CN111575187A - Multi-organ chip and using method thereof - Google Patents

Abstract

The invention belongs to the field of organ chips, and particularly discloses a multi-organ chip and a using method thereof, wherein the chip consists of three parts, including a main body structure (100) capable of being inserted into the chip, a cover structure (200), and at least one sheet-shaped chip (300) capable of being inserted into the main body structure. Wherein the body structure (100) and the lid structure (200) are combinable and form a sealed chamber therein; meanwhile, the chip (300) in sheet form can be inserted into the sealed chamber and divide the chamber into a plurality of independent spaces. The chip designed by the invention is flexible and changeable, and the cells cultured in each independent space can exchange substances through the porous membrane, so that the chip can be applied to research works in the fields of biomedicine, new drug research and development, toxicology and the like.

Description

Multi-organ chip and using method thereof

Technical Field

The invention belongs to the field of organ chips, and particularly discloses a multi-organ chip and a using method thereof.

Background

Researchers are currently developing human organ micro-models on plastic chips and this technique, called organ chips, is considered as a substitute for animal models. While this goal remains remote, the technology is gaining increasing attention as large pharmaceutical companies begin to use these in vitro systems in drug development. The support of organ chips alleges that the chips are a more realistic model of the human body than flat thin layer cells grown in culture dishes and are more useful for drug development and testing than animal models. For example, a pulmonary chip is composed of a layer of cells, one side bathed in a blood-like culture medium and the other side exposed to air, and connected to a machine that stretches and compresses the cells to mimic respiratory motion. As research progresses, a variety of different organ chips have been developed, including, liver, intestine, lung, blood vessels, blood brain barrier, kidney, etc. Based on the above, more integrated multi-organ chips, even human body chips, have become one of the main development directions of organ chips.

Although researchers have designed a variety of multi-organ chips, these chips still dominate the flow channel, and thus require the injection of cell suspension into the microchannel during cell seeding. This operation adds some operational complexity to the user, which causes problems such as uneven cell seeding and cross contamination. In addition, at present, the organ chip mainly uses elastic material PDMS as a main material, the production period is long, the cost is high, meanwhile, the chip also needs complex operations such as sealing, punching and intubation, and is not friendly to users.

The existing organ chips are basically developed in a targeted way, the flexibility of experimental design is greatly limited by the special chip, and meanwhile, the cost of chip design and preparation is increased.

Disclosure of Invention

In view of the above disadvantages, the present invention discloses a multi-organ chip and a method for using the same, wherein a sheet-like chip can be embedded in an existing culture dish or well plate without changing the conventional cell culture operation habit. The various designs on the surface of the sheet can meet the culture modes of different cells, the existence of the porous membrane can realize the material exchange among different cells, and the plug-in assembly mode is flexible and changeable and can meet various experimental designs.

The technical scheme of the invention is as follows:

a multi-organ chip is composed of three parts, including a main structure which can be inserted into the chip, a cover structure at the side of the main structure, and at least one sheet chip which can be inserted into the main structure; the body structure and the lid structure may be combined and form a sealed chamber therein; the sheet chip can be inserted into the sealed cavity and divides the cavity into a plurality of independent spaces; the main body structure and the cover structure are respectively provided with a plurality of corresponding main body side through holes and cover side through holes, and the through holes can be used for connecting an inflow end and an outflow end of a culture medium; the main body structure and the cover structure are internally provided with a plurality of corresponding main body side clamping groove structures and cover side clamping groove structures, and the clamping groove structures are used for inserting the sheet-shaped chips.

Further, in the multi-organ chip, the sheet-like chip is circular, square or rectangular; the closed cavity structure formed by combining the main body structure and the cover structure is matched with the sheet-shaped chips, so that independent spaces formed among the sheet-shaped chips are closed; the clamping groove structure is a hard structure or an elastic structure.

Further, according to the multi-organ chip, the combination mode of the main body structure and the cover structure is selected from a snap-fit mode, a screw nut and stud connection mode or an external clamp matched with a sealing gasket.

Furthermore, in the multi-organ chip, the diameter of the through hole is 100-2000 microns; the outer side of the through hole is provided with a corresponding joint structure, the outer diameter of the joint structure is 1-3 mm, and the joint can be a luer joint or a pagoda-like joint.

Furthermore, in the multi-organ chip, the outer edge of the inner part of the chip is provided with a protruding structure corresponding to the slot structure.

Further, in the above multi-organ-chip, when the chip is circular, the diameter of the chip is 9 to 70 mm; when the sheet chip is square or rectangular, the side length is 9-70 mm, the thickness of the sheet chip is 200-2000 microns, the width of the projection structure is 200-500 microns, and the height is 200-2000 microns.

Further, in the multi-organ-chip, the surface inside the chip is of a micropore array structure, a porous membrane structure or a solid structure.

Furthermore, in the multi-organ chip, the micropores in the micropore array structure are columnar or conical, the aperture of the micropore is 1500 microns, and the depth is 200 microns 1000 microns.

Further, in the above multiple organ chip, the porous membrane in the porous membrane structure has a thickness of 10 to 150 μm and a pore size of 0.45 to 10 μm.

Further, the multi-organ-chip is made of one or more materials selected from the group consisting of PC, PMMA, PS and PET.

Further, the method for using the multi-organ chip comprises the following steps:

(1) subjecting each component of the multi-organ-chip to a sterilization process, including, but not limited to, the following: alcohol, sterilized water, ray sterilization, gas sterilization;

(2) placing the sheet chip in a cell culture dish or a pore plate, and then inoculating the required cells, wherein the chip containing the micropore array and the solid structure can only be inoculated with one cell, and the chip containing the porous membrane structure can be respectively inoculated with two cells on two sides of the chip;

(3) after the cells are well inoculated, removing the culture medium, taking out the sheet chip, and then inserting the sheet chip into the clamping groove structure of the main body structure;

(4) connecting the lid structure to the body structure;

(5) the fluid conduit is connected to the junction of the combined through-hole and the desired perfusion culture is started.

According to the technical scheme, the invention has the following beneficial effects: when the multi-organ chip disclosed by the invention is used for inoculating cells, the sheet-shaped chip can be placed in a cell culture dish or a pore plate. After the inoculation of the cells is completed, the sheet-shaped chips containing the cells are inserted into the main body structure according to the sequence required to be designed, and then the cover structures are combined to form independent culture spaces of different cells. Finally, the medium conduit is connected with the through hole, and perfusion culture is started. The chip designed by the invention is flexible and changeable, and the cells cultured in each independent space can exchange substances through the porous membrane, so that the chip can be applied to research works in the fields of biomedicine, new drug research and development, toxicology and the like.

Drawings

FIG. 1 is a side view of a body structure of a multi-organ chip prior to assembly;

FIG. 2 is a schematic side view of a multiple organ chip pre-assembly lid configuration;

FIG. 3 is a schematic side view of a multi-organ-chip pre-assembly laminar chip;

FIG. 4 is a schematic diagram of a multi-organ chip after assembly;

FIG. 5 is a schematic view of a multi-organ-chip having a thin-plate-like chip with a micro-well array structure;

FIG. 6 is a schematic view of a multi-organ chip having a thin sheet-like chip with a porous membrane structure;

FIG. 7 is a schematic diagram of a multi-organ chip having a solid structure of a thin sheet chip;

wherein: 100 body structure, 101 body side via, 102 body side card slot structure, 200 lid structure, 201 lid side via, 202 lid side card slot structure, 300 sheet chip, 301 chip internal, 302 convex structure, 303 micropore array structure, 304 porous membrane structure, 305 solid structure.

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

As shown in fig. 1 to 4, a multi-organ chip is composed of three parts, including a body structure 100 into which the chip is inserted, a lid structure 200, and at least one sheet-like chip 300 that can be inserted into the body structure 100; the body structure 100 and the lid structure 200 may be combined and form a sealed chamber therein; the chip 300 can be inserted into the sealed chamber and divided into several independent spaces; the main body structure 100 and the cover structure 200 are respectively provided with a plurality of corresponding main body side through holes 101 and cover side through holes 201, and the through holes can be used for connecting an inflow end and an outflow end of a culture medium; the main body structure 100 and the cover structure 200 contain a plurality of corresponding main body side card slot structures 102 and cover side card slot structures 202, and the card slot structures are used for inserting the sheet-like chips 300; further, the sheet-like chip 300 is circular, square or rectangular; the closed cavity structure formed by combining the main body structure 100 and the cover structure 200 is matched with the sheet-shaped chips 300, so that independent spaces formed between the sheet-shaped chips 300 are closed; the clamping groove structure is a hard structure or an elastic structure; preferably, the combination of the main body structure 100 and the cover structure 200 is selected from a snap-fit type, a nut-bolt connection or an external clamp matching with a sealing gasket; in particular, the diameter of the through hole is 100-2000 microns; the outer side of the through hole is provided with a corresponding joint structure, the outer diameter of the joint structure is 1-3 mm, and the joint can be a luer joint or a pagoda-like joint; preferably, the sheet-like chip 300 includes a chip interior 301, and a protrusion structure 302 corresponding to the card slot structure is disposed on an outer edge of the chip interior 301; further, when the sheet-like chip 300 is circular, the diameter thereof is 9 to 70 mm; when the sheet-like chip 300 is square or rectangular, the side length is 9-70 mm, the thickness of the sheet-like chip 300 is 200-; further, the surface of the chip interior 301 is a micropore array structure 303, a porous membrane structure 304 or a solid structure 305.

In operation, the unassembled parts are sterilized, cells are seeded on the sheet-like chips 300, the seeded sheet-like chips 300 are inserted into the body structure 100, the cap structure 200 is connected, and the culture medium is perfused and discharged through the body-side through-holes 101 and the cap-side through-holes 201 to form a plurality of relatively independent culture spaces, thereby forming a multi-organ chip.

Example 2

With reference to fig. 5, in the multi-organ-chip of the embodiment 1, the surface of the chip interior 301 is a micro-pore array structure 303, and the micro-pores in the micro-pore array structure 303 are columnar or conical; the aperture of the micropore is 1500 microns, and the depth is 200 microns and 1000 microns.

Example 3

With reference to fig. 6, the multi-organ-chip described in example 1, wherein the surface of the chip interior 301 is a porous membrane structure 304, the thickness of the porous membrane in the porous membrane structure 304 is 10-150 μm, and the pore size is 0.45-10 μm.

Example 4

With reference to fig. 7, a multi-organ-chip as described in example 1, wherein the surface of the chip interior 301 is a solid structure 305.

Example 5

In this embodiment, in order to construct the application method of the brain-like chip, three kinds of cells are cultured in this embodiment, and two independent spaces are required to complete the culture, so that one sheet of the sheet-like chip 300 having the micro-pore array structure 303 on the surface thereof as described in embodiment 2, one sheet of the sheet-like chip 300 having the porous membrane structure 304 as described in embodiment 3, and one sheet of the sheet-like chip 300 having the solid structure 305 as described in embodiment 4 are selected.

The method specifically comprises the following steps:

(1) performing ultraviolet sterilization treatment on the selected sheet-like chip 300;

(2) placing a sheet-like chip 300 having a microwell array structure 303 on its surface in a well plate;

(3) inoculating pluripotent stem cells into micropores of the sheet-like chip 300, inducing after cell balls are formed, and forming a brain-like tissue after differentiation;

(4) after the brain-like tissue is induced to form, the sheet-like chip 300 containing the porous membrane structure 304 is placed in another well plate; firstly, inoculating astrocytes on one side of a porous membrane, and then inoculating vascular endothelial cells on the other side of the porous membrane;

(5) chip assembly, removing all culture medium, firstly inserting the sheet chip 300 cultured with brain-like tissue into the bottom layer of the main structure 100, then inserting the sheet chip 300 inoculated with two kinds of cells and containing the porous membrane structure 304 into the second layer of clamping grooves of the main structure 100 from bottom to top, and paying attention to keep the astrocyte face down; then the sheet chip 300 with the solid structure 305 without cells is inserted into the third layer of card slots from the bottom up, and finally the cover structure 200 is connected;

(6) the fluid conduit is connected to the main body side through hole 101 and the cover side through hole 201 of the main body structure 100 and the cover structure 200, and the required perfusion culture is started, namely a chip combining the brain-like organ and the blood brain barrier is formed, and the chip can be used for observing the process that drug molecules penetrate the blood brain barrier and act on the brain.

Example 6

In this embodiment, two sheets 300 of the sheet-like chip 300 with the porous membrane structure 304 described in embodiment 3 and two sheets 300 of the sheet-like chip with the solid structure 305 described in embodiment 4 are selected and specifically performed according to the following steps:

(1) subjecting the selected chip component to an ultraviolet sterilization treatment;

(2) placing two sheet-shaped chips 300 containing porous membrane structures 304 and one sheet-shaped chip 300 containing solid structures 305 in a cell culture pore plate for cell inoculation, wherein one side of one sheet-shaped chip 300 containing solid structures 305 is inoculated with ovarian cancer cells, two sides of the porous membrane of one sheet-shaped chip 300 containing porous membrane structures 304 are respectively inoculated with intestinal cells Caco2 and vascular endothelial cells, and two sides of the porous membrane of the other sheet-shaped chip containing porous membrane structures 304 are respectively inoculated with hepatic cells HepG2 and vascular endothelial cells;

(3) assembly is performed after all cells have been seeded. Inserting the sheet-like chip 300 seeded with ovarian cancer cells into the lowermost layer of the main structure 100 with the cell-containing side facing upward; inserting the lamellar chip 300 inoculated with the hepatic cells and the vascular endothelial cells into a second layer of clamping grooves of the main structure 100 from the bottom to the top, with the inoculated vascular endothelial cell face down, inserting the lamellar chip 300 inoculated with the intestinal cells and the vascular endothelial cells into a third layer of clamping grooves of the main structure 100 from the bottom to the top, with the inoculated vascular endothelial cell face down, then inserting the lamellar chip 300 of the solid structure 305 without the cells into a fourth layer of clamping grooves from the bottom to the top, and finally connecting the cover structure 200 with the cover structure;

(4) the fluid conduit is connected to the main body side through hole 101 and the cover side through hole 201 of the main body structure 100 and the cover structure 200, and the required perfusion culture is started, namely, an intestine-liver-tumor organ combined chip is formed, and can be used for observing the absorption, metabolism and effect evaluation of tumor treatment.

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 (10)

1. A multiple organ chip, wherein said chip is composed of three parts, including a body structure (100) insertable into the chip, a cover structure (200) on the side of the body structure (100), and at least one laminar chip (300) insertable into the body structure (100); the body structure (100) and the lid structure (200) may be combined and form a sealed chamber therein; the sheet-like chip (300) can be inserted into the sealed chamber and divide the chamber into a plurality of independent spaces; the main body structure (100) and the cover structure (200) are respectively provided with a plurality of corresponding main body side through holes (101) and cover side through holes (201), and the through holes can be used for connecting an inflow end and an outflow end of a culture medium; the main body structure (100) and the cover structure (200) are internally provided with a plurality of corresponding main body side slot structures (102) and cover side slot structures (202), and the slot structures are used for inserting the sheet-shaped chips (300).

2. A multi-organ-chip according to claim 1, wherein the lamellar chip (300) is circular, square or rectangular; the closed cavity structure formed by combining the main body structure (100) and the cover structure (200) is matched with the sheet-shaped chips (300), so that independent spaces formed between the sheet-shaped chips (300) are closed; the clamping groove structure is a hard structure or an elastic structure.

3. A multiple organ chip according to claim 1, wherein the body structure (100) and the lid structure (200) are combined in a manner selected from the group consisting of snap-fit, screw-nut stud connection, and external clamp-fit sealing gasket.

4. The multi-organ chip of claim 1, wherein the diameter of said through-hole is 100-2000 μm; the outer side of the through hole is provided with a corresponding joint structure, the outer diameter of the joint structure is 1-3 mm, and the joint can be a luer joint or a pagoda-like joint.

5. A multi-organ chip according to claim 1, wherein the sheet-like chip (300) comprises a chip interior (301), the outer edge of the chip interior (301) being provided with a protruding structure (302) corresponding to the card-slot structure.

6. A multi-organ-chip according to claim 5, wherein the sheet-like chip (300) has a diameter of 9-70 mm when it is circular; when the sheet-like chip (300) is square or rectangular, the side length is 9-70 mm, the thickness of the sheet-like chip (300) is 200-.

7. A multi-organ-chip according to claim 5, characterized in that the surface of the chip interior (301) is a micro-porous array structure (303), a porous membrane structure (304) or a solid structure (305).

8. A multi-organ-chip according to claim 7, wherein the micro-wells in the micro-well array structure (303) are columnar or pyramidal; the aperture of the micropore is 1500 microns, and the depth is 200 microns and 1000 microns; the porous membrane in the porous membrane structure (304) has a thickness of 10-150 microns and a pore size of 0.45-10 microns.

9. A multi-organ-chip according to any one of claims 1 to 8, wherein the multi-organ-chip is made of a material selected from one or more of PC, PMMA, PS and PET.

10. Method of use of a multi-organ-chip according to claim 7 or 8, comprising the steps of:

(1) subjecting each component of the multi-organ-chip to a sterilization process, including, but not limited to, the following: alcohol, sterilized water, ray sterilization, gas sterilization;

(2) placing the sheet chip (300) in a cell culture dish or a pore plate, and then inoculating the required cells, wherein the chip containing the micropore array and the solid structure can only be inoculated with one cell, and the chip containing the porous membrane structure can be respectively inoculated with two cells on two sides of the chip;

(3) after the cells are well inoculated, removing the culture medium, taking out the sheet chip (300), and then inserting the sheet chip into the clamping groove structure of the main body structure (100);

(4) connecting the lid structure (200) with the body structure (100);

(5) the fluid conduit is connected to the junction of the combined through-hole and the desired perfusion culture is started.

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