Organ chip for constructing tissue barrier model and model construction method

Abstract

The invention relates to the technical field of biology, in particular to an organ chip for constructing a tissue barrier model and a model construction method. The organ chip comprises a culture assembly, and the culture assembly comprises a platy first culture layer, a platy second culture layer and a thin-film-shaped exchange membrane connected between the platy first culture layer and the platy second culture layer. An upper-layer groove and a lower-layer groove are formed in the side surfaces, close to the exchange membrane, of the first culture layer and the second culture layer respectively. And the first exchange section of the upper-layer tank corresponds to the second exchange section of the lower-layer tank in position. An upper layer inlet, an upper layer outlet, a lower layer inlet and a lower layer outlet which are in a through hole shape are formed in the first culture layer. The upper-layer inlet and the upper-layer outlet are respectively communicated with two ends of the upper-layer groove, and the lower-layer inlet and the lower-layer outlet are respectively corresponding to two ends of the lower-layer groove. The exchange membrane comprises a liquid inlet corresponding to the lower layer inlet and a liquid outlet corresponding to the lower layer outlet, and a plurality of micropores for substance exchange are formed in the surface of the exchange membrane. The structure is convenient for automatic and continuous dynamic culture of cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an organ chip for constructing a tissue barrier model and a model construction method. Background Art

[0002] An organ chip is a microfluidic cell culture device integrating multiple technologies such as microfluidics, tissue engineering, microelectronics, stem cells, and detection technologies. By culturing cells in an environment close to the microenvironment in the human body, it can simulate the physiological function requirements of human organs, thereby promoting the research of tissue development, organ physiology, and disease etiology. The organ chip has significant advantages compared with traditional cell culture methods. It can simulate the human microenvironment, including fluid flow, mechanical stress, and cell-cell interactions, thus more realistically reproducing the functions and physiological characteristics of organs. Secondly, the organ chip can achieve co-culture of multiple cell types, simulating complex tissue structures and organ functions, which is particularly important for studying cell-cell interactions and disease mechanisms. In addition, the organ chip technology has high throughput and repeatability, enabling large-scale drug screening and toxicity testing, improving the experimental efficiency and the reliability of results. Moreover, the organ chip can replace animal experiments to a certain extent, reducing the use of animals, meeting ethical requirements, and at the same time reducing research costs.

[0003] Tissue barriers are important protective barriers for the human body to defend against the invasion of foreign substances. Before a drug acts on its target after entering the body, it often needs to pass through tissue barriers. The ability of the drug to penetrate through tissue barriers and its destructiveness to tissue barriers are important indicators that need to be focused on for drugs, and have important guiding significance for determining the administration route, dosage, and dosage form of drugs. Therefore, tissue barrier models are widely used in drug trials. Constructing a tissue barrier model with an organ chip has a good effect of simulating human organs and helps to obtain more accurate experimental results.

[0004] Existing organ chips are mostly connected through external pipelines. To cooperate with the external pipelines, the culture area of existing organ chips is usually designed to be open, so as to facilitate cell inoculation and the connection of external pipelines. This will lead to relatively complex operations and a high risk of cell contamination, which is not conducive to model construction. Summary of the Invention

[0005] The purpose of the present invention is to provide an organ chip for constructing a tissue barrier model that is not easily contaminated, and a model construction method.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] An organ chip for constructing a tissue barrier model, comprising a culture component, and the culture component includes:

[0008] The first culture layer is constructed as a plate-like structure and has an upper-layer inlet, an upper-layer outlet, a lower-layer inlet, and a lower-layer outlet in the shape of through holes. An upper-layer groove is formed on the surface of the first culture layer, with two ends respectively communicating with the upper-layer inlet and the upper-layer outlet. The upper-layer groove is used for culturing first cells and has a first exchange section;

[0009] The second culture layer is constructed as a plate-like structure and is arranged on one side of the first culture layer close to the upper-layer groove. A lower-layer groove for culturing second cells is formed on the side surface of the second culture layer close to the first culture layer. One end of the lower-layer groove communicating with the lower-layer inlet is the liquid inlet end, and one end communicating with the lower-layer outlet is the liquid outlet end. The lower-layer groove has a second exchange section corresponding to the position of the first exchange section;

[0010] The exchange membrane is connected between the first culture layer and the second culture layer and includes a liquid inlet for communicating the lower-layer inlet and the lower-layer groove, and a liquid outlet for communicating the lower-layer outlet and the lower-layer groove. The exchange membrane has micropores communicating the two side surfaces thereof, and the micropores are used for substance exchange.

[0011] Optionally, the upper-layer groove further includes two first arc sections connected to both ends of the linear first exchange section, and the lower-layer groove further includes two second arc sections connected to both ends of the linear second exchange section.

[0012] Optionally, the central angles of the first arc section and the second arc section are both any value in the range of 45° to 90°, the ratio of the radius of the inner circle of the first arc section to the width of the upper-layer groove is any value in the range of 1 to 10, and the ratio of the radius of the inner circle of the second arc section to the width of the lower-layer groove is any value in the range of 1 to 10.

[0013] Optionally, the material of the exchange membrane is polycarbonate or polyethylene terephthalate, the pore diameter of the micropores is any value in the range of 0.02 μm to 100 μm, and the thickness of the exchange membrane is any value in the range of 5 μm to 50 μm;

[0014] The material of the first culture layer and / or the second culture layer is one or more of polymethyl methacrylate, polycarbonate, polyethylene terephthalate, polypropylene, polyvinyl chloride, acrylonitrile-butadiene-styrene copolymer, thermoplastic polyurethane elastomer rubber, polystyrene, polysulfone, and nylon.

[0015] Optionally, the organ-on-a-chip further includes a housing detachably connected to the culture assembly. The housing includes a first pre-storage tank and a second pre-storage tank separated from each other. The first pre-storage tank is used to accommodate a first culture medium for the first cells and is connected to the upper inlet. The second pre-storage tank is used to accommodate a second culture medium for the second cells and is connected to the lower inlet.

[0016] Optionally, the housing includes a liquid storage tank with an open top and an upper cover covering the opening of the liquid storage tank. The interior of the liquid storage tank is partitioned to form the mutually separated first pre-storage tank, a first waste liquid tank, the second pre-storage tank, and a second waste liquid tank. The first pre-storage tank is only connected to the outside through a first through-hole and a first pressure hole. The first through-hole is used to connect to the upper inlet, and the first pressure hole is used for pressurization. The first waste liquid tank is only connected to the outside through a second through-hole and a first air outlet hole. The second through-hole is used to connect to the upper outlet, and the first air outlet hole is used to connect to the atmosphere. The first pre-storage tank is only connected to the outside through a third through-hole and a second pressure hole. The third through-hole is used to connect to the lower inlet, and the second pressure hole is used for pressurization. The second waste liquid tank is only connected to the outside through a fourth through-hole and a second air outlet hole. The fourth through-hole is used to connect to the lower outlet, and the second air outlet hole is used to connect to the atmosphere.

[0017] Optionally, the housing further includes a first flow stabilization channel and a second flow stabilization channel. The two ends of the first flow stabilization channel are respectively connected to the upper inlet and the first through-hole, and the two ends of the second flow stabilization channel are respectively connected to the lower inlet and the third through-hole.

[0018] Optionally, the organ-on-a-chip includes multiple groups of model units, and each group of model units includes the upper layer tank and the lower layer tank.

[0019] In a second aspect, the present invention also provides a method for constructing a model based on the above organ-on-a-chip, including:

[0020] Set the culture assembly with the first culture layer facing upward, add a suspension of the first cells into the upper layer tank from the upper inlet, and let it stand to make the first cells adhere to the exchange membrane.

[0021] Add a suspension of the second cells into the lower layer tank from the lower inlet, flip the culture assembly and let it stand to make the second cells adhere to the exchange membrane.

[0022] Introduce a first culture medium into the upper layer tank through the upper inlet and introduce a second culture medium into the lower layer tank through the lower inlet, and perform cell culture to obtain a model.

[0023] Optionally, the seeding density of the first cell and / or the second cell is any value from 10 5 cells / mL to 10 8 cells / mL. When the method of cell culture is dynamic culture, the fluid shear force in both the first exchange section and the second exchange section is any value from 0.01 dyn / cm 2 to 50 dyn / cm 2 .

[0024] According to the first aspect of the present invention: The first cell is inoculated into the upper layer tank through the upper inlet, so that the first cell grows and proliferates on the surface of the exchange membrane. The second cell is inoculated into the lower layer tank through the lower inlet and the liquid inlet, so that the second cell grows and proliferates on the surface of the exchange membrane. Since the positions of the first exchange section in the upper layer tank and the second exchange section in the lower layer tank correspond to each other, the growth positions of the first cell and the second cell are opposite to each other and are only separated by the exchange membrane. Through the self-arrangement and assembly of the cells, a barrier structure widely existing in the body is simulated. By continuously injecting the culture medium from the upper inlet, flowing through the upper layer tank and then flowing out from the upper outlet, the dynamic culture of the first cell is realized. By continuously injecting the culture medium from the lower inlet, flowing through the lower layer tank and then flowing out from the lower outlet, the dynamic culture of the second cell is realized. The distances between the upper layer tank and the lower layer tank and the outside are relatively large, reducing the risk of external environment contaminating the cells. After the cell inoculation is completed, only by continuously injecting the two culture media through the external power device, the construction of the barrier model can be autonomously realized, which helps to reduce the difficulty of model construction.

[0025] According to the second aspect of the present invention: Only through simple operations, a barrier model with high biomimicry can be obtained, which helps to improve the experimental efficiency and reduce the operation difficulty.

[0026] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it according to the content of the specification, the following takes the preferred embodiments of the present invention and combines with the drawings to describe in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic structural diagram of the organ chip shown in Embodiment 1 of the present invention;

[0028] Figure 2 is an exploded schematic structural diagram of the organ chip shown in Embodiment 1 of the present invention;

[0029] Figure 3 is a schematic structural diagram of the back surface of the first culture layer shown in Embodiment 1 of the present invention;

[0030] Figure 4 is a schematic structural diagram of the back surface of the liquid storage tank shown in Embodiment 1 of the present invention;

[0031] Figure 5 It is a flowchart of the method for constructing a dynamic culture model shown in the first embodiment of the present invention;

[0032] Figure 6 It is an immunofluorescence characterization diagram and a 3D reconstruction diagram of the static culture model shown in the first embodiment of the present invention;

[0033] Figure 7 It is an immunofluorescence characterization diagram and a 3D reconstruction diagram of the dynamic culture model shown in the first embodiment of the present invention;

[0034] Figure 8 It is a graph showing the change of the transepithelial electrical resistance of the static culture model and the dynamic culture model shown in the first embodiment of the present invention with the culture time.

