An organ-on-a-chip and method for organo-ide detection and electrical stimulation

Abstract

The application relates to an organ chip and a method for detecting and electrically stimulating an organoid, wherein a chip upper layer, an organoid culture layer and a chip base layer are stacked in a top-to-bottom mode, a culture chamber is formed in the organoid culture layer, a liquid flow channel is arranged in the chip upper layer and the organoid culture layer, the culture chamber is communicated with the liquid flow channel, a pressure chamber is formed in the chip base layer, a gas flow channel is arranged in the chip upper layer, the organoid culture layer and the chip base layer, the pressure chamber is communicated with the gas flow channel, the gas flow channel is connected with a gas pressure supply instrument, a signal transmission layer is embedded in the chip base layer, the outer edge of the signal transmission layer extends out of the organ chip, a conductor is arranged on the signal transmission layer, one end of the conductor is located in the culture chamber and above the pressure chamber, the other end of the conductor extends out of the organ chip and is connected with an electric signal instrument. Compared with the prior art, the application constructs a multifunctional organoid chip platform with high-sensitivity electric signal detection, piezoelectric response identification and electric stimulation application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, and relates to an organ-on-a-chip for detecting and stimulating organoid piezoelectric signals and a method for detecting and stimulating organoid piezoelectric signals. Background Technology

[0002] Human tissues exhibit complex bioelectrical signaling activities under physiological conditions. These signals not only directly reflect cellular function but also serve as crucial physical clues for regulating tissue development, maintaining homeostasis, and responding to external stimuli. In brain organoids, the spontaneous firing and synchronous oscillation of neurons reflect the maturity and functional connectivity of neural networks. In pancreatic organoids, the electrical activity rhythms of α and β cells directly determine the precision and dynamic response of hormone secretion. Meanwhile, the piezoelectric signals generated by mechanically sensitive tissues such as cartilage and bone under mechanical loads are endogenous regulatory mechanisms for maintaining cell metabolism and matrix synthesis. Simultaneously, exogenous electrical stimulation has been proven to effectively regulate stem cell differentiation, promote tissue maturation, accelerate damage repair, and even reshape the pathological microenvironment. Therefore, real-time monitoring of endogenous physiological electrical signals and piezoelectric response signals, as well as the precise application and dynamic regulation of exogenous electrical stimulation in organoid culture systems, are of significant scientific value and application prospects for a deeper understanding of tissue development mechanisms, the construction of functionally mature organoid models, disease modeling and drug screening, and the advancement of regenerative medicine clinical translation.

[0003] Despite significant progress in organoid technology in mimicking the structure and function of human tissues, existing organoid culture chips still face considerable limitations in electrophysiological characterization and regulation. While traditional two-dimensional multi-electrode arrays can be used for recording electrical signals in monolayer cell cultures, the contact area between the electrodes and tissue is extremely limited when dealing with the irregular surface morphology and internal heterogeneous structure of three-dimensional organoids. This results in low signal acquisition efficiency, insufficient spatial resolution, and difficulty in capturing the true electrical activity characteristics of different cell subpopulations within the organoid. Although there have been reports in recent years of using flexible mesh electronic devices to achieve passive integration during organoid development, these strategies often disrupt the three-dimensional self-assembly process of organoids and lack adaptability to mature organoids. More critically, existing systems generally lack the ability to detect piezoelectric signals, failing to distinguish and quantify the piezoelectric responses of mechanically sensitive tissues such as cartilage and bone under mechanical stimulation from the spontaneous physiological electrical signals of neural and endocrine organoids such as the brain and pancreas, thus limiting research on the mechano-electric coupling mechanism. Furthermore, most chips only possess a single signal monitoring function and lack an electrical stimulation application module, making it impossible to achieve closed-loop intervention of monitoring-feedback-regulation. This hinders the ability to guide stem cell differentiation, promote organoid functional maturation, or simulate abnormal pathological electrical activity through exogenous electric fields. Simultaneously, the existing electrode materials and chip structures suffer from insufficient biocompatibility, long-term stability, and multi-channel parallel detection capabilities, limiting the continuous tracking and systematic analysis of electrical activity across the entire organoid development cycle and multiple cell types.

[0004] Patent CN115612609A discloses an integrated organ-on-a-chip and its fabrication method. The integrated organ-on-a-chip is used to apply electrical and mechanical stimulation as needed during the culture of cells, cell clusters, or organoids. The integrated organ-on-a-chip includes: an electrode layer for providing electrical stimulation, a microporous layer above the electrode layer for culture, a flow chamber layer for providing mechanical stimulation and interacting with an external circuit; and peripheral circuitry. The electrode layer includes: a substrate, and bare electrodes corresponding to the micropores in the microporous layer on the substrate. The microporous layer includes: a structure with multiple independent single-well chambers attached to the electrode layer, each single pore being permeable and having a single bare electrode or multiple electrodes forming a multi-electrode structure corresponding to the bottom of the electrode layer. The flow chamber layer is a structure with multiple single cavities attached above the microporous layer for carrying the culture medium to which the culture belongs. Although this patent uses a flow chamber layer to provide mechanical stimulation to organoids, the mechanical stimulation method is the fluid shear force generated by liquid flow, which cannot apply pressure stimulation and cannot detect piezoelectric signals.

