CN115612609A

CN115612609A - Integrated organ chip and preparation method thereof

Info

Publication number: CN115612609A
Application number: CN202211177557.3A
Authority: CN
Inventors: 朱楚洪; 柯明
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2022-09-26
Filing date: 2022-09-26
Publication date: 2023-01-17
Also published as: WO2024066251A1

Abstract

The invention relates to an integrated organ chip and a preparation method thereof, wherein the integrated organ chip is used for applying electrical stimulation and mechanical stimulation according to requirements in the process of culturing cells, cell clusters or organoids; the integrated organ chip includes: an electrode layer for providing electrical stimulation, a microporous layer for culturing positioned above the electrode layer, a flow cavity layer for providing mechanical stimulation and interacting with an external connector; a peripheral circuit; the electrode layer includes: the substrate is positioned on the substrate and corresponds to the bare electrode of each micropore in the micropore layer; the microporous layer includes: the structure is adhered on the electrode layer and is provided with a plurality of independent single-hole cavities, each single hole is a transparent single electrode, and the bottom of each single hole corresponds to a bare electrode of the electrode layer or a plurality of electrodes to form a multi-electrode; the flow cavity layer is a structure which is adhered above the micropore layer and is provided with a plurality of single cavities for bearing the culture medium to which the culture belongs. The above methods may combine a variety of regulatory techniques to meet organoid or cell mass culture and promote functional maturation.

Description

Integrated organ chip and preparation method thereof

Technical Field

The invention relates to biomedicine and microflow technology, in particular to an integrated organ chip and a preparation method thereof.

Background

When organoids or cell clusters are cultured in vitro, the cells may need external electrical or mechanical stimulation to regulate and control the cells so that the functions of the cells tend to be mature. The integrated organ chip which takes cells as main culture can realize the function by multi-factor stimulation regulation and control based on the micro-fluidic technology in vitro. The integrated organ chip system assembled by the microfluidic devices with single functions can meet the requirements of dynamic and static culture of cultures and electrical stimulation and mechanical stimulation in the culture process, and the integrated microfluidic chip is widely applied to culture and regulation of cells, cell clusters and organoids at present.

Patent application with prior publication number CN113667603A discloses a liver organoid culture chip, its preparation method and application, the liver organoid culture chip comprises: a cell culture plate; a biomaterial having an array of microwells; the culture method comprises digesting human embryonic stem cells or human induced multifunctional stem cells into single cells, inoculating the single cells into a culture medium, and culturing to obtain foregut embryonic cells; and digesting the foregut embryonic cells into single cells, inoculating the single cells into the micropore array in the liver organoid culture chip, and culturing to obtain the liver organoid. While the above methods can be used for uniform and high throughput liver organoid culture from different sources or of different tissue types, they do not incorporate regulatory techniques to promote organoid maturation and require external perfusion devices. In practical applications, promoting functional maturation of organoids cultured in vitro is critical for further organoid culture and development.

Therefore, how to combine multiple control technologies to satisfy organoid culture and promote functional maturation of the integrated organ chip is a technical problem that needs to be solved at present.

Disclosure of Invention

Technical problem to be solved

In view of the above-mentioned drawbacks and deficiencies of the prior art, the present invention provides an integrated organ chip and a method for manufacturing the same.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

in a first aspect, embodiments of the present invention provide an integrated organ chip for applying electrical and mechanical stimuli on demand during the culture of cells, cell clusters or organoids; the integrated organ chip includes:

the micro-porous electrode comprises an electrode layer for providing electrical stimulation, a micro-porous layer positioned above the electrode layer, and a flow cavity layer for providing mechanical stimulation and interacting with an external connector; a peripheral circuit;

the electrode layer includes: the substrate is positioned on the substrate and corresponds to the bare electrode of each micropore in the micropore layer; the bare electrode is electrically connected with a peripheral circuit by means of a conductive component nested on the surface of the substrate; the non-bare electrode area on the surface of the substrate is an insulating area;

the microporous layer includes: the structure is adhered to the electrode layer and is provided with a plurality of independent single-hole chambers, the single hole in each single-hole chamber is a through bare electrode, the bottom of the bare electrode corresponds to the electrode layer, and the electrified bare electrode applies adjustable electrical stimulation to the culture in the single-hole chamber;

the flowing cavity layer is a structure which is adhered above the micropore layer and is provided with a plurality of cavities and used for bearing a culture medium to which the culture belongs, the culture medium flows in the respective cavity structure under the assistance of auxiliary equipment so as to obtain controllable fluid shear stress, and the cavity of each cavity structure is communicated with a plurality of single-pore chambers of the micropore layer and provides the culture medium for the culture in the single-pore chambers.

Optionally, each bare electrode is a sheet-shaped circular bare electrode; each bare electrode on the substrate is mutually independent and connected with a peripheral circuit; the diameter of the round bare electrode is smaller than the aperture of each single hole in the microporous layer;

the substrate includes: the glass substrate, the conductive component layer laminated on the glass substrate and the insulating layer of the non-bare electrode area; the conductive assembly of the conductive assembly layer is a metal laminated conducting wire, one end of the conducting wire is connected with the bare electrode, and the other end of the conducting wire is provided with a metal bonding pad with a mark for being electrically connected with a peripheral circuit.

Optionally, the glass base is 4-inch circular high-light-transmittance glass with the thickness of 500+ -20um;

the diameter of the round bare electrode is 70+ -10um;

the insulating layer is made of polyimide and has a thickness less than or equal to 2um;

and/or the materials of the microporous layer and the flow cavity layer are consistent.

Optionally, the microporous layer is a PDMS porous membrane layer formed by punching holes after curing polydimethylsiloxane polymer PDMS;

the aperture of each single hole is 1mm to 6mm;

the microporous layer is characterized in that a plurality of single-hole chambers of the microporous layer are arranged in a plurality of rows, each row is provided with N single-hole chambers, N is greater than or equal to 2, and the bottom of each single-hole chamber corresponds to one or more bare electrodes.

Optionally, the cavity structure of the flow cavity layer is a single cavity structure having a rectangular parallelepiped shape; each cuboid-shaped single cavity structure corresponds to one row of the single-hole chambers; the end of the cuboid single cavity structure is provided with a culture medium inlet, and the end tail is provided with a culture medium outlet;

or,

the flowing cavity layer is a female die of a single cavity structure with a plurality of cuboid shapes formed by film pouring and curing of PDMS prepolymer on a tungsten steel bar male die; each cuboid-shaped single cavity structure corresponds to one row of the single-hole chambers; and the end of the single cavity structure in the shape of a cuboid is provided with a culture medium inlet, and the end tail is provided with a culture medium outlet.

Optionally, the cuboid shaped single cavity structure is 15mm long, 6mm wide, 1mm deep;

the aperture of the culture medium inlet/the culture medium outlet is 1mm-5mm;

or,

the auxiliary device includes: peripheral perfusion equipment or a warped plate shaker for realizing the flowing of the culture medium.

In a second aspect, an embodiment of the present invention provides a method for preparing an integrated organ chip according to any one of the first aspect, including:

s01, sequentially manufacturing the electrode layer, the microporous layer and the flow cavity layer;

s02, bonding the microporous layer and the flow cavity layer together, and enabling the culture medium of the flow cavity layer to correspond to each single-hole chamber of the microporous layer;

s03, laminating and splicing the bonded microporous layer and the flow cavity on the electrode layer, and electrically connecting the electrode layer with a peripheral circuit to obtain the integrated organ chip.

Optionally, the fabricating the microporous layer in S01 includes:

curing the liquid polydimethylsiloxane prepolymer in a 60 ℃ oven for 4 hours or an 85 ℃ oven for 2 hours to form a PDMS polymer, and then punching the PDMS polymer according to a preset structure by using a microfluidic puncher to form a matrix type single-hole chamber with a plurality of single-hole structures;

more than one round bare electrode of the electrode layer is exposed on the bottom surface of the single-hole cavity of each single-hole structure;

the fabricating of the flow cavity layer in S01 includes:

covering the PDMS prepolymer with bubbles removed on a tungsten steel bar male mold, curing for 4 hours in a 60 ℃ oven or 2 hours at 85 ℃ to form a plurality of cuboid-shaped single cavity structures,

and respectively drilling a hole at two ends of each cuboid single cavity structure to form a culture medium inlet and a culture medium outlet.

Optionally, the S02 includes:

coating a PDMS prepolymer with bubbles removed on the upper surface of the microporous layer, covering the surface of the microporous layer with the flow cavity layer, putting the microporous layer into a 60 ℃ oven, and performing hot-pressing curing overnight in a hot-pressing manner to perform packaging, so that the microporous layer and the flow cavity layer are bonded together to form a reversible packaging structure.

Optionally, the fabricating the electrode layer in S01 includes:

selecting a high-transmittance 4-inch glass sheet with the thickness of 500 mu m, and sequentially cleaning the surface of the glass with acetone, pure water, absolute ethyl alcohol and pure water for not less than 5 minutes;

forming a metal conducting layer with the thickness of 150nm on the surface of the first layer of glass through magnetron sputtering of a metal layer;

spin-coating insulation material polyimide with the thickness less than or equal to 2 microns outside the non-circular metal points and the metal bonding pad on the surface of the first layer of metal layer by a spin-coating method, and baking to form an insulation layer to obtain an intermediate structure;

and cutting the intermediate structure into a single piece containing 16 bare electrodes and bonding pads according to a preset size to obtain the electrode layer.

(III) advantageous effects

The integrated organ chip of the invention can realize the electrical stimulation and the mechanical stimulation to the culture and realize the culture of proper culture according to the experimental requirements. In addition, each single pore cavity of the microporous layer can independently perform dynamic suspension and static culture on organoids or cell groups, and each single pore cavity is provided with one or more electrode points, so that current or voltage stimulation can be accurately performed on the culture.

Furthermore, the electrode layer is made of high-light-transmission glass, has higher hardness than stainless steel or plastic, has good light transmission, is convenient for observation under a microscope and high-temperature sterilization, and is more suitable for cell culture.

In addition, the flow chamber layer in a particular application may be free of peripheral perfusion equipment, and a steady flow may be created in the microcavities by the rocker shaker, and a shear force of the fluid around the surface of the suspended culture may be created in a single lumen.

Drawings

FIGS. 1A and 1B are schematic top views of an integrated organ chip according to an embodiment of the invention;

FIG. 2 is a schematic top view of the second microporous layer of FIG. 1;

FIG. 3 is a schematic top view of the third flow chamber layer of FIG. 1;

FIG. 4 is a schematic top view of the first electrode layer of FIG. 1;

FIG. 5 is a schematic cross-sectional view of an integrated organ-on-chip electro-stimulation spherical culture according to the present invention;

FIG. 6 is a schematic cross-sectional view of the dynamic suspension culture in an integrated organ-on-chip according to the present invention.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

In order to better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

At present, there are also reports in the literature on organoid chips combining single regulatory techniques with culture, such as: organoid chip for regulating and controlling induced myocardial cell maturation and promoting vascular organoid lumen-like structure maturation. However, no integrated organ chip has been reported which combines various control technologies to satisfy organoid culture and promote functional maturation without external perfusion equipment. In order to solve the above defects, the invention provides an integrated organ chip and a preparation method thereof, which can be used for in vitro experimental organoid dynamic and static culture and regulation.

Example one

The present embodiment provides an integrated organ chip for applying electrical and mechanical stimuli as required during the culture of cells, cell clusters or organoids.

Wherein the integrated organ chip includes: the micro-porous culture medium comprises an electrode layer for providing electrical stimulation, a micro-porous layer positioned above the electrode layer for culture, and a flow cavity layer for providing mechanical stimulation and interacting with an external connector; a peripheral circuit;

the electrode layer includes: the substrate is provided with a bare electrode corresponding to each micropore in the micropore layer; the bare electrode is electrically connected with a peripheral circuit by means of a conductive component nested on the surface of the substrate; the non-naked electrode area on the surface of the substrate is an insulating area; the peripheral circuit of the present embodiment may be a printed circuit board, as shown in fig. 1A.

The microporous layer includes: the electrode layer is adhered with a structure with a plurality of independent single-hole chambers, each single hole is transparent, and the bottom of each single hole corresponds to one or more bare electrodes of the electrode layer, so that the electrified bare electrodes apply adjustable electrical stimulation to the culture in the single-hole chambers;

the flowing cavity layer is a single cavity structure (such as a plurality of cuboid-shaped single cavity structures) which is adhered above the microporous layer and used for bearing a culture medium of a culture, the culture medium flows in the respective cavity structure (namely flows above each microporous layer) with the aid of an auxiliary device so as to obtain controllable fluid shear stress, and each cavity structure is communicated with a plurality of single-hole chambers of the microporous layer and provides the culture medium for the culture in the single-hole chamber.

In practical applications, the cavity structures of the flow cavity layer may be configured as single cavity structures having a rectangular parallelepiped shape, so that each single cavity structure having a rectangular parallelepiped shape corresponds to one row of the single-hole chambers; and the end of the single cavity structure in the shape of a cuboid is provided with a culture medium inlet, and the end tail is provided with a culture medium outlet. Certainly, the flow cavity layer in this embodiment is not limited to a rectangular parallelepiped shape, and the rectangular parallelepiped shape is illustrated in this embodiment, and is selected according to actual needs in actual applications.

The integrated organ chip of the embodiment can realize electrical stimulation and mechanical stimulation to the culture, and realize culture of a proper culture according to experimental requirements. In addition, each single cavity chamber in the microporous layer can independently carry out dynamic suspension and static culture on organoids or cell groups, and each single cavity chamber is provided with one or more electrode points, namely bare electrodes, so that current or voltage stimulation can be accurately carried out on a culture.

Typically, each single-pore chamber in the microporous layer corresponds to a bare electrode; in other embodiments, one single-hole chamber may correspond to three bare electrodes, and the following drawings and embodiments are described with the single-hole chamber corresponding to one bare electrode.

A specific integrated organ chip will be described below with reference to fig. 1A to 4. In fig. 1B, 111 is a first glass substrate, 112 is a metal pad, 113 is a metal extension line, 114 is a metal circular electrode, i.e., a circular bare electrode/electrode point, 211 is a second microporous layer, 311 is a third microporous layer, i.e., a flow cavity layer, 312 is a rectangular single cavity structure, and 313 is an inlet and an outlet of a culture medium;

for the electrode layer, as shown in fig. 4, each bare electrode is a sheet-shaped circular bare electrode; each bare electrode on the substrate is mutually independent and connected with a peripheral circuit; the diameter of the round bare electrode is smaller than the aperture of each single hole in the microporous layer; in fig. 4, a circle represents a circular metal electrode point or a circular bare electrode or an electrode of an electrode layer, a line of the circle is a metal extension line, and a rectangle at the other end of the metal extension line represents a metal pad.

It is understood that the substrate of the present embodiment may include: the glass substrate, the conductive component layer laminated on the glass substrate and the insulating layer of the non-bare electrode area; the conductive component of the conductive component layer is a wire (such as a metal extension wire/metal laminated wire), one end of the wire is connected with the bare electrode, and the other end of the wire is provided with a metal bonding pad with an identifier for electrically connecting with a peripheral circuit.

Since the transparent glass facilitates observation of the growth state of the culture, a glass base can be selected from the electrode layers shown in fig. 1 and 4, and in this embodiment, the glass base can be 4-inch or 6-inch circular high-light-transmittance glass with a thickness of 500+ -50um; the circular glass can be cut according to the structure of the electrode layer to form a plurality of electrode layers, and the size of each electrode layer is about 4cm × 5cm after cutting.

The diameter of the round bare electrode is 70um; the insulating layer can be made of polyimide, and the thickness of the insulating layer is less than or equal to 2 micrometers; generally, 16 circular metal dots (more than 16, such as 16 to 400, can be designed with adjustable design size) can be disposed on the substrate, and the conductive wires, such as metal extension wires, can be extended to the edge of the glass as required, and rectangular metal pads are disposed on the edge of the glass to be connected with each metal extension wire. The peripheral circuit may be a PCB board electrically connected, e.g. soldered, to the metal pads through the bus pins on the PCB board.

In practical application, the insulating layer can adopt the coating film mode to cover the surface of the glass base and realize the insulation of the non-bare electrode area, the material of the insulating layer is polyimide, and the thickness is less than or equal to 2um. In the present embodiment, the polyimide is not limited and is selected according to actual needs.

As shown in fig. 2, fig. 2 is a schematic top view of a microporous layer, and the microporous layer of this embodiment may be a Polydimethylsiloxane (PDMS) porous membrane layer formed by drilling a PDMS polymer after being cured by PDMS; the PDMS polymer has good biocompatibility and high light transmittance, and is used for the growth and observation of cells; the porous structure (i.e. a plurality of single-hole chambers) is arranged in a row to cover the electrode layer, each row is provided with 4 single-hole chambers, a round metal point, namely a bare electrode, is exposed at the bottom surface of each hole, and the aperture of each single hole is 1mm to 6mm, such as 3mm. In the embodiment, the bare electrode can be directly contacted with the culture in the single hole, so that current or voltage stimulation on the culture is realized.

The porous structure of the microporous layer shown in fig. 2 may be arranged in multiple rows, each row having N (N is greater than or equal to 2) individual single-pore chambers, and the bottom of each single-pore chamber corresponds to a bare electrode. In other embodiments, the arrangement and the number of the porous structures are not limited, and can be selected according to actual needs.

As shown in fig. 3, fig. 3 is a schematic top view of a flow cavity layer, the flow cavity layer of this embodiment may be formed by covering a metal male mold (e.g., a tungsten steel bar male mold) with a liquid Polydimethylsiloxane (PDMS) prepolymer, and curing the liquid PDMS prepolymer to form a plurality of single cavity structures having a rectangular parallelepiped shape, where each single cavity structure has a length of 15mm, a width of 6mm, and a depth of 1mm. Two ends of each single cavity structure are respectively provided with a hole as an inlet and an outlet, namely, the end of each cuboid-shaped single cavity structure is provided with a culture medium inlet, and the end tail is provided with a culture medium outlet; the aperture of the culture medium inlet/outlet is 5mm.

Since 4 single cavities shown in fig. 2 make up a row, each single cavity structure of a rectangular parallelepiped shape corresponds to a row of single-hole chambers in fig. 3. Of course, in other embodiments, the cavity structure of the flow chamber layer may not be limited to a rectangular parallelepiped shape, but may be other shapes, and is selected and arranged according to actual needs, such as being capable of corresponding to more than one single-pore chamber of the microporous layer and realizing the provision of the culture medium and the flow shear force.

In this embodiment, the third flow chamber layer is adhered to the second porous layer, and the three layers are stacked and encapsulated to form an integrated organ chip. In order to better realize the preparation or reversible packaging of the integrated organ chip, the materials of the microporous layer and the flow cavity layer are made to be the same in this embodiment, and in other embodiments, the materials of the two layers are not limited, and are selected according to actual needs.

In this example, when the organoid or cell mass is cultured dynamically suspended and statically using the integrated organ chip, the organoid or cell mass is placed in the single-pore chamber of the second microporous layer, the first electrode layer can apply voltage or current-adjustable electrical stimulation to the culture, and the flow of the culture medium in the third flow chamber layer can apply controllable fluid shear stress to the culture.

It should be noted that the auxiliary device of the present embodiment may include: peripheral perfusion equipment or a warped plate shaker for realizing the flowing of the culture medium. The use of a rocker is preferred, whereby the cultivation of the culture is preferably achieved without the need for a line-limiting structure such as a connecting line. Namely, the whole culture process can be carried out without connecting peripheral perfusion equipment, and the chip can be placed on a warped plate shaker to realize the flow culture.

The chip can perform precise electrical stimulation (including current and voltage) and stable fluid shear force stimulation on organoid or cell mass. The first layer adopts high light-transmitting glass, has higher hardness than stainless steel or plastic, has good light transmission, is convenient for observation under a microscope and high-temperature sterilization, and is more suitable for cell culture. Each single-cavity chamber in the second layer can independently carry out dynamic suspension and static culture on organoids or cell groups. Each single-cavity chamber is provided with one or more electrode points, and the current or voltage stimulation can be accurately carried out on the culture. The third layer may be free of peripheral perfusion apparatus, allowing for a steady flow in the microcavity by a rocking platform and a shear force of the fluid around the surface of the suspended culture within the single-well chamber.

Example two

This example provides a method for preparing an integrated organ chip, which is mainly illustrative of the process for preparing an integrated organ chip shown in example one.

The method for preparing the integrated organ chip of the embodiment comprises the following steps:

s01, sequentially manufacturing the electrode layer, the microporous layer and the flow cavity layer.

For example, fabricating the microporous layer can include:

curing the liquid Polydimethylsiloxane (PDMS) prepolymer in an oven at 60 ℃ for 4 hours, and punching the PDMS polymer according to a preset structure by using a universal puncher for the microfluidic chip to form a plurality of single-hole chambers; each single-hole cavity bottom surface exposes one or more circular bare electrodes of the electrode layer.

Additionally, fabricating the flow chamber layer may include:

and (3) pouring a film on the liquid polydimethylsiloxane prepolymer on a tungsten steel bar male die, and curing to form a female die with a single cavity structure and a plurality of cuboid shapes. Specifically, the PDMS prepolymer without bubbles is covered on a tungsten steel strip male mold, membrane inversion and solidification are carried out for 4 hours at the temperature of 60 ℃, a plurality of single cavity structures with cuboid structures are formed, two ends of each cavity structure are respectively provided with a hole, and a culture medium inlet and a culture medium outlet are formed.

And S02, bonding the micropore layer and the flow cavity layer together, and enabling the culture medium of the flow cavity layer to correspond to each single-pore chamber of the micropore layer.

The step may specifically comprise: coating PDMS prepolymer on the upper surface of the microporous layer, covering the surface of the microporous layer with the flow cavity layer, putting the microporous layer into a 60 ℃ oven, and performing hot-pressing curing overnight in a hot-pressing manner for packaging, so that the microporous layer and the flow cavity layer are bonded together. At this time, the formed encapsulation structure is a reversible structure, and it should be noted that the reversible encapsulation structure can be used to remove the flow cavity layer after the experiment is completed, so as to facilitate the extraction of the experimental culture, and can be used repeatedly after the microporous layer and the flow cavity layer are cleaned.

The splicing and stacking manner of the microporous layer and the flow cavity layer after bonding and stacking on the electrode layer is the same as the bonding process of the microporous layer and the flow cavity layer in S02. Namely, PDMS prepolymer is coated on the upper surface of an electrode layer, a microporous layer is covered on the surface of the electrode layer, the electrode layer is placed in a 60 ℃ oven, and hot-pressing curing is carried out overnight in a hot-pressing mode for packaging, so that the bonded microporous layer and the flow cavity are laminated and spliced on the electrode layer. At this time, the package structure formed there is also a reversible structure.

That is, the first layer, the second layer and the third layer of the integrated organ chip are sequentially stacked and adhered together to realize reversible packaging.

For better understanding, the following provides a method for preparing the integrated organ chip shown in fig. 1, which comprises the following specific steps:

m01: a high transmission 4 inch/6 inch glass sheet with a thickness of 500 μm was selected.

M02: and sequentially cleaning the surface of the glass by using acetone, pure water, absolute ethyl alcohol and pure water for not less than 5 minutes.

M03: and forming a metal conducting layer on the surface of the first layer of glass by magnetron sputtering of a metal layer (Au) with the thickness of 150 nm.

M04: and spin-coating an insulating material polyimide on the surface of the first metal layer (except for the positions of the circular metal points and the metal pads) by using a spin coating method, wherein the thickness of the insulating material polyimide is not more than 2 mu m, and forming an insulating layer after baking.

M05: and cutting the first glass metal conducting layer structure into electrode layer sheets containing 16 electrode points and welding pads according to the designed external dimension, wherein the size range of the electrode layer sheets is about 4cm × 5 cm.

Then, preparing a microporous layer, specifically comprising the steps of:

m06: preparing PDMS prepolymer solution, pouring into a glass dish, and removing bubbles in a vacuum box with vacuum degree of-15 kPa for at least 30 minutes.

M07: the PDMS prepolymer was cured at 85 ℃ for 2 hours or 60 ℃ for 4 hours.

M08: and cutting off the strip PDMS by using a knife, and punching holes by using a puncher corresponding to the arrangement positions of the circular metal points to form a plurality of microporous structures, namely a plurality of independent single-hole chambers.

M09: and adhering the PDMS with the micropore structure on the second layer to the surface of the first layer to expose the circular electrode points and the metal bonding pad.

Then, preparing a flow cavity layer, which specifically comprises the following steps:

m10: covering the smooth tungsten steel alloy male die with the bubble-removed PDMS prepolymer, and curing the PDMS prepolymer at 85 ℃ for 2 hours or 60 ℃ for 4 hours.

M11: and cutting off the PDMS female die which is covered on the male die and has a cuboid cavity structure by using a knife.

M12: two ends of a cuboid cavity structure (namely a cuboid single cavity structure) are respectively punched with a hole, and then one surface of the cavity is adhered to the second microporous layer.

M13: the metal bonding pads on the organ chip are connected with the flat cable pins on the printed circuit board in a one-to-one correspondence manner by welding the single-stranded superfine copper wires, and finally, the integrated organ chip can be obtained by testing the welding points with the aid of a universal meter.

The integrated organ chip prepared by the method mainly comprises a first electrode layer, a second microporous layer and a third flow cavity layer. When the chip is used for dynamically suspending and statically culturing organoids or cell clusters, the organoids or cell clusters are placed in the single-hole chamber of the second microporous layer, the first electrode layer can apply voltage or current-adjustable electric stimulation to the culture, and the flow of the culture medium in the third flowing chamber layer can apply controllable fluid shear stress to the culture; thereby, precise electrical stimulation (including current and voltage) and stable fluid shear force stimulation of organoids or cell clusters is achieved. And the whole culture process can be carried out without connecting peripheral perfusion equipment, and the chip can be placed on a warped plate shaking table to realize the flow culture.

In practical use, when one organoid or cell mass is inoculated in the single pore of the second layer for static culture, through an external electrical stimulation signal, the circular metal electrode point of the first layer can carry out accurate current or voltage stimulation on the organoid or cell mass, so that the culture in each single pore can obtain a consistent stimulation signal, and errors caused by experimental conditions in the same experimental batch can be reduced.

When carrying out the dynamic suspension culture to the culture in single aperture chamber, place equipment on the wane shaking table, set up different rocking frequency and range, the liquid flow in the third layer microcavity can form stable fluid shear force around the culture surface in single micropore.

The static electric stimulation experiment and the dynamic suspension culture can be carried out simultaneously or step by step according to the experiment requirement. When single suspension culture is carried out independently, the whole experimental device can be not connected with external perfusion equipment, is convenient to put into an incubator for a long time to carry out flowing experiments, and avoids the pollution caused by the external equipment.

As shown in fig. 5 and 6. Wherein the circular metal electrode point can contact with the bottom surface of the circular culture with the diameter of 200 μm-2mm, and the electrode point electrically stimulates the culture. The microporous structure is designed into a single-pore cavity structure which is not communicated with each other, when in flow culture, the culture solution can flow while the culture is in a suspension state, stable wall surface shearing force is formed on the surface of the culture, and the magnitude of the wall surface shearing force can be adjusted by adjusting the flow speed. In this embodiment, each of the 16 circular metal electrode points in the electrode layer is illustrated, the embodiment is not limited thereto, and the structure of each chamber in the microporous layer and the flow cavity layer is also illustrated, and the technology in this embodiment is extended to more, the size and shape of the whole device can be changed according to the experimental requirements, and the peripheral circuit can be extended according to the number of the metal pads.

It should be noted that in the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. The word "comprising" does not exclude the presence of elements or steps not listed in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the claims enumerating several means, several of these means may be embodied by one and the same item of hardware. The use of the terms first, second, third, etc. are used for convenience only and do not denote any order. These words are to be understood as part of the name of the component.

Furthermore, it should be noted that in the description of the present specification, the description of the term "one embodiment", "some embodiments", "examples", "specific examples" or "some examples", etc., means that a specific feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Moreover, various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without being mutually inconsistent.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, the claims should be construed to include preferred embodiments and all changes and modifications that fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention should also include such modifications and variations.

Claims

1. An integrated organ chip for applying electrical and mechanical stimuli on demand during the culture of cells, cell clusters or organoids; the integrated organ chip includes:

the electrode layer includes: the substrate is positioned on the substrate and corresponds to the bare electrode of each micropore in the micropore layer; the bare electrode is electrically connected with a peripheral circuit by means of a conductive component nested on the surface of the substrate; the non-naked electrode area on the surface of the substrate is an insulating area;

2. The integrated organ chip according to claim 1,

each bare electrode is a sheet-shaped circular bare electrode; each bare electrode on the substrate is mutually independent and connected with a peripheral circuit; the diameter of the round bare electrode is smaller than the aperture of each single hole in the micropore layer;

3. The integrated organ chip according to claim 2,

the glass base is 4-inch round high-light-transmittance glass with the thickness of 500+ -20um;

the diameter of the round bare electrode is 70+ -10um;

4. The integrated organ chip according to claim 1,

the microporous layer is a PDMS porous membrane layer formed by punching after polydimethylsiloxane polymer PDMS is cured;

the aperture of each single hole is 1mm to 6mm;

5. The integrated organ chip according to claim 1 or 4,

the cavity structure of the flow cavity layer is a single cavity structure with a cuboid shape; each cuboid-shaped single cavity structure corresponds to one row of the single-hole chambers; the end of the single cavity structure in the cuboid shape is provided with a culture medium inlet, and the end tail is provided with a culture medium outlet;

or,

6. The integrated organ-chip according to claim 5,

the length of the cuboid single cavity structure is 15mm, the width is 6mm, and the depth is 1mm;

the aperture of the culture medium inlet/the culture medium outlet is 1mm-5mm;

or,

7. A method for preparing an integrated organ chip according to any one of claims 1 to 6, comprising:

8. The method according to claim 7, wherein the step of fabricating the microporous layer in S01 includes:

the fabricating of the flow cavity layer in S01 includes:

9. The method according to claim 7, wherein the S02 includes:

coating a PDMS prepolymer with bubbles removed on the upper surface of the microporous layer, covering the flow cavity layer on the surface of the microporous layer, putting the microporous layer into a 60 ℃ oven, and performing hot-pressing curing overnight in a hot-pressing manner for packaging, so that the microporous layer and the flow cavity layer are bonded together and form a reversible packaging structure.

10. The method according to any one of claims 7 to 9, wherein the fabricating the electrode layer in S01 includes: