Organ chip for constructing tissue barrier model

Abstract

The utility model relates to the technical field of biology, in particular to an organ chip for constructing a tissue barrier model, which comprises a culture component, and the culture component comprises a platy first culture layer, a platy second culture layer and a thin-film 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 utility model relates to the field of biotechnology, especially relate to a kind of organ chip for constructing tissue barrier model. BACKGROUND

[0002] Organ chip is a kind of microfluidic cell culture equipment that integrates microfluidic, tissue engineering, microelectronics, stem cell, detection technology and other technologies, which simulates the physiological function of human organs by culturing cells in an environment close to the human body microenvironment, and thus promotes the research of tissue development, organ physiology and disease etiology. Compared with traditional cell culture methods, organ chip has significant advantages, can simulate the human microenvironment, including fluid flow, mechanical stress and cell-cell interaction, thus more truly reproducing the function and physiological characteristics of organs. Secondly, organ chip can realize multi-cell type co-culture, simulate complex tissue structure and organ function, which is particularly important for studying cell-cell interaction and disease mechanism. In addition, organ chip technology has high throughput and repeatability, can perform large-scale drug screening and toxicity testing, improve experimental efficiency and reliability of results. In addition, organ chip can replace animal experiments to some extent, reduce animal use, meet ethical requirements, and reduce research costs.

[0003] Tissue barrier is an important protective barrier for the human body to defend against invasion of foreign substances. Before drugs act on target points in the body, they often need to pass through tissue barriers, and the ability of drugs to penetrate through tissue barriers and the destructiveness of drugs to tissue barriers are important indicators that need to be investigated, which has important guiding significance for determining the drug administration route, dose and dosage form. Therefore, tissue barrier models are widely used in drug testing. Organ chip constructed tissue barrier model has good human organ simulation effect, which helps to obtain more accurate experimental results.

[0004] The culture area of the existing organ chip is usually designed to be open, so as to facilitate cell inoculation and external pipeline connection. This will lead to more complex operation and greater risk of cell contamination, which is not conducive to model construction. UTILITY MODEL CONTENT

[0005] The utility model aims at providing an organ chip for constructing tissue barrier model, which is not easy to be contaminated.

[0006] To achieve the above-mentioned purpose, the utility model provides the following technical scheme:

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

[0008] The first culture layer is configured as a plate structure, and has a through-hole shaped upper layer inlet, an upper layer outlet, a lower layer inlet and a lower layer outlet; a surface of the first culture layer is formed with an upper layer groove having two ends respectively connected to the upper layer inlet and the upper layer outlet, the upper layer groove being used for culturing first cells and having a first exchange section;

[0009] The second culture layer is configured as a plate structure, and is arranged on a side of the first culture layer close to the upper layer groove; the second culture layer is formed with a lower layer groove close to a side surface of the first culture layer, the lower layer groove being used for culturing second cells and having one end connected to the lower layer inlet as a liquid inlet end and one end connected to the lower layer outlet as a liquid outlet end, the lower layer groove having 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 connecting the lower layer inlet and the lower layer groove, and a liquid outlet for connecting the lower layer outlet and the lower layer groove; the exchange membrane has micropores connecting two side surfaces thereof, and the micropores are used for material exchange.

[0011] Optionally, the upper layer groove further includes two first circular arc sections connected to two ends of the first exchange section in a straight line shape, and the lower layer groove further includes two second circular arc sections connected to two ends of the second exchange section in a straight line shape.

[0012] Optionally, a central angle of each of the first circular arc section and the second circular arc section is any value in a range from 45° to 90°; a ratio of a radius of an inner circle of the first circular arc section to a width of the upper layer groove is any value in a range from 1 to 10; and a ratio of a radius of an inner circle of the second circular arc section to a width of the lower layer groove is any value in a range from 1 to 10.

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

[0014] Optionally, a 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 chip further comprises a housing, the housing comprising a top-opened liquid storage tank and a cover cap arranged at the opening of the liquid storage tank, the liquid storage tank being internally partitioned into a first pre-storage tank, a first waste liquid tank, a second pre-storage tank and a second waste liquid tank, the first pre-storage tank being in communication with the outside only through a first through hole and a first pressure hole, the first through hole being used for connecting the upper inlet, the first pressure hole being used for pressurization, the first waste liquid tank being in communication with the outside only through a second through hole and a first air outlet hole, the second through hole being used for connecting the upper outlet, the first air outlet hole being used for connecting the atmosphere, the first pre-storage tank being in communication with the outside only through a third through hole and a second pressure hole, the third through hole being used for connecting the lower inlet, the second pressure hole being used for pressurization, the second waste liquid tank being in communication with the outside only through a fourth through hole and a second air outlet hole, the fourth through hole being used for connecting the lower outlet, the second air outlet hole being used for connecting the atmosphere.

[0016] Optionally, the housing further comprises a lower cover cap, the lower cover cap being connected to a side of the liquid storage tank away from the cover cap, and being used for clamping the first culture layer, the exchange membrane and the second culture layer between the lower cover cap and the liquid storage tank.

[0017] Optionally, the housing further comprises a first steady flow channel and a second steady flow channel, two ends of the first steady flow channel being connected to the upper inlet and the first through hole respectively, two ends of the second steady flow channel being connected to the lower inlet and the third through hole respectively.

[0018] Optionally, the organ chip comprises a plurality of groups of model units, each group of the model units comprising the upper tank and the lower tank.

[0019] The organ chip has the advantages that: the first cells are inoculated into the upper tank through the upper inlet, and the first cells grow and proliferate on the surface of the exchange membrane; the second cells are inoculated into the lower tank through the lower inlet and the liquid inlet, and the second cells grow and proliferate on the surface of the exchange membrane; the first exchange section of the upper tank and the second exchange section of the lower tank are in corresponding positions, so that the positions where the first cells and the second cells grow are opposite to each other and are separated only by the exchange membrane; the barrier structure widely existing in the body is simulated through self-arrangement and assembly of the cells; the first cells are dynamically cultured by continuously injecting the culture medium into the upper inlet, flowing through the upper tank and flowing out from the upper outlet; the second cells are dynamically cultured by continuously injecting the culture medium into the lower inlet, flowing through the lower tank and flowing out from the lower outlet; the distance between the upper tank and the lower tank and the outside is large, so that the risk of pollution of the cells caused by the external environment is reduced; after the inoculation of the cells is completed, the construction of the barrier model is automatically realized by continuously injecting the two culture media through the external power device, and the difficulty of model construction is reduced.

[0020] The above description is only a summary of the technical scheme of the present application, in order to more clearly understand the technical means of the present application, and can be implemented according to the content of the specification, the following is the preferred embodiment of the present application and the detailed description of the drawings as follows. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 A structure diagram of an organ chip shown in the present application embodiment one;

[0022] Figure 2 A structure exploded diagram of the organ chip shown in the present application embodiment one;

[0023] Figure 3 A structure diagram of the back of the first culture layer shown in the present application embodiment one;

[0024] Figure 4 A structure diagram of the back of the liquid storage tank shown in the present application embodiment one;

[0025] Figure 5 A flow chart of the dynamic culture model construction method shown in the present application embodiment one;

[0026] Figure 6 An immunofluorescence characterization diagram and a 3D reconstruction diagram of the static culture model shown in the present application embodiment one;

[0027] Figure 7 An immunofluorescence characterization diagram and a 3D reconstruction diagram of the dynamic culture model shown in the present application embodiment one;

[0028] Figure 8 A diagram of the transepithelial electrical resistance of the static culture model and the dynamic culture model changing with culture time shown in the present application embodiment one.

[0029] Legend: 1 - upper cover, 11 - first pressure hole, 12 - first gas outlet hole, 13 - second pressure hole, 14 - second gas outlet hole, 2 - sealing 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 circular 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 circular arc section, 8 - lower cover, 81 - male buckle. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be described clearly and completely below in conjunction with the drawings. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0031] In the description of the present application, it should be noted that the orientations or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as limiting the devices or elements indicated to have a specific orientation, to be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third" are only for the purpose of description, and cannot be understood as indicating or implying relative importance.

[0032] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meanings of the above terms in the present application can be understood according to the specific circumstances.

[0033] In addition, the technical features involved in the different embodiments of the utility model described below can be combined with each other as long as they do not conflict with each other.

[0034] Please see Figure 1 , Figure 2 and Figure 3 The organ chip for constructing tissue barrier model protected by the utility model application comprises a culture assembly, and the culture assembly comprises a first culture layer 5, a second culture layer 7 and an exchange film 6 arranged in sequence. The exchange film 6 in the form of a film is connected between the first culture layer 5 and the second culture layer 7 in the form of a plate. The first culture layer 5 is formed with an upper layer inlet 51, an upper layer outlet 52, a lower layer inlet 53 and a lower layer outlet 54 in the form of through holes. The surface of the first culture layer 5 close to the exchange film 6 is formed with an upper layer groove 55 for culturing first cells. The two ends of the upper layer groove 55 are respectively communicated with the upper layer inlet 51 and the upper layer outlet 52, and the upper layer groove 55 has a first exchange section 551. The exchange film 6 comprises a liquid inlet 61 corresponding to the lower layer inlet 53 and a liquid outlet 62 corresponding to the lower layer outlet 54, and the exchange film 6 has micropores communicating the two side surfaces thereof, and the micropores are used for material exchange. The second culture layer 7 is formed with a lower layer groove 71 for culturing second cells close to the side surface of the exchange film 6. One end of the lower layer groove 71 communicated with the liquid inlet 61 is a liquid inlet end 711, and one end of the lower layer groove 71 communicated with the liquid outlet 62 is a liquid outlet end 712. The lower layer groove 71 has a second exchange section 713 corresponding to the position of the first exchange section 551.

[0035] The first cells are inoculated into the upper layer groove 55 through the upper layer inlet 51, so that the first cells grow and proliferate on the surface of the exchange film 6. The second cells are inoculated into the lower layer groove 71 through the lower layer inlet 53 and the liquid inlet 61, so that the second cells grow and proliferate on the surface of the exchange film 6. Since the first exchange section 551 of the upper layer groove 55 and the second exchange section 713 of the lower layer groove 71 correspond in position, the positions where the first cells and the second cells grow are opposite to each other, and are only separated by the exchange film 6. Through self-arrangement and assembly of the cells, a barrier structure widely existing in the body is simulated. Through continuous injection of the culture medium from the upper layer inlet 51, the culture medium flows through the upper layer groove 55 and then flows out from the upper layer outlet 52, so that dynamic culture of the first cells is realized. Through continuous injection of the culture medium from the lower layer inlet 53, the culture medium flows through the lower layer groove 71 and then flows out from the lower layer outlet 54, so that dynamic culture of the second cells is realized. After the inoculation of the cells is completed, the injection of the two culture media is continuously carried out only through an external power device, so that the construction of the barrier model is autonomously realized, and the difficulty of model construction is reduced.

[0036] In some embodiments, the upper layer groove 55 further comprises two first circular arc segments 552 connected to two ends of the linear first exchange segment 551, and the lower layer groove 71 further comprises two second circular arc segments 714 connected to two ends of the linear second exchange segment 713. The circular arc structure helps to reduce the impact force of the liquid directly impacting the cells and helps to ensure the uniformity of the fluid velocity distribution, thereby stabilizing the fluid flow and ensuring the stability and bionics of the cell growth environment.

[0037] In some embodiments, the central angle of the first circular arc segment 552 and the second circular arc segment 714 is any value in the range of 45° to 90°, such as any value in the range of 45°, 60°, 75°, and 90°, which helps to prevent excessive fluid impact force due to a too small central angle, thereby damaging the cells due to excessive local pressure, and helps to prevent uneven fluid velocity distribution when the fluid is in a straight line due to a too large central angle, thereby forming a dead zone or vortex near the entrance of the straight line portion, causing a drastic change in the growth environment. The ratio of the radius of the inner circle of the first circular arc segment 552 to the width of the upper layer groove 55 is any value in the range of 1 to 10, such as any value in the range of 1, 3, 5, 7, 9, and 10; the ratio of the radius of the inner circle of the second circular arc segment 714 to the width of the lower layer groove 71 is any value in the range of 1 to 10, such as any value in the range of 1, 3, 5, 7, 9, and 10. This prevents the generation of excessive dead volume due to a too large ratio, resulting in waste of cell samples, and prevents a decrease in the buffering effect of the fluid at the entrance region of the straight line portion due to a too small ratio, resulting in unstable fluid flow.

[0038] In some embodiments, the material of the exchange membrane 6 is polycarbonate or polyethylene terephthalate, and the pore size of the micropores is any value in the range of 0.02 μm to 100 μm, such as any value in the range of 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, such as any value in the range of 5 μm, 20 μm, 35 μm, and 50 μm, which helps to simulate tissues and prevent damage to the exchange membrane 6.

[0039] In some embodiments, 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 transparent and high-bio-compatible hard materials, which can reduce the difficulty of preparation, and can be mass-produced precisely by machining, 3D printing, injection molding, and laser engraving, etc. under the premise of ensuring the convenience of observation and the stable adhesion growth of cells, thereby helping to improve the practicability of the organ chip.

[0040] In some embodiments, the organ chip further comprises a housing, the housing comprising a top-opened liquid storage tank 3 and a cover 1 covering the opening of the liquid storage tank 3, the liquid storage tank 3 being divided into 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 which are separated from each other, the first pre-storage tank 31 being in communication with the outside only through a first through hole 311 and a first pressure hole 11, the first through hole 311 being used for connecting the upper layer inlet 51, and the first pressure hole 11 being used for pressurization, the first waste liquid tank 32 being in communication with the outside only through a second through hole 321 and a first gas outlet hole 12, the second through hole 321 being used for connecting the upper layer outlet 52, and the first gas outlet hole 12 being used for connecting the atmosphere, the first pre-storage tank 31 being in communication with the outside only through a third through hole 331 and a second pressure hole 13, the third through hole 331 being used for connecting the lower layer inlet 53, and the second pressure hole 13 being used for pressurization, the second waste liquid tank 34 being in communication with the outside only through a fourth through hole 341 and a second gas outlet hole 14, the fourth through hole 341 being used for connecting the lower layer outlet 54, and the second gas outlet hole 14 being used for connecting the atmosphere. The liquid storage tank 3 provides the culture medium required for the growth of the first cells and the second cells, respectively, and collects the waste liquid generated by dynamic culture, which helps to continue the dynamic culture, and helps to ensure the sterility and stable flow of the culture medium entering the culture assembly, thereby improving the simulation capability of the organ chip to the internal environment of the human body and realizing the autonomous operation of the organ chip.

[0041] In some embodiments, the housing further comprises a lower cover 8 connected to the side of the liquid storage tank 3 away from the cover 1, for clamping 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 to realize the autonomous operation of the organ chip.

[0042] In some embodiments, the housing further comprises a first flow stabilizing channel 35 and a second flow stabilizing channel 36, the two ends of the first flow stabilizing channel 35 being connected to the upper layer inlet 51 and the first through hole 311, respectively, and the two ends of the second flow stabilizing channel 36 being connected to the lower layer inlet 53 and the third through hole 331, respectively, so that the culture medium entering the culture assembly flows stably through the flow stabilizing channels, thereby keeping the fluid shear stress at a stable level and maintaining the stability of the cell growth environment.

[0043] In some embodiments, the organ chip comprises multiple sets of model units, each set of model units comprising an upper layer of channels 55 and a lower layer of channels 71, so that multiple sets of experiments can be performed simultaneously and variables can be strictly controlled between different experiments.

[0044] For details, see the following examples.

[0045] Example 1

[0046] For details, see the following examples. Figure 1 and Figure 2 The organ chip shown in a preferred embodiment of the present application comprises a housing and a culture assembly. The housing comprises an upper cover 1, a sealing gasket 2, a liquid storage tank 3, a liquid sealing plate 4 and a lower cover 8 connected in sequence. The culture assembly is arranged between the liquid sealing plate 4 and the lower cover 8, and comprises 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 chip in this embodiment comprises three model units arranged in sequence and having the same structure, and the construction of three models can be performed simultaneously.

[0047] For details, see the following examples. Figure 2 and Figure 3The first culture layer 5 and the second culture layer 7 are integrally configured in a rectangular plate structure, and are both prepared by 3D printing using polyethylene terephthalate (PET). The first culture layer 5 is formed with three groups of structure groups arranged in sequence and used for forming model units. Each structure group includes a through-hole-shaped upper-layer inlet 51, an upper-layer outlet 52, a lower-layer inlet 53, and a lower-layer outlet 54 arranged in a rectangular direction, and further includes an upper-layer groove 55 formed on a side surface of the first culture layer 5. The upper-layer groove 55 is a symmetrical structure including a first exchange section 551 in a linear shape and two first circular arc sections 552 connected to both ends of the first exchange section 551. The two first circular arc sections 552 are both configured in a circular arc shape and curved toward the same side of the first exchange section 551. The two first circular arc sections 552 extend toward the upper-layer inlet 51 and the upper-layer outlet 52, respectively, from one end away from the first exchange section 551. In the 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 circular arc section 552 is 7000 μm, and the central angle is 90°. The second culture layer 7 is formed with a lower-layer groove 71 close to the side surface of 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 configured in a blind hole shape. The lower-layer groove 71 includes a second exchange section 713 and a second circular arc section 714. The structure of the second exchange section 713 is coincident with the structure of the first exchange section 551 and is the same as the structure of the first exchange section 551. The structure of the second circular arc section 714 is the same as the structure of the first circular arc section 552, but the bending direction is opposite to that of the first circular arc section 552. The two second circular arc sections 714 extend to form the liquid inlet end 711 and the liquid outlet end 712, respectively, from one end away from the second exchange section 713. An exchange membrane 6 is disposed between the first culture layer 5 and the second culture layer 7. The exchange membrane 6 is formed with a liquid inlet 61 corresponding to the lower-layer inlet 53 and a liquid outlet 62 corresponding to the lower-layer outlet 54. In the 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 permeation of molecules.

[0048] The first culture layer 5, the exchange film 6 and the second culture layer 7 are sealed by glue liquid, so that the sealed culture assembly is obtained. The liquid entering the culture assembly from the upper layer inlet 51 flows through the flow channel surrounded by the upper layer groove 55 and the exchange film 6, and exits the culture assembly from the upper layer outlet 52. The liquid entering the culture assembly from the lower layer inlet 53 reaches the liquid inlet end 711 of the lower layer groove 71 through the liquid inlet 61, and then flows in the flow channel surrounded by the lower layer groove 71 and the exchange film 6, and finally reaches the liquid outlet end 712 of the lower layer groove 71 through the liquid outlet 62, and exits the culture assembly from the lower layer outlet 54. Since the central angles, radii and widths of the first circular arc segment 552 and the second circular arc segment 714 are all constrained, the liquid flowing through the upper layer groove 55 and the lower layer groove 71 is uniformly distributed in speed, no dead zone or vortex is formed, and the impact of the liquid on the surfaces of the upper layer groove 55, the lower layer groove 71 and the corresponding exchange film 6 can be ensured to be small, and the pressure is uniform and stable, so that the orientation of the cultured cells is consistent.

[0049] The liquid storage tank 3 is configured as a rectangular box structure with an open top, and the inside is partitioned into three regions of the same structure, which are used to form each model unit. Each region is partitioned to form a plurality of chambers, four of which 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. The bottom of the liquid storage tank 3 is formed with 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 configured as a symmetrical structure as a whole. The upper cover 1 is configured as a cover-shaped structure with an open bottom and is matched with the liquid storage tank 3, and is detachably connected to the opening of the liquid storage tank 3, so that the top of the liquid storage tank 3 is closed. The upper cover 1 is formed with a first pressure hole 11 corresponding to the first pre-storage tank 31, a first gas 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 gas outlet hole 14 corresponding to the second waste liquid tank 34. In this embodiment, the first pressure hole 11, the first gas outlet hole 12, the second pressure hole 13 and the second gas outlet hole 14 are arranged in a rectangular shape. The flexible sealing 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 gas outlet hole 12, the second pressure hole 13 and the second gas outlet hole 14 are arranged on the sealing gasket 2. The sealing gasket 2 is arranged between the upper cover 1 and the liquid storage tank 3 to improve the sealing of the connection between the upper cover 1 and the liquid storage tank 3. In this embodiment, the liquid storage tank 3 and the upper cover 1 are both made of PET material and are prepared by 3D printing, and the sealing 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.

[0050] The first pre-stored tank 31 pre-stores the first culture medium. After pressure is applied to the first pre-stored tank 31 through the first pressurizing hole, the first culture medium flows out of the first through hole 311, enters the culture assembly through the upper layer inlet 51, and flows through the upper layer tank 55. The first culture medium then flows into the second through hole 321 from the upper layer outlet 52, enters the first waste liquid tank 32, and the first waste liquid tank 32 balances the atmospheric pressure through the first gas outlet hole 12. The second pre-stored tank 33 pre-stores the second culture medium. After pressure is applied to the second pre-stored tank 33 through the second pressurizing hole, the second culture medium flows out of the third through hole 331, enters the culture assembly through the lower layer inlet 53, and flows through the lower layer tank 71. The second culture medium then flows into the fourth through hole 341 from the lower layer outlet 54, enters the second waste liquid tank 34, and the second waste liquid tank 34 balances the atmospheric pressure through the second gas outlet hole 14. Through this structure, only by connecting the external pressurizing device to the first pressurizing hole and the second pressurizing hole, the culture assembly can be stably provided with clean first culture medium and second culture medium, which helps the organ-on-a-chip to run autonomously and continuously and stably, and facilitates the construction of the model.

[0051] Please refer to Figure 2 and Figure 4 The first stable flow channel 35 and the second stable flow channel 36 are formed on the side of the liquid storage tank 3 away from the upper cover 1. The first stable flow channel 35 includes a first flow channel 351 and a first blind hole 352, and the second stable flow 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 the first blind hole 352 and the second blind hole 362 are arranged in a rectangular shape along the second through hole 321 and the fourth through hole 341. The first flow channel 351 is configured as an elongated slot extending in a serpentine direction, with one end connected to the first through hole 311 and the other end connected to 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 the first stable flow channel 35 and the second stable flow channel 36 are both 200 μm, and the widths of the first flow channel 351 and the second flow channel 361 are both 200 μm. The liquid sealing plate 4 is arranged on the side of the liquid storage tank 3 away from the upper cover 1, and a plurality of through 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 are arranged on the liquid sealing plate 4. The liquid storage tank 3 and the liquid sealing plate 4 are sealed together as a whole by an adhesive, so that the first stable flow channel 35 and the second stable flow 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 liquid sealing plate 4, the first stable flow channel 35 and the second stable flow channel 36, the first culture medium and the second culture medium flowing out of the liquid storage tank 3 are stabilized before entering the culture assembly, so that the fluid is more stable, which helps to reduce the risk of damage to cells by the fluid and further simulates the human body environment.

[0052] In the embodiment, the first culture layer 5 is formed with a plurality of circular sealing grooves 56 on the side close to the liquid reservoir 3. Each sealing groove 56 is inlaid 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. The sealing groove 56 is used to accommodate a circular ring-shaped sealing ring matched with the sealing groove 56, so as to ensure the sealing between the liquid sealing plate 4 and the culture assembly while the liquid flows.

[0053] Please refer to Figure 1 and Figure 2 , the side of the liquid reservoir 3 is formed with a female buckle 37 protruding outside the liquid reservoir 3. The lower cover 8 is a box-shaped structure matched with the culture assembly and the liquid reservoir 3, and the side of the lower cover 8 is formed with a male buckle 81 matched with the female buckle 37, so as to be sleeved outside the liquid reservoir 3 and the culture assembly and detachably connected to the liquid reservoir 3. The culture assembly is installed in the shell only through the clamping action between the lower cover 8 and the liquid sealing plate 4. When the type of the structure model is the same, the shell can be repeatedly used, and this structure facilitates the installation and disassembly of the culture assembly. In the embodiment, the length of the organ chip is 90 cm, the width is 30 cm, and the height is 30 cm.

[0054] Please refer to Figure 5 , the method for constructing a model by dynamic culture based on the organ chip in the embodiment includes:

[0055] S100, arranging the culture assembly with the first culture layer 5 on the top, adding a suspension of first cells into the upper layer groove 55 from the upper layer inlet 51, and standing to make the first cells adhere to the exchange membrane 6.

[0056] S200, adding a suspension of second cells into the lower layer groove 71 from the lower layer inlet 53, turning over the culture assembly and standing to make the second cells adhere to the exchange membrane 6.

[0057] S300, introducing a first culture medium into the upper layer groove 55 through the upper layer inlet 51, and introducing a second culture medium into the lower layer groove 71 through the lower layer inlet 53, and culturing the cells to obtain a model.

[0058] In step S100, the first cells are intestinal epithelial cells, and the cell density is 10 7 cells / mL.

[0059] In step S200, the second cells are also intestinal epithelial cells, and the cell density is also 10 7 cells / mL.

[0060] Step S300 includes:

[0061] S310, install the culture assembly inoculated with the first cell and the second cell into the housing, add the first culture medium matched with the first cell into the first pre-storage groove 31, and add the second culture medium matched with the second cell into the second pre-storage groove 33.

[0062] S320, connect the first pressure hole 11 and the second pressure hole 13 to an external pressure device, which is a pressure pump in this embodiment. In this embodiment, the fluid shear force in the upper groove 55 and the lower groove 71 is about 0.1 dyn / cm 2 .

[0063] S330, after 3 to 7 days of culture, a relatively dense tissue barrier model can be formed.

[0064] Please refer to Figure 6 and Figure 7 , respectively, to construct the model in a static culture and a dynamic culture, obtain an immunofluorescence characterization diagram, and obtain a reconstruction diagram through 3D reconstruction. By comparison, it can be seen that dynamic culture can promote cell growth and proliferation, and the tissue barrier model formed under dynamic culture will have more dense intercellular connections.

[0065] Please refer to Figure 8 , detect the trans-epithelial electrical resistance (TEER) values of the static culture model and the dynamic culture model at different culture times, and verify the integrity of the barrier function.

[0066] Embodiment Two:

[0067] The difference between this embodiment and Embodiment One is that:

[0068] In this embodiment, the organ chip is entirely constructed of polycarbonate (PC) material, including four model units. The depth of the upper groove 55 of each model unit is 200 μm, the width is 1000 μm, the inner radius of the first circular segment 552 is 2500 μm, the central angle is 70°, the thickness of the exchange membrane 6 is 10 μm, the pore size of the micropore is 2 μm, the depth of the first steady flow channel 35 and the second steady flow channel 36 is 100 μm, and the width of the first flow channel 351 and the second flow channel 361 is 100 μm, and the length, width and height of each model unit are all 45 cm.

[0069] 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 both 10 6 cells / mL. The external pressure device is a peristaltic pump, and the fluid shear force in the upper groove 55 and the lower groove 71 is about 40 dyn / cm 2 .

[0070] In this embodiment, bright field optical characterization, secretion function characterization and molecular permeability characterization are also performed, which prove that the model obtained in this embodiment has complete barrier function.

[0071] Embodiment three:

[0072] The difference between this embodiment and embodiment one is that the first steady flow channel 35 and the second steady flow channel 36 in this embodiment are both formed on the side of the liquid sealing plate 4 close to the liquid storage groove 3.

[0073] The technical features of the above embodiments can be combined in any manner. In order to make the description simple, all possible combinations of the technical features in the above embodiments are not described, however, as long as the combinations of the technical features do not exist contradictory, they should be considered as the scope of the present disclosure.

[0074] The above embodiments only express several implementation manners of the present application, the description is relatively specific and detailed, but it should not be understood as the limitation of the scope of the present application. It should be pointed out that for those skilled in the art, without departing from the concept of the present application, some modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. An organ-on-a-chip for constructing a tissue barrier model, characterized in that, Includes a culture component, the culture component comprising: The first culture layer (5) is constructed into a plate-like structure and has a through-hole-like upper inlet (51), upper outlet (52), lower inlet (53) and lower outlet (54). The surface of the first culture layer (5) has an upper groove (55) with its two ends connected to the upper inlet (51) and the upper outlet (52) respectively. The upper 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 located on the side of the first culture layer (5) near the upper tank (55). The side of the second culture layer (7) near the first culture layer (5) has a lower tank (71) for culturing second cells. The lower tank (71) is connected to the lower inlet (53) as the liquid inlet end (711) and connected to the lower outlet (54) as the liquid outlet end (712). The lower tank (71) has a second exchange section (713) corresponding to the position of the first exchange section (551). An exchange membrane (6) is connected between the first culture layer (5) and the second culture layer (7), including a liquid inlet (61) for connecting the lower inlet (53) and the lower tank (71), and a liquid outlet (62) for connecting the lower outlet (54) and the lower tank (71). The exchange membrane (6) has micropores connecting its two sides for material exchange.

2. The organ-on-a-chip as described in claim 1, characterized in that, The upper groove (55) further includes two first arc segments (552) connected to both ends of the straight first exchange section (551), and the lower groove (71) further includes two second arc segments (714) connected to both ends of the straight second exchange section (713).

3. The organ-on-a-chip as described in claim 2, characterized in that, The central angles of the first arc segment (552) and the second arc segment (714) are both any values ​​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. 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-on-a-chip as described in 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 from 0.02μm to 100μm, and the thickness of the exchange membrane (6) is any value from 5μm to 50μm.

5. The organ-on-a-chip as described in claim 1, characterized in that, 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.

6. The organ-on-a-chip as described in claim 1, characterized in that, It also includes an outer casing, which includes a liquid storage tank (3) with a top opening and a top cover (1) covering the opening of the liquid storage tank (3). The liquid storage tank (3) is internally divided into 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), which are separated from each other. The first pre-storage tank (31) is connected to the outside only through a first through hole (311) and a first pressure hole (11). The first through hole (311) is used to connect to the upper inlet (51), and the first pressure hole (11) is used for pressurization. The first waste liquid tank (32) is connected only through a second through hole (321) and a first vent hole (14). 12) Connected to the outside world, the second through hole (321) is used to connect to the upper outlet (52), the first vent hole (12) is used to connect to the atmosphere, the first pre-storage tank (31) is connected to 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 to the lower inlet (53), the second pressure hole (13) is used for pressurization, the second waste liquid tank (34) is connected to the outside world only through the fourth through hole (341) and the second vent hole (14), the fourth through hole (341) is used to connect to the lower outlet (54), and the second vent hole (14) is used to connect to the atmosphere.

7. The organ-on-a-chip as described in claim 6, characterized in that, The outer shell also includes a lower cover (8), which is connected to the side of the liquid storage tank (3) away from the upper cover (1) and is used to hold 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).

8. The organ-on-a-chip as described in claim 6, characterized in that, The outer casing also 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 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 inlet (53) and the third through hole (331).

9. The organ-on-a-chip according to any one of claims 1 to 8, characterized in that, It includes multiple sets of model units, each set of model units including the upper groove (55) and the lower groove (71).

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