[0005] Patent CN116814424A discloses a dual-chamber three-dimensional biochip and its detection method. The chip includes a first channel, a second channel, a fourth channel, and a third channel arranged in parallel. The first and third channels are strip-shaped. The second channel includes interconnected first and second upper culture chambers. The fourth channel includes interconnected first and second lower culture chambers. From top to bottom, the chip includes a connecting layer, an upper culture layer, a lower culture layer, a sealing layer, and a circuit layer. The connecting layer and the circuit layer are located on the upper and lower surfaces of the chip, respectively. The upper culture layer, lower culture layer, and sealing layer are located between the connecting layer and the circuit layer. Although this patent uses a bubble removal structure with through-holes, allowing bubbles to rise to the surface of the bubble removal chamber during passage, this method still cannot directly apply mechanical stimulation to the organoid and lacks the ability to simulate the microenvironment of mechanical pressure within the organ. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one of the defects of the prior art and provide an organ-on-a-chip and method for organoid piezoelectric signal detection and electrical stimulation. This invention constructs a multifunctional organoid chip platform that combines high-sensitivity electrical signal detection, piezoelectric response recognition, and electrical stimulation application.

[0007] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide an organ-on-a-chip for detecting and stimulating piezoelectric signals in organoids. The organ-on-a-chip includes a top layer, an organoid culture layer, a signal transmission layer, and a base layer, wherein the top layer, the organoid culture layer, and the base layer are stacked vertically. A culture chamber is formed through the organoid culture layer, and liquid channels are formed between the upper layer of the chip and the organoid culture layer. The culture chamber is connected to the liquid channels. A pressure chamber is formed within the chip substrate layer. Gas channels are formed within the chip upper layer, organoid culture layer, and chip substrate layer. The pressure chamber is connected to the gas channels, and gas pressure supply equipment is connected to the gas channels. The signal transmission layer is located between the organoid culture layer and the chip substrate layer. The signal transmission layer is embedded in the chip substrate layer, and the outer edge of the signal transmission layer extends out of the organ chip. A conductor is disposed on the signal transmission layer. One end of the conductor is located inside the culture chamber and above the pressure chamber. The signal transmission layer isolates the culture chamber and the pressure chamber. The other end of the conductor extends out of the organ chip and is connected to an external electrical signal instrument. The electrical signal instrument is selected from one or more of electrical signal measuring instruments and electrical signal excitation instruments.

[0008] Furthermore, the upper layer of the chip is provided with an upper liquid inlet port and an upper liquid outlet port respectively. The organoid culture layer is provided with a culture layer liquid inlet port and a culture layer liquid outlet port. The organoid culture layer is provided with a liquid channel, which includes a liquid inlet channel and a liquid outlet channel. The culture layer liquid inlet port and the culture chamber are connected through the liquid inlet channel. The culture layer liquid outlet port and the culture chamber are connected through the liquid outlet channel. The upper liquid inlet port and the culture layer liquid inlet port are connected to form a liquid inlet channel. The upper liquid outlet port and the culture layer liquid outlet port are connected to form a liquid outlet channel.

[0009] Furthermore, an upper-layer gas port is provided through the upper layer of the chip; The organoid culture layer has a through-hole gas port. The chip substrate has a substrate gas port and a gas channel. The substrate gas port and the pressure chamber are connected through the gas channel. The upper gas port, the culture layer gas port and the substrate gas port are connected to form a gas flow channel. The upper gas port is connected to an external gas pressure supply instrument.

[0010] Furthermore, a culture chamber top block is provided on the upper layer of the chip, and the culture chamber top block is embedded in and seals the culture chamber.

[0011] Furthermore, each culture chamber is equipped with a number of conductors, including culture chamber conductors, connecting conductors, and conductors for electrical signal instruments and equipment. One end of the conductor is a culture chamber conductor, which is located inside the culture chamber and above the pressure chamber, and is cone-shaped. The other end of the conductor is a conductor for electrical signal instruments and equipment, and the conductor for electrical signal instruments and equipment is externally connected to electrical signal instruments and equipment. The conductors of the culture chamber and the conductors of the electrical signal instruments and equipment are connected by a connecting conductor, the surface of which is coated with an insulating protective material.

[0012] Furthermore, the signal transmission layer is plate-shaped; A positioning groove is formed on the chip substrate layer, and the signal transmission layer is embedded in the positioning groove. While the size of the signal transmission layer is adapted to the size of the positioning groove, the outer edge of the signal transmission layer containing the conductor of the electrical signal instrument extends out of the chip substrate layer.

[0013] Furthermore, the projection of the culture chamber coincides with that of the pressure chamber, and the projection of the signal transmission layer covers and extends beyond the pressure chamber; Several culture chambers and several pressure chambers are provided, with fewer pressure chambers than culture chambers.

[0014] One of the technical solutions of the present invention is to provide a method for detecting and stimulating organoid piezoelectric signals. This method uses the aforementioned organ-on-a-chip to detect and stimulate organoid piezoelectric signals, and includes the following steps: S1, chip assembly and organoid injection S1.1 Embed the signal transmission layer into the chip substrate layer. At this time, one end of the conductor of the signal transmission layer covers the pressure chamber of the chip substrate layer, while the other end of the conductor of the signal transmission layer extends out of the chip substrate layer. S1.2 Cover the assembled signal transduction layer and chip substrate layer with the organoid culture layer. At this time, the culture chamber of the organoid culture layer contains one end of the conductor of the signal transduction layer, while the other end of the conductor of the signal transduction layer is not blocked by the organoid culture layer, and the culture chamber of the organoid culture layer corresponds to the pressure chamber of the chip substrate layer. S1.3. Inject the organoids into the culture chamber of the organoid culture layer; S1.4. Cover the upper layer of the chip onto the organoid culture layer. At this time, the liquid flow channel is connected and the gas flow channel is connected. S2. Pressure stimulation, electrical signal detection, or electrical stimulation during organoid culture. After the organoids are injected, the culture medium flows in and out through the liquid channels; Gas is pressurized into the pressure chamber through a gas flow channel using gas supply equipment, and the pressure is transmitted to stimulate the organoids in the culture chamber. Electrical signals within the organoids in the culture chamber are detected using electrical signal measuring instruments and equipment through the conductor of the signal transmission layer. The organoids in the culture chamber are stimulated by an instrument that uses electrical signals to conduct electrical signals through a conductor in the signal transmission layer.

[0015] As a preferred technical solution, in step S1.1, the signal transmission layer is embedded into the chip substrate layer, that is, embedded into the positioning groove of the chip substrate layer. At this time, the growth chamber conductor of the signal transmission layer just covers the pressure chamber of the chip substrate layer, while the electrical signal instrument conductor of the signal transmission layer grows out of the chip substrate layer.

[0016] As a preferred technical solution, in step S1.2, the organoid culture layer is covered on the assembled combination of the signal transmission layer and the chip substrate layer. At this time, the culture chamber of the organoid culture layer can contain all the conductors of the culture chamber of the signal transmission layer, while the conductors of the electrical signal instruments and equipment of the signal transmission layer are not blocked by the organoid culture layer. Furthermore, the culture chamber of the organoid culture layer corresponds to the pressure chamber of the chip substrate layer, and the gas port of the culture layer of the organoid culture layer is connected to the gas port of the substrate layer of the chip substrate layer.

[0017] As a preferred technical solution, in step S1.4, the upper layer of the chip is covered on the organoid culture layer. At this time, the top block of the culture chamber of the upper layer of the chip is embedded in the culture chamber of the organoid culture layer, which plays a role in positioning and limiting. The upper liquid inlet port of the upper layer of the chip is connected to the culture layer liquid inlet port of the organoid culture layer, the upper liquid outlet port of the upper layer of the chip is connected to the culture layer liquid outlet port of the organoid culture layer, and the upper gas port of the upper layer of the chip is connected to the culture layer gas port of the organoid culture layer.

[0018] As a preferred technical solution, after the organoid is injected in step S2, the culture medium flows in from the upper liquid inlet port of the chip and flows out from the upper liquid outlet port of the chip.

[0019] As a preferred technical solution, in step S2, a gas pressurization instrument is used to pressurize gas into the pressure chamber through the upper gas port on the chip, and the pressure is transmitted to stimulate the organoids in the culture chamber.

[0020] Furthermore, when conducting organoid pressure stimulation and electrical signal detection, different control experiments were carried out by changing the culture conditions in each culture chamber, namely organoids, culture medium and pressure. By applying different pressure stimuli, namely with and without pressure, and different pressure levels, the electrical signal response was detected to study the effect of this condition on organoids.

