Multi-organ chip as well as preparation method and use method thereof

Abstract

The invention provides a multi-organ chip as well as a preparation method and a use method thereof. The multi-organ chip comprises a structural layer and an insulating substrate, wherein the structural layer is provided with a culture chamber, an perfusion channel and a hydrogel perfusion pipeline; a microelectrode array is arranged on the insulating substrate; the limiting part extending from the bottom of the culture chamber to the top of the culture chamber is arranged in the culture chamber, so that the accommodating cavity for accommodating the cell mass or the organoid is formed between the outer side wall of the limiting part and the inner side wall of the culture chamber, and the cell mass or the organoid is limited in the accommodating cavity and is in contact with the hydrogel barrier; due to the affinity between the hydrogel barrier and the cell mass or the organoid, the cell mass or the organoid can be firmly attached to the hydrogel barrier, and the electrode sites of the microelectrodes in the microelectrode array are located at the bottom of the containing cavity, so that the shape and the function of the cell mass or the organoid can be maintained, and the cell mass or the organoid can be prevented from being damaged. And a stable basis is provided for subsequent electrophysiological signal detection.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and particularly relates to a multi-organ chip and a preparation method and a usage method thereof. Background Art

[0002] In the field of biomedical research, the organ chip technology, as a new technical platform, provides a powerful tool for simulating the functions of human organs, disease research, drug screening, etc. Among them, the microelectrode array, as a key component in the organ chip, is widely used in detecting the discharge behaviors of biological tissues such as neurons and muscle cells, providing an important means for understanding bioelectrophysiological phenomena.

[0003] For brain chips, most of the existing detection methods rely on culturing neurons on the microelectrode arrays in the chip. By culturing neurons from different brain regions in different culture chambers respectively, and allowing the axons of neurons to pass through while preventing the cell bodies from entering through the microchannels, the accurate simulation and detection of the neuron network are achieved. Although this method has made remarkable progress in the research of neuron networks, the research on using integrated planar microelectrode arrays for directly detecting brain organoids (i.e., highly biomimetic brain tissue models) is relatively rare.

[0004] In the research of myocardial or muscle tissues, the traditional detection method is usually to culture myocardial or muscle tissues on a cantilever beam containing electrodes. When the myocardial or muscle tissues pulsate, the elastic cantilever beam deforms accordingly, and then changes the resistance of the electrodes, thereby achieving the detection of pulsation signals. In recent years, there have also been studies attempting to culture cardiac organoids (i.e., highly biomimetic heart tissue models) in chambers on the surface of microelectrode arrays, and reflecting the electrophysiological activities of cardiac organoids by real-time detecting the changes in field potential caused by discharges.

[0005] However, there is a major difficulty in using planar microelectrode arrays to detect the electrophysiological activities of electricity-producing organoids (such as brain organoids and cardiac organoids), that is, the fixation problem of organoids. Since the highly biomimetic brain organoids and cardiac organoids are approximately spherical or ellipsoidal in shape and usually grow suspended in the culture medium, it is very difficult to achieve stable and reliable fixation on the microelectrode arrays. In addition, in order to maintain a good metabolic balance of the organoids, it is usually necessary to keep the solution shaken to a certain extent, which further increases the position change of the organoids during the detection process, bringing difficulties to signal analysis. Summary of the Invention

[0006] In view of the defects and deficiencies of the above-mentioned prior art, the present application provides a multi-organ chip and its preparation method and usage method. In this multi-organ chip, a limiting member extending from the bottom to the top of the culture chamber is provided in the culture chamber. An accommodation cavity for accommodating cell clusters or organoids is formed between the outer sidewall of the limiting member and the inner sidewall of the culture chamber, so that the cell clusters or organoids are restricted within the accommodation cavity and contact with the hydrogel barrier. Due to the affinity between the hydrogel barrier and the cell clusters or organoids, the cell clusters or organoids can firmly adhere to the hydrogel barrier. Thus, it not only helps to maintain the morphology and function of the cell clusters or organoids, but also provides a stable basis for subsequent electrophysiological signal detection.

[0007] An embodiment of the present application provides a multi-organ chip, including:

[0008] A structural layer, on which at least two culture chambers, a perfusion channel, and a hydrogel perfusion pipeline corresponding to the culture chambers are provided; different culture chambers are respectively used for culturing different cell clusters or organoids. A limiting member is provided in the culture chamber, and the limiting member extends from the bottom of the culture chamber to the top of the culture chamber. An accommodation cavity for accommodating cell clusters or organoids is formed between the outer sidewall of the limiting member and the inner sidewall of the culture chamber; the perfusion channel is a closed pipeline with openings at both ends, used to connect different culture chambers; the perfusion channel is connected to the culture chamber through the hydrogel perfusion pipeline, and a hydrogel barrier is constructed in the hydrogel perfusion pipeline, and the hydrogel barrier is used for culturing vascular endothelial cells to form a vascular endothelial barrier and a capillary network;

[0009] An insulating substrate, on which at least two groups of microelectrode arrays are provided, and each group of microelectrode arrays includes a plurality of microelectrodes, and the electrode sites of the microelectrodes are located at the bottom of the accommodation cavity.

