Multi-organ chip capable of monitoring electrophysiological signals

Abstract

The invention relates to a multi-organ chip capable of monitoring electrophysiological signals. The multi-organ chip sequentially comprises a cover plate (1), a multi-organ interconnection chip substrate (3), a printed circuit board (4) and a glass substrate (5) integrated with a microelectrode array from top to bottom, the multi-organ interconnection chip base body (3) is provided with a small chamber insertion hole (3-4), and the organ culture small chamber (2) can be inserted into and pulled out of the small chamber insertion hole (3-4). The multi-organ chip provided by the invention can simulate interaction and material exchange among organs in a body, and provides an innovative platform for drug screening and disease model research.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological device manufacturing, and particularly relates to a multi-organ chip capable of monitoring electrophysiological signals. Background Art

[0002] An organ chip is a culture device that reproduces the complex physiological functions of organs on a chip based on microfluidic technology. A multi-organ chip integrates multiple organ models through microfluidic technology and can simulate the functions of different organs on a single platform. Such a chip usually includes microfluidic channels and cell culture chambers and can reconstruct the physiological interactions between organs in vitro. Researchers can use multi-organ chips to explore the effects of drugs on multiple organs, evaluate toxic reactions, and study disease mechanisms.

[0003] Electrophysiology is a discipline that studies the electrical properties of biological cells and tissues, mainly involving the measurement of voltage and current. Generally, electrophysiological measurement techniques include clamping techniques, patch clamp techniques, multi-electrode arrays, in vitro tissue slices, biosensor techniques, etc. Electrophysiological measurement techniques have important applications in the fields of neuroscience, cardiac physiology, and muscle physiology.

[0004] Although multi-organ chips can simulate the microenvironments of multiple organs, how to maintain cell diversity and function in the chips remains a major challenge. Different types of cells may lose their specific functions during in vitro culture, which may affect the true reliability of experimental results. Therefore, the ability to monitor organ behavior in real time is a major challenge for multi-organ chip technology. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a multi-organ chip capable of monitoring electrophysiological signals, which can simulate the interactions and material exchanges between organs in vivo and provides an innovative platform for drug screening and disease model research.

[0006] The present invention provides a multi-organ chip capable of monitoring electrophysiological signals, which successively includes a cover plate, a multi-organ interconnected chip substrate, a printed circuit board, and a glass substrate integrated with a microelectrode array from top to bottom; the multi-organ interconnected chip substrate is provided with small chamber insertion holes, and organ culture chambers can be inserted into and removed from the small chamber insertion holes.

[0007] Preferably, the cover plate is embedded in the upper part of the multi-organ interconnected chip substrate. The cover plate is colorless and transparent for convenient observation, and can be placed or removed by various methods such as manual operation or a negative pressure suction cup.

[0008] Preferably, the cover plate and the multi-organ interconnected chip substrate can be formed by various processing methods such as mechanical engraving, laser engraving, 3D printing, and casting.

[0009] Preferably, the organ culture chamber is a processed Transwell chamber analogue or a commercially available Transwell chamber is directly used.

[0010] Preferably, the organ culture chamber includes a top, a side wall and a bottom; the top is provided with a buckle; the side wall is provided with at least one positioning post; the bottom is provided with a porous structure.

[0011] Preferably, the organ culture chamber is embedded and rotationally snapped into the multi-organ interconnected chip substrate, and the precise positioning of the organ culture chamber is achieved by the positioning posts. According to needs, the interconnection between different organs can be realized through fluid perfusion.

[0012] Preferably, each organ culture chamber can culture one or more organs, and the organs can exist in the form of aggregates of cells such as cell spheres, organoids, living tissues, etc. inside or outside the chamber. The cell culture methods include attaching to the porous structure at the bottom of the chamber, suspending in a liquid culture medium or culturing in a three-dimensional matrix.

[0013] Preferably, the porous structure is a material or structure with porous permeability such as microchannels, porous membranes, microgrids or hydrogels. This not only prevents the cells in the cell culture chamber from entering the interconnected substrate or the cells in the substrate from entering the organ culture chamber, but also allows liquids, cytokines, exosomes, drugs or cell metabolites to pass through, realizing organ interconnection.

[0014] Preferably, the multi-organ interconnected chip substrate has an external liquid connection hole, a fluid inlet chip substrate channel, an internal fluid perfusion hole, a chamber insertion hole, a chamber insertion bayonet, an organ communication flow channel and a fluid outlet chip channel; the fluid inlet chip substrate channel and the fluid outlet chip channel are respectively embedded in the long axis direction of the organ interconnected chip substrate, and both ends are connected to the external liquid connection hole.

[0015] Preferably, the chamber insertion holes in the multi-organ interconnected chip substrate are consistent with the center positions of the culture holes of commercial cell culture 6-well plates, 12-well plates, 24-well plates, 48-well plates and 96-well plates, and can be compatible with a variety of automated pipetting devices and a variety of detection devices such as microplate readers and microscopes.

[0016] Preferably, the positioning posts on the side of the organ culture chamber match the chamber insertion bayonets of the multi-organ interconnected chip substrate. After the organ culture chamber is vertically inserted into the multi-organ interconnected chip substrate and rotated by a certain angle, the chamber can be fixed in the chip substrate for easy culture or observation.

[0017] Preferably, the organ culture chambers are interconnected through the organ communication channels at the bottom of the multi-organ interconnected chip substrate, allowing arbitrary communication between two or more identical or different organs, communicating each organ through fluid perfusion, and providing a shear force culture environment for the organs.

[0018] Preferably, the height of the fluid entering the chip substrate channel is higher than that of the fluid flowing out of the chip channel in the horizontal direction.

[0019] Preferably, the glass substrate of the integrated microelectrode array is connected to the printed circuit board through an adhesive, which is used to monitor and regulate the electrophysiological activities of the multi-organ chip unit.

[0020] Preferably, the microelectrode array on the glass substrate of the integrated microelectrode array is fabricated by ultrasonic cleaning, spin coating, photolithography, metal sputtering, stripping, insulating layer coating, and electroplating on the glass substrate. The number of microarray electrode channels is 16*n (n = 1, 2, 3...); the diameter of the electrode points is 10 - 1000 μm, and the coverage area is 0.1 mm × 0.1 mm to 10 mm × 10 mm. It can be adjusted according to different experimental requirements and application scenarios.

[0021] Preferably, the printed circuit board provides the connection between the sites of the microarray electrode array and the leads, which not only provides mechanical support but also realizes the effective transmission of electrophysiological signals through precise point alignment.

[0022] Beneficial effects

[0023] (1) The multi-organ chip of the present invention can be used for perfusion culture. The overall size of the chip is the same as that of a commercial cell culture multi-well plate and is compatible with a variety of culture or detection devices. The organ culture chambers can also be inserted and removed according to a certain program by an automated device to meet the requirements of modern industrial automated operations.

[0024] (2) The multi-organ chip of the present invention can perform various forms of organ culture. Organs can be constructed according to the physiological characteristics of different organs; at the same time, each culture chamber is relatively independent, and independent culture can be carried out using the culture medium required by the organs.

[0025] (3) The connection method of the organ culture chambers of the present invention is through the channels at the bottom of the chip. The chip can be used for circulating perfusion culture by using an external pipeline or a pump device. At the same time, the internal channels are lined with vascular endothelial cells, which can simulate organ perfusion in the blood circulation system in vitro.

[0026] (4) The organ culture chambers of the present invention can be assembled with any number of organs according to requirements. The number of co-cultured organs is scalable, and two or more organs can be connected for co-culture according to requirements.

[0027] (5) The glass substrate of the integrated microelectrode array proposed by the present invention is connected to the PCB layer and is used to monitor and regulate the electrophysiological activities of the multi-organ chip unit. The design of the microelectrode array allows for precise monitoring of electrophysiological signals of the organ chip unit, providing important data support for the study of neural activities and organ functions.

[0028] (6) The multi-organ chip proposed by the present invention can simulate the interactions and material exchanges between organs in vivo, providing an innovative platform for drug screening and disease model research. Description of the Drawings

[0029] Figure 1 It is a component diagram of the multi-organ chip of the present invention.

[0030] Figure 2 It is a schematic diagram of the organ culture chamber in the multi-organ chip of the present invention.

[0031] Figure 3 It is a top view of the multi-organ interconnected chip substrate in the multi-organ chip of the present invention.

[0032] Figure 4 It is a top view of the printed circuit board in the multi-organ chip of the present invention.

[0033] Figure 5 It is a side view of the multi-organ interconnected chip substrate in the multi-organ chip of the present invention.

[0034] Figure 6 It is a schematic diagram of the glass substrate microelectrode array in the multi-organ chip of the present invention.

[0035] Figure 7 It is a photo of a cardiac organoid cultured in the multi-organ chip of the present invention. Detailed Embodiments

[0036] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0037] Embodiment 1

[0038] As Figure 1 shown, a multi-organ chip capable of monitoring electrophysiological signals provided in this embodiment includes, from top to bottom, a cover plate 1, a multi-organ interconnected chip substrate 3, a printed circuit board 4, and a glass substrate 5 with an integrated microelectrode array; the multi-organ interconnected chip substrate 3 is provided with a chamber insertion hole 3-4, and the organ culture chamber 2 can be inserted and removed from the chamber insertion hole 3-4.

[0039] Preferably, the cover plate 1 and the multi-organ interconnected chip substrate 3 can be formed by various processing methods such as mechanical engraving, laser engraving, 3D printing, and casting molding; polymethyl methacrylate, polystyrene, or some 3D printing materials with high biocompatibility can be used.

[0040] Preferably, the cover plate 1 is colorless and transparent and is embedded in the upper part of the multi-organ interconnected chip substrate 3 for easy observation. The cover plate 1 can be placed or removed by various methods such as manual operation or a negative pressure suction cup.

[0041] Preferably, the organ culture chamber 2 is a processed Transwell chamber analog or directly uses a commercial Transwell chamber insert. The organ culture chamber 2 includes a top, side walls, and a bottom; the top is provided with a snap 2-1; the side walls are provided with at least one positioning post 2-2; the bottom is provided with a porous structure 2-3. The porous structure 2-3 can be a material or structure with porous permeability such as a microchannel, porous membrane, microfence, or hydrogel;

[0042] Furthermore, at least one positioning post 2-2 is provided on the outer wall surface of the organ culture chamber 2. Preferably, two rectangular parallelepiped or cylindrical positioning posts 2-2 are symmetrically distributed outside the side walls of the organ culture chamber, with a protruding height of 0.5 - 3 mm, as Figure 2 shown. The organ culture chamber 2 is snapped into the multi-organ chip substrate by various methods such as embedding and rotation, and the positioning post 2-2 realizes the precise positioning of the organ culture chamber 2. Therefore, according to needs, any interconnection between different organs can be achieved through fluid perfusion.

[0043] Preferably, the organ culture chamber 2 can culture one or more organs, and the organs can exist in the form of aggregates of cells such as cell spheres, organoids, and living tissues inside or outside the organ culture chamber 2. The cell culture methods include attaching to the porous structure 2-4 at the bottom of the organ culture chamber 2, suspending in a liquid culture medium, or culturing in a three-dimensional matrix.

[0044] As Figure 3 and Figure 5 shown, the multi-organ interconnected chip substrate 3 has various structures such as an external liquid connection hole 3-1, a fluid inlet chip substrate channel 3-2, an internal fluid perfusion hole 3-3, a chamber insertion hole 3-4, a chamber insertion snap 3-5, an organ communication flow channel 3-6, and a fluid outlet chip channel 3-7.

[0045] Preferably, the external liquid connection hole 3-1 is connected to an external diaphragm pump, peristaltic pump, pneumatic pump, or syringe pump through a micro-pipeline such as a Luer connector to pump the culture medium into the chip.

[0046] Preferably, the multi-organ interconnected chip substrate 3 has two or more fluid perfusion channels, one of which is the fluid inlet channel 3-2 of the chip substrate, and the other is the fluid outlet channel 3-7 of the chip. The diameter of the fluid channel is 1-5 mm; the two liquid perfusion channels are respectively embedded in the long axis direction of the multi-organ interconnected chip substrate 3, 4-10 mm away from the bottom of the chip substrate, and both ends are connected to the external liquid connection holes 3-1 in the vertical direction. The diameter of the external liquid connection holes 3-1 is 2-5 mm, and they are used in cooperation with the Luer connectors of commercial fluid components.

[0047] Preferably, the fluid inlet channel 3-2 of the chip substrate is slightly higher than the fluid inlet and outlet channel 3-7 of the chip substrate by 0.5-3 mm in the horizontal height, so that the height difference of the liquid level inside the chip substrate can drive the fluid flow.

[0048] Preferably, the multi-organ interconnected chip substrate 3 has a number of small chamber insertion holes 3-4 for connecting with the organ culture chambers 2, and different arrays are formed according to the number of co-cultured organs, which has scalability.

[0049] Preferably, the side of the small chamber insertion hole 3-4 has symmetric small chamber insertion chucks 3-5. After the organ culture chamber 2 is vertically inserted into the substrate and rotated by 1-90°, the organ culture chamber 2 can be fixed inside the chip substrate for easy culture or observation.

[0050] Preferably, the small chamber insertion holes 3-4 are connected to each other through the organ communication channels 3-6 at the bottom of the multi-organ interconnected chip substrate 3, allowing any connection between two or more identical or different organs, connecting each organ through fluid perfusion, and providing a shear force culture environment for the organs;

[0051] Preferably, the glass substrate 5 of the integrated microelectrode array is connected to the printed circuit board 4 (PCB layer) through an adhesive for monitoring and regulating the electrophysiological activities of the organ chip unit.

[0052] Preferably, the microarray electrodes are fabricated by semiconductor microfabrication processes such as ultrasonic cleaning, spin coating, photolithography, metal sputtering, stripping, insulating layer coating, electroplating, etc. on the glass substrate. In this embodiment, the number of microarray electrode channels is 32; the diameter of the electrode points is 20 μm, and the coverage area is 1 mm×1 mm.

[0053] Preferably, as Figure 4 shown, the printed circuit board 4 provides the connection between the microarray electrode sites and the leads, not only providing mechanical support, but also achieving the effective transmission of electrophysiological signals through precise point alignment.

[0054] This embodiment also provides a preparation method for microarray electrodes on a glass substrate:

[0055] The microarray electrode is fabricated by semiconductor microfabrication processes such as ultrasonic cleaning, spin coating, photolithography, metal sputtering, stripping, insulating layer coating, electroplating, etc. on a glass substrate. As Figure 6 shown, the number of channels of the microarray electrode is 32; the diameter of the electrode point is 20 μm, and the coverage area is 1 mm × 1 mm.

[0056] This embodiment also provides an implementation method for co-culturing heart-lung-brain organoids:

[0057] 1) Prepare a type I collagen PBS solution with a concentration of 50 - 500 μg / mL, add 50 μL to each organ culture chamber 2, and coat the bottom porous material 2-3 of the organ culture chamber 2 at a density of 5 - 20 μg / m 2 overnight at 4°C.

[0058] 2) The next day, wash it 1 - 3 times with clean PBS. Invert the organ culture chamber 2, dilute it with PBS, use a matrix gel with a concentration of 5 - 30%, and add 10 - 50 μL of the coating solution to each porous material 2-drop, incubate at room temperature for 1 h, and rinse it once with clean PBS.

[0059] 3) Add 50 - 200 μL of a vascular endothelial cell suspension with a concentration of 0.1 - 5×10 6 cells / mL outside the porous material 2-3, and keep it static at 37°C for at least 2 hours to allow the cells to adhere and grow.

[0060] 4) Invert the organ culture chamber 2, add the prepared cell suspension with a concentration of 0.1 - 5×10 6 cells / mL to the non-vascular side of the porous material 2-3, add 50 - 200 μL to each organ culture chamber 2, and add a sufficient amount of culture medium, keep it static for at least 2 hours, and finally obtain the heart organoids as shown in Figure 7 Figure.

Claims

1. A multi-organ chip capable of monitoring electrophysiological signals, characterized in that: The multi-organ chip comprises, from top to bottom, a cover plate (1), a multi-organ interconnected chip substrate (3), a printed circuit board (4), and a glass substrate (5) with an integrated microelectrode array; the multi-organ interconnected chip substrate (3) is provided with a chamber insertion hole (3-4), and the organ culture chamber (2) can be inserted into and removed from the chamber insertion hole (3-4).

2. The multi-organ chip according to claim 1, characterized in that: The cover plate (1) is embedded in the upper part of the multi-organ interconnection chip substrate (3).

3. The multi-organ chip according to claim 1, characterized in that: The organ culture chamber (2) comprises a top, a side wall and a bottom; the top is provided with a buckle (2-1); the side wall is provided with at least one positioning column (2-2); and the bottom is provided with a porous structure (2-3).

4. The multi-organ chip according to claim 3, characterized in that: The organ culture chamber (2) is embedded and rotated into the multi-organ interconnected chip matrix (3), and the positioning column (2-2) realizes the precise positioning of the organ culture chamber (2).

5. The multi-organ chip according to claim 3, characterized in that: The porous structure (2-3) is a microchannel, a porous membrane, a microfence or a hydrogel.

6. The multi-organ chip according to claim 1, characterized in that: The multi-organ interconnected chip substrate (3) comprises an external liquid connection hole (3-1), a fluid inlet chip substrate channel (3-2), an internal fluid perfusion hole (3-3), a chamber insertion hole (3-4), a chamber insertion bayonet (3-5), an organ communication channel (3-6) and a fluid outflow chip channel (3-7); the fluid inlet chip substrate channel (3-2) and the fluid outflow chip channel (3-7) are respectively embedded in the long axis direction of the organ interconnected chip substrate (3), and both ends are connected to the external liquid connection hole; the organ culture chambers (2) are connected to each other through the organ communication channel (3-6) at the bottom of the multi-organ interconnected chip substrate (3).

7. The multi-organ chip according to claim 6, characterized in that: The fluid inlet chip substrate channel (3-2) is higher in level than the fluid outlet chip channel (3-7).

8. The multi-organ chip according to claim 1, characterized in that: The glass substrate (5) of the integrated microelectrode array is connected to the printed circuit board (4) via an adhesive and is used to monitor and regulate the electrophysiological activities of the multi-organ chip.

9. The multi-organ chip according to claim 1, characterized in that: The microelectrode array on the glass substrate (5) of the integrated microelectrode array is manufactured by ultrasonic cleaning, photolithography, metal sputtering, stripping, insulating layer coating and electroplating on the glass substrate.

10. The multi-organ chip according to claim 1, characterized in that: The printed circuit board (4) provides connection between the sites of the microarray electrode array and the leads.

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