[0035] Legend: 1 - upper cover, 11 - first pressure hole, 12 - first air outlet hole, 13 - second pressure hole, 14 - second air outlet hole, 2 - gasket, 21 - communication hole, 3 - liquid storage tank, 31 - first pre - storage tank, 311 - first through hole, 32 - first waste liquid tank, 321 - second through hole, 33 - second pre - storage tank, 331 - third through hole, 34 - second waste liquid tank, 341 - fourth through hole, 35 - first steady - flow channel, 351 - first flow channel, 352 - first blind hole, 36 - second steady - flow channel, 361 - second flow channel, 362 - second blind hole, 37 - female buckle, 4 - liquid - sealing plate, 41 - channel hole, 5 - first culture layer, 51 - upper - layer inlet, 52 - upper - layer outlet, 53 - lower - layer inlet, 54 - lower - layer outlet, 55 - upper - layer tank, 551 - first exchange section, 552 - first arc section, 56 - sealing groove, 6 - exchange membrane, 61 - liquid inlet, 62 - liquid outlet, 7 - second culture layer, 71 - lower - layer tank, 711 - liquid inlet end, 712 - liquid outlet end, 713 - second exchange section, 714 - second arc section, 8 - lower cover, 81 - male buckle. Detailed implementation manners

[0036] Next, the technical solutions of the present invention will be described clearly and completely in conjunction with the accompanying drawings. Obviously, the described embodiments are some, rather than all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0037] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention. In addition, the terms "first", "second", "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0038] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, the terms "installed", "connected", "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0039] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0040] Please refer to Figure 1 、 Figure 2 and Figure 3 , the organ-on-a-chip for constructing a tissue barrier model protected by the present invention application includes a culture component, and the culture component includes a first culture layer 5, a second culture layer 7 and an exchange membrane 6 stacked in sequence. The thin-film exchange membrane 6 is connected between the plate-shaped first culture layer 5 and the second culture layer 7. Through-hole-shaped upper inlets 51, upper outlets 52, lower inlets 53 and lower outlets 54 are formed on the first culture layer 5. An upper groove 55 for culturing the first cells is formed on the surface of the first culture layer 5 close to the exchange membrane 6. Both ends of the upper groove 55 communicate with the upper inlet 51 and the upper outlet 52 respectively, and have a first exchange section 551. The exchange membrane 6 includes a liquid inlet 61 corresponding to the lower inlet 53 and a liquid outlet 62 corresponding to the lower outlet 54, and the exchange membrane 6 has micropores connecting its two side surfaces, and the micropores are used for material exchange. A lower groove 71 for culturing the second cells is formed on the side surface of the second culture layer 7 close to the exchange membrane 6. One end of the lower groove 71 communicating with the liquid inlet 61 is the liquid inlet end 711, and one end communicating with the liquid outlet 62 is the liquid outlet end 712. And the lower groove 71 has a second exchange section 713 corresponding to the position of the first exchange section 551.

[0041] The first cells are inoculated into the upper slot 55 through the upper inlet 51, enabling the first cells to grow and proliferate on the surface of the exchange membrane 6. The second cells are inoculated into the lower slot 71 through the lower inlet 53 and the liquid inlet 61, enabling the second cells to grow and proliferate on the surface of the exchange membrane 6. Since the first exchange section 551 of the upper slot 55 and the second exchange section 713 of the lower slot 71 are in corresponding positions, the growth positions of the first cells and the second cells are opposite to each other, and are only separated by the exchange membrane 6. Through the self-arrangement and assembly of the cells, a barrier structure widely existing in the body is simulated. By continuously injecting the culture medium from the upper inlet 51, flowing through the upper slot 55 and then flowing out from the upper outlet 52, the dynamic culture of the first cells is achieved. The distances between the upper slot and the lower slot and the outside are relatively large, reducing the risk of external environment contaminating the cells. By continuously injecting the culture medium from the lower inlet 53, flowing through the lower slot 71 and then flowing out from the lower outlet 54, the dynamic culture of the second cells is achieved. After the cell inoculation is completed, only by continuously injecting the two culture media through the external power device, the construction of the barrier model can be autonomously realized, which helps to reduce the difficulty of model construction.

[0042] In some embodiments, the upper slot 55 further includes two first arc segments 552 connected to both ends of the linear first exchange section 551, and the lower slot 71 further includes two second arc segments 714 connected to both ends of the linear second exchange section 713. The arc-shaped structure is used to introduce and extract the liquid for the first cells and the second cells growing towards the membrane, which helps to reduce the impact force of the liquid directly hitting the cells, and helps to ensure the uniformity of the fluid velocity distribution, thereby correcting the stability of the fluid flow direction and velocity, and further ensuring the stability and biomimetic degree of the cell growth environment.

[0043] In some embodiments, the central angles of both the first arc segment 552 and the second arc segment 714 are any value in the range of 45° to 90°, for example, they can be any value among 45°, 60°, 75° and 90°. This helps to prevent the excessive impact force of the fluid caused by too small a central angle, and further damage the cells due to excessive local pressure, and helps to prevent the uneven velocity distribution when the fluid is in the linear part due to too large a central angle, resulting in the formation of dead zones or eddies near the inlet of the linear part, causing drastic changes in the growth environment. The ratio of the radius of the inner circle of the first arc segment 552 to the width of the upper slot 55 is any value in the range of 1 to 10, for example, it can be any value among 1, 3, 5, 7, 9 and 10; the ratio of the radius of the inner circle of the second arc segment 714 to the width of the lower slot 71 is any value in the range of 1 to 10, for example, it can be any value among 1, 3, 5, 7, 9 and 10. This prevents the generation of excessive dead volume due to too large a ratio, resulting in waste of cell samples, and prevents the reduction of the buffering effect of the linear part inlet area on the fluid due to too small a ratio, generating unstable liquid flow.