[0010] As an implementation manner, the perfusion channel is provided with a perfusion channel inlet and a perfusion channel outlet, and at least two reference electrodes are further provided on the insulating substrate, and the electrode sites of one of the reference electrodes are respectively provided at the bottom of the perfusion channel inlet and the bottom of the perfusion channel outlet.

[0011] As an implementation manner, the hydrogel perfusion pipeline includes an introduction section communicated with the hydrogel perfusion pipeline injection port and a diffusion section surrounding the bottom of the outer sidewall of the culture chamber. Each end of the diffusion section is provided with an opening. The outlet of the introduction section is communicated with the openings at both ends of the diffusion section. The construction raw materials of the hydrogel barrier flow out from the introduction section and enter the diffusion section from the openings at both ends of the diffusion section respectively, and converge at a certain place in the diffusion section.

[0012] As an implementation manner, along the circumference of the outer sidewall of the culture chamber, the length of the diffusion section is 3 / 4 of the circumference of the outer sidewall of the culture chamber.

[0013] As an implementation manner, the construction raw material of the hydrogel barrier is a biohydrogel for cell culture.

[0014] As an implementation manner, the biohydrogel includes a natural hydrogel or an engineered active hydrogel;

[0015] As an implementation manner, the natural hydrogel is selected from human fibrinogen, bovine fibrinogen, or type I rat tail collagen, or, the engineered active hydrogel is selected from methacrylated gelatin.

[0016] As an implementation manner, a part of the outer sidewall of the culture chamber is surrounded by the perfusion channel, and the surrounded part of the culture chamber and the perfusion channel are communicated through the hydrogel perfusion pipeline.

[0017] As an implementation manner, the perfusion channel includes an annular section surrounding the outer sidewall of the culture chamber and a straight section connected to the annular section, and the included angle between the annular section and the straight section is not less than 60°.

[0018] As an implementation manner, the bottom of the limiting member is located at the central position of the bottom of the culture chamber, and the bottom of the limiting member is circular.

[0019] As an implementation manner, the bottom diameter of the limiting member is between 1 mm and 3 mm;

[0020] As an implementation manner, the height of the limiting member is between 2 mm and 5 mm.

[0021] As an implementation manner, the culture chamber is a circular chamber open at the top.

[0022] As an implementation manner, the diameter of the culture chamber is between 3 mm and 5 mm.

[0023] As an implementation manner, the multi-organ chip further includes a liquid storage hole layer and a transparent substrate. The liquid storage hole layer is arranged above the structure layer. The liquid storage hole layer is provided with a plurality of liquid storage holes penetrating through its upper surface and its lower surface. The centers of the perfusion channel inlet, the perfusion channel outlet, the injection port of the hydrogel perfusion pipeline, and the culture chamber of the structure layer respectively have the centers of the corresponding liquid storage holes aligned. The hole spacing between adjacent liquid storage holes is 9 mm;

[0024] The transparent substrate is arranged below the insulating substrate.

[0025] As an implementation manner, the electrode sites of the plurality of microelectrodes are distributed annularly along the accommodation cavity.

[0026] Another embodiment of the present application provides a method for preparing a multi-organ chip, including the following steps:

[0027] Fabricate a structural layer and a limiting member; the structural layer includes at least two culture chambers, a perfusion channel, and a hydrogel perfusion pipeline corresponding to the culture chambers;

[0028] Fabricate at least two groups of microelectrode arrays on an insulating substrate, each group of the microelectrode arrays including a plurality of microelectrodes;

[0029] Bond the structural layer to the insulating substrate, and bond the limiting member to the insulating substrate, so as to form an accommodation cavity between the outer sidewall of the limiting member and the inner sidewall of the culture chamber, and the electrode sites of the microelectrodes are located at the bottom of the accommodation cavity.

[0030] As an implementation manner, fabricating the structural layer includes the following steps:

[0031] Fabricate a mold with a microfluidic structure;

[0032] Based on the above mold, perform a casting on the structural layer to obtain a microfluidic structure.

[0033] As an implementation manner, fabricating the limiting member includes the following steps: three-dimensionally print a casting mold of the limiting member, and cast out the limiting member.

[0034] As an implementation manner, fabricating at least two groups of microelectrode arrays on an insulating substrate includes the following steps:

[0035] Clean the insulating substrate;

[0036] Fabricate a microelectrode array on the insulating substrate;

[0037] Fabricate an electrode insulating layer.

[0038] Another embodiment of the present application provides a method for using a multi-organ chip, including the following steps:

[0039] Construct a hydrogel barrier in the hydrogel perfusion pipeline;

[0040] Inject vascular endothelial cells into the perfusion channel of the multi-organ chip;

[0041] Add cell clusters or organoids and their specific culture media into the culture chambers of the multi-organ chip for culture;

[0042] Connect the perfusion channel of the multi-organ chip to an external driving pump for perfusion culture;

[0043] Detect the discharge behavior of cell clusters or organoids in the culture chamber.

