CN111363682A - Organ chip with multilayer combined structure and use method thereof - Google Patents

Abstract

The invention belongs to the field of organ chips, and particularly discloses an organ chip with a multilayer combined structure and a using method thereof. After the cells are adhered and grown, the bottom layer, the middle layer and the top layer cover are combined, and an independent culture chamber is formed between the layers, so that each chamber can be subjected to dynamic perfusion culture, and meanwhile, substance exchange can be carried out between the chambers through a porous membrane. Can be applied to the research works in the fields of biomedicine, new drug research and development, toxicology and the like.

Description

Organ chip with multilayer combined structure and use method thereof

Technical Field

The invention belongs to the field of organ chips, and particularly discloses an organ chip with a multilayer combined structure and a using method thereof.

Background

In recent decades, with the convergence of microfluidic technology and biotechnology, organ chip technology formed by tissue organ culture based on microfluidic technology has become one of the hot spots in the fields of biomedicine, drug research and development, etc. The organ chip technology can realize the construction of the tissue organ module with bionic results in the chip by accurately controlling the space, fluid and the like in the chip. Currently, researchers have developed a variety of organ chips, including: liver, intestine, lung, blood vessels, Blood Brain Barrier (BBB), kidney, etc. Therefore, the development of organ chip technology will contribute to the fields of biomedicine, drug development, and the like.

However, the current organ chip still mainly has a fluid channel structure, so that a cell suspension needs to be injected into the channel during cell seeding. The operation increases the operation difficulty of a user, thereby causing the problems of uneven cell inoculation, cross contamination and the like. In addition, at present, the organ chip mainly uses the elastic material PDMS as a main material, so that the production period is long, the cost is high, and meanwhile, the chip also needs to be subjected to complex operations such as punching, intubation and the like.

Disclosure of Invention

Aiming at the defects, the invention discloses an organ chip with a multilayer combined structure and a using method thereof, which can be used for conveniently and independently carrying out cell inoculation and short-term static culture on a bottom layer and a middle layer in a split state, and then carrying out combined culture, and have simple and convenient operation.

The technical scheme of the invention is as follows:

an organ chip with a multilayer combined structure at least comprises a three-layer combined structure from bottom to top, and comprises a bottom-layer substrate structure, at least 1 number of first middle layers and a top-layer cover structure; the bottom layer base structure and the first middle layer structure are both uncovered hollow containers with certain wall thickness, and the top layer cover structure is composed of a top cover and a plug below the top cover; the first middle layer can be embedded on the bottom layer base structure, and a first culture space is formed at the bottom of the first middle layer; the top layer cover structure can be nested on the middle layer through a plug, and a second culture space is formed at the bottom; two bottom layer wall grooves are symmetrically formed in the wall of the bottom layer substrate structure; the wall of the first middle layer is symmetrically provided with first middle layer wall grooves; the top wall opening part of the first middle layer is provided with a circle of first annular structure which is larger than the diameter of the container; the first annular structure is provided with a first middle layer through hole group, and when the first middle layer is nested on the bottom layer substrate structure, the first middle layer through hole group can be communicated with the bottom layer wall groove; a top cover on the top cover structure is provided with top cover through holes which are in one-to-one correspondence with the first middle layer wall grooves of the first middle layer and the first middle layer through hole groups; when the top cover structure is embedded into the first middle layer, the plug enters the container of the first middle layer, the top cover seals the top of the first middle layer, and the top cover through holes are respectively communicated with the first middle layer wall groove and the first middle layer through hole group. According to the scheme, the organ chip disclosed by the invention can conveniently add the number of layers, so that the number of cells cultured at the same time can be flexibly changed, meanwhile, the shapes of the bottom layer substrate structure and the middle layer are not limited, the cross section of the bottom layer substrate structure and the middle layer can be circular or rectangular or other shapes, and the purpose of the invention can be realized as long as the upper layer and the lower layer keep the same shape and gradually reduce the size; meanwhile, the fluid passage may not only pass through the fluid directly, but also pass through the through hole by a hose or the like to enter the culture space to add and reduce the culture medium, and those skilled in the art can make various selections based on the solution.

Further, in the organ chip with a multilayer combined structure, a second intermediate layer is further arranged on the first intermediate layer; the second intermediate layer can be embedded on the first intermediate layer, and a third culture space is formed at the bottom; the top layer cover structure covers the second middle layer; the wall of the second middle layer is symmetrically provided with second middle layer wall grooves; the top wall opening part of the second middle layer is provided with a circle of second annular structure with the diameter larger than that of the container; the second annular structure is provided with a first middle layer through hole group and a second middle layer through hole group; when a second intermediate layer is nested on the first intermediate layer, the first intermediate layer through hole group is communicated with the first intermediate layer wall groove of the first intermediate layer, and the second intermediate layer through hole group is communicated with the first intermediate layer through hole group; when the top cover structure is embedded into the second middle layer, the plug enters the container of the second middle layer, the top cover seals the top of the second middle layer, and the top cover through hole is respectively communicated with the second middle layer wall groove, the first second middle layer through hole group and the second middle layer through hole group.

Further, in the organ chip with a multi-layer combined structure, a third intermediate layer is further arranged on the second intermediate layer, and the third intermediate layer can be embedded on the second intermediate layer and forms a fourth culture space at the bottom; the top layer cover structure covers the third middle layer; third middle layer wall grooves are symmetrically formed in the wall of the second middle layer; the top wall opening part of the second middle layer is provided with a circle of third annular structure which is larger than the diameter of the container; the third annular structure is provided with a first middle layer through hole group, a second middle layer through hole group and a third middle layer through hole group; when a third intermediate layer is nested on the second intermediate layer, the first intermediate layer through hole group is communicated with a second intermediate layer wall groove of the second intermediate layer, the second intermediate layer through hole group is communicated with the first intermediate layer through hole group, and the third intermediate layer through hole group is communicated with the second intermediate layer through hole group; when the top layer cover structure is nested in the third middle layer, the plug enters the container of the third middle layer, the top cover seals the top of the third middle layer, and the top cover through holes are respectively communicated with the third middle layer wall groove, the third middle layer through hole group I, the third middle layer through hole group II and the third middle layer through hole group III.

Further, in the organ chip with the multilayer combined structure, the inner diameter of the bottom layer substrate structure and the first middle layer is 0.5-5 cm, the depth is 1-5 cm, and the wall thickness is 0.5-5 mm; the width of the bottom layer wall groove or the first middle layer wall groove is 0.5-1 mm, and the depth is 0.2-1 mm; the outer diameter of the bottom plug of the top layer cover structure is 0.5-5 cm, and the depth is 1-5 cm.

Further, the organ chip with the multilayer combined structure is cylindrical as a whole, and the bottom of the middle layer is of a porous membrane structure. When the middle bottom part adopts a porous membrane structure, the upper culture space and the lower culture space can carry out material flow and exchange, and during drug screening, the multi-organ and multi-cell environment in a human body can be fully simulated, so that the drug screening accuracy is improved.

Further, the above organ chip with multilayer composite structure, the pore size of the porous membrane structure is 0.22-10 μm. The pore size of the porous membrane can be freely selected according to the cultured cells of the upper layer and the lower layer.

Further, the organ chip with the multilayer combined structure is made of one or more materials selected from PC, PMMA and PET. The high polymer materials such as polycarbonate, polymethyl methacrylate, polyester resin and the like are used, and the high polymer materials can be prepared and molded through 3D printing, so that the preparation is convenient, and the cost is low. The production of the chip can also be carried out in a mode of carrying out injection molding processing by opening the mold.

Further, in the above organ chip with multilayer combined structure, the bottom of the bottom layer substrate structure 100 is a plane or a surface with an array micro-pore structure; the pore diameter of the array micropore structure is 100-. The planar structure may be used to culture cells grown on a mucosal wall; the microporous structure may be used to form 3D cell spheres.

Further, in the organ chip with the multilayer combined structure, a joint is arranged on a top cover through hole of the top cover structure; the joint is a luer joint or a pagoda-like joint. The connector is connected with the through hole of the top cover, so that the pipeline, the peristaltic pump and the like can be conveniently connected, and accurate computer-controlled perfusion can be carried out.

Further, when the number of the intermediate layers is greater than one, the length of each additional intermediate layer is reduced by 200 and 1000 microns, and the outer diameter of each additional intermediate layer is matched with the inner diameter of the previous intermediate layer. The space for cell growth is reserved between the newly added middle layer and the previous layer, and the expansibility is strong.

Further, the method for using the organ chip with the multilayer combined structure comprises the following steps:

(1) sterilizing each layer of the organ chip with the multilayer combined structure by the following methods: alcohol, sterilized water, ultraviolet rays, gas sterilization, etc.;

(2) inoculating different required cells in different layers of the chip, wherein the upper side and the lower side of the porous membrane at the bottom of each middle layer can be inoculated with the cells;

(3) assembling different layers inoculated with cells, removing the culture medium before assembling, inserting the middle layer into the bottom layer in sequence, paying attention to the alignment of the groove structure and the through hole, and finally inserting the cover on the top layer, and simultaneously ensuring that all the through holes are connected correctly;

(4) the fluid conduit is connected to the connector on the cap and the desired perfusion culture is started.

According to the technical scheme, the invention has the following beneficial effects: the invention discloses an organ chip with a multilayer combined structure and a using method thereof, the organ chip with the multilayer structure designed by the invention can not change the operation habit of the existing cell inoculation when in use, and can adopt a method of inoculation first and combination later to culture cells, thereby reducing the complexity of operation; meanwhile, the organ chip with the multilayer structure is made of hard materials, is suitable for rapid production and has low cost; in addition, the design of the chip is flexible and changeable, and the requirement of joint culture of various organs can be met.

Drawings

FIG. 1 is a schematic view of an organ chip of a multi-layered composite structure of example 1 before assembly;

FIG. 2 is a schematic view of an assembled organ chip of a multi-layered combined structure according to example 1;

FIG. 3 is a top view of an intermediate layer in an organ chip of a multi-layered composite structure according to example 1;

FIG. 4 is a schematic view of an organ chip of a multi-layered combined structure of example 2 before assembly, excluding a top cover structure;

FIG. 5 is a combination diagram of the top cap structure and the second intermediate layer of the organ chip of the multi-layer composite structure according to example 2;

FIG. 6 is a combination diagram of a second intermediate layer and a third intermediate layer in an organ chip of a multi-layered composite structure according to example 3;

FIG. 7 is a schematic view of the assembled brain chip with a multi-layer structure of example 4, in which cells are grown on the bottom layer and the top layer of the porous membrane, respectively;

FIG. 8 is a schematic view of the multi-layered combined structure of the entero-hepato-tumor chip of example 5 before the assembly, wherein the bottom and top layers of the porous membrane have cells grown thereon;

wherein: 10 first culture space, 20 second culture space, 30 third culture space, 40 fourth culture space, 100 bottom floor structure, 101 bottom wall groove, 200 top lid structure, 211 top lid through hole, 210 top lid, 220 plug, 230 connector, 300 first middle layer, 301 first middle layer wall groove, 310 first ring structure, 311 first middle layer through hole group, 400 second middle layer, 401 second middle layer wall groove, 410 second ring structure, 411 second middle layer through hole group one, 412 second middle layer through hole group two, 500 third middle layer, 501 third middle layer wall groove, 510 third ring structure, 511 third middle layer through hole group one, 512 third middle layer through hole group two, 513 third middle layer through hole group three.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "clockwise", "counterclockwise", and the like, indicate orientations and positional relationships based on those shown in the drawings, and are used only for convenience of description and simplicity of description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be considered as limiting the present invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, unless otherwise specified, "a plurality" means two or more unless explicitly defined otherwise.

In the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

In the present invention, unless otherwise expressly stated or limited, "above" or "below" a first feature means that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact with each other via another feature therebetween. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly under and obliquely below the second feature, or simply meaning that the first feature is at a lesser elevation than the second feature.

Example 1

1-3, the organ chip comprises a three-layer combined structure from bottom to top, including a bottom base structure 100, a first middle layer 300 and a top cover structure 200; the bottom layer base structure 100 and the first middle layer structure 300 are both uncovered hollow containers with certain wall thickness, and the top layer cover structure 200 is composed of a top cover 210 and a plug 220 below the top cover 210; the first intermediate layer 300 may be nested on the underlying base structure 100 and the bottom forms a first culture space 10; the top lid structure 200 can be nested on the middle layer 300 by a plug 220 and form a second culture space 20 at the bottom; two bottom layer wall grooves 101 are symmetrically formed in the wall of the bottom layer substrate structure 100; the wall of the first middle layer 300 is symmetrically provided with first middle layer wall grooves 301; the top wall mouth portion of the first intermediate layer 300 has a ring of first annular structures 310 larger than the vessel diameter thereof; the first ring structure 310 is provided with a first middle layer through hole group 311, and when the first middle layer 300 is nested on the bottom layer substrate structure 100, the first middle layer through hole group 311 can be communicated with the bottom layer wall groove 101; the top cover 210 of the top cover structure 200 is provided with top cover through holes 211 corresponding to the first middle layer wall grooves 301 of the first middle layer 300 and the first middle layer through hole groups 311 one by one; when the top lid arrangement 200 is nested in the first middle tier 300, the plug 220 enters the receptacle of the first middle tier 300, the top cover 210 seals the top of the first middle tier 300, and the top cover through holes 211 communicate with the first middle tier wall slots 301 and the first middle tier through hole set 311, respectively.

When in use: firstly, the three-layer combined structure is in a separated state, different cells are respectively inoculated on the bottom of the bottom-layer substrate structure 100 and the two sides of the bottom porous membrane of the first middle layer 300, and after the cells are adhered and grown, the bottom-layer substrate structure 100, the middle layer 300 and the top-layer cover structure 200 are combined; firstly, sleeving the middle layer 300 on the bottom layer substrate structure 100, so that the first middle layer through hole group 311 on the first annular structure 310 of the middle layer 300 is aligned with the bottom layer wall groove 101 of the bottom layer substrate structure 100, and then plugging the plug 220 on the top layer cover structure 200 into the middle layer 300, so that the top cover through hole 211 on the top cover 210 is communicated with the first middle layer wall groove 301 and the first middle layer through hole group 311 of the middle layer 300; that is, the bottom wall groove 101 of the bottom base structure 100 communicates with the first middle layer through hole group 311 and then with the top cover through hole 211 to form a fluid path for an inlet liquid and a fluid path for an outlet liquid to be supplied to the first culture space 10; meanwhile, the first middle layer wall groove 301 of the middle layer 300 is communicated with the top cover through hole 211 on the top cover structure 200 to form a liquid inlet fluid passage and a liquid outlet fluid passage of the second culture space 20; so that each culture space can be independently fed with liquid and discharged with liquid.

Example 2

4-5, a multilayered composite structure organ chip, which is based on the structure of example 1, and has a second intermediate layer 400 on the first intermediate layer 300; the second intermediate layer 400 may be nested on the first intermediate layer 300 and form a third culture space 30 at the bottom; the top cover structure 200 covers the second middle layer 400; a second middle layer wall groove 401 is symmetrically formed in the wall of the second middle layer 400; the top wall mouth portion of the second intermediate layer 400 has a ring of second annular structures 410 larger than the vessel diameter thereof; a first middle layer through hole group 411 and a second middle layer through hole group 412 are arranged on the second annular structure 410; when a second intermediate layer 400 is nested on the first intermediate layer 300, the first second intermediate layer set of through holes 411 is in communication with the first intermediate layer wall grooves 301 of the first intermediate layer 300, and the second intermediate layer set of through holes 412 is in communication with the first intermediate layer set of through holes 311; when the top lid structure 200 is nested in the second middle tier 400, the plug 220 enters the container of the second middle tier 400, the top lid 210 seals the top of the second middle tier 400, and the top lid through hole 211 communicates with the second middle tier wall slot 401 and the second middle tier through hole set one 411 and the second middle tier through hole set two 412, respectively; preferably, the top cover through hole 211 of the top cover structure 200 is provided with a joint 230; the connector 230 is a pagoda-like connector.

When in use: referring to the manner of use of example 1, 3 to 5 kinds of cells can be cultured simultaneously with one more third culture space than in example 1.

Example 3

As shown in FIG. 6, in the organ-chip with multi-layer composite structure, in addition to the structure of example 2, a third intermediate layer 500 is further provided on the second intermediate layer 400, and the third intermediate layer 500 can be embedded on the second intermediate layer 400 and form a fourth culture space 40 at the bottom; the top cap structure 200 covers the third middle layer 500; third interlayer wall grooves 501 are symmetrically formed in the wall of the second interlayer 500; the top wall mouth portion of the second intermediate layer 400 has a ring of third ring-shaped formations 510 larger than the vessel diameter thereof; a third middle layer through hole group I511, a third middle layer through hole group II 512 and a third middle layer through hole group III 513 are arranged on the third annular structure 510; when a third interlayer 500 is nested on the second interlayer 400, the first interlayer through-hole set 511 is in communication with the second interlayer wall groove 401 of the second interlayer 400, the second interlayer through-hole set 512 is in communication with the first interlayer through-hole set 411, and the third interlayer through-hole set 513 is in communication with the second interlayer through-hole set 412; when the top lid arrangement 200 is nested in the third middle tier 500, the plug 220 enters the container of the third middle tier 500, the top lid 210 seals the top of the third middle tier 500, and the top lid through hole 211 communicates with the third middle tier wall channel 501 and the third middle tier through hole set one 511, the third middle tier through hole set two 512, and the third middle tier through hole set three 513, respectively; preferably, the organ chip is cylindrical as a whole, and the bottom of the middle layer is of a porous membrane structure; in particular, the pore size of the porous membrane structure is 0.22 microns; further, the organ chip is made of PMMA; further, the bottom of the bottom substrate structure 100 is a surface with an array micro-pore structure; the average pore size of the microporous structure of the array was 500 micrometers and the average depth was 500 micrometers.

When in use: referring to the manner of use of example 1, 4 to 7 kinds of cells can be cultured simultaneously with one more third culture space 30 and one more fourth culture space 40 than in example 1.

Example 4.

The brain-like chip is constructed by using the chip in the embodiment 1, referring to the attached figures 1, 2, 3 and 7, and the method specifically comprises the following steps:

(1) each layer of the organ chip of the multilayer composite structure of example 1 was subjected to ultraviolet sterilization treatment;

(2) inoculating pluripotent stem cells into a bottom wall groove 101 of a bottom substrate structure 100, and inducing after cell balls are formed to form a brain-like body;

(3) after the brain-like cells are induced to form, the astrocytes are inoculated on the porous membrane bottom layer of the first middle layer 300, after the astrocytes grow in an adhesion manner, the vascular endothelial cells are inoculated on the porous membrane upper layer of the first middle layer 300, and a blood brain barrier structure is formed on the porous membrane;

(4) assembling different parts inoculated with cells, removing the culture medium before assembling, inserting the middle layer structure into the substrate layer, paying attention to the alignment of the groove structure and the through hole, and finally inserting the cover on the top layer, and simultaneously ensuring that all the through holes are connected correctly;

(5) the fluid conduit is connected to the top cover through hole 211 of the top cover structure 200, and the required perfusion culture is started, so that a brain-like organ and blood brain barrier combined chip is formed, and the chip can be used for investigating the process that drug molecules penetrate through the blood brain barrier and act on the brain.

Example 5

The entero-hepato-tumor chip was constructed using the chip of example 2, as shown in fig. 4, 5 and 8, and specifically according to the following steps:

(1) each layer of the organ chip of the multilayer composite structure of example 2 was subjected to ultraviolet sterilization treatment;

(2) seeding ovarian cancer cells on the basal structure floor 100; vascular endothelial cells were seeded on the lower surface of the porous membrane of the first intermediate layer 300 and the lower surface of the porous membrane of the second intermediate layer 400, and hepatic cells (HepG 2) were seeded on the upper surface of the porous membrane of the first intermediate layer 300 and intestinal cells (Caco 2) were seeded on the upper surface of the porous membrane of the second intermediate layer 400 after the vascular endothelial cells were adherently grown.

(3) Assembling different parts inoculated with cells, removing the culture medium before assembling, inserting the two middle layer structures into the substrate layer in sequence, paying attention to the alignment of the groove structures and the through holes, and finally inserting the cover on the top layer, and simultaneously ensuring that all the through holes are correctly connected;

(4) the fluid conduit is connected to the connector 230 on the top cover through hole 211 of the top cover structure 200, and the required perfusion culture is started, so that an intestine-liver-tumor organ combined chip is formed, and the chip can be used for observing the absorption, metabolism and effect evaluation of tumor treatment of the medicament.

The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.

Claims (11)

1. An organ chip with a multilayer combined structure is characterized in that the organ chip at least comprises a three-layer combined structure from bottom to top, and comprises a bottom-layer base structure (100), at least 1 number of first middle layers (300) and top-layer cover structures (200); the bottom layer base structure (100) and the first middle layer structure (300) are both uncovered hollow containers with certain wall thickness, and the top layer cover structure (200) is composed of a top cover (210) and a plug (220) below the top cover (210); the first intermediate layer (300) may be nested on the underlying base structure (100) and bottom forms a first culture space (10); the top cover structure (200) can be nested on the first middle layer (300) by a plug (220) and forms a second culture space (20) at the bottom; two bottom layer wall grooves (101) are symmetrically formed in the wall of the bottom layer substrate structure (100); the wall of the first middle layer (300) is symmetrically provided with first middle layer wall grooves (301); the top wall mouth portion of the first intermediate layer (300) has a ring of first annular structure (310) larger than the container diameter thereof; the first annular structure (310) is provided with a first middle layer through hole group (311), and when the first middle layer (300) is nested on the bottom layer substrate structure (100), the first middle layer through hole group (311) can be communicated with the bottom layer wall groove (101); the top cover (210) on the top cover structure (200) is provided with top cover through holes (211) which are in one-to-one correspondence with the first middle layer wall groove (301) of the first middle layer (300) and the first middle layer through hole group (311); when the top cover structure (200) is nested into the first intermediate layer (300), the plug (220) enters the container of the first intermediate layer (300), the top cover (210) seals the top of the first intermediate layer (300), and the top cover through holes (211) are respectively communicated with the first intermediate layer wall groove (301) and the first intermediate layer through hole group (311).

2. The organ chip of a multilayer composite structure according to claim 1, wherein a second intermediate layer (400) is further provided on the first intermediate layer (300); the second intermediate layer (400) can be nested on the first intermediate layer (300) and forms a third culture space (30) at the bottom; the top layer cover structure (200) covers the second middle layer (400); a second middle layer wall groove (401) is symmetrically formed in the wall of the second middle layer (400); the top wall opening part of the second middle layer (400) is provided with a ring of second annular structures (410) which are larger than the container diameter; a first middle layer through hole group (411) and a second middle layer through hole group (412) are formed in the second annular structure (410); when a second intermediate layer (400) is nested on the first intermediate layer (300), the first second intermediate layer through hole group (411) is in communication with the first intermediate layer wall groove (301) of the first intermediate layer (300), and the second intermediate layer through hole group (412) is in communication with the first intermediate layer through hole group (311); when the top lid structure (200) is nested into the second intermediate level (400), the plug (220) enters the container of the second intermediate level (400), the top lid (210) seals the top of the second intermediate level (400), and the top lid through holes (211) communicate with the second intermediate level wall groove (401) and the first (411) and the second (412) sets of intermediate level through holes, respectively.

3. The organ chip of a multilayer composite structure according to claim 2, characterized in that a third intermediate layer (500) is further provided on the second intermediate layer (400), the third intermediate layer (500) can be embedded on the second intermediate layer (400) and forms a fourth culture space (40) at the bottom; the top layer cover structure (200) covers the third middle layer (500); third interlayer wall grooves (501) are symmetrically formed in the wall of the second interlayer (500); the top wall mouth portion of the second intermediate layer (400) has a ring of third annular formations (510) larger than the vessel diameter thereof; a third middle layer through hole group I (511), a third middle layer through hole group II (512) and a third middle layer through hole group III (513) are formed in the third annular structure (510); when a third interlayer (500) is nested on the second interlayer (400), the first interlayer through hole group (511) is communicated with the second interlayer wall groove (401) of the second interlayer (400), the second interlayer through hole group (512) is communicated with the first interlayer through hole group (411), and the third interlayer through hole group (513) is communicated with the second interlayer through hole group (412); when top layer lid structure (200) nests in third intermediate level (500), stopper (220) get into in the container of third intermediate level (500), top cap (210) seals third intermediate level (500) top, top cap through-hole (211) communicate with third intermediate level wall groove (501) and third intermediate level through-hole group one (511), third intermediate level through-hole group two (512) and third intermediate level through-hole group three (513) respectively.

4. The organ chip of a multilayer composite structure according to claim 1, wherein the bottom base structure (100) and the first intermediate layer (200) have an inner diameter of 0.5 to 5 cm, a depth of 1 to 5 cm and a wall thickness of 0.5 to 5 mm; the width of the bottom layer wall groove (101) or the first middle layer wall groove (301) is 0.5-1 mm, and the depth is 0.2-1 mm; the bottom plug (220) of the top layer cover structure (200) has an outer diameter of 0.5-5 cm and a depth of 1-5 cm.

5. The organ chip with multi-layered combined structure according to any one of claims 1 to 4, wherein the organ chip has an overall cylindrical shape, and the bottom of the intermediate layer has a porous membrane structure.

6. The organ chip of multilayer composite structure according to claim 5, wherein the pore size of said porous membrane structure is 0.22 to 10 μm.

7. The organ chip of multi-layered combined structure according to any one of claims 1 to 4, wherein the organ chip is made of a material selected from one or more of PC, PMMA and PET.

8. The organ chip with multi-layered combined structure as claimed in any one of claims 1 to 4, wherein the bottom of the underlying substrate structure 100 is planar or has a surface with an array of micro-porous structures; the pore diameter of the array micropore structure is 100-.

9. The organ chip of a multilayer combined structure according to any one of claims 1 to 4, wherein the top cover through hole (211) of the top cover structure (200) is provided with a joint (230); the connector (230) is a luer connector or a pagoda-like connector.

10. The organ chip with multi-layered combined structure as claimed in any one of claims 1 to 4, wherein when the number of said intermediate layers is more than one, the length is reduced by 200 and 1000 μm for every additional intermediate layer with an outer diameter matching the inner diameter of the previous intermediate layer.

11. The method of using the multilayer composite structured organ chip according to claim 9, comprising the steps of:

(1) sterilizing each layer of the organ chip with the multilayer combined structure by the following methods: alcohol, sterilized water, ultraviolet rays, gas sterilization, etc.;

(2) inoculating different required cells in different layers of the chip, wherein the upper side and the lower side of the porous membrane at the bottom of each middle layer can be inoculated with the cells;

(3) assembling different layers inoculated with cells, removing the culture medium before assembling, inserting the middle layer into the bottom layer in sequence, paying attention to the alignment of the groove structure and the through hole, and finally inserting the cover on the top layer, and simultaneously ensuring that all the through holes are connected correctly;

(4) the fluid conduit is connected to the connector on the cap and the desired perfusion culture is started.

Images