[0044] In some embodiments, the material of the exchange membrane 6 is polycarbonate or polyethylene terephthalate. The pore diameter of the micropores is any value in the range of 0.02 μm to 100 μm, for example, it can be any value among 0.02 μm, 0.05 μm, 0.1 μm, 1 μm, 10 μm, 60 μm, and 100 μm, which helps to intercept cells and allow some molecules to pass through. The thickness of the exchange membrane 6 is any value in the range of 5 μm to 50 μm, for example, it can be any value among 5 μm, 20 μm, 35 μm, and 50 μm, which helps to simulate tissues and prevent the exchange membrane 6 from being damaged. The material of the first culture layer 5 and / or the second culture layer 7 is one or more of polymethyl methacrylate, polycarbonate, polyethylene terephthalate, polypropylene, polyvinyl chloride, acrylonitrile-butadiene-styrene copolymer, thermoplastic polyurethane elastomer rubber, polystyrene, polysulfone, and nylon. The first culture layer 5 and the second culture layer 7 are constructed with a transparent and highly biocompatible rigid material, which reduces the preparation difficulty on the premise of ensuring the convenience of observation and the stable adherent growth of cells, and is mass-produced precisely by means of machining, 3D printing, injection molding, laser engraving, etc., which helps to improve the practicality of the organ chip.

[0045] In some embodiments, the organ chip further includes a housing, which is detachably connected to the culture assembly. The housing includes a mutually separated first pre-storage groove 31 and a second pre-storage groove 33. The first pre-storage groove 31 is used to accommodate the first culture medium for the first cells and is connected to the upper layer inlet 51. The second pre-storage groove 33 is used to accommodate the second culture medium for the second cells and is connected to the lower layer inlet 53. By providing the first pre-storage groove 31 and the second pre-storage groove 33, the culture medium is accommodated inside the organ chip and is directly connected to the culture assembly, reducing the risk of cell contamination in the organ chip, overcoming the problems of complex external pipelines and inconvenient operation, improving the single culture throughput, and preventing the influence of external factors such as the length and diameter of the pipeline on the flow rate stability of the culture medium during cultivation under low flow rate conditions.

[0046] In some embodiments, the outer shell includes a liquid storage tank 3 with an open top and an upper cover 1 covering the opening of the liquid storage tank 3. The interior of the liquid storage tank 3 is partitioned to form a first pre-storage tank 31, a first waste liquid tank 32, a second pre-storage tank 33, and a second waste liquid tank 34 that are separated from each other. The first pre-storage tank 31 is only connected to the outside through a first through hole 311 and a first pressure hole 11. The first through hole 311 is used to connect to the upper layer inlet 51, and the first pressure hole 11 is used for pressurization. The first waste liquid tank 32 is only connected to the outside through a second through hole 321 and a first air outlet hole 12. The second through hole 321 is used to connect to the upper layer outlet 52, and the first air outlet hole 12 is used to connect to the atmosphere. The first pre-storage tank 31 is only connected to the outside through a third through hole 331 and a second pressure hole 13. The third through hole 331 is used to connect to the lower layer inlet 53, and the second pressure hole 13 is used for pressurization. The second waste liquid tank 34 is only connected to the outside through a fourth through hole 341 and a second air outlet hole 14. The fourth through hole 341 is used to connect to the lower layer outlet 54, and the second air outlet hole 14 is used to connect to the atmosphere. Collecting the waste liquid generated by dynamic culture through the liquid storage tank 3 helps the continuous progress of dynamic culture, and helps ensure the sterility and flow stability of the culture medium entering the culture component, thereby improving the simulation ability of the organ chip for the human internal environment and realizing the autonomous operation of the organ chip.

[0047] In some embodiments, the outer shell further includes a first flow stabilizing channel 35 and a second flow stabilizing channel 36. The two ends of the first flow stabilizing channel 35 are respectively connected to the upper layer inlet 51 and the first through hole 311, and the two ends of the second flow stabilizing channel 36 are respectively connected to the lower layer inlet 53 and the third through hole 331. The culture medium entering the culture component flows stably through the flow stabilizing channels, so that the fluid shear force is maintained at a stable level and the cell growth environment is kept stable.

[0048] In some embodiments, the outer shell further includes a lower cover 8. The lower cover 8 is connected to the side of the liquid storage tank 3 away from the upper cover 1 and is used to clamp the first culture layer 5, the exchange membrane 6, and the second culture layer 7 between the lower cover 8 and the liquid storage tank 3, which helps the autonomous operation of the organ chip.

[0049] In some embodiments, the organ chip includes multiple groups of model units. Each group of model units includes an upper layer tank 55 and a lower layer tank 71, so as to conduct multiple groups of experiments simultaneously and strictly control variables between different experiments.

[0050] In a second aspect, the present invention also claims protection for a method for constructing a model based on the above organ chip, including:

[0051] S100: Set the culture component with the first culture layer 5 facing up, add a suspension of the first cells into the upper layer tank 55 from the upper layer inlet 51, and let it stand to make the first cells adhere to the exchange membrane 6.

[0052] S200. Add the suspension of the second cells into the lower layer tank 71 from the lower layer inlet 53, turn over the culture assembly and let it stand still to allow the second cells to adhere to the exchange membrane 6.

[0053] S300. Pass the first culture medium into the upper layer tank 55 through the upper layer inlet 51, and pass the second culture medium into the lower layer tank 71 through the lower layer inlet 53 for cell culture to obtain a model.

[0054] By simply operating, a barrier model with high mimetic property can be obtained, which helps to improve the experimental efficiency and reduce the operation difficulty.

[0055] In some embodiments, the seeding density of the first cells and / or the second cells is any value from 10 5 cells / mL to 10 8 cells / mL, for example, it can be 10 5 cells / mL, 5×10 5 cells / mL, 10 6 cells / mL, 5×10 6 cells / mL, 10 7 cells / mL, 5×10 7 cells / mL and 10 8 cells / mL. When the cell culture method is dynamic culture, the fluid shear force is any value from 0.01 dyn / cm 2 to 50 dyn / cm 2 , for example, it can be 0.01 dyn / cm 2 , 0.1 dyn / cm 2 , 1 dyn / cm 2 , 10 dyn / cm 2 , 30 dyn / cm 2 and 50 dyn / cm 2 . By restricting the seeding density and the fluid shear force, the human body internal environment is simulated and the stable growth of cells is ensured.

[0056] For details, please refer to the following embodiments.

[0057] Example 1:

[0058] Please refer to Figure 1 and Figure 2, the organ-on-a-chip shown in a preferred embodiment of the present application includes a housing and a culture component. The housing includes an upper cover 1, a gasket 2, a liquid reservoir 3, a liquid sealing plate 4, and a lower cover 8 that are stacked and connected in sequence. The culture component is disposed between the liquid sealing plate 4 and the lower cover 8, and includes a first culture layer 5 close to the liquid sealing plate 4, a second culture layer 7 close to the lower cover 8, and an exchange membrane 6 connected between the first culture layer 5 and the second culture layer 7. The organ-on-a-chip in this embodiment includes three model units with the same structure arranged in sequence, and three models can be constructed simultaneously.

[0059] Please refer to Figure 2 and Figure 3 , the first culture layer 5 and the second culture layer 7 are integrally constructed into a rectangular plate-like structure, and are both prepared by 3D printing of polyethylene terephthalate (PET). Three sets of structural groups are formed on the first culture layer 5, which are arranged in sequence and are respectively used to form the model units. Each structural group includes an upper layer inlet 51, an upper layer outlet 52, a lower layer inlet 53, and a lower layer outlet 54 in the shape of through holes arranged in the rectangular direction, and also includes an upper layer groove 55 formed on one side surface of the first culture layer 5. The upper layer groove 55 is a symmetric structure, including a linear first exchange section 551 and two first arc sections 552 connected to both ends of the first exchange section 551. Both of the two first arc sections 552 are constructed in an arc shape and bend toward the same side of the first exchange section 551. The ends of the two first arc sections 552 away from the first exchange section 551 extend toward the upper layer inlet 51 and the upper layer outlet 52 respectively. In this embodiment, the depth of the upper layer groove 55 is 300 μm, the width is 2000 μm, the radius of the inner circle of the first arc section 552 is 7000 μm, and the central angle is 90°. A lower layer groove 71 is formed on the side surface of the second culture layer 7 close to the first culture layer 5. The lower layer groove 71 has a liquid inlet end 711 corresponding to the lower layer inlet 53 and a liquid outlet end 712 corresponding to the lower layer outlet 54, and both the liquid inlet end 711 and the liquid outlet end 712 are constructed in a blind hole shape. The lower layer groove 71 includes a second exchange section 713 and a second arc section 714. The position of the structure of the second exchange section 713 coincides with that of the first exchange section 551, and the structure is the same as that of the first exchange section 551. The structure of the second arc section 714 is the same as that of the first arc section 552, but the bending direction is opposite to that of the first arc section 552. The ends of the two second arc sections 714 away from the second exchange section 713 extend to form the liquid inlet end 711 and the liquid outlet end 712 respectively. The exchange membrane 6 is separated between the first culture layer 5 and the second culture layer 7. A liquid inlet 61 is formed at a position corresponding to the lower layer inlet 53 thereon, and a liquid outlet 62 is formed at a position corresponding to the lower layer outlet 54. In this embodiment, the exchange membrane 6 is a PET microporous membrane with a thickness of 40 μm and a pore diameter of 10 μm, which is used for cell blocking and selective molecular permeation.

[0060] The first culture layer 5, the exchange membrane 6, and the second culture layer 7 are sealed by glue to obtain a sealed culture assembly. The liquid entering the culture assembly from the upper inlet 51 flows through the flow channel formed by the upper tank 55 and the exchange membrane 6 and leaves the culture assembly from the upper outlet 52. The liquid entering the culture assembly from the lower inlet 53 reaches the liquid inlet end 711 of the lower tank 71 through the liquid inlet 61, passes through the flow channel formed by the lower tank 71 and the exchange membrane 6, and then reaches the lower outlet 54 from the liquid outlet end 712 of the lower tank 71 through the liquid outlet 62 and leaves the culture assembly. Since the central angles, radii, and widths of the first arc segment 552 and the second arc segment 714 are constrained, it helps to make the liquid velocity distributions in the upper tank 55 and the lower tank 71 uniform, without forming dead zones or vortices, and can ensure that the impact of the liquid on the surfaces of the upper tank 55, the lower tank 71, and the corresponding exchange membrane 6 is small, and the pressure is relatively uniform and stable, so that the cultured cells have a consistent orientation.

[0061] The liquid storage tank 3 is constructed as a rectangular box-like structure with an open top, and its internal partition is divided into three regions with the same structure, which are respectively used to form each model unit. Each region is further partitioned to form a plurality of chambers, among which four chambers have the same volume and are completely separated from each other, namely the first pre-storage tank 31, the first waste liquid tank 32, the second pre-storage tank 33, and the second waste liquid tank 34. At the bottom of the liquid storage tank 3, there are formed a first through hole 311 corresponding to the first pre-storage tank 31, a second through hole 321 corresponding to the first waste liquid tank 32, a third through hole 331 corresponding to the second pre-storage tank 33, and a fourth through hole 341 corresponding to the second waste liquid tank 34. In this embodiment, the first through hole 311 and the third through hole 331 are arranged outside the second through hole 321 and the fourth through hole 341, and the liquid storage tank 3 is integrally constructed as a symmetric structure. The upper cover 1 is constructed as a cover-like structure with an open bottom and is fitted to the liquid storage tank 3, and is detachably connected to the opening of the liquid storage tank 3 to close the top of the liquid storage tank 3. On the upper cover 1, there are formed a first pressure hole 11 corresponding to the first pre-storage tank 31, a first air outlet hole 12 corresponding to the first waste liquid tank 32, a second pressure hole 13 corresponding to the second pre-storage tank 33, and a second air outlet hole 14 corresponding to the second waste liquid tank 34. In this implementation, the first pressure hole 11, the first air outlet hole 12, the second pressure hole 13, and the second air outlet hole 14 are arranged in a rectangular pattern. The flexible gasket 2 is attached to the inner side of the upper cover 1, and a plurality of communication holes 21 corresponding to the first pressure hole 11, the first air outlet hole 12, the second pressure hole 13, and the second air outlet hole 14 are provided thereon. The gasket 2 is arranged between the upper cover 1 and the liquid storage tank 3 to improve the sealing performance of the connection between the upper cover 1 and the liquid storage tank 3. In this embodiment, both the liquid storage tank 3 and the upper cover 1 are made of PET material and are prepared by 3D printing, and the gasket 2 is made of medical silicone material and is obtained by machining and cutting. In this embodiment, the two corners of the liquid storage tank 3 on the same side are chamfered to facilitate the identification of the direction.

[0062] The first culture medium is pre-stored in the first pre-storage tank 31. After applying pressure to the first pre-storage tank 31 through the first pressure application hole, the first culture medium flows out from the first through-hole 311, enters the culture assembly through the upper-layer inlet 51, and after flowing through the upper-layer tank 55, the first culture medium flows into the second through-hole 321 from the upper-layer outlet 52 and enters the first waste liquid tank 32. The first waste liquid tank 32 balances the atmospheric pressure through the first air outlet hole 12. The second culture medium is pre-stored in the second pre-storage tank 33. After applying pressure to the second pre-storage tank 33 through the second pressure application hole, the second culture medium flows out from the third through-hole 331, enters the culture assembly through the lower-layer inlet 53, and after flowing through the lower-layer tank 71, the second culture medium flows into the fourth through-hole 341 from the lower-layer outlet 54 and enters the second waste liquid tank 34. The second waste liquid tank 34 balances the atmospheric pressure through the second air outlet hole 14. With this structure, only by means of an external pressure application device connected to the first pressure application hole and the second pressure application hole, clean first culture medium and second culture medium can be stably provided to the culture assembly, which helps the organic chip to operate autonomously, continuously and stably, and is convenient for model construction.

[0063] Please refer to Figure 2 and Figure 4 On the side of the liquid storage tank 3 away from the upper cover 1, a first flow stabilization channel 35 and a second flow stabilization channel 36 are formed. The first flow stabilization channel 35 includes a first flow channel 351 and a first blind hole 352, and the second flow stabilization channel 36 includes a second flow channel 361 and a second blind hole 362. The diameters of the circular first blind hole 352 and the second blind hole 362 are the same as those of the second through-hole 321 and the fourth through-hole 341, and they are arranged in a rectangle along with the second through-hole 321 and the fourth through-hole 341. The first flow channel 351 is configured as a long strip-shaped channel extending in a serpentine direction, with one end communicating with the first through-hole 311 and the other end communicating with the first blind hole 352. The second flow channel 361 is symmetrically arranged with the first flow channel 351. In this embodiment, the depths of both the first flow stabilization channel 35 and the second flow stabilization channel 36 are 200 μm, and the widths of both the first flow channel 351 and the second flow channel 361 are 200 μm. The sealing liquid plate 4 is arranged on the side of the liquid storage tank 3 away from the upper cover 1, and is provided with a plurality of channel holes 41 corresponding to the first blind hole 352, the second blind hole 362, the second through-hole 321 and the fourth through-hole 341 respectively, and is sealed with the liquid storage tank 3 as a whole through an adhesive, so that the first flow stabilization channel 35 and the second flow stabilization channel 36 are closed. The position of the upper-layer inlet 51 of the first culture layer 5 corresponds to the first blind hole 352, the position of the upper-layer outlet 52 corresponds to the second through-hole 321, the position of the lower-layer inlet 53 corresponds to the second blind hole 362, and the position of the lower-layer outlet 54 corresponds to the fourth through-hole 341. Through the sealing liquid plate 4, the first flow stabilization channel 35 and the second flow stabilization channel 36, the first culture medium and the second culture medium flowing out from the liquid storage tank 3 enter the culture assembly after flow stabilization, so that the fluid is more stable, which helps to reduce the risk of damage to cells caused by the fluid and further simulate the human body environment.