[0021] Furthermore, when performing organoid electrical stimulation and electrical signal detection, different control experiments were conducted by changing the culture conditions in each culture chamber, namely organoids, culture medium, and electrical signals. By providing different electrical signal stimulation, namely with and without electrical signals, as well as electrical signals with different waveforms, amplitudes, and frequencies, the electrical signal response was detected, and the effects of these conditions on organoids were studied.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention proposes a multifunctional organ-on-a-chip for organoid piezoelectric signal detection and electrical stimulation. Through hierarchical modular design and conductive material interface engineering, it systematically solves the core defects of existing technologies in electrophysiological monitoring and regulation, and realizes the functional integration of culture microenvironment construction, electrical signal transmission and mechanical stimulation application. (2) The core innovation of the signal transmission layer in this invention is the use of a conical contact conductor array design. Through precise spatial layout, the conductor tip forms close contact with the organoids and culture medium at the bottom of the culture chamber, maximizing the electrode-tissue interface area and ensuring high sensitivity capture of weak electrical signals inside the organoids. (3) The chip substrate layer of the present invention is designed with a cavity pressure chamber structure corresponding to the culture chamber. Controllable pressure can be applied by gas supply pressure instrument equipment to simulate the mechanical load borne by tissues such as cartilage and bone in the body, thereby inducing and detecting the electrical signal response generated by piezoelectric materials or piezoelectric sensitive organoids. For the first time, the synchronous coupling of mechanical stimulation and electrical signal detection has been realized. (4) What makes this invention more versatile is that it can not only detect pressure-induced piezoelectric signals, but also capture spontaneous physiological electrical activity of brain organoids, pancreatic organoids and other tissues through parallel culture chambers corresponding to the cavity-free pressure chamber structure, or cavity pressure chamber structure without pressure, thereby achieving a unified platform characterization of the electrophysiological characteristics of different types of organoids. (5) In this invention, the contact conductor array is connected to the interface of external electrical signal instruments and equipment through embedded wires, which supports multi-channel parallel signal acquisition and real-time monitoring, that is, it supports bidirectional electrical signal transmission. It can apply electrical stimulation of different waveforms, amplitudes and frequencies to the culture chamber through electrical signal excitation instruments and equipment, thereby realizing active regulation of organoid development process, and providing experimental tools for studying biological problems such as electric field-guided stem cell differentiation, neural network synchronization, and hormone secretion rhythm regulation. (6) The present invention adopts a multi-pore culture chamber design, which supports the parallel development of multiple control experiments. It can systematically evaluate the effects of different organoid types, culture medium formulations, pressure parameters and electrical stimulation protocols on electrophysiological behavior. It not only provides a quantitative means for analyzing the electromaturation mechanism of organoids and optimizing culture protocols, but also opens up new technical paths for constructing biomimetic organoids with precise electrophysiological functions, advancing disease modeling and drug screening, and exploring electrical stimulation-mediated regenerative medicine treatment strategies. Attached Figure Description

[0023] Figure 1 This is a three-dimensional structural schematic diagram of the organ-on-a-chip for organoid piezoelectric signal detection and electrical stimulation in an embodiment of the present invention; Figure 2 This is a schematic diagram of the exploded structure of the organ-on-a-chip for organoid piezoelectric signal detection and electrical stimulation in an embodiment of the present invention; Figure 3 This is a schematic diagram of the upper layer of the chip in an embodiment of the present invention; Figure 4 This is a schematic diagram of the structure of the organoid culture layer in an embodiment of the present invention; Figure 5 This is a schematic diagram of the signal transmission layer in an embodiment of the present invention; Figure 6 This is a schematic diagram of the chip substrate layer in an embodiment of the present invention; Figure 7 This is a three-dimensional structural diagram of the chip after the signal transmission layer is assembled in an embodiment of the present invention; Figure 8 This is a top view of the chip structure after the signal transmission layer is assembled in an embodiment of the present invention; Figure 9 This is a schematic diagram of the three-dimensional structure of the chip after assembling the organoid culture layer in an embodiment of the present invention; Figure 10 This is a top view of the chip structure after assembling the organoid culture layer in an embodiment of the present invention; Figure 11 This is a schematic diagram of the organoid injection process in an embodiment of the present invention; Figure 12 This is a schematic diagram of the three-dimensional structure of the chip after the upper layer of the chip is assembled in an embodiment of the present invention; Figure 13 This is a top view of the chip structure after the upper layer of the chip is assembled in an embodiment of the present invention; Figure 14 This is a waveform diagram of the sinusoidal electrical stimulation signal in an embodiment of the present invention; Figure 15 This is a waveform diagram of the square wave electrical stimulation signal in an embodiment of the present invention; Figure 16 This is a waveform diagram of the triangular wave electrical stimulation signal in an embodiment of the present invention; Figure 17 The waveform diagram is shown for the sawtooth wave electrical stimulation signal in an embodiment of the present invention.

[0024] Explanation of markings in the diagram: 1-Chip top layer, 2-Organoid culture layer, 3-Signal transduction layer, 4-Chip base layer; 11-Upper liquid inlet port, 12-Upper liquid outlet port, 13-Upper gas port, 14-Cultivation chamber top block; 21-Culture layer liquid inlet port, 22-Culture layer liquid outlet port, 23-Culture layer gas port, 24-Culture chamber, 241-First culture chamber, 242-Second culture chamber, 243-Third culture chamber, 244-Fourth culture chamber; 35 - Conductor; 351 - Incubation chamber conductor; 352 - Connecting conductor; 353 - Electrical signal instrument and equipment conductor; 43-Base layer gas port, 44-Pressure chamber, 46-Positioning groove. Detailed Implementation

[0025] The present invention will now be described in detail with reference to specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0026] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. Furthermore, the terms "first," "second," "third," etc., used to describe a common object only indicate different instances of the same object, and do not imply that the objects described in this way must be in a given order, whether temporally, spatially, sequentially, or in any other way.

[0027] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0028] Example 1: An organ-on-a-chip for detecting piezoelectric signals and electrical stimulation in organoids, such as Figure 1 and Figure 2 As shown, it includes a chip upper layer 1, an organoid culture layer 2, a signal transduction layer 3, and a chip base layer 4, which are stacked one on top of the other. A culture chamber 24 is formed through the organoid culture layer 2. Liquid channels are formed between the upper chip layer 1 and the organoid culture layer 2, and the culture chamber 24 is connected to the liquid channels. A pressure chamber 44 is formed within the chip substrate layer 4. Gas channels are formed within the chip upper layer 1, the organoid culture layer 2, and the chip substrate layer 4. The pressure chamber 44 is connected to the gas channels, and gas pressure supply equipment is connected to the outside of the gas channels. The signal transmission layer 3 is located between the organoid culture layer 2 and the chip substrate layer 4. The signal transmission layer 3 is embedded in the chip substrate layer 4, and the outer edge of the signal transmission layer 3 extends out of the organ chip. A conductor 35 is provided on the signal transmission layer 3. One end of the conductor 35 is located inside the culture chamber 24 and above the pressure chamber 44. The signal transmission layer 3 isolates the culture chamber 24 and the pressure chamber 44. The other end of the conductor 35 extends out of the organ chip and is connected to an external electrical signal instrument. The electrical signal instrument is selected from one or more of electrical signal measuring instruments and electrical signal excitation instruments. In this embodiment, the substrate material of the signal transmission layer 3 is polydimethylsiloxane (PDMS), which is highly flexible, biocompatible, and easy to withstand the pressure of the pressure chamber 44 on the culture chamber 24 and recover in time under pressureless conditions. In this embodiment, the conductor 35 is made of platinum-iridium alloy, which is extremely resistant to electrochemical corrosion, has high charge injection capacity, and suitable rigidity. It can be made into a three-dimensional conical electrode for dual purposes of electrical signal detection and electrical stimulation. In this embodiment, the gas pressure supply instrument is a gas pump, the electrical signal measurement instrument is a multimeter, oscilloscope or electrophysiological recorder, preferably an oscilloscope, and the electrical signal excitation instrument is a signal generator or a bioelectric stimulator, preferably a signal generator. like Figure 3 As shown, the upper layer 1 of the chip has an upper liquid inlet port 11 and an upper liquid outlet port 12 respectively. A gas port 13 is provided through the upper layer 1 of the chip. A culture chamber top block 14 is provided on the upper layer 1 of the chip, and the culture chamber top block 14 is embedded in and seals the culture chamber 24; like Figure 4 As shown, the organoid culture layer 2 is provided with a culture layer liquid inlet port 21 and a culture layer liquid outlet port 22. The organoid culture layer 2 is provided with a liquid channel, which includes a liquid inlet channel and a liquid outlet channel. The culture layer liquid inlet port 21 and the culture chamber 24 are connected through the liquid inlet channel. The culture layer liquid outlet port 22 and the culture chamber 24 are connected through the liquid outlet channel. The upper liquid inlet port 11 and the culture layer liquid inlet port 21 are connected to form a liquid inlet channel. The upper liquid outlet port 12 and the culture layer liquid outlet port 22 are connected to form a liquid outlet channel. The organoid culture layer 2 has a gas port 23 that runs through it. like Figure 5 As shown, signal transmission layer 3 is plate-shaped; A culture chamber 24 is provided with several conductors 35, including culture chamber conductors 351, connecting conductors 352, and electrical signal instrument and equipment conductors 353. One end of conductor 35 is a culture chamber conductor 351, which is located inside culture chamber 24 and above pressure chamber 44, and is cone-shaped. The other end of conductor 35 is conductor 353 for electrical signal instruments and equipment, and conductor 353 is connected to external electrical signal instruments and equipment. The culture chamber conductor 351 and the electrical signal instrument conductor 353 are connected by a connecting conductor 352. The surface of the connecting conductor 352 is coated with an insulating protective material to prevent electrolyte short circuits. If the surface of the connecting conductor 352 is not coated with an insulating protective material, the collected electrical signals are prone to superimposed interference signals. When an electrical signal is applied, the electrical signal will cause a short circuit through the culture medium and other liquids, and the electrical signal cannot be transmitted to the organoid. In this embodiment, the surface of the culture chamber conductor 351 is modified with poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonate) (PEDOT:PSS). This material has a modulus close to that of soft tissue, is cell-friendly, has good biocompatibility, can adapt to dynamic deformation, and the mixed conduction of ions and electrons can reduce interfacial impedance. In this embodiment, the insulating protective material is selected from atomic layer deposition (ALD) alumina (Al2O3) film, silicon nitride (Si3N4) film or polyurethane (PU) coating, preferably atomic layer deposition alumina film; like Figure 6 As shown, a base layer gas port 43 is provided in the chip base layer 4, and a gas channel is provided in the chip base layer 4. The base layer gas port 43 and the pressure chamber 44 are connected through the gas channel. The upper gas port 13, the culture layer gas port 23 and the base layer gas port 43 are connected to form a gas flow channel. The upper gas port 13 is connected to an external gas supply pressure instrument. A positioning groove 46 is provided on the chip substrate 4, and the signal transmission layer 3 is embedded in the positioning groove 46. While the size of the signal transmission layer 3 is adapted to the size of the positioning groove 46, the outer edge of the signal transmission layer 3 containing the electrical signal instrument conductor 353 extends out of the chip substrate 4. The projection of the culture chamber 24 is consistent with that of the pressure chamber 44, and the projection of the signal transmission layer 3 covers and extends beyond the pressure chamber 44. Several culture chambers 24 and several pressure chambers 44 are provided, but the number of pressure chambers 44 is less than that of culture chambers 24. In this embodiment, the organ-on-a-chip is equipped with four through-slots serving as culture chambers 24: a first culture chamber 241, a second culture chamber 242, a third culture chamber 243, and a fourth culture chamber 244. Each culture chamber 24 is equipped with a pair of liquid inlet channels and a pair of liquid outlet channels. The liquid inlet channel is formed by a through-hole serving as the upper liquid inlet port 11 and a through-hole serving as the culture layer liquid inlet port 21. The liquid outlet channel is formed by a through-hole serving as the upper liquid outlet port 12 and a through-hole serving as the culture layer liquid outlet port 22. The organ-on-a-chip has two blind grooves on the upper surface of the chip substrate 4, which serve as pressure chambers 44. That is, pressure chambers 44 are located below the second culture chamber 242 and the fourth culture chamber 244, which constitute half of the culture chambers 24. All culture chambers 24 share a gas channel, which is composed of through holes as upper gas ports 13, through holes as culture layer gas ports 23, and blind holes on the upper surface of the chip substrate 4 as base layer gas ports 43. There are four sets of conductors 35 on the upper surface of the signal transmission layer 3 on the blind slot on the upper surface of the chip substrate layer 4, which serves as the positioning slot 46, with five conductors in each set.

[0029] Example 2: A method for detecting and stimulating organoid piezoelectric signals, using the organ-on-a-chip described in Example 1, comprises the following steps: S1, chip assembly and organoid injection S1.1, such as Figure 7 and Figure 8 As shown, the signal transmission layer 3 is embedded into the chip substrate layer 4, that is, embedded into the positioning groove 46 of the chip substrate layer 4. At this time, it can be seen that the incubation chamber conductor 351 of the signal transmission layer 3 covers the pressure chamber 44 of the chip substrate layer 4, while the electrical signal instrument conductor 353 of the signal transmission layer 3 grows out of the chip substrate layer 4. S1.2, such as Figure 9 and Figure 10 As shown, the organoid culture layer 2 is covered on the assembled signal transmission layer 3 and chip substrate layer 4. At this time, it can be seen that the culture chamber 24 of the organoid culture layer 2 can completely contain the culture chamber conductor 351 of the signal transmission layer 3, while the electrical signal instrument conductor 353 of the signal transmission layer 3 is not blocked by the organoid culture layer 2. The culture chamber 24 of the organoid culture layer 2 corresponds to the pressure chamber 44 of the chip substrate layer 4, and the culture layer gas port 23 of the organoid culture layer 2 is connected to the substrate gas port 43 of the chip substrate layer 4. S1.3, such as Figure 11 As shown, organoids are injected into the culture chamber 24 of organoid culture layer 2; S1.4, such as Figure 12 As shown, the upper chip layer 1 is placed on the organoid culture layer 2. At this time, the top block 14 of the culture chamber of the upper chip layer 1 is embedded in the culture chamber 24 of the organoid culture layer 2, which plays a role in positioning and limiting. The upper liquid inlet port 11 of the upper chip layer 1 is connected to the culture layer liquid inlet port 21 of the organoid culture layer 2, the upper liquid outlet port 12 of the upper chip layer 1 is connected to the culture layer liquid outlet port 22 of the organoid culture layer 2, and the upper gas port 13 of the upper chip layer 1 is connected to the culture layer gas port 23 of the organoid culture layer 2. S2. Pressure stimulation, electrical signal detection, or electrical stimulation during organoid culture. like Figure 13 As shown, after the organoid is injected, the culture medium flows in from the upper liquid inlet port 11 of the upper layer 1 of the chip and flows out from the upper liquid outlet port 12 of the upper layer 1 of the chip. Gas can be pressurized into the pressure chamber 44 through the upper gas port 13 of the chip 1 using a gas supply instrument, and the pressure can be transmitted to stimulate the organoid in the culture chamber 24. Electrical signals within the organoids in the culture chamber 24 can be detected using electrical signal measuring instruments and equipment through the conductor 35 of the signal transmission layer 3; The organoids in the culture chamber 24 can be stimulated by an electrical signal excitation device through the conductor 35 of the signal transmission layer 3.

[0030] Example 3: A method for detecting piezoelectric signals and stimulating organoids is basically the same as in Example 2, except that the organ-on-a-chip described in Example 1 is used for organoid pressure stimulation and electrical signal detection. The specific differences are as follows: By changing the culture conditions in each culture chamber 24, namely organoids, culture medium and pressure, different control experiments can be conducted. By applying different pressure stimuli, i.e. with or without pressure, and different pressure levels, and detecting the electrical signal response, the effects of these conditions on organoids can be studied.

[0031] Example 4: A method for detecting and stimulating organoid piezoelectric signals is basically the same as in Example 2, except that the organ-on-a-chip described in Example 1 is used for organoid electrical stimulation and signal detection. The specific differences are as follows: By changing the culture conditions in each culture chamber 24, namely organoids, culture medium, and electrical signals, different control experiments can be conducted. By applying different electrical signal stimuli, i.e., with or without electrical signals, as well as electrical signals with different waveforms, amplitudes, and frequencies, and detecting the electrical signal response, the effects of these conditions on organoids can be studied.

[0032] like Figures 14 to 17 As shown, some common electrical stimulation signals can be sine waves, square waves, triangular waves, or sawtooth waves. Sine wave electrical signals provide gentle and continuous stimulation, suitable for simulating natural bioelectrical signals in the body. The rising and falling edges of square wave electrical signals can effectively induce action potentials in cells. The linearly changing voltages of triangular and sawtooth wave electrical signals can simulate the slow depolarization process of chemical synapses. These basic waveform electrical signals can be effectively arranged and combined to generate new arbitrary waveform electrical signals, which can be used to study the effects of different morphological electrical signals on organoid maturation.

[0033] The waveform, amplitude, and frequency of the signal can be set according to the actual experimental requirements. The detected electrical signal is generally a potential difference.

[0034] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An organ-on-a-chip for detecting piezoelectric signals and stimulating organs, characterized in that, The organ-on-a-chip comprises a chip top layer (1), an organoid culture layer (2), a signal transduction layer (3), and a chip base layer (4), wherein the chip top layer (1), the organoid culture layer (2), and the chip base layer (4) are stacked one on top of the other. A culture chamber (24) is provided through the organoid culture layer (2). A liquid flow channel is provided through the upper chip layer (1) and the organoid culture layer (2). The culture chamber (24) is connected to the liquid flow channel. A pressure chamber (44) is provided inside the chip substrate layer (4). Gas channels are provided inside the chip upper layer (1), the organoid culture layer (2), and the chip substrate layer (4). The pressure chamber (44) is connected to the gas channels. Gas pressure supply equipment is connected to the gas channels. The signal transmission layer (3) is located between the organoid culture layer (2) and the chip substrate layer (4). The signal transmission layer (3) is embedded in the chip substrate layer (4), and the outer edge of the signal transmission layer (3) extends out of the organ chip. A conductor (35) is provided on the signal transmission layer (3). One end of the conductor (35) is located inside the culture chamber (24) and above the pressure chamber (44). The signal transmission layer (3) isolates the culture chamber (24) and the pressure chamber (44). The other end of the conductor (35) extends out of the organ chip and is connected to an external electrical signal instrument. The electrical signal instrument is selected from one or more of electrical signal measuring instruments and electrical signal excitation instruments.

2. The organ-on-a-chip for detecting piezoelectric signals and electrical stimulation of organoids according to claim 1, characterized in that, The upper layer (1) of the chip is provided with an upper liquid inlet port (11) and an upper liquid outlet port (12). The organoid culture layer (2) is provided with a culture layer liquid inlet port (21) and a culture layer liquid outlet port (22) respectively. The organoid culture layer (2) is provided with a liquid channel, which includes a liquid inlet channel and a liquid outlet channel. The culture layer liquid inlet port (21) and the culture chamber (24) are connected through the liquid inlet channel. The culture layer liquid outlet port (22) and the culture chamber (24) are connected through the liquid outlet channel. The upper liquid inlet port (11) and the culture layer liquid inlet port (21) are connected to form a liquid inlet channel. The upper liquid outlet port (12) and the culture layer liquid outlet port (22) are connected to form a liquid outlet channel.

3. The organ-on-a-chip for detecting piezoelectric signals and electrical stimulation of organoids according to claim 1, characterized in that, The upper layer (1) of the chip has an upper gas port (13) that runs through it. The organoid culture layer (2) has a gas port (23) that runs through it. The chip substrate (4) has a substrate gas port (43) and a gas channel. The substrate gas port (43) and the pressure chamber (44) are connected through the gas channel. The upper gas port (13), the culture layer gas port (23) and the substrate gas port (43) are connected to form a gas flow channel. The upper gas port (13) is connected to an external gas pressure supply instrument.

4. The organ-on-a-chip for detecting piezoelectric signals and electrical stimulation of organoids according to claim 1, characterized in that, The upper layer (1) of the chip is provided with a culture chamber top block (14), which is embedded in and seals the culture chamber (24).

5. An organ-on-a-chip for detecting piezoelectric signals and electrical stimulation of organoids according to claim 1, characterized in that, A culture chamber (24) is provided with a number of conductors (35), the conductors (35) including culture chamber conductors (351), connecting conductors (352) and electrical signal instrument equipment conductors (353). One end of the conductor (35) is a culture chamber conductor (351), which is located inside the culture chamber (24) and above the pressure chamber (44), and is cone-shaped. The other end of the conductor (35) is an electrical signal instrument conductor (353), which is externally connected to an electrical signal instrument. The culture chamber conductor (351) and the electrical signal instrument equipment conductor (353) are connected by a connecting conductor (352), the surface of which is coated with an insulating protective material.

6. An organ-on-a-chip for detecting piezoelectric signals and electrical stimulation of organoids according to claim 5, characterized in that, The signal transmission layer (3) is plate-shaped; The chip substrate (4) has a positioning groove (46) and the signal transmission layer (3) is embedded in the positioning groove (46). The size of the signal transmission layer (3) is adapted to the size of the positioning groove (46), and the outer edge of the signal transmission layer (3) containing the electrical signal instrument conductor (353) extends out of the chip substrate (4).

7. An organ-on-a-chip for detecting piezoelectric signals and electrical stimulation of organoids according to claim 1, characterized in that, The projection of the culture chamber (24) is consistent with the pressure chamber (44), and the projection of the signal transmission layer (3) covers and extends beyond the pressure chamber (44). The culture chamber (24) and the pressure chamber (44) are provided in several quantities, and the number of pressure chambers (44) is less than that of culture chambers (24).

8. A method for detecting and electrically stimulating organoid piezoelectric signals, characterized in that, This method uses an organ-on-a-chip as described in any one of claims 1 to 7 to perform organoid piezoelectric signal detection and electrical stimulation, the method comprising the following steps: S1, chip assembly and organoid injection S1.

1. Embed the signal transmission layer (3) into the chip substrate layer (4). At this time, one end of the conductor (35) of the signal transmission layer (3) covers the pressure chamber (44) of the chip substrate layer (4), while the other end of the conductor (35) of the signal transmission layer (3) extends out of the chip substrate layer (4). S1.2 Cover the assembled signal transmission layer (3) and chip substrate layer (4) with the organoid culture layer (2). At this time, the culture chamber (24) of the organoid culture layer (2) includes one end of the conductor (35) of the signal transmission layer (3), while the other end of the conductor (35) of the signal transmission layer (3) is not blocked by the organoid culture layer (2), and the culture chamber (24) of the organoid culture layer (2) corresponds to the pressure chamber (44) of the chip substrate layer (4). S1.3, inject the organoids into the culture chamber (24) of the organoid culture layer (2); S1.

4. Cover the upper layer (1) of the chip onto the organoid culture layer (2), at which point the liquid flow channel is connected and the gas flow channel is connected. S2. Pressure stimulation, electrical signal detection, or electrical stimulation during organoid culture. After the organoids are injected, the culture medium flows in and out through the liquid channels; Gas is pressurized through a gas flow channel into the pressure chamber (44) using a gas pressurization instrument, and the pressure is transmitted to stimulate the organoids in the culture chamber (24); Electrical signals within the organoids in the culture chamber (24) are detected using electrical signal measuring instruments through the conductor (35) of the signal transmission layer (3); The organoids in the culture chamber (24) are stimulated by an electrical signal excitation device through the conductor (35) of the signal transmission layer (3).

9. The method for detecting and electrically stimulating organoid piezoelectric signals according to claim 8, characterized in that, When performing organoid pressure stimulation and electrical signal detection, different control experiments were conducted by changing the culture conditions in each culture chamber (24), namely organoids, culture medium and pressure. By giving different pressure stimulation, namely with or without pressure and different pressure levels, the electrical signal response was detected to study the effect of this condition on organoids.

10. The method for detecting and electrically stimulating organoid piezoelectric signals according to claim 8, characterized in that, When performing organoid electrical stimulation and electrical signal detection, different control experiments were conducted by changing the culture conditions in each culture chamber (24), namely organoids, culture medium and electrical signals. By giving different electrical signal stimulation, namely with and without electrical signals, and electrical signals with different waveforms, amplitudes and frequencies, the electrical signal response was detected, and the effect of this condition on organoids was studied.

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