourth layers 600, each of the fourth layers 600 has a group of liquid inlet holes 610 and a group of liquid outlet holes 620, and the two fourth layers 600 are responsible for supplying materials to the first culture chamber 310 and the second culture chamber 410 respectively.

[0095] Correspondingly, the organ chip is formed with a first flow channel 320 and a second flow channel 330, wherein the first flow channel 320 connects the liquid inlet 610 and the first culture chamber 310, and the second flow channel 330 connects the liquid outlet 620 and the first culture chamber 310. The organ chip is also formed with a third flow channel 420 and a fourth flow channel 430, wherein the third flow channel 420 connects the liquid inlet 610 and the second culture chamber 410, and the fourth flow channel 430 connects the liquid outlet 620 and the second culture chamber 410.

[0096] For example, refer to Figure 3 In an embodiment of the second aspect, there is only one fourth layer 600, and the fourth layer 600 is attached to the side of the first layer 300 away from the culture membrane 100, and the fourth layer 600 has two groups of liquid inlet holes 610 and two groups of liquid outlet holes 620, the two groups of liquid inlet holes 610 are respectively connected to the first culture chamber 310 and the second culture chamber 410, and the two groups of liquid outlet holes 620 are respectively connected to the first culture chamber 310 and the second culture chamber 410. At the same time, the first layer 300 forms a first flow channel 320 and a second flow channel 330, and the second layer 400 forms a third flow channel 420 and a fourth flow channel 430.

[0097] In this case, the first layer 300 also needs to be formed with a fifth flow channel 340 and a sixth flow channel 350, and the liquid inlet 610 and the liquid outlet 620 are respectively connected to the second culture chamber 410 located in the second layer 400 through the fifth flow channel 340 and the sixth flow channel 350. It goes without saying that the fifth flow channel 340 and the sixth flow channel 350 are not connected to the first culture chamber 310 located in the first layer 300 to prevent cross contamination of cells.

[0098] For ease of processing, the first flow channel 320 and the second flow channel 330 can pass through the first layer 300 in the stacking direction (i.e., longitudinal direction) of the organ chip, and the third flow channel 420 and the fourth flow channel 430 can pass through the second layer 400 in the stacking direction of the organ chip. At this time, the first flow channel 320 and the second flow channel 330 located in the first layer 300 and the third flow channel 420 and the fourth flow channel 430 located in the second layer 400 need to be staggered to avoid mutual connection between different flow channels.

[0099] In order to ensure the light transmittance of the organ chip, the materials of the first layer 300 , the second layer 400 , the third layer 500 , and the fourth layer 600 can all be polymethyl methacrylate.

[0100] For example, the actuating film 200 made of polydimethylsiloxane material can be combined with the second layer 400 and the third layer 500 by means of a plasma cleaning process. The specific assembly process is: a thin layer of polydimethylsiloxane is spin-coated on the second layer 400 and the third layer 500, and after curing at 60°C, the spin-coated polydimethylsiloxane surface of the second layer 400 and the actuating film 200 are bonded after plasma surface treatment, and the actuating film 200 is bonded to the spin-coated polydimethylsiloxane surface of the third layer 500 in the same way. The assembly order of the second layer 400 and the third layer 500 can be interchanged.

[0101] For example, the culture membrane 100 of the hydrogel material can be combined with the first layer 300 and the second layer 400 by means of tape, and the culture membrane 100 can be flattened on the first layer 300 covered with tape; or the culture membrane 100 can be bonded after applying glue (such as AB glue, UV-curable glue, and polydimethylsiloxane prepolymer, etc.) on the surface of the first layer 300. After the culture membrane 100 is combined with the first layer 300, the culture membrane 100 is combined with the second layer 400 in the same manner. It goes without saying that the order of combining the first layer 300 and the second layer 400 can be exchanged.

[0102] For example, the first layer 300 and the fourth layer 600 may be bonded together by means of a hot pressing method, or by coating the surfaces with a strong glue and then bonding them together.

[0103] The organ chip can achieve a good sealing effect by gluing it. However, when gluing, it is necessary to first apply glue to the surface of the first layer 300 to be glued, and then press the first layer 300 and the fourth layer 600 together. The undried glue may flow into the fifth flow channel 340 and the sixth flow channel 350 when squeezed, causing the fluidity of the fifth flow channel 340 and the sixth flow channel 350 to deteriorate or even become blocked. Therefore, the first layer 300 can further be formed with overflow glue grooves 360, and two groups of overflow glue grooves 360 surround the fifth flow channel 340 and the sixth flow channel 350 respectively. The overflow glue grooves 360 are used to accommodate the excess glue generated when the first layer 300 is bonded.