[0064] In this embodiment, a plurality of circular sealing grooves 56 are formed on the side of the first culture layer 5 close to the liquid storage tank 3. Each of the sealing grooves 56 is embedded in the first culture layer 5 and is concentrically arranged with the upper layer inlet 51, the upper layer outlet 52, the lower layer inlet 53 and the lower layer outlet 54 respectively. The sealing groove 56 is used to accommodate an annular sealing ring that fits into the sealing groove 56, ensuring the sealing between the liquid sealing plate 4 and the culture assembly while allowing liquid to flow.

[0065] Please refer to Figure 1 and Figure 2 , a male buckle 37 protruding from the outside of the liquid storage tank 3 is formed on the side of the liquid storage tank 3. The lower cover 8 is a box-shaped structure that structurally fits the culture assembly and the liquid storage tank 3. A female buckle 81 that fits the male buckle 37 is formed on its side, so that it is sleeved on the outside of the liquid storage tank 3 and the culture assembly and is detachably connected to the liquid storage tank 3. The culture assembly is installed in the outer shell only through the clamping action between the lower cover 8 and the liquid sealing plate 4. When the types of the constructed models are the same, the outer shell can be reused multiple times, and this structure facilitates the installation and disassembly of the culture assembly. In this embodiment, the length of the organ chip is 90 cm, the width is 30 cm, and the height is 30 cm.

[0066] Please refer to Figure 5 , the method for constructing a model by dynamic culture based on the organ chip in this embodiment includes:

[0067] S100. Set the culture assembly with the first culture layer 5 on the top, add a suspension of the first cells into the upper layer tank 55 from the upper layer inlet 51, and let it stand to allow the first cells to adhere to the exchange membrane 6.

[0068] S200. Add a suspension of the second cells into the lower layer tank 71 from the lower layer inlet 53, flip the culture assembly and let it stand to allow the second cells to adhere to the exchange membrane 6.

[0069] S300. Pass a first culture medium into the upper layer tank 55 through the upper layer inlet 51, and pass a second culture medium into the lower layer tank 71 through the lower layer inlet 53 to perform cell culture and obtain a model.

[0070] In step S100, the first cells are intestinal epithelial cells, and the cell density is 10 7 cells / mL.

[0071] In step S200, the second cells are also intestinal epithelial cells, and the cell density is also 10 7 cells / mL.

[0072] Step S300 includes:

[0073] S310. Install the culture component inoculated with the first cell and the second cell into the outer shell, add the first culture medium compatible with the first cell into the first pre-storage tank 31, and add the second culture medium compatible with the second cell into the second pre-storage tank 33.

[0074] S320. Connect the first pressure hole 11 and the second pressure hole 13 to an external pressurizing device. In this embodiment, the external pressurizing device is a pressure pump. In this embodiment, the fluid shear stress in the upper layer tank 55 and the lower layer tank 71 is about 0.1 dyn / cm 2 .

[0075] S330. After culturing for 3 to 7 days, a relatively dense tissue barrier model can be formed.

[0076] Please refer to Figure 6 and Figure 7 , and construct the model in static culture and dynamic culture modes respectively to obtain immunofluorescence characterization diagrams, and obtain the reconstruction diagrams through 3D restoration. It can be seen by comparison that dynamic culture can promote cell growth and proliferation, and the tissue barrier model formed under dynamic culture will have denser intercellular connections.

[0077] Please refer to Figure 8 , and detect the trans-epithelial electrical resistance (TEER) values of the static culture model and the dynamic culture model at different culture times to verify the integrity of the barrier function.

[0078] Example 2:

[0079] The difference between this embodiment and Example 1 is that:

[0080] In this embodiment, the whole organ chip is constructed of polycarbonate (PC) material and includes four model units. Among them, the depth of the upper layer tank 55 of each model unit is 200 μm, the width is 1000 μm, the radius of the inner circle of the first arc segment 552 is 2500 μm, the central angle is 70°, the thickness of the exchange membrane 6 is 10 μm, the pore diameter of the micropores is 2 μm, the depths of the first steady flow channel 35 and the second steady flow channel 36 are both 100 μm, and the widths of the first flow channel 351 and the second flow channel 361 are both 100 μm, and the length, width and height of each model unit are all 45 cm.

[0081] In this embodiment, the first cell is a trophoblast cell, the second cell is a human umbilical vein endothelial cell, and the cell density is 10 6 cells / mL. The external pressurizing device is a peristaltic pump, and the fluid shear stress in the upper layer tank 55 and the lower layer tank 71 is controlled to be about 40 dyn / cm 2 .

[0082] In this embodiment, bright-field optical characterization, secretion function characterization, and molecular permeability characterization were also carried out, demonstrating that the model obtained in this embodiment has a complete barrier function.

[0083] Embodiment Three:

[0084] The difference between this embodiment and Embodiment One lies only in that: both the first steady-flow channel 35 and the second steady-flow channel 36 in this embodiment are formed on the side of the liquid-sealing plate 4 close to the liquid storage tank 3.

[0085] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0086] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. An organ chip for constructing a tissue barrier model, characterized in that: The invention comprises a culture component, wherein the culture component comprises: The first culture layer (5) is constructed in a plate-like structure and has a through-hole-shaped upper layer inlet (51), an upper layer outlet (52), a lower layer inlet (53) and a lower layer outlet (54); an upper layer groove (55) is formed on the surface of the first culture layer (5) with two ends respectively connected to the upper layer inlet (51) and the upper layer outlet (52); the upper layer groove (55) is used to culture the first cell and has a first exchange section (551); The second culture layer (7) is constructed in a plate-like structure and is arranged on a side of the first culture layer (5) close to the upper groove (55); a lower groove (71) for culturing second cells is formed on the side of the second culture layer (7) close to the first culture layer (5); one end of the lower groove (71) connected to the lower layer inlet (53) is a liquid inlet end (711), and one end of the lower groove (71) connected to the lower layer outlet (54) is a liquid outlet end (712); the lower groove (71) has a second exchange section (713) corresponding to the position of the first exchange section (551); The exchange membrane (6) is connected between the first culture layer (5) and the second culture layer (7), and comprises a liquid inlet (61) for connecting the lower layer inlet (53) and the lower layer groove (71), and a liquid outlet (62) for connecting the lower layer outlet (54) and the lower layer groove (71). The exchange membrane (6) has micropores connecting the two side surfaces thereof, and the micropores are used for material exchange.

2. The organ chip according to claim 1, characterized in that: The upper layer groove (55) further includes two first arc segments (552) connected to the two ends of the first linear exchange segment (551), and the lower layer groove (71) further includes two second arc segments (714) connected to the two ends of the second linear exchange segment (713).

3. The organ chip according to claim 2, characterized in that: The central angles of the first arc segment (552) and the second arc segment (714) are both any value between 45° and 90°, the ratio of the radius of the inner circle of the first arc segment (552) to the width of the upper groove (55) is any value between 1 and 10, and the ratio of the radius of the inner circle of the second arc segment (714) to the width of the lower groove (71) is any value between 1 and 10.

4. The organ chip according to claim 1, characterized in that: The material of the exchange membrane (6) is polycarbonate or polyethylene terephthalate, the pore size of the micropores is any value between 0.02 μm and 100 μm, and the thickness of the exchange membrane (6) is any value between 5 μm and 50 μm; The material of the first culture layer (5) and / or the second culture layer (7) is one or more of polymethyl methacrylate, polycarbonate, polyethylene terephthalate, polypropylene, polyvinyl chloride, acrylonitrile-butadiene-styrene copolymer, thermoplastic polyurethane elastomer rubber, polystyrene, polysulfone and nylon.

5. The organ chip according to claim 1, characterized in that: The invention also comprises a shell, which is detachably connected to the culture component, and comprises a first pre-storage tank (31) and a second pre-storage tank (33) which are separated from each other, wherein the first pre-storage tank (31) is used to accommodate a first culture medium matched to the first cells and is connected to the upper layer inlet (51), and the second pre-storage tank (33) is used to accommodate a second culture medium matched to the second cells and is connected to the lower layer inlet (53).

6. The organ chip according to claim 5, characterized in that: The housing comprises a liquid storage tank (3) with an opening at the top and an upper cover (1) covering the opening of the liquid storage tank (3); the liquid storage tank (3) is internally divided into the first pre-storage tank (31), the first waste liquid tank (32), the second pre-storage tank (33) and the second waste liquid tank (34) which are separated from each other; the first pre-storage tank (31) is connected to the outside only through the first through hole (311) and the first pressure hole (11); the first through hole (311) is used to connect to the upper layer inlet (51); the first pressure hole (11) is used to pressurize; the first waste liquid tank (32) is connected to the outside only through the second through hole (321) and the first air outlet (11); 2) is connected with the outside world, the second through hole (321) is used to connect with the upper layer outlet (52), the first air outlet (12) is used to connect with the atmosphere, the first pre-storage tank (31) is connected with the outside world only through the third through hole (331) and the second pressure hole (13), the third through hole (331) is used to connect with the lower layer inlet (53), the second pressure hole (13) is used for pressurization, the second waste liquid tank (34) is connected with the outside world only through the fourth through hole (341) and the second air outlet (14), the fourth through hole (341) is used to connect with the lower layer outlet (54), and the second air outlet (14) is used to connect with the atmosphere.

7. The organ chip according to claim 5, characterized in that: The shell further comprises a first steady flow channel (35) and a second steady flow channel (36), wherein two ends of the first steady flow channel (35) are respectively connected to the upper layer inlet (51) and the first through hole (311), and two ends of the second steady flow channel (36) are respectively connected to the lower layer inlet (53) and the third through hole (331).

8. The organ chip according to any one of claims 1 to 7, characterized in that: It comprises a plurality of groups of model units, and each group of the model units comprises the upper layer groove (55) and the lower layer groove (71).

9. A method for constructing a model based on an organ chip according to any one of claims 1 to 8, characterized in that: include: The culture component is arranged in a direction where the first culture layer (5) is on top, a suspension of the first cells is added into the upper tank (55) from the upper inlet (51), and the suspension is allowed to stand to allow the first cells to adhere to the exchange membrane (6); Adding the second cell suspension into the lower tank (71) from the lower inlet (53), turning over the culture component and leaving it to stand, so that the second cells adhere to the exchange membrane (6); A first culture medium is introduced into the upper tank (55) through the upper inlet (51), and a second culture medium is introduced into the lower tank (71) through the lower inlet (53) to perform cell culture to obtain a tissue barrier model.

10. The method according to claim 9, characterized in that The seeding density of the first cell and / or the second cell is 10 5 / mL~10 8 When the cell culture method is dynamic culture, the shear force of the fluid in the first exchange section (551) and the second exchange section (713) is 0.01 dyn / cm 2 ~50dyn / cm 2 Any value in .

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