[0044] As an implementation manner, the method for constructing the hydrogel barrier is the spontaneous capillary injection method, including the following steps: dropping the hydrogel construction raw materials into the main inlet of the hydrogel perfusion pipeline, and completing the injection of the hydrogel construction raw materials by spontaneous driving of capillary force, and curing to form the hydrogel barrier.

[0045] As described above, the multi-organ chip, its preparation method and usage method of the present application have the following beneficial effects:

[0046] The multi-organ chip of the present application is provided with a limiting member extending from the bottom of the culture chamber to the top of the culture chamber in the culture chamber. An accommodation cavity is formed between the outer side wall of the limiting member and the inner side wall of the culture chamber. The accommodation cavity is used to accommodate cell clusters or organoids, effectively promoting the contact between the cell clusters or organoids and the hydrogel barrier. Since the surface of the hydrogel barrier has good affinity with the cell clusters or organoids, it can ensure that the cell clusters or organoids are firmly attached to the hydrogel barrier, which not only helps to maintain the morphology and function of the cell clusters or organoids, but also provides a stable basis for subsequent electrophysiological signal detection; at the same time, the cell clusters or organoids are fixed by the hydrogel barrier from the side, which will not affect the contact area between the cell clusters or organoids and the microelectrodes, ensuring efficient signal acquisition and accuracy; by optimizing the contact between the cell clusters or organoids and the electrode sites, it can effectively reduce signal interference caused by position changes or unstable contacts, improve the signal-to-noise ratio of electrophysiological signals, and thus improve the reliability of experimental data. Description of the Drawings

[0047] Figure 1 It shows a schematic structural diagram of the multi-organ chip provided in Embodiment 1 of the present application.

[0048] Figure 2 It shows Figure 1 The exploded structural diagram of

[0049] Figure 3 It shows a schematic structural diagram of the structural layer in the multi-organ chip provided in Embodiment 1 of the present application.

[0050] Figure 4 It shows Figure 3 The cross-sectional view along the A-A direction in

[0051] Figure 5 It shows a schematic structural diagram of the structural layer in the multi-organ chip provided in Embodiment 2 of the present application.

[0052] Figure 6 It shows a schematic structural diagram of the multi-organ chip provided in Embodiment 2 of the present application.

[0053] Figure 7 It showsFigure 6 Explosion structure schematic diagram.

[0054] Figure 8 Shown as Figure 6 Top view structure schematic diagram.

[0055] Component label description

[0056] 100, structural layer; 110, culture chamber; 120, perfusion channel; 121, perfusion channel inlet; 122, perfusion channel outlet; 123, annular section; 124, straight section; 130, hydrogel perfusion pipeline; 131, hydrogel perfusion pipeline injection port; 132, introduction section; 133, diffusion section; 140, limiting member; 200, insulating substrate; 210, microelectrode array; 211, microelectrode; 220, reference electrode; 300, liquid storage hole layer; 310, liquid storage hole; 400, transparent substrate; 2110, electrode site of microelectrode; 2210, electrode site of reference electrode. Specific implementation mode

[0057] The following uses specific specific examples to illustrate the implementation mode of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. For the examples without specific experimental conditions or operation conditions noted, they are made according to conventional conditions or according to the conditions recommended by the material suppliers.

[0058] In addition, it should be understood that one or more method steps mentioned in this application do not exclude the existence of other method steps before and after the combined steps or the insertion of other method steps between these clearly mentioned steps, unless otherwise stated; it should also be understood that the combined connection relationship between one or more devices or apparatuses mentioned in this application does not exclude the existence of other devices or apparatuses before and after the combined devices or apparatuses or the insertion of other devices or apparatuses between these two clearly mentioned devices or apparatuses, unless otherwise stated. Moreover, unless otherwise stated, the numbers of each method step are only convenient tools for identifying each method step, rather than restricting the arrangement order of each method step or limiting the scope of implementation of this application. The change or adjustment of their relative relationship, without substantial change in technical content, should also be regarded as the scope of implementation of this application.

[0059] In the following examples, the reagents, materials, and instruments used, if not specifically stated, can be obtained commercially.

[0060] In the field of biomedical research, the organ-on-a-chip technology, as an emerging technology platform, provides a powerful tool for simulating the functions of human organs, disease research, and drug screening. Among them, the microelectrode array, as a key component in the organ-on-a-chip, is widely used to detect the electrical discharge behaviors of biological tissues such as neurons and muscle cells, providing an important means for understanding bioelectrophysiological phenomena. However, there is a major difficulty in using planar microelectrode arrays to detect the electrophysiological activities of electricity-producing organoids (such as brain organoids and heart organoids), that is, the fixation problem of organoids. Due to the highly biomimetic brain organoids and heart organoids being morphologically similar to spheres or ellipsoids and usually growing suspended in a culture medium, it is very difficult to achieve stable and reliable fixation on the microelectrode array. In addition, in order to maintain a good metabolic balance of the organoids, it is usually necessary to keep the solution shaking to a certain extent, which further increases the position change of the organoids during the detection process, bringing difficulties to signal analysis.

[0061] In view of the above defects, the present application provides a multi-organ chip and its preparation method and usage method. The following examples will be used for detailed description.

[0062] Example 1

[0063] This example provides a multi-organ chip, as Figure 1 and Figure 2 shown, the multi-organ chip includes a structural layer 100 and an insulating substrate 200.

[0064] At least two culture chambers 110, at least one perfusion channel 120, and at least two hydrogel perfusion pipes 130 are arranged on the structural layer 100. Figure 1 and Figure 2The multi-organ chip therein is provided with two culture chambers 110, one perfusion channel 120 and two hydrogel perfusion pipes 130. For example only, the number of culture chambers 110, the number of perfusion channels 120 and the number of hydrogel perfusion pipes 130 can be adjusted according to actual needs, and no specific limitation is made in this embodiment. Among them, the culture chamber 110 is a circular chamber open at the top, which is convenient for placing cell clusters or organoids. Different culture chambers 110 are used to culture different cell clusters or organoids. Thus, different culture chambers 110 can separately provide corresponding culture conditions for different cell clusters or organoids. A limiting member 140 is arranged in the culture chamber 110. The limiting member 140 extends from the bottom of the culture chamber 110 to the top of the culture chamber 110. An accommodation cavity (not marked in the figure) is formed between the outer side wall of the limiting member 140 and the inner side wall of the culture chamber 110. The cell cluster or organoid is cultured in the accommodation cavity. The accommodation cavity provides a stable microenvironment for the cell cluster or organoid, and can effectively fix the cell cluster or organoid to prevent the cell cluster or organoid from shifting during the culture process. The perfusion channel 120 is a closed pipe with openings at both ends. The perfusion channel 120 can simulate the blood flow and circulation system in the human body to provide a dynamic culture environment for the cell clusters or organoids on the multi-organ chip; at the same time, the perfusion channel 120 is used to connect different culture chambers 110 to simulate the interaction and signal transmission between organs. The perfusion channel 120 is connected to the culture chamber 110 through the hydrogel perfusion pipe 130. A hydrogel barrier is constructed in the hydrogel perfusion pipe 130. The hydrogel barrier is used to culture vascular endothelial cells to form a vascular endothelial barrier and a capillary network. Moreover, in this embodiment, by arranging the limiting member 140 in the culture chamber 110, an accommodation cavity is formed between the outer side wall of the limiting member 140 and the inner side wall of the culture chamber 110. After research, after a period of culture, the cell cluster or organoid in the accommodation cavity will firmly adhere to the hydrogel barrier, which can further improve the position fixation of the cell cluster or organoid. The hydrogel perfusion pipe 130 in this embodiment is used to connect each culture chamber 110 with the perfusion channel 120. Therefore, the hydrogel perfusion pipe 130 is correspondingly arranged with the culture chamber 110, that is, the number of hydrogel perfusion pipes 130 is the same as that of the culture chambers 110.

[0065] Such as Figure 1 and Figure 2As shown, at least two groups of microelectrode arrays 210 are provided on the insulating substrate 200. Each group of microelectrode arrays 210 includes a plurality of microelectrodes 211, and the electrode sites 2110 of the microelectrodes are located at the bottom of the accommodating cavity. The electrode sites 2110 of the microelectrodes are arranged in a ring shape at the bottom of the accommodating cavity. Since there is an affinity between the cell mass or organoid and the hydrogel barrier, it can be fixed on the hydrogel barrier, realizing the fixation of the cell mass or organoid, which is convenient for microelectrode detection. Since the hydrogel barrier fixes the cell mass or organoid from the side, it will not affect the close contact between the cell mass or organoid and the electrode sites 2110 of the microelectrodes, ensuring the acquisition of signals. The diameter and number of the electrode sites 2110 of the microelectrodes in each culture chamber 110 can be adjusted according to requirements. The electrode sites 2110 of the microelectrodes can also be fabricated into different three-dimensional structures according to requirements.

[0066] The multi-organ chip provided in this embodiment forms an accommodating cavity between the outer sidewall of the limiting member 140 and the inner sidewall of the culture chamber 110 by providing the limiting member 140 in the culture chamber 110, facilitating the arrangement and growth of cell masses or organoids at the edge of the culture chamber 110. At the same time, the electrode sites 2110 of the microelectrodes are arranged at the bottom of the accommodating cavity, which is crucial for maintaining the stability of the cell mass or organoid during the detection of the microelectrode array, helping to improve the accuracy and repeatability of signal detection. While not affecting the detection of the microelectrode array, the fixation of the cell mass or organoid is realized, enabling the real-time monitoring of the discharging behaviors of power-generating organoids such as brain organoids, heart organoids, and muscle organoids in the multi-organ chip.

[0067] In an alternative embodiment, as Figure 3 and Figure 4 shown, the perfusion channel 120 is provided with a perfusion channel inlet 121 and a perfusion channel outlet 122. Figure 3 The Figure 4 positions of the perfusion channel inlet 121 and the perfusion channel outlet 122 can be interchanged. The positions of the perfusion channel inlet 121 and the perfusion channel outlet 122 depend on the flow direction of the medium in the perfusion channel 120, and no specific limitation is made in this embodiment. As Figure 2 shown, at least two reference electrodes 220 are further provided on the insulating substrate 200, and electrode sites 2210 of a reference electrode are respectively provided at the bottom of the perfusion channel inlet 121 and the bottom of the perfusion channel outlet 122.

[0068] In an alternative embodiment, the construction raw material of the hydrogel barrier is a bio-hydrogel for cell culture. Optionally, the bio-hydrogel includes a natural hydrogel or an engineered active hydrogel. The natural hydrogel is selected from human fibrinogen, bovine fibrinogen, or type I rat tail collagen. The engineered active hydrogel is selected from methacrylated gelatin.

[0069] In an alternative embodiment, the diameter of the culture chamber 110 is between 3 mm and 5 mm. Specifically, the diameter of the culture chamber 110 can be, for example, 3 mm, 4 mm, or 5 mm. This is to simulate the microenvironment of in-vivo organs, enabling cell clusters or organoids to grow and differentiate in an environment closer to physiological conditions, thereby improving the accuracy and reliability of experiments.

[0070] In an alternative embodiment, as Figure 4 shown, the bottom of the limiting member 140 is located at the central position of the bottom of the culture chamber 110, and the bottom of the limiting member 140 is circular. Optionally, the bottom diameter of the limiting member 140 is between 1 mm and 3 mm. Specifically, the bottom diameter of the limiting member 140 can be, for example, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, etc. The bottom diameter of the limiting member 140 can be adjusted according to the size of the cell cluster or organoid. Optionally, the height of the limiting member 140 is between 2 mm and 5 mm. Specifically, the height of the limiting member 140 can be, for example, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, etc. The height of the limiting member 140 can be determined according to the size of the cell cluster or organoid and the difficulty level of the actual manufacturing process. Optionally, the shape of the limiting member 140 can be a conical cylinder, a cylinder, etc.

[0071] Embodiment Two

[0072] This embodiment also provides a multi-organ chip. As Figure 5 shown, this multi-organ chip also includes a structural layer 100 and an insulating substrate 200. The difference between this embodiment and Embodiment One is as follows:

[0073] As Figure 5 shown, the hydrogel perfusion pipeline 130 includes an introduction section 132 communicating with the hydrogel perfusion pipeline injection port 131 and a diffusion section 133 surrounding the bottom of the outer wall of the culture chamber 110. Each end of the diffusion section 133 is provided with an opening (not labeled in the figure). The outlet of the introduction section 132 communicates with the openings at both ends of the diffusion section 133. The raw materials for constructing the hydrogel barrier flow out from the introduction section 132 and enter the diffusion section 133 through the openings at both ends of the diffusion section 133 respectively, and converge at a certain place within the diffusion section 133. In the multi-organ chip provided in this embodiment, after combining the diffusion section 133 and the introduction section 132 in the hydrogel perfusion pipeline 130, it can completely surround the bottom of the outer wall of the culture chamber 110 in a circle, and the raw materials for constructing the hydrogel barrier enter the diffusion section 133 from two directions from the introduction section 132, forming a complete hydrogel barrier surrounding the culture chamber 110. By dropping a fixed volume of the raw materials for constructing the hydrogel barrier into the hydrogel perfusion pipeline injection port 131, the construction of the hydrogel barrier is completed by spontaneous capillary injection. The operation difficulty is low, and the success rate can almost reach 100%, improving the success rate of constructing the hydrogel barrier.

[0074] In an alternative embodiment, as Figure 5 shown, a portion of the outer wall of the culture chamber 110 is surrounded by a perfusion channel 120. The surrounded portion of the culture chamber 110 and the perfusion channel 120 are connected through a hydrogel perfusion pipeline 130, specifically through a diffusion section 133 of the hydrogel perfusion pipeline 130.

[0075] In an alternative embodiment, as Figure 5 shown, along the circumferential direction of the outer wall of the culture chamber 110, the length of the diffusion section 133 is 3 / 4 of the circumference of the outer wall of the culture chamber 110. Thus, a complete hydrogel barrier surrounding the culture chamber 110 is formed, which is beneficial to fixing the cell mass or organoid from the side by the hydrogel barrier and does not affect the close contact between the cell mass or organoid and the electrode site 2110 of the microelectrode at the bottom of the culture chamber 110, improving the accuracy and reliability of microelectrode detection.

[0076] In an alternative embodiment, as Figure 5 shown, the perfusion channel 120 includes an annular section 123 surrounding the outer wall of the culture chamber 110 and a straight section 124 connected to the annular section 123. The annular section 123 is completely attached to the diffusion section 133. There is an included angle β between the annular section 123 and the straight section 124, and the included angle β is not less than 60°. Specifically, the included angle β can be, for example, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, etc. It has been experimentally proven that within the above range, the larger the included angle β between the annular section 123 and the straight section 124, the less the amount of the hydrogel barrier overflowing into the perfusion channel.

[0077] In an alternative embodiment, as Figures 6 to 8As shown in the figure, the multi-organ chip of this embodiment further includes a liquid storage hole layer 300 and a transparent substrate 400. The liquid storage hole layer 300 is disposed above the structure layer 100. A plurality of liquid storage holes 310 are provided on the liquid storage hole layer 300, and the liquid storage holes 310 penetrate from the upper surface of the liquid storage hole layer 300 to the lower surface of the liquid storage hole layer 300. Among them, the perfusion channel inlet 121, the perfusion channel outlet 122, the hydrogel perfusion pipeline injection port 131, and the culture chamber 110 in the structure layer 100 respectively have corresponding liquid storage holes 310 on the liquid storage hole layer 300, and the centers of the corresponding liquid storage holes 310 are aligned with the centers of the above-mentioned structures. Optionally, the hole pitch between adjacent liquid storage holes 310 is 9 mm. The material of the liquid storage hole layer 300 can be, for example, polycarbonate (PC), or polymethyl methacrylate (PMMA), etc. The thickness of the liquid storage hole layer 300 is between 8 mm and 10 mm. The positions of the plurality of liquid storage holes 310 provided on the liquid storage hole layer 300 can correspond to the perfusion channel inlet 121, the perfusion channel outlet 122, the hydrogel perfusion pipeline injection port 131, and the culture chamber 110 provided on the structure layer 100. The liquid storage hole layer 300 can be a 96-well plate, which is compatible with commercial devices for the well plate, such as an automatic pipetting workstation, a shaking shaker, etc. The transparent substrate 400 plays a role of mechanical support for the insulating substrate 200 and the structure layer 100.

[0078] Embodiment 3

[0079] This embodiment provides a method for preparing a multi-organ chip, including the following steps:

[0080] S1. Fabricate the structure layer and the limiting member; the fabricated structure layer includes at least two culture chambers, a perfusion channel, and at least two hydrogel perfusion pipelines, and the hydrogel perfusion pipelines are correspondingly arranged with the culture chambers;

[0081] S2. Fabricate at least two groups of microelectrode arrays on the insulating substrate, and each group of microelectrode arrays includes a plurality of microelectrodes;

[0082] S3. Bond the structure layer to the insulating layer substrate, and bond the limiting member to the insulating substrate, so as to form an accommodation cavity between the outer side wall of the limiting member and the inner side wall of the culture chamber, and the electrode sites of the microelectrodes are located at the bottom of the accommodation cavity.

[0083] Among them, step S1 includes fabricating the structure layer and the limiting member according to the soft lithography process. The structure layer is a PDMS structure layer.

[0084] According to the designed chip structure, use the standard photolithography process to fabricate a mold with two layers of structures on the silicon wafer:

[0085] First, transfer a 300-μm-thick SU8 dry film onto the silicon wafer, and perform ultraviolet exposure to obtain the first layer of pattern;

[0086] After the post-baking of SU8, transfer the 500-μm-thick SU8 dry film onto the first layer of SU8, and then perform ultraviolet exposure, post-baking, and development to obtain an SU8 mold with a two-layer structure.

[0087] Based on the SU8 mold, cast a microfluidic structure using PDMS, with a thickness ranging from 1 mm to 2 mm.

[0088] Drill through holes, which include a culture chamber, an inlet for the hydrogel perfusion pipeline, an inlet for the perfusion channel, and an outlet for the perfusion channel.

[0089] 3D print a limiting part mold for the PDMS casting, such as a conical mold, and cast out the limiting part.

[0090] In step S2, the layout of the microelectrode array sites can refer to Figure 2 . Fabricate the microelectrode array base layer according to the microelectrode array manufacturing process. Specifically, it includes the following steps:

[0091] Clean the substrate: Clean the quartz wafer or borosilicate glass wafer using a Phiranha solution and perform plasma treatment for 1 minute.

[0092] Fabricate the microelectrode array using the Lift-off process: Spin-coat AZ4620 on the substrate, perform patterning through photolithography, then sputter a titanium / gold metal layer, and remove the photoresist with acetone to leave the metal layer.

[0093] Fabricate the electrode insulation layer: Deposit a silicon dioxide insulation layer by chemical vapor deposition and etch the microelectrode sites and gold electrode pins through reactive ion etching.

[0094] In step S3, it specifically includes the following steps:

[0095] After plasma treatment (200 W, 2 min) of the PDMS structural layer and the insulating substrate, perform irreversible alignment bonding.

[0096] After plasma treatment (200 W, 1 min) of the bonded above structure and the limiting part, irreversibly align and bond the limiting part onto the insulating substrate.

[0097] To assist alignment, corresponding alignment mark patterns can be fabricated on the insulating substrate. The pins of the microelectrodes on the insulating substrate are connected to the PCB board through the gold wire ball bonding process, and interfaces adapted to the commercial brain-computer interface system are fabricated on the PCB board. The chip connected to the PCB board is sterilized using ethylene oxide. During use, sterilize the liquid storage hole layer containing the liquid storage holes by ultraviolet irradiation, align the culture chamber, the inlet for the hydrogel perfusion pipeline, the inlet for the perfusion channel, and the outlet for the perfusion channel on the structural layer, and make the chip fit tightly to complete the assembly of the chip.

[0098] Example 4

[0099] This embodiment provides a method for using a multi-organ chip, including the following steps:

[0100] S100. Construct a hydrogel barrier in the hydrogel perfusion pipeline;

[0101] S200. Inject vascular endothelial cells into the perfusion channels of the multi-organ chip;

[0102] S300. Add cell clusters or organoids and their specific culture media into the culture chambers of the multi-organ chip for culture;

[0103] S400. Connect the perfusion channels of the multi-organ chip to an external driving pump for perfusion culture;

[0104] S500. Detect the discharging behavior of the cell clusters or organoids in the culture chambers.

[0105] The hydrogel barrier in step S100 can be a fibrin hydrogel barrier or a type I rat tail collagen hydrogel barrier.

[0106] The formation of the fibrin hydrogel barrier includes the following steps:

[0107] Prepare a fibrinogen working solution with a concentration of 10 mg / ml and a thrombin working solution with an activity of 20 U / ml in advance;

[0108] Perform plasma treatment (200 W, 2 min) on the assembled multi-organ chip, and then place it on ice for 2 min;

[0109] Mix the thrombin and fibrinogen solutions in a ratio of 1:4, and drop 10 μl of the mixed solution into the inlet of each hydrogel perfusion pipeline;

[0110] Place the multi-organ chip in an incubator at 37 °C for 30 min to allow the hydrogel to polymerize completely;

[0111] After polymerization, add phosphate buffered saline (PBS) to the multi-organ chip, and then store the multi-organ chip in a clean and humid environment until it is taken out when needed.

[0112] The formation of the type I rat tail collagen hydrogel barrier includes the following steps:

[0113] Perform plasma treatment (200 W, 2 min) on the assembled multi-organ chip, and then place it on ice for 2 min;

[0114] Mix type I rat tail collagen (5 mg / ml), PBS and NaHCO 3 (37 g / L) evenly in a ratio of 8:1:1, and drop 10 μl of the mixed solution into the inlet of each hydrogel perfusion pipeline;

[0115] Place the multi-organ chip in an incubator at 37 °C for 1 h to allow the hydrogel to fully polymerize;

[0116] After polymerization, add phosphate buffered saline (PBS) to the multi-organ chip, and then store the multi-organ chip in a clean and humid environment until it is taken out when needed.

[0117] The formation of the biomimetic endothelial barrier in step S200 includes the following steps:

[0118] Use human umbilical vein endothelial cells (HUVEC) with passage numbers 4 - 8 and culture them in EGM-2 (C3162, LONZA) at an incubator temperature of 37 °C with 5% CO 2 ; Trypsinize the cells, centrifuge, and resuspend them at a concentration of 1×10 7 cells / ml;

[0119] Remove the PBS from the multi-organ chip with the pre-constructed fibrin hydrogel barrier or type I rat tail collagen hydrogel barrier, and then inject 50 μl of the cell suspension into the perfusion channel; Place the multi-organ chip in an incubator at 37 °C with 5% CO 2 for culture; After 12 hours, add 200 μl of EGM-2 to the inlet of the perfusion channel; Then place the multi-organ chip on a rocking shaker for perfusion culture; The parameters of the shaker are set as follows: tilt angle 10°, rocking speed 20 times / minute, rocking interval 2 seconds; Change the culture medium every day and observe and record the cell growth and behavior.

[0120] Step S500 specifically includes the following steps:

[0121] For the multi-organ chip inoculated with HUVEC, after three days of perfusion culture, the endothelial barrier is completely formed;

[0122] Cerebral organoids are induced and differentiated from human induced pluripotent stem cells (iPSC) according to STEMdiff TM Cerebral Organoid Kit (08570, 08571, STEMCELL), and cardiac organoids are obtained from iPSC according to Human iPSC-Derived Cardiac Organoid Differentiation Kit (RIPO-HWM002K, AC-RObiosystems);

[0123] Place the two types of organoids in two culture chambers respectively, and position the organoids along the limiting parts in the culture chambers. Add 200 μl each of brain maturation medium, cardiac organoid medium M-M, and EGM-2 medium to the brain culture chamber, cardiac culture chamber, and the inlet of the perfusion channel respectively; Perform perfusion culture with a shear stress of 1.5 - 2.5 dynes / cm -2, change the culture medium in the culture wells every day;

[0124] After culturing for 2 days, when the organoids adhere to the hydrogel barrier, the discharge behavior of the organoids can be detected using a brain-computer interface detection system.

[0125] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the relevant technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A multi-organ chip, characterized in that: include: A structural layer, wherein at least two culture chambers, a perfusion channel and a hydrogel perfusion pipeline corresponding to the culture chambers are arranged on the structural layer; different culture chambers are used to culture different cell clusters or organoids respectively, and a limiter is arranged in the culture chamber, and the limiter extends from the bottom of the culture chamber to the top of the culture chamber, and a receiving cavity for accommodating cell clusters or organoids is formed between the outer wall of the limiter and the inner wall of the culture chamber; the perfusion channel is a closed pipeline with openings at both ends, which is used to connect different culture chambers; the perfusion channel is connected with the culture chamber through the hydrogel perfusion pipeline, and a hydrogel barrier is constructed in the hydrogel perfusion pipeline, and the hydrogel barrier is used to culture vascular endothelial cells to form a vascular endothelial barrier and a capillary network; An insulating substrate is provided with at least two groups of microelectrode arrays, each group of the microelectrode arrays includes a plurality of microelectrodes, and the electrode sites of the microelectrodes are located at the bottom of the accommodating cavity.

2. The multi-organ chip according to claim 1, characterized in that: The perfusion channel is provided with a perfusion channel inlet and a perfusion channel outlet. At least two reference electrodes are also provided on the insulating substrate. An electrode site of the reference electrode is respectively provided at the bottom of the perfusion channel inlet and the bottom of the perfusion channel outlet.

3. The multi-organ chip according to claim 1, characterized in that: The hydrogel perfusion pipeline includes an introduction section connected to the injection port of the hydrogel perfusion pipeline, and a diffusion section surrounding the bottom of the outer wall of the culture chamber, an opening is provided at each end of the diffusion section, and the outlet of the introduction section is connected to the openings at both ends of the diffusion section. After flowing out of the introduction section, the construction materials of the hydrogel barrier enter the diffusion section from the openings at both ends of the diffusion section respectively, and converge somewhere in the diffusion section; Preferably, along the circumference of the outer wall of the culture chamber, the length of the diffusion section is 3 / 4 of the circumference of the outer wall of the culture chamber.

4. The multi-organ chip according to claim 1 or 3, characterized in that: The raw material for constructing the hydrogel barrier is biological hydrogel used for cell culture; Preferably, the biohydrogel comprises a natural hydrogel or an engineered active hydrogel; Preferably, the natural hydrogel is selected from human fibrinogen, bovine fibrinogen, or type I rat tail collagen, or the engineered active hydrogel is selected from methacryloyl gelatin.

5. The multi-organ chip according to claim 1 or 3, characterized in that: A portion of the outer wall of the culture chamber is surrounded by the perfusion channel, and a portion of the culture chamber surrounded by the perfusion channel is communicated with the perfusion channel through the hydrogel perfusion pipe.

6. The multi-organ chip according to claim 5, characterized in that: The perfusion channel comprises an annular segment surrounding the outer wall of the culture chamber and a straight segment connected to the annular segment, and the angle between the annular segment and the straight segment is not less than 60°.

7. The multi-organ chip according to claim 1, characterized in that: The bottom of the limiting member is located at the center of the bottom of the culture chamber, and the bottom of the limiting member is circular; Preferably, the bottom diameter of the stopper is between 1 mm and 3 mm; Preferably, the height of the limiting member is between 2 mm and 5 mm.

8. The multi-organ chip according to claim 1, characterized in that: The culture chamber is a circular chamber open at the top; Preferably, the diameter of the culture chamber is between 3 mm and 5 mm.

9. The multi-organ chip according to claim 1, further comprising a liquid reservoir layer and a transparent substrate, wherein the liquid reservoir layer is disposed above the structural layer, and the liquid reservoir layer is provided with a plurality of liquid reservoirs penetrating its upper surface and its lower surface, and the center of the inlet of the perfusion channel of the structural layer, the center of the outlet of the perfusion channel, the center of the injection port of the hydrogel perfusion pipeline, and the center of the culture chamber are respectively aligned with the center of the corresponding liquid reservoir, and the hole spacing between adjacent liquid reservoirs is 9 mm; The transparent substrate is arranged below the insulating substrate.

10. A method for preparing a multi-organ chip, characterized in that: The following steps are involved: Making a structural layer and a stopper; the structural layer comprises at least two culture chambers, a perfusion channel, and a hydrogel perfusion pipeline corresponding to the culture chambers; Making at least two groups of microelectrode arrays on an insulating substrate, each group of the microelectrode arrays comprising a plurality of microelectrodes; The structural layer is bonded to the insulating substrate, and the limiting member is bonded to the insulating substrate, so that a receiving cavity is formed between the outer wall of the limiting member and the inner wall of the culture chamber, and the electrode site of the microelectrode is located at the bottom of the receiving cavity.

11. A method for using a multi-organ chip, characterized in that: The following steps are involved: constructing a hydrogel barrier within the hydrogel perfusion channel; Injecting vascular endothelial cells into the perfusion channel of the multi-organ chip; Adding cell clusters or organoids and their specific culture medium into a culture chamber of a multi-organ chip for culture; Connect the perfusion channel of the multi-organ chip to an external driving pump for perfusion culture; The discharge behavior of the cell cluster or organoid in the culture chamber is detected.

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