[0104] After the organ chip is manufactured, the deformation effect of the culture membrane 100 can be evaluated to ensure that the organ chip meets the design requirements. For example, the culture membrane 100 can be observed under a microscope under pressure, and the deformation effect can be evaluated based on the observation results; or fluorescent microspheres can be added to the culture membrane 100 in advance, and after the organ chip is assembled, the displacement process of the fluorescent microspheres can be observed under excitation light, and the deformation effect can be evaluated based on the observation results.

[0105] Reference Figure 7 The organ chip of the present application may further include a clamp 700, wherein the two clamps 700 are respectively located at the two ends of the organ chip in the longitudinal direction, and the clamps 700 clamp the rest of the organ chip (at Figure 7 The splint 700 is specifically a culture membrane 100, an actuating membrane 200, a first layer 300, a second layer 400, a third layer 500, and a fourth layer 600). The addition of the splint 700 can facilitate the carrying of the organ chip, and different positioning structures can be designed on the splint 700 as needed to facilitate the positioning and installation of the organ chip on other equipment. In the embodiment of the organ chip using a layered structure, the splint 700 can also provide a clamping force to prevent the layers of the organ chip from slipping and dislocating.

[0106] The present application also provides a method for preparing a pulmonary gas exchange model. The method for preparing a pulmonary gas exchange model comprises the following steps:

[0107] Providing the organ chip of the present application;

[0108] Inoculating and culturing lung epithelial cells on one side of the culture membrane 100;

[0109] Vascular endothelial cells are seeded and cultured on the other surface of the culture membrane 100 .

[0110] Exemplarily, the lung epithelial cells are inoculated and cultured, comprising:

[0111] The surface of the culture membrane 100 to be inoculated is facing upward, and a solution carrying lung epithelial cells is introduced into the lung epithelial cell culture chamber;

[0112] The organ chip is placed in a cell culture incubator to allow the lung epithelial cells to settle and adhere to the surface of the culture membrane 100;

[0113] Supply flowing cell culture medium to the lung epithelial cell culture chamber.

[0114] Seeding and culturing endothelial cells, including:

[0115] The surface of the culture membrane 100 to be inoculated is facing upward, and a solution carrying vascular endothelial cells is introduced into the vascular endothelial cell culture chamber;

[0116] 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 100;

[0117] Supply flowing cell culture medium to the endothelial cell culture chamber.

[0118] The static time can be set to 0.5 h to 24 h to ensure that sufficient cells are deposited on the surface of the culture membrane 100 .

[0119] For a pulmonary gas exchange model in which the culture membrane 100 uses a degradable hydrogel porous membrane, the preparation method of the pulmonary gas exchange model can be designed to culture pulmonary epithelial cells and vascular endothelial cells until the culture membrane 100 is degraded to a thickness less than or equal to 100 nm. Further, it can be designed to culture pulmonary epithelial cells and vascular endothelial cells until the culture membrane 100 is completely degraded.

[0120] Correspondingly, since the degradation of the culture membrane 100 takes a long time, in the preparation method of the pulmonary gas exchange model, it is also necessary to detect and evaluate the degradation status of the culture membrane 100 and the growth status of the pulmonary epithelial cells and vascular endothelial cells.

[0121] For example, fluorescence staining can be used to observe and evaluate the growth of lung epithelial cells and vascular endothelial cells. The specific implementation of fluorescence staining observation can refer to related technologies, such as staining and observing dead cells and living cells separately, staining and observing under pressure, etc., which will not be repeated here.

[0122] For example, the culture membrane 100 may be fluorescently labeled, so that the integrity and thickness of the culture membrane 100 can be monitored according to the fluorescent signal, thereby evaluating the degradation status of the culture membrane 100 .

[0123] In order to form an environment suitable for cell growth and simulate the intercellular matrix more realistically, before inoculating lung epithelial cells and vascular endothelial cells, the preparation method of the lung gas exchange model further includes: impregnating the entire culture membrane 100 with biomacromolecules.

[0124] Exemplarily, the entire culture membrane 100 is impregnated with biomacromolecules, including:

[0125] Passing a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide solution with a concentration ranging from 0.1% to 1% into a lung epithelial cell culture chamber and a vascular endothelial cell culture chamber;

[0126] A composite solution consisting of collagen, fibronectin and laminin is introduced into a lung epithelial cell culture chamber and a vascular endothelial cell culture chamber, wherein the collagen is type I collagen or type IV collagen;

[0127] The organ chip is left to stand and the solution is allowed to react.

[0128] A buffer solution is introduced into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber to flush out the molecules that have not stained the culture membrane 100 and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide;

[0129] The cell culture medium is introduced into the lung epithelial cell culture chamber and the vascular endothelial cell culture chamber to infiltrate the culture membrane 100 .

[0130] The present application also provides a three-dimensional deformation method based on the lung gas exchange model, which uses the lung gas exchange model of the present application. The three-dimensional deformation method includes the following steps: adjusting the pressure of the actuation chamber 510 so that the pressure of each actuation chamber 510 changes periodically synchronously according to a set curve, thereby causing the culture membrane 100 to expand and contract periodically.

[0131] The periodic expansion and contraction of the culture membrane 100 can realistically simulate the physiological activities of the alveoli, thereby allowing cells to grow and be observed in a more realistic environment, which helps to improve the accuracy of the experiment.

[0132] 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.

[0133] 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.

[0134] 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 culture chamber and at least two actuation chambers, and the organ chip comprises: a culture membrane, wherein the culture membrane separates the culture chamber into a first culture chamber and a second culture chamber; an actuating membrane, the actuating membrane separates the second culture chamber and each of the actuating chambers, the actuating chambers define a deformable region of the actuating membrane, at least two of the actuating chambers are equidistantly arranged around the axis of the culture chamber along a circle, the actuating membrane located in the actuating chamber is deformable in response to a pressure change in the actuating chamber to transmit the pressure change between the actuating chamber and the second culture chamber, and the culture membrane is deformable in response to a pressure change in the second culture chamber.

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

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

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

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

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

7. The organ chip according to claim 6, characterized in that: The cross section of the first culture chamber and / or the second culture chamber gradually increases from a side close to the culture membrane to a side away from the culture membrane.

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

9. The organ chip according to claim 8, characterized in that: The pressure holes are connected to the actuating chambers respectively through symmetrical pressure channels.

10. The organ chip according to claim 1, characterized in that: The actuating film is a polydimethylsiloxane film.

11. The organ chip according to any one of claims 1 to 10, characterized in that: The first culture chamber is a lung epithelial cell culture chamber, and the second culture chamber is a vascular endothelial cell culture chamber.

12. The organ chip according to any one of claims 1 to 10, characterized in that: The organ chip comprises a first layer and a second layer, wherein the first layer and the second layer are respectively attached to two sides of the culture membrane in the longitudinal direction, the first culture chamber is located in the first layer, and the second culture chamber is located in the second layer.

13. The organ chip according to claim 12, characterized in that: The actuating membrane is attached to a side of the second layer away from the culture membrane. The organ chip comprises a third layer. The actuating chamber is located in the third layer. The third layer is attached to a side of the actuating membrane away from the culture membrane.

14. The organ chip according to claim 13, characterized in that: The organ chip includes a fourth layer, which is attached to the side of the first layer away from the culture membrane, and the fourth layer is formed with a liquid inlet and a liquid outlet, two groups of the liquid inlet are respectively connected to the first culture chamber and the second culture chamber, and two groups of the liquid outlet are respectively connected to the first culture chamber and the second culture chamber.

15. The organ chip according to claim 14, characterized in that: The first layer is formed with at least one first flow channel and at least one second flow channel, the first flow channel is connected with the liquid inlet and the first culture chamber, and the second flow channel is connected with the liquid outlet and the first culture chamber; The second layer is formed with at least one third flow channel and at least one fourth flow channel, the third flow channel connects the liquid inlet and the second culture chamber, and the fourth flow channel connects the liquid outlet and the second culture chamber.

16. The organ chip according to claim 15, characterized in that: The first layer is also formed with at least one fifth flow channel and at least one sixth flow channel, the liquid inlet is connected to the third flow channel through the fifth flow channel, and the liquid outlet is connected to the fourth flow channel through the sixth flow channel.

17. The organ chip according to claim 16, characterized in that: The first layer is formed with glue overflow grooves, and two groups of the glue overflow grooves surround the fifth flow channel and the sixth flow channel respectively. The glue overflow grooves are used to accommodate excess glue generated when the first layer is bonded.

18. The organ chip according to claim 15, characterized in that: The first flow channel and the second flow channel penetrate the first layer in the longitudinal direction, the third flow channel and the fourth flow channel penetrate the second layer in the longitudinal direction, and the first flow channel and the second flow channel located on the first layer and the third flow channel and the fourth flow channel located on the second layer are staggered with each other.

19. The organ chip according to claim 14, characterized in that: The organ chip comprises a clamping plate, wherein two clamping plates are respectively located at two ends of the organ chip in the longitudinal direction, and the clamping plates clamp the rest of the organ chip.

20. A pulmonary gas exchange model, characterized in that: The pulmonary gas exchange model comprises the organ chip according to any one of claims 1 to 19.

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