Neuromuscular multi-element coupled organoid micro-fluidic chip, device and method

Abstract

The invention provides a neuromuscular multi-element coupled organoid micro-fluidic chip, a device and a method. Relates to the technical field of organoid micro-fluidic chips. A first culture chamber, a second culture chamber and a third culture chamber are arranged in a culture layer of the chip; the first culture room is used for culturing nerve cells; the second culture room is used for culturing muscle cells; a microfluidic channel for communicating the first culture chamber with the second culture chamber is arranged between the first culture chamber and the second culture chamber; the third culture room is arranged on the micro-fluidic channel and is used for culturing a neuromuscular joint formed by convergence of axons of nerve cells and muscle cells; a microelectrode array plate is arranged in the circuit layer and is used for performing electrical stimulation and electrophysiological function detection on neuromuscular cells. Through the chip, co-culture of in-vitro nerves and muscle organs can be realized, a complex interaction relationship in a neuromuscular system can be accurately simulated, and the chip can also be used for constructing various neuromuscular related disease models.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid microfluidic chips, and in particular to a neuromuscular multi-coupled organoid microfluidic chip, device and method. Background Art

[0002] The neuromuscular system is composed of neurons and muscle cells, and their coordinated function is essential for the physiological functions of organisms such as movement, sensation, and response. Figure 1 As shown in Figure a, the interaction between neurons and muscle cells is achieved through the neuromuscular junction (NMJ), which is a complex synaptic structure responsible for transmitting nerve signals to muscle cells and triggering muscle contraction. Figure 1 As shown in Figure b, the neuromuscular junction consists of the presynaptic membrane (nerve terminal membrane), the synaptic cleft, and the postsynaptic membrane (sarcolemma). When the nerve impulse reaches the nerve terminal, the presynaptic membrane depolarizes, causing the voltage-gated calcium channel to open, and calcium ions to flow into the nerve terminal, triggering the exocytosis of synaptic vesicles and releasing neurotransmitters (such as acetylcholine). Neurotransmitters diffuse through the synaptic cleft to the postsynaptic membrane, bind to receptors on the postsynaptic membrane, cause the postsynaptic membrane to depolarize, and then trigger the excitation and contraction of muscle cells.

[0003] Traditional neuromuscular research methods, such as animal models and two-dimensional cell culture, are difficult to accurately simulate the complex interactions in the neuromuscular system in vitro. Although animal models can provide a certain degree of physiological authenticity, their results are often affected by species differences and cannot fully reflect the situation in the human body. Two-dimensional cell culture lacks three-dimensional structure and natural interactions between cells, and cannot fully reproduce the functional and pathological characteristics of the neuromuscular system. In recent years, with the development of organoid technology and microfluidic technology, a new platform has been provided for the study of the neuromuscular system. Organoid chips can generate three-dimensional models of human organs or tissues in vitro to simulate the physiological structure in vivo. Microfluidic chips can provide a precise cell culture environment, simulate the microenvironment conditions in the body, and improve the physiological authenticity of organoids, but the manufacturing precision is high and the operation is complicated.

[0004] Multilayer microfluidic chip structures are relatively common. Generally, the upper layer is the culture layer, including the culture chamber and fluid channels, the middle layer includes the channel isolation structure and membrane structure, and the bottom layer is the circuit layer, including various sensors and electronic components.

[0005] In order to effectively simulate the interaction between nerves and muscles, comprehensively study the physiological, pathological, regenerative and repair mechanisms between nerves and muscles, and achieve high-throughput drug screening and other application purposes, the industry urgently needs to develop neuromuscular organoid microfluidic chips with simple structure and reliable performance. Summary of the invention

[0006] The object of the present invention is to provide a neuromuscular multi-coupled organoid microfluidic chip, device and method to solve at least one technical problem existing in the above-mentioned background technology.

[0007] In a first aspect, the present invention provides a neuromuscular multi-coupled organoid microfluidic chip, comprising: In the culture layer, a first culture chamber, a second culture chamber and a third culture chamber are provided. The first culture chamber is used to culture nerve cells, and the second culture chamber is used to culture muscle cells. A microfluidic channel for connecting the first culture chamber and the second culture chamber is provided between the first culture chamber and the second culture chamber. A liquid inlet and a liquid outlet are provided at both ends of the microfluidic channel, respectively, and the liquid inlet is provided at one end close to the first culture chamber. The third culture chamber is provided on the microfluidic channel, and is used to culture the neuromuscular junction formed by the axons of nerve cells and the convergence of muscle cells. Through the above chip, independent culture chambers can be provided for nerve cells and muscle cells, and nutrient solution can be input and metabolic waste can be discharged through the microfluidic channel, thereby reliably simulating the complex interaction relationship between blood circulation in the body and multiple organ systems.

[0008] In a feasible embodiment, the microfluidic channel includes a plurality of axial separation zones, and the separation zones are provided with permeable holes, so that the axons of the nerve cells can grow along the microfluidic channel to the muscle area, and the fluids on both sides of the separation zones can flow to each other, thereby facilitating the penetration of nutrient solution and drugs.

[0009] In a feasible embodiment, the number of the first culture chamber is 1, the number of the second culture chamber is 2, the number of the liquid inlets is 2, and the number of the liquid outlet is 1; the first culture chamber and the liquid outlet are arranged at both ends of the microfluidic channel; the second culture chamber and the liquid inlet are symmetrically arranged on both sides of the microfluidic channel. This structure of multiple independent but connected culture chambers facilitates the co-culture of multiple organoids on the same microfluidic chip.

[0010] In a feasible embodiment, a microfluidic module is arranged in the center of the third culture chamber; an electrode plate is embedded in the bottom surface of the microfluidic module; a microelectrode array plate is arranged in the circuit layer below the electrode plate; the gap between the electrode plate and the microelectrode array plate constitutes a discharge space, which is used to electrically stimulate cells entering the discharge space and perform electrophysiological function detection; a number of vertical channels are opened on the side walls of the microfluidic module to connect the third culture chamber with the discharge space, and at the same time, on the basis that the third culture chamber is connected to the microfluidic channel, nerve cells and muscle cells can smoothly enter the discharge space, and then the electrical stimulation provided by the electrode plate and the microelectrode array plate promotes synapse formation, thereby realizing the functionalization of the neuromuscular junction; and then the electrophysiological activity between nerve cells and muscle cells is recorded through electrophysiological function detection, thereby evaluating the function of the neuromuscular junction.

[0011] In a feasible embodiment, corresponding sensing points are evenly distributed around the microelectrodes of the microelectrode array board, and the microelectrodes and the sensing points are connected by a one-to-one corresponding first wire for transmitting electrical signals from the microelectrodes, so that the integrated circuit matrix can control the microelectrode array board and collect data, thereby electrically stimulating cells and recording electrical activities at multiple locations at the same time, accurately capturing electrical signals between cells and cell networks; the integrated circuit matrix and second wires connecting the integrated circuit matrix are evenly distributed around the sensing points, which are used to control the chip and process electrical signals.

[0012] In a feasible embodiment, the chip substrate includes polydimethylsiloxane, which has the characteristics of good light transmittance, good flexibility and biocompatibility, and is convenient for optical imaging observation and oxygen penetration.

[0013] In a feasible implementation, the material of the microelectrodes in the microelectrode array plate is TiN, which has excellent electrical conductivity, long-term stability, good biocompatibility and cell adhesion.

[0014] In the second aspect, based on the same inventive concept, the present invention also provides a microfluidic device using the neuromuscular multi-coupled organ-on-chip as described in the first aspect, which also includes an integrated circuit matrix, a microenvironment detection sensor, and a dynamic fluid transmission unit.

[0015] The microenvironment detection sensor comprises a first culture chamber detection sensor, a second culture chamber detection sensor and a third culture chamber detection sensor; the first culture chamber detection sensor is arranged between the culture layer and the circuit layer at the first culture chamber, and is used to detect the microenvironment data of the first culture chamber; the second culture chamber detection sensor is arranged between the culture layer and the circuit layer at the second culture chamber, and is used to detect the microenvironment data of the second culture chamber; the third culture chamber detection sensor is arranged between the culture layer and the circuit layer at the third culture chamber, and is used to detect the microenvironment data of the third culture chamber; the microenvironment data includes fluid flow rate, chemical gradient, temperature and dissolved oxygen level, etc.; in this way, an independent detection sensor can be configured for each culture chamber, and the microenvironment of each culture chamber can be monitored in real time to ensure the stability of the microenvironment, thereby maintaining the activity of nerve cells and muscle cells and promoting the formation of synapses and neuromuscular junctions; The dynamic fluid transmission part includes a plurality of micro pumps and valves, which are respectively arranged at the liquid inlet and the liquid outlet, and are used to programmatically input nutrient solution and / or drugs into the microfluidic channel and output metabolic waste of cells; in this way, the blood circulation in the body can be simulated, and the required amount of nutrient solution and / or drugs can be automatically pumped into each organ and / or tissue continuously or in time periods, so as to ensure that each organoid grows and develops under the most suitable conditions, thereby improving the repeatability and reliability of the experiment, as well as the co-culture of multiple organoids; The microfluidic module, the microenvironment detection sensor and the dynamic fluid transmission unit are electrically connected to regulate the microenvironment in the chip; The integrated circuit matrix is ​​electrically connected to the microfluidic module and the microelectrode array board, and is used to control the chip to perform data acquisition, electrical stimulation, and microfluidic management.

[0016] In a feasible implementation manner, the film structure of the middle layer of the chip adopts a PETE (polyethylene terephthalate) porous film.

[0017] In a feasible embodiment, a tension sensor can be provided in the PETE porous membrane below the second culture chamber to monitor muscle contraction force, thereby observing abnormalities in muscle tissue function; an electrophysiological sensor can be provided in the PETE porous membrane below the first culture chamber to monitor the conduction of nerve signals; an optical sensor can be integrated in the PETE porous membrane below any culture chamber to monitor oxygen and / or pH in the culture chamber.

[0018] In a feasible implementation manner, the electrical stimulation parameters include: the current range is 0.1 to 10 microamperes; the voltage range is 0.05 to 10 volts; the pulse is a biphasic square wave with a frequency range of 20 to 100 Hz; and the current density range is 0 to 1 mA / cm².

[0019] In a third aspect, based on the same inventive concept, the present invention further provides an application method of the neuromuscular multi-coupled organ-on-a-chip as described in the first aspect, specifically comprising: Step a1, obtaining skin cells or blood cells from patients with amyotrophic lateral sclerosis (ALS), reprogramming to obtain induced pluripotent stem cells with the mutated gene characteristics of the disease, and differentiating them into motor nerve cells and muscle cells; Step a2, introducing the motor nerve cells into the first culture chamber; introducing the muscle cells into the second culture chamber; co-culturing into motor neuron organoids and muscle organoids, forming neuromuscular junctions in the third culture chamber through microfluidic channels, and obtaining an amyotrophic lateral sclerosis disease model; Step a3, observing abnormal phenomena of motor neurons, muscle contraction and synaptic function in the disease model, and recording electrophysiological data through a microelectrode array plate; Step a4, using the disease model to perform drug screening, testing the improvement effects of antioxidant drugs, neurotrophic factors, etc. on motor nerve cell survival and muscle function.

[0020] In a feasible implementation manner, the application method further includes: Step b1, obtaining induced pluripotent stem cells from patients with spinal muscular atrophy, and differentiating motor nerve cells and muscle cells, wherein the induced pluripotent stem cells carry an SMN1 gene mutation; Step b2, introducing the motor nerve cells into the first culture chamber; introducing the muscle cells into the second culture chamber; co-culturing into motor neuron organoids and muscle organoids, forming neuromuscular junctions in the third culture chamber through microfluidic channels, and obtaining a spinal muscular atrophy disease model; Step b3, using electrophysiological recording and molecular markers to detect motor neuron signal transduction, including synaptic protein levels and neuronal connectivity levels, to evaluate the structure and function of neuronal synapses; Step b4, testing compounds or genes that enhance SMN protein expression in the disease model to evaluate the effects of the compounds or genes on improving the interaction between neurons and muscles or delaying the progression of neuronal degeneration.

[0021] In a feasible implementation manner, the application method further includes: Step c1, obtaining induced pluripotent stem cells with dystrophin gene mutation from patients with Duchenne muscular dystrophy, and differentiating motor nerve cells and muscle cells; Step c2, introducing the motor nerve cells into the first culture chamber; introducing the muscle cells into the second culture chamber; co-culturing into motor neuron organoids and muscle organoids, forming neuromuscular junctions in the third culture chamber through microfluidic channels, and obtaining a Duchenne muscular dystrophy disease model; Step c3, setting a tension sensor in the second culture chamber to measure muscle contraction force; Step c4, conducting muscle contraction response test through electrophysiological stimulation; Step c5, testing gene therapy, expression effect of exogenous dystrophin protein and drug response through the disease model.

[0022] In a feasible implementation manner, the application method further includes: Step d1, obtaining induced pluripotent stem cells from a healthy body and differentiating them into motor neurons and muscle cells; Step d2, introducing the motor nerve cells into the first culture chamber; introducing the muscle cells into the second culture chamber; co-culturing into motor neuron organoids and muscle organoids, and forming neuromuscular junctions in the third culture chamber through microfluidic channels; Step d3, adding specific antibodies into the third culture chamber to simulate the attack of autologous antibodies on the neuromuscular junction to obtain a myasthenia gravis disease model; Step d4, observing the effect of the antibody on muscle contraction, and recording abnormal data of the response of muscle organoids to neuronal signals, for evaluating the expression level of acetylcholine receptors at synapses; Step d5, testing antibody inhibitors, immunosuppressive drugs or neurotransmitter enhancing drugs through the disease model, and recording signal transduction or muscle function data.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention provides a neuromuscular multi-coupled organoid microfluidic chip, device and method. Through the structural design of the microfluidic chip, a co-culture environment of multiple neuromuscular organoids is provided, which can effectively simulate the physiological structure of coupling between nerves and muscles; through microfluidic technology, the microenvironment of organoid culture in the chip is accurately controlled, which can effectively simulate the physiological environment in the body; through the integrated electrode plate and microelectrode array plate, electrical stimulation is provided to the organoid, which can promote the development of the organoid and the formation and functional differentiation of neuromuscular connections, and at the same time, electrophysiological signals are collected for data analysis. This scheme can not only realize the construction of multiple coupled organoid systems on the same microfluidic chip, accurately simulate the interaction between neurons and muscle cells in vitro, so as to study the physiological mechanism of the neuromuscular system and the problem of organ or tissue regeneration, but also construct a variety of disease models, thereby providing a convenient and reliable research platform for multiple application fields such as pathological research and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 Schematic diagram of the physiological structure of nerve cells, muscle cells and neuromuscular junctions: Figure a is an overall diagram; Figure b is an enlarged view of the synapse site; Figure 2 A top view of a neuromuscular multi-coupled organoid microfluidic chip provided in an embodiment of the present invention; Figure 3 An enlarged structural illustration of a neuromuscular multi-coupled organoid microfluidic chip provided in an embodiment of the present invention; Figure 4 A schematic diagram of the structural decomposition of a neuromuscular multi-coupled organoid microfluidic chip provided by an embodiment of the present invention; Figure 5 A flow chart of the application method of constructing an amyotrophic lateral sclerosis disease model using a neuromuscular multi-coupled organoid microfluidic chip provided in an embodiment of the present invention; Figure 6 Flow chart of the application method of the neuromuscular multi-coupled organoid microfluidic chip provided in an embodiment of the present invention to construct a spinal muscular atrophy disease model.

[0026] Figure 7 A flow chart of the application method of the neuromuscular multi-coupled organoid microfluidic chip provided in an embodiment of the present invention to construct a Duchenne muscular dystrophy disease model; Figure 8 Flowchart of the application method of the neuromuscular multi-coupled organoid microfluidic chip provided in an embodiment of the present invention to construct a myasthenia gravis disease model.

[0027] Reference numerals: 1-first culture chamber; 2-second culture chamber; 3-third culture chamber; 4-liquid inlet; 5-liquid outlet; 6-electrode plate; 7-microfluidic module; 8-first microfluidic subchannel; 9-second microfluidic subchannel; 10-microelectrode array plate; 11-first wire; 12-sensing point; 13-integrated circuit matrix; 14-second wire; 15-circuit layer bottom film; 16-culture layer bottom film; 31-chamber wall; 17-vertical channel; 18-microelectrode; 19-dividing zone; 20-permeation hole. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The present invention is further explained below in conjunction with specific implementation modes.

[0030] Embodiment 1: like Figure 2-4 As shown, the neuromuscular multi-coupled organoid microfluidic chip provided in this embodiment includes a culture layer and a circuit layer; A first culture chamber 1 is arranged at one end of the culture layer, and two second culture chambers 2 are arranged at the other end of the culture layer; the first culture chamber 1 is used to culture nerve cells, and the second culture chamber 2 is used to culture muscle cells; a microfluidic channel for connecting the first culture chamber 1 and the second culture chamber 2 is arranged between the two; the third culture chamber 3 is arranged in the center of the microfluidic channel, and is used to culture the neuromuscular junction formed by the axons of nerve cells and the fusion of muscle cells; two liquid inlets 4 are symmetrically arranged at one end of the microfluidic channel close to the first culture chamber 1, and a liquid outlet 5 is arranged at one end of the microfluidic channel close to the second culture chamber 2; a third culture chamber 3 is arranged in the middle part of the microfluidic channel, and is used to culture the neuromuscular junction formed by the fusion of the axons of nerve cells and the fusion of muscle cells.

[0031] The microfluidic channel is divided into four sub-channels by three axially arranged dividing strips 19, namely: the two symmetrical outer strips are the first microfluidic sub-channels 8, and the two symmetrical inner strips are the second microfluidic sub-channels 9; a plurality of permeation holes 20 are evenly distributed on the dividing strips 19; One end of the two second microfluidic sub-channels 9 is connected to the first culture chamber 1 , and the other end is connected to the liquid outlet 5 ; One end of a first microfluidic subchannel 8 is connected to a second culture chamber 2, and the other end is connected to a liquid inlet 4; one end of another first microfluidic subchannel 8 is connected to another second culture chamber 2, and the other end of this first microfluidic subchannel 8 is connected to another liquid inlet 4; In this way, nutrients or drugs can be delivered to the first culture chamber 1, the second culture chamber 2 and the third culture chamber 3 by pumping or osmosis, and the corresponding waste liquid can be discharged, thereby simulating a stable neuromuscular growth environment. Through the mutually spaced but connected structural layout of microfluidic channels, the signal transmission process between nerve cells and muscle cells can also be simulated, ensuring that signal molecules can be effectively transmitted while avoiding interference from different signals.

[0032] The third culture chamber 3 includes a chamber wall 31, a microfluidic module 7 and an electrode plate 6; the chamber wall 31 is a ring-shaped structure fixed on the culture layer bottom film 16, and is provided with an opening connected to the microfluidic channel; the microfluidic module 7 is arranged in the center of the third culture chamber 3; a card slot is arranged at the bottom of the microfluidic module 7 for embedding the electrode plate 6; a microelectrode array plate 10 is arranged in the circuit layer below the electrode plate 6; the gap between the electrode plate 6 and the microelectrode array plate 10 constitutes a discharge space; four vertical channels 17 are opened on the side wall of the microfluidic module 7 for connecting the third culture chamber 3 and the discharge space; the microelectrode array plate 10 is arranged on the upper surface of the circuit layer bottom film 15, and a plurality of microelectrodes 18 are arranged in the center of the microelectrode array plate 10; Multi-electrode Array (MEA) is an existing technology, which includes a number of tiny electrodes with a dense honeycomb structure. It is usually used to measure electrophysiological activities in cells and tissues. The microelectrode array records the electrical signals of cells by directly contacting or approaching the cells with each tiny electrode.

[0033] In this way, through the third culture chamber 3, the microfluidic module 7, the microfluidic channel and the microelectrode array plate 10, on the one hand, the physiological structure of the neuromuscular junction can be simulated to facilitate the formation of the neuromuscular junction, and the axons of the nerve cells gradually grow and extend, merging with the muscle cells to form the neuromuscular junction; on the other hand, the electrode plate 6 and the microelectrode array plate 10 can also provide electrical stimulation function to promote the development of nerve cells, synapse formation and the functionalization of the neuromuscular junction, and test, record and evaluate electrophysiological activities (such as the propagation of action potentials at the neuromuscular junction).

[0034] Furthermore, the electrode plate 6 is a flexible electrode, which is convenient for microstructure design and manufacturing to provide electrical stimulation functions that promote neuron development, synapse formation and optimization of electrophysiological characteristics, and can monitor the corresponding electrical activities in real time.

[0035] Furthermore, in the circuit layer, the microelectrodes 18 of the microelectrode array board 10 are evenly distributed with corresponding sensing points 12 for transmitting electrical signals of the microelectrodes 18; the microelectrodes 18 and the sensing points 12 are connected by one-to-one corresponding first wires 11; the integrated circuit matrix 13 and the second wires 14 for interconnecting the integrated circuit matrix 13 are evenly distributed around the sensing points 12, which are used to control the chip and process the electrical signals.

[0036] Furthermore, the material of the microelectrode 18 is TiN, which has excellent electrical conductivity, long-term stability, good biocompatibility and cell adhesion, as well as high-resolution and multi-channel recording capabilities.

[0037] Furthermore, the diameter of the inscribed circle of the microelectrode array plate 10 is about 5 mm; the microelectrodes 18 are arranged in a dot matrix, with a number of 8x8; in this way, a microelectrode array recording system can be subsequently formed through the microelectrode array plate 10, the integrated circuit matrix 13 and the second wire 14. When nerve cells and muscle cells grow on the microelectrodes 18, the system can synchronously collect and record extracellular field potential signals at least 60 points, and can also electrically stimulate the cells to promote synapse formation.

[0038] Furthermore, the integrated circuit matrix 13 includes a signal amplifier, a filter, and a connector, etc., which are used to collect the electrophysiological signals captured by the microelectrode array board 10, and provide them to the external system for storage, processing and analysis after amplification, noise filtering and other processing. In this process, the time pattern, frequency and other electrical signals of nerve cell activity can be extracted, and the electrical signals can be digitized for analysis and storage, which is helpful for subsequent research on electrophysiological information such as the firing pattern, network activity, and response characteristics of nerve cells.

[0039] Furthermore, the circuit layer film 15, the PETE porous membrane and the culture layer film 16 are sealed as a whole in sequence by plasma bonding; the circuit layer film 15 is mainly used to carry the microelectrode array plate 10 and the integrated circuit matrix 13, and the culture layer film 16 is mainly used to carry each culture chamber, the microfluidic module 7, the microfluidic channel, the liquid inlet 4 and the liquid outlet 5.

[0040] Preferably, the chip substrate (circuit layer substrate 15, culture layer substrate 16, microelectrode array plate 10 and chamber wall 31, etc.) uses polydimethylsiloxane (PDMS), which has the advantages of good flexibility, high biocompatibility, breathability, transparency, etc., which is conducive to the exchange of oxygen and carbon dioxide and the long-term growth of nerve cells, and is convenient for optical imaging and microscopic observation of cell growth and activity.

[0041] Furthermore, collagen, laminin or poly-D-lysine may be coated on the inner wall surface of each culture chamber and microfluidic channel to promote the attachment, differentiation and elongation of nerve cells.

[0042] Furthermore, a micro-patterned three-dimensional scaffold made of biological materials such as sodium alginate and gelatin is placed in the first culture chamber 1, and astrocytes, microglia, glial cells and / or endothelial cells are introduced for co-culture, which is conducive to enhancing the close interaction between cells and matrix, constructing the real environment of nerve cells in the body, and thus helping to form a more complex neural network. Among them, glial cells can significantly improve the survival rate, stability and synaptic activity of nerve cells.

[0043] Furthermore, by adding chemical inducers or growth factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), etc., to the first culture chamber 1, the differentiation of nerve cells and the formation of synaptic connections can be promoted; especially in the early differentiation stage, designing a chemical gradient in the culture chamber can guide the growth direction of synapses, help form a specific neural network structure, and simulate the growth pattern of nerve cells in vivo.

[0044] Furthermore, the width of each microfluidic subchannel is generally set between tens and hundreds of microns, with a typical range of 50-500 microns, and a height of about 100 microns; the width of the permeation hole 20 is usually 10-50 microns, which helps to simulate the size of blood vessels or tissue gaps so as to accurately control the effective exchange of fluids such as nutrient solutions and metabolic waste; the size of the microelectrode 18 ranges from 10-30 microns, with a minimum spacing of 30 microns to ensure the sensitivity of neural signal detection; the area of ​​each culture chamber is generally 1-2 square millimeters, and the depth is 100-500 microns, so that it can meet the space required for cell growth without affecting the effective control of fluid dynamics, which is conducive to the formation of three-dimensional tissues; the overall size of the microfluidic chip is controlled at 15-30 mm to adapt to microscopes and standard experimental equipment.

[0045] Furthermore, the method of using the neuromuscular multi-coupled organoid microfluidic chip is as follows; Step 1, chip preparation; The chip is made of polydimethylsiloxane (PDMS) material through photolithography, molding and other technologies; Step 2, obtaining nerve cells and muscle cells; Motor neurons and muscle progenitor cells differentiated from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) (or directly extracted primary muscle cells); Step 3, neuromuscular co-culture; The nerve cells and muscle cells are introduced into the corresponding culture chambers of the chip respectively, the microfluidic technology is used to provide the microenvironment required for cell growth, and the optimized culture medium and growth factors are used to promote the formation of neuromuscular junctions; Step 4, electrical stimulation and chemical induction; The microelectrode array plate 10 and the electrode plate 6 integrated on the chip are used to electrically stimulate nerve cells to promote the growth of nerve cell axons and the formation of neuromuscular junctions. At the same time, chemical inducers such as acetylcholine are used to further promote the maturation and functionalization of neