An integrated organ chip and a method of manufacturing the same

Abstract

The present application relates to an integrated organ chip and a preparation method thereof, 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 comprises: an electrode layer for providing electrical stimulation, a microwell layer for culture located above the electrode layer, a flow cavity layer for providing mechanical stimulation and interacting with an external connection; a peripheral circuit; the electrode layer comprises: a substrate, a bare electrode corresponding to each microwell in the microwell layer located on the substrate; the microwell layer comprises: a structure with a plurality of independent single-hole cavities pasted on the electrode layer, each single hole is permeable and the bottom corresponds to a bare electrode or a plurality of electrodes consisting of a multi-electrode of the electrode layer; the flow cavity layer is a plurality of single cavity structures for carrying the culture medium of the culture pasted above the microwell layer. The above method can combine various regulation technologies to meet the culture and promote the functional maturation of organoids or cell clusters.

Description

TECHNICAL FIELD

[0001] The present application relates to biomedical and microfluidic technology, and in particular to an integrated organ chip and a preparation method thereof. BACKGROUND

[0002] When culturing organoids or cell clusters in vitro, the cells may need external electrical or mechanical stimulation to regulate the cells and make their functions mature. An integrated organ chip based on microfluidic technology can achieve this function. The integrated organ chip system assembled by microfluidic devices capable of achieving a single function can meet the dynamic and static culture of the culture and the electrical and mechanical stimulation during the culture process. At present, integrated microfluidic chips have been widely used in the culture and regulation of cells, cell clusters and organoids.

[0003] The existing patent application with publication number CN113667603A discloses a liver organoid culture chip and a preparation method and application thereof. The liver organoid culture chip comprises: a cell culture plate; a biological material with a micropore array; the culture method comprises: digesting human embryonic stem cells or human induced pluripotent stem cells into single cells, inoculating the single cells in a culture medium for culture to obtain foregut embryonic cells; and then digesting the foregut embryonic cells into single cells, inoculating the single cells in the micropore array in the liver organoid culture chip for culture to obtain liver organoids. Although the above method can be used for uniform and high-throughput liver organoid culture of different sources or different tissue types, it does not combine the regulation technology for promoting the maturation of organoids, and needs an external perfusion device. In actual application, promoting the functional maturation of in vitro cultured organoids is the key to further culture and development of organoids.

[0004] Therefore, how to combine multiple regulation technologies to meet the integrated organ chip for organoid culture and promote the functional maturation of organoids has become a technical problem to be solved at present. SUMMARY

[0005] (I) Technical problems to be solved

[0006] In view of the above shortcomings and deficiencies of the prior art, the present application provides an integrated organ chip and a preparation method thereof.

[0007] (II) Technical solutions

[0008] In order to achieve the above-mentioned purpose, the main technical solutions adopted by the present application include:

[0009] In a first aspect, the present application provides an integrated organ chip for applying electrical and mechanical stimulation as required during the culture of cells, cell clusters or organoids; the integrated organ chip comprises:

[0010] An electrode layer for providing electrical stimulation, a microwell layer above the electrode layer, a flow chamber layer for providing mechanical stimulation and interacting with an external circuit; a peripheral circuit;

[0011] The electrode layer comprises: a substrate, bare electrodes corresponding to each microwell in the microwell layer above the substrate; the bare electrodes are electrically connected to the peripheral circuit by means of conductive components embedded in the surface of the substrate; the non-bare electrode area of the substrate surface is an insulating area;

[0012] The microwell layer comprises: a structure with a plurality of independent single-well chambers adhered to the electrode layer, the single-well in each single-well chamber is permeable and the bottom corresponds to one or more bare electrodes of the electrode layer, so that the bare electrodes after being electrified apply adjustable electrical stimulation to the culture in the single-well chamber;

[0013] The flow chamber layer is a plurality of cavity structures for carrying the culture medium of the culture adhered above the microwell layer, the culture medium flows in each cavity structure with the assistance of auxiliary equipment to obtain controllable fluid shear stress, and the cavity of each cavity structure communicates with a plurality of single-well chambers of the microwell layer and provides the culture in the single-well chamber with the culture medium.

[0014] Optionally, each bare electrode is a sheet-like circular bare electrode; each bare electrode on the substrate is independently connected to the peripheral circuit; and the diameter of the circular bare electrode is smaller than the pore size of each single-well in the microwell layer.

[0015] The substrate comprises: a glass base, a conductive component layer stacked on the glass base, and an insulating layer of the non-bare electrode area; the conductive components of the conductive component layer are metal stacked wires, one end of the wire is connected to the bare electrode, and the other end is provided with a metal pad with an identifier for electrical connection with the peripheral circuit.

[0016] Optionally, the glass base is a 4-inch circular high-transmittance glass with a thickness of 500+-20um;

[0017] The diameter of the circular bare electrode is 70+-10um;

[0018] The insulating layer is made of polyimide with a thickness less than or equal to 2um;

[0019] And / or, the materials of the microwell layer and the flow chamber layer are consistent.

[0020] Optionally, the microwell layer is a PDMS porous membrane layer formed by punching after curing of a polydimethylsiloxane polymer PDMS;

[0021] The pore size of each single-well is 1mm to 6mm;

[0022] The plurality of single-hole chambers of the microporous layer are arranged in multiple rows, each row having N individual single-hole chambers, N being greater than or equal to 2, and the bottom of each single-hole chamber corresponding to one or more bare electrodes.

[0023] Optionally, the cavity structure of the flow cavity layer is a single cavity structure in the shape of a cuboid; each single cavity structure in the shape of a cuboid corresponds to a row of single-hole chambers; and the head of the single cavity structure in the shape of a cuboid is provided with a culture medium inlet, and the tail is provided with a culture medium outlet.

[0024] Alternatively,

[0025] The flow cavity layer is a negative mold formed by pouring and curing PDMS pre-polymer on a tungsten steel strip positive mold to form a plurality of single cavity structures in the shape of a cuboid; each single cavity structure in the shape of a cuboid corresponds to a row of single-hole chambers; and the head of the single cavity structure in the shape of a cuboid is provided with a culture medium inlet, and the tail is provided with a culture medium outlet.

[0026] Optionally, the single cavity structure in the shape of a cuboid is 15 mm long, 6 mm wide, and 1 mm deep.

[0027] The pore size of the culture medium inlet / culture medium outlet is 1 mm-5 mm.

[0028] Alternatively,

[0029] The auxiliary equipment includes a peripheral perfusion device or a shaker for realizing culture medium flow.

[0030] In a second aspect, the embodiments of the present application provide a preparation method of the integrated organ chip according to any one of the first aspect, which comprises:

[0031] S01, sequentially manufacturing the electrode layer, the microporous layer, and the flow cavity layer;

[0032] S02, bonding the microporous layer and the flow cavity layer together, and corresponding the culture medium of the flow cavity layer to each single-hole chamber of the microporous layer;

[0033] S03, stacking and splicing the bonded microporous layer and flow cavity layer on the electrode layer, and electrically connecting the electrode layer with the peripheral circuit to obtain the integrated organ chip.

[0034] Optionally, the manufacturing of the microporous layer in S01 comprises:

[0035] Curing the liquid polydimethylsiloxane pre-polymer in a 60-degree Celsius oven for 4 hours or in a 85-degree Celsius oven for 2 hours to form a PDMS polymer, and then punching the PDMS polymer according to a preset structure to form a plurality of single-hole structures of single-hole chambers in a matrix form by using a microfluidic puncher;

[0036] The single-hole cavity of each single-hole structure exposes one or more circular bare electrodes of the electrode layer;

[0037] The manufacturing of the flow cavity layer in S01 comprises:

[0038] The bubble-removed PDMS prepolymer is coated on the tungsten steel strip male die, and is cured in a 60 DEG C oven for 4 hours or a 85 DEG C oven for 2 hours to form a plurality of cuboid-shaped single cavity structures,

[0039] A hole is punched at each end of each cuboid-shaped single cavity structure to form a culture medium inlet and a culture medium outlet.

[0040] Optionally, the S02 comprises:

[0041] The bubble-removed PDMS prepolymer is coated on the microporous layer, the flow cavity layer is covered on the surface of the microporous layer, and the packaging is performed by heat pressing and curing overnight in a 60 DEG C oven, so that the microporous layer and the flow cavity layer are bonded together and a reversible packaging structure is formed.

[0042] Optionally, the manufacturing of the electrode layer in S01 comprises:

[0043] A 4-inch glass sheet with a thickness of 500 mu m and high light transmission is selected, and the surface of the glass is sequentially cleaned with acetone, pure water, anhydrous ethanol and pure water, and the cleaning time is not less than 5 minutes;

[0044] A metal conductive layer with a thickness of 150 nm is formed on the surface of the first layer of glass by magnetron sputtering of a metal layer;

[0045] An insulating layer is formed by spin coating an insulating material polyimide with a thickness of less than or equal to 2 mu m on the non-circular metal points and the metal pad positions on the surface of the first layer of metal layer after baking, and an intermediate structure is obtained.

[0046] The intermediate structure is cut into a single piece containing 16 bare electrodes and pads according to a predetermined size, and an electrode layer is obtained.

[0047] (III) beneficial effects

[0048] The integrated organ chip can realize electrical stimulation and mechanical stimulation of the culture, and realize the culture of suitable culture according to experimental requirements. In addition, the micro-porous layer can dynamically suspend and statically culture the organoids or cell clusters in each single cavity, and one or more electrode points are arranged in each single cavity, so that the culture can be precisely stimulated by current or voltage.

[0049] Further, the electrode layer adopts high-transmittance glass, which has larger hardness and better light transmittance than stainless steel or plastic, is convenient for observation under a microscope and high-temperature sterilization, and is more suitable for cell culture.

[0050] In addition, the flow cavity layer in the specific application can not be connected to the peripheral perfusion device, and stable flow can be formed in the microcavity through the rocker bed, and fluid shear force around the surface of the suspension culture is formed in the single hole cavity. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1A and Figure 1B respectively, are a top view schematic diagram of an integrated organ chip provided by an embodiment of the present application;

[0052] Figure 2 is a top view schematic diagram of the second layer of micro-hole layer in Figure 1;

[0053] Figure 3 is a top view schematic diagram of the third layer of flow cavity layer in Figure 1;

[0054] Figure 4 is a top view schematic diagram of the first layer of electrode layer in Figure 1;

[0055] Figure 5 is a schematic diagram of the profile of electrically stimulating the spherical culture of the integrated organ chip of the present application;

[0056] Figure 6 is a schematic diagram of the profile of the dynamic suspension culture in the integrated organ chip of the present application. DETAILED DESCRIPTION

[0057] In order to better explain the present application, the following will be described in detail in combination with the accompanying drawings and specific embodiments.

[0058] In order to better understand the above technical solutions, the following will describe the exemplary embodiments of the present application in more detail with reference to the accompanying drawings. Although the exemplary embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a clearer, more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0059] Currently, there are also documents reporting organoid chips combining single regulation technology and culture, such as organoid chips regulating and inducing maturation of cardiomyocytes and promoting maturation of lumen-like structures of vascular organoids. However, there is no report on integrated organ chips combining multiple regulation technologies to meet the culture and promote the functional maturation of organoids without external perfusion devices. To solve the above problems, the present application provides an integrated organ chip and a preparation method thereof, which can dynamically culture and regulate organoids in vitro.

[0060] Example 1

[0061] The embodiment provides an integrated organ chip, and 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.

[0062] The integrated organ chip comprises an electrode layer for providing electrical stimulation, a microwell layer above the electrode layer for culture, a flow cavity layer for providing mechanical stimulation and interacting with an external connection, and a peripheral circuit.

[0063] The electrode layer comprises a substrate and bare electrodes corresponding to microwells in the microwell layer on the substrate; the bare electrodes are electrically connected to the peripheral circuit through conductive components embedded in the surface of the substrate; and the non-bare electrode area of the surface of the substrate is an insulating area; the peripheral circuit can be a printed circuit board, as shown in the figure. Figure 1A

[0064] The microwell layer comprises a structure with a plurality of independent single-well cavities attached to the electrode layer; each single well is permeable and the bottom corresponds to one or more bare electrodes of the electrode layer, so that the bare electrodes after being electrified can apply adjustable electrical stimulation to the culture in the single-well cavity.

[0065] The flow cavity layer is a plurality of cavity structures (such as a plurality of cuboid-shaped single cavity structures) for carrying the culture medium of the culture attached above the microwell layer; the culture medium flows in the respective cavity structures (i.e., flows above the microwell layer) with the assistance of auxiliary equipment to obtain controllable fluid shear stress; and each cavity structure is connected to a plurality of single-well cavities of the microwell layer and provides the culture medium for the culture in the single-well cavity.

[0066] In actual application, the cavity structure of the flow cavity layer can be set as a cuboid-shaped single cavity structure, so that each cuboid-shaped single cavity structure corresponds to a row of single-well cavities; and the end of the cuboid-shaped single cavity structure is provided with a culture medium inlet and the tail end is provided with a culture medium outlet. Of course, the flow cavity layer in the embodiment is not limited to a cuboid shape, and the cuboid shape is used for illustration in the embodiment, which can be selected according to actual needs in actual application.

[0067] The integrated organ chip can realize electrical stimulation and mechanical stimulation of the culture, and realize the culture of suitable culture according to experimental requirements. In addition, each single-well cavity in the microwell layer can be used for dynamic suspension and static culture of organoids or cell clusters, and each single-well cavity has one or more electrode points, i.e., bare electrodes, which can accurately stimulate the culture with current or voltage.

[0068] ​Typically, each single-pore chamber in the microporous layer corresponds to one bare electrode; in other embodiments, one single-pore chamber may correspond to three bare electrodes. The following figures and embodiments will be described with one single-pore chamber corresponding to one bare electrode.

[0069] The following combination Figures 1A to 4 The image illustrates a specific example of an integrated organ-on-a-chip. Figure 1B In the diagram, 111 is the first glass base, 112 is the metal pad, 113 is the metal extension line, 114 is the metal circular electrode (i.e., the circular bare electrode / electrode point), 211 is the second layer (i.e., the microporous layer), 311 is the third layer (i.e., the flow chamber layer), 312 is a single cavity structure in the shape of a cuboid, and 313 is the inlet and outlet of the culture medium.

[0070] For the electrode layer, such as Figure 4 As shown, each bare electrode is a sheet-like circular bare electrode; each bare electrode on the substrate is independently connected to the peripheral circuit; and the diameter of the circular bare electrode is smaller than the pore diameter of each single pore in the microporous layer. Figure 4 The middle circle represents a circular metal electrode point, a circular bare electrode, or an electrode layer. The line of the circle is a metal extension line, and the rectangle at the other end of the metal extension line represents a metal pad.

[0071] It is understood that the substrate of this embodiment may include: a glass base, a conductive component layer laminated on the glass base, and an insulating layer for non-bare electrode areas; 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 to the bare electrode, and the other end is provided with a marked metal pad for electrical connection with the peripheral circuit.

[0072] Because transparent glass facilitates observation of the growth status of the culture, it is used in Figure 1 and... Figure 4 The electrode layer shown can be a glass base. In this embodiment, the glass base can be a 4-inch or 6-inch circular high-transmittance glass with a thickness of 500 ± 50 μm. The circular glass can be cut according to the structure of the electrode layer to form multiple electrode layers. The size of each cut electrode layer is about 4 cm * 5 cm.

[0073] The diameter of the circular bare electrode is 70µm; the insulating layer can be made of polyimide with a thickness of 2µm or less; typically, 16 circular metal dots can be set on the substrate (in practice, the design size can be adjusted to set more than 16, such as 16 to 400), and the wires, such as metal extension lines, extend to the edge of the glass as needed. Rectangular metal pads are set on the edge of the glass to connect with each metal extension line. The peripheral circuit can be a PCB board, which is electrically connected to the metal pads through ribbon cable pins on the PCB board, such as by soldering.

[0074] In practical applications, the insulating layer can be used to cover the surface of the glass base to insulate the non-bare electrode area. The insulating layer is made of polyimide and has a thickness of less than or equal to 2 um. In this embodiment, the polyimide is not limited and can be selected according to actual needs.

[0075] As shown in Figure 2 , Figure 2 A top view of a microporous layer is shown. The microporous layer of this embodiment can be a PDMS porous membrane layer formed by punching a PDMS polymer after curing a polydimethylsiloxane (PDMS) prepolymer. The PDMS polymer has good biocompatibility and high light transmittance, and is used for cell growth and observation. The porous structure (i.e., multiple single-hole chambers) is arranged in a row on the electrode layer, each row has 4 single-hole chambers, and each hole chamber exposes a circular metal point, i.e., a bare electrode, on the bottom surface. The diameter of each single-hole is 1 mm to 6 mm, such as 3 mm. In this embodiment, the bare electrode can directly contact the culture in the single-hole to achieve current or voltage stimulation of the culture.

[0076] Figure 2 The porous structure of the microporous layer shown in

[0077] As shown in Figure 3 , Figure 3 A top view of a flow cavity layer is shown. The flow cavity layer of this embodiment can be formed by covering a liquid polydimethylsiloxane (PDMS) prepolymer on a metal male die (such as a tungsten steel strip male die) and curing it to form multiple single-cavity structures with a cuboid shape. Each single-cavity structure is 15 mm long, 6 mm wide, and 1 mm deep. Each single-cavity structure has a hole at each end as an inlet and an outlet, i.e., each cuboid-shaped single-cavity structure has a medium inlet at the head and a medium outlet at the tail. The diameter of the medium inlet / medium outlet is 5 mm.

[0078] Because Figure 2 four single-hole chambers form a row, in Figure 3 each cuboid-shaped single-cavity structure corresponds to a row of single-hole chambers. Of course, in other embodiments, the cavity structure of the flow cavity layer can not be limited to a cuboid shape, but can also be other shapes, which can be selected and set according to actual needs, such as being able to correspond to more than one single-hole chamber of the microporous layer and providing medium and flow shear force.

[0079] The third flow cavity layer is pasted on the surface of the second porous layer in this embodiment, and the three layers are stacked and packaged to form the integrated organ chip. In order to better realize the preparation or reversible packaging of the integrated organ chip, the material of the microporous layer and the flow cavity layer is consistent in this embodiment. In other embodiments, the material of the two layers is not limited, and can be selected according to actual needs.

[0080] In this embodiment, when the organoid or cell cluster is dynamically suspended and statically cultured using the integrated organ chip, the organoid or cell cluster is 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 flow cavity layer can apply controllable fluid shear stress to the culture.

[0081] It should be noted that the auxiliary equipment of this embodiment can include a peripheral perfusion device or a shaker for realizing the flow of the culture medium. Preferably, a shaker is used, so that the culture of the culture can be better realized without the limitation of connecting pipelines and the like. That is, the entire culture process can be realized by placing the chip on the shaker without connecting the peripheral perfusion device.

[0082] The above chip can precisely stimulate the organoid or cell cluster with electric stimulation (including current and voltage) and stable fluid shear force. The first layer adopts high-transmittance glass, which has greater hardness than stainless steel or plastic and good transmittance, is convenient for observation under a microscope and high-temperature sterilization, and is more suitable for cell culture. The second layer can individually dynamically suspend and statically culture the organoid or cell cluster in each single-hole chamber. One or more electrode points are arranged in each single-hole chamber, so that the culture can be precisely stimulated with current or voltage. The third layer can not be connected to a peripheral perfusion device, and stable flow can be formed in the microcavity through the shaker, and fluid shear force around the surface of the suspended culture can be formed in the single-hole chamber.

[0083] Embodiment Two

[0084] This embodiment provides a preparation method of an integrated organ chip.

[0085] The preparation method of the integrated organ chip of this embodiment comprises:

[0086] S01, sequentially manufacturing the electrode layer, the microporous layer and the flow cavity layer.

[0087] For example, manufacturing the microporous layer can comprise:

[0088] After the liquid polydimethylsiloxane (PDMS) prepolymer is cured in an oven at 60°C for 4 hours, the PDMS polymer is punched according to a preset structure by a microfluidic chip general puncher to form a plurality of single-hole chambers; the bottom surface of each single-hole chamber is exposed to one or more circular bare electrodes of the electrode layer.

[0089] In addition, the preparation of the flow cavity layer can include:

[0090] After the liquid polydimethylsiloxane prepolymer is cured by pouring on a tungsten steel strip male mold, a female mold with a single cavity structure of a plurality of cuboid shapes is formed. Specifically, the PDMS prepolymer with bubbles removed is covered on the tungsten steel strip male mold, and is cured by pouring at 60°C for 4 hours to form a single cavity structure of a plurality of cuboid structures. A hole is punched at each end of each cavity structure to form a culture medium inlet and a culture medium outlet.

[0091] S02, bonding the micropore layer and the flow cavity layer together, and corresponding the culture medium of the flow cavity layer to each single-hole chamber of the micropore layer.

[0092] This step can specifically include: using PDMS prepolymer to coat the upper surface of the micropore layer, covering the flow cavity layer on the surface of the micropore layer, and placing it in an oven at 60°C. The packaging is performed by heat pressing and curing overnight to bond the micropore layer and the flow cavity layer together. At this time, the packaging structure formed is a reversible structure. It should be noted that this reversible packaging structure can remove the flow cavity layer after the experiment is completed, which is convenient for taking out the experimental culture, and the micropore layer and the flow cavity layer can be cleaned and reused.

[0093] S03, stacking the bonded micropore layer and flow cavity layer on the electrode layer, and electrically connecting the electrode layer with the peripheral circuit to obtain an integrated organ chip.

[0094] It should be noted that the stacking method of the bonded micropore layer and the flow cavity layer on the electrode layer is the same as the bonding process of the micropore layer and the flow cavity layer in S02. That is, PDMS prepolymer is used to coat the upper surface of the electrode layer, the micropore layer is covered on the surface of the electrode layer, and the packaging is performed by placing it in an oven at 60°C and heat pressing and curing overnight to bond the micropore layer and the flow cavity layer on the electrode layer. At this time, the packaging structure formed at this time is also a reversible structure.

[0095] That is, the first layer, the second layer, and the third layer of the integrated organ chip are stacked and bonded in sequence to achieve reversible packaging.

[0096] For better understanding, a preparation method of the integrated organ chip shown in FIG. 1 is provided as follows:

[0097] M01: Select a 4-inch / 6-inch glass sheet with a thickness of 500 μm and high light transmission.

[0098] M02: Clean the glass surface with acetone, pure water, anhydrous ethanol, and pure water in sequence, with a cleaning time of no less than 5 minutes.

[0099] M03: Form a metal conductive layer by magnetron sputtering a metal layer (Au) on the surface of the first layer of glass, with a thickness of 150 nm.

[0100] M04: Form an insulating layer by spin coating an insulating material, polyimide, on the surface of the first layer of metal (except for the positions of the circular metal points and metal pads), with a thickness of no more than 2 μm, and baking after spin coating.

[0101] M05: Cut the first layer of glass metal conductive layer structure into electrode layer pieces containing 16 electrode points and pads according to the designed outer dimensions, with the size of the electrode layer pieces ranging around 4 cm * 5 cm.

[0102] Next, a micropore layer is prepared, specifically including the following steps:

[0103] M06: Prepare a PDMS prepolymer solution and pour it into a glass dish to degas in a vacuum box, with a vacuum degree of -15 kPa and a pumping time of no less than 30 minutes.

[0104] M07: Cure the PDMS prepolymer at 85°C for 2 hours or at 60°C for 4 hours.

[0105] M08: Cut the long strip of PDMS with a knife, and punch holes in the corresponding circular metal point arrangement position to form multiple micropore structures, i.e., multiple independent single-hole chambers.

[0106] M09: Paste the second layer of PDMS with micropore structure on the surface of the first layer, exposing the circular electrode points and metal pads.

[0107] Then, a flow chamber layer is prepared, specifically including the following steps:

[0108] M10: Cover the PDMS prepolymer that has been degassed on a smooth tungsten steel alloy male mold, and cure the PDMS prepolymer at 85°C for 2 hours or at 60°C for 4 hours.

[0109] M11: Cut the female mold with the cuboid cavity structure of the PDMS covering the male mold with a knife.

[0110] M12: Punch one hole at each end of the cuboid cavity structure (i.e., a single cavity structure in the shape of a cuboid), and then paste the cavity side of the structure on the second layer of micropore layer.

[0111] M13: The integrated organ chip is obtained by soldering single-strand ultra-fine copper wires to connect the metal pads on the organ chip to the ribbon cable pins on the printed circuit board. Finally, the solder joints are tested with a multimeter.

[0112] The integrated organ-on-a-chip prepared by the above method mainly comprises a first electrode layer, a second microporous layer, and a third flow chamber layer. When using this chip for dynamic suspension and static culture of organoids or cell clusters, the organoids or cell clusters are placed in the single-well chamber of the second microporous layer. The first electrode layer can apply adjustable voltage or current 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. This achieves precise electrical stimulation (including current and voltage) and stable fluid shear force stimulation to the organoids or cell clusters. Furthermore, the entire culture process can be performed without connecting to external perfusion equipment; the chip can be placed on a rocker bed for flow culture.

[0113] In practical use, when an organoid or cell cluster is seeded and statically cultured in a single well in the second layer, the circular metal electrode points in the first layer can precisely stimulate the organoid or cell cluster with current or voltage through external electrical stimulation signals, ensuring that the culture in each single well receives a consistent stimulation signal, which can reduce the error caused by experimental conditions between the same experimental batches.

[0114] When performing dynamic suspension culture on a culture in a single micro-cavity, the device is placed on a rocker bed and different rocking frequencies and amplitudes are set. The liquid flow in the third layer of microcavities can form a stable fluid shear force around the surface of the culture within the single micro-pore.

[0115] Static electrical stimulation experiments and dynamic suspension cultures can be performed simultaneously or in stages, depending on the experimental requirements. When conducting a single suspension culture, the entire experimental setup can be placed in an incubator without external perfusion equipment, facilitating long-term flow experiments and avoiding contamination from external equipment.

[0116] like Figure 5 and Figure 6 As shown, the circular metal electrode points can contact the bottom surface of a circular culture medium with a diameter of 200μm-2mm, providing electrical stimulation to the culture. The microporous structure is designed as a non-interconnected single-cavity structure. During flow culture, the flow of the culture medium keeps the culture in a suspended state, forming a stable wall shear force on the culture surface. The magnitude of the wall shear force can be adjusted by changing the flow rate. This embodiment uses 16 circular metal electrode points in the electrode layer as an example, but this embodiment is not limited to this. The structure of each chamber in the microporous layer and flow cavity layer is also illustrative; the technology in this embodiment can be extended to more chambers. The size and shape of the entire device can be changed according to experimental needs, and the peripheral circuitry can be expanded based on the number of metal pads.

[0117] It should be noted that the description uses the term "comprising" not to mean "consisting only of" but to mean "including, permitting also of". It should be noted that in the claims the word "comprising" does not exclude other elements or steps than the ones stated in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. It should be noted that the disclosure can be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the claims, the word "comprising" does not exclude other elements than the ones stated in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. It should be noted that the word "comprising" does not exclude other elements or steps than those listed in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The word "first", "second", "third", etc. does not imply any order but is used for distinguishing between two or more elements. The word "one" does not exclude the presence of more than one element but signifies that at least one of the element is present.

[0118] Furthermore, it is noted that the described features and characteristics can be combined in all possible technically feasible ways unless expressly stated otherwise. Numerous specific details of the application have been set forth in this description in order to provide a thorough understanding of the application. However, it will be apparent to those skilled in the art that the application can be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in order to avoid unnecessarily obscuring the application. Therefore, the scope of the application should not be construed as limited to the particular forms described herein, but should be understood to include all possible variations and modifications.

[0119] While the application has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations using the principles of the application. Therefore, to the extent there are any inconsistencies between the description and the claims, the claims should control.

[0120] Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. An integrated organ chip, characterized by, The integrated organ chip is used for applying electrical stimulation and mechanical stimulation to cells, cell clusters or organoids in vitro culture process according to requirements; The integrated organ chip is used for regulating and promoting the maturation of functions, and comprises: An electrode layer for providing electrical stimulation, a microwell layer above the electrode layer, a flow chamber layer for providing mechanical stimulation and interacting with an external circuit, and a peripheral circuit; The electrode layer comprises: a substrate, and bare electrodes corresponding to each microwell in the microwell layer on the substrate; the bare electrodes are electrically connected to the peripheral circuit through conductive components embedded in the surface of the substrate; the non-bare electrode area of the substrate surface is an insulating area; each bare electrode is independently connected to the peripheral circuit; The microwell layer comprises: a structure with a plurality of independent single-well chambers adhered to the electrode layer, and a single well in each single-well chamber is permeable and the bottom corresponds to more than one sheet-shaped bare electrode of the electrode layer, so that the bare electrode after being electrified applies adjustable electrical stimulation to the culture in the single-well chamber; the diameter of the bare electrode is smaller than the pore size of each single well, and directly contacts the culture in the single well for current or voltage stimulation; each single-well chamber in the microwell layer can independently dynamically suspend and statically culture cell clusters or organoids; The flow chamber layer is a plurality of cavity structures for carrying culture medium of the culture above the microwell layer, and the culture medium flows in the respective cavity structures with the aid of auxiliary equipment to obtain controllable fluid wall shear stress around the surface of the culture; the cavity of each cavity structure is connected to a plurality of single-well chambers of the microwell layer and provides culture medium for the culture in the single-well chamber; the flow chamber layer is not connected to the peripheral perfusion equipment, and the culture medium forms stable flow in the microcavity through the shaker. When the integrated organ chip is used to dynamically suspend and statically culture organoids or cell clusters, the organoids or cell clusters are placed in the single-well chamber of the second microwell layer, the first electrode layer applies voltage or current adjustable electrical stimulation to the culture, and the flow of the culture medium in the third flow chamber layer applies controllable fluid shear stress to the culture.

2. The integrated organ chip according to claim 1, wherein: each bare electrode is a sheet-shaped circular bare electrode; each bare electrode on the substrate is independently connected to the peripheral circuit; and the diameter of the circular bare electrode is smaller than the pore size of each single well in the microwell layer; the substrate comprises a glass base, a conductive component layer stacked on the glass base, and an insulating layer of the non-bare electrode area; the conductive components of the conductive component layer are metal stacked wires, one end of the wire is connected to the bare electrode, and the other end is provided with a metal pad with an identifier for electrical connection with the peripheral circuit.

3. The integrated organ chip according to claim 2, wherein: the glass base is a 4-inch circular high-transmittance glass with a thickness of 500+-20um; the diameter of the circular bare electrode is 70+-10um; the insulating layer is made of polyimide with a thickness less than or equal to 2um; and / or, the material of the microwell layer and the flow chamber layer is consistent.

4. The integrated organ chip according to claim 1, wherein: The microporous layer is a PDMS porous membrane layer formed by punching after curing of a polydimethylsiloxane (PDMS) polymer; Each single-hole has a pore size of 1 mm to 6 mm; The single-hole chambers of the microporous layer are arranged in multiple rows, each row having N single-hole chambers, N being greater than or equal to 2, and the bottom of each single-hole chamber corresponding to one or more bare electrodes.

5. The integrated organ chip according to claim 1 or 4, wherein, The cavity structure of the flow cavity layer is a single cavity structure in the shape of a cuboid, each single cavity structure in the shape of a cuboid corresponds to a row of single-hole chambers, and the head of the single cavity structure in the shape of a cuboid is provided with a culture medium inlet and the tail is provided with a culture medium outlet. Alternatively, The flow cavity layer is a negative mold formed by pouring and curing of a PDMS prepolymer on a tungsten steel strip positive mold, and the flow cavity layer has multiple single cavity structures in the shape of a cuboid, each single cavity structure in the shape of a cuboid corresponds to a row of single-hole chambers, and the head of the single cavity structure in the shape of a cuboid is provided with a culture medium inlet and the tail is provided with a culture medium outlet.

6. The integrated organ chip according to claim 5, wherein, The single cavity structure in the shape of a cuboid has a length of 15 mm, a width of 6 mm, and a depth of 1 mm. The pore size of the culture medium inlet / culture medium outlet is 1 mm-5 mm. Alternatively, The auxiliary equipment includes a peripheral perfusion device or a shaker for realizing culture medium flow.

7. A method for producing an integrated organ chip according to any one of claims 1 to 6, characterized in that Including: S01, sequentially fabricating the electrode layer, the microporous layer, and the flow cavity layer; S02, bonding the microporous layer and the flow cavity layer together, and making the culture medium of the flow cavity layer correspond to each single-hole chamber of the microporous layer; S03, stacking the bonded microporous layer and the flow cavity layer on the electrode layer, and electrically connecting the electrode layer with a peripheral circuit to obtain an integrated organ chip.

8. The preparation method according to claim 7, characterized in that, The fabrication of the microporous layer in S01 includes: Curing the liquid PDMS prepolymer in a 60-degree Celsius oven for 4 hours or in an 85-degree Celsius oven for 2 hours to form a PDMS polymer, and then punching the PDMS polymer according to a preset structure to form a matrix of multiple single-hole structures of single-hole chambers using a microfluidic puncher; The bottom surface of each single-hole structure of single-hole chambers exposes one or more circular bare electrodes of the electrode layer; The fabrication of the flow cavity layer in S01 includes: Covering the PDMS prepolymer without bubbles on a tungsten steel strip positive mold, and curing in a 60-degree Celsius oven for 4 hours or in an 85-degree Celsius oven for 2 hours to form multiple single cavity structures in the shape of a cuboid, Punching one hole at each end of each single cavity structure in the shape of a cuboid to form a culture medium inlet and a culture medium outlet.

9. The preparation method according to claim 7, characterized in that, The S02 includes: Using the PDMS prepolymer without bubbles to coat the upper surface of the microporous layer, covering the flow cavity layer on the surface of the microporous layer, and placing it in a 60-degree Celsius oven for heat pressing and curing overnight to form a reversible packaging structure by heat pressing, so that the microporous layer and the flow cavity layer are bonded together.

10. The production method according to any one of claims 7 to 9, characterized by, The fabrication of the electrode layer in S01 includes: Select a 4-inch glass sheet with a thickness of 500 μm and high light transmission, and sequentially clean the glass surface with acetone, pure water, anhydrous ethanol, and pure water, with a cleaning time of no less than 5 minutes; Form a metal conductive layer with a thickness of 150 nm on the surface of the first layer of glass by magnetron sputtering of a metal layer; Form an insulating layer by spin coating an insulating material, polyimide, on the non-circular metal points and the positions outside the metal pads on the surface of the first layer of metal, with a thickness of less than or equal to 2 μm, and baking to obtain an intermediate structure; Cut the intermediate structure into a single piece containing 16 bare electrodes and pads according to a predetermined size to obtain an electrode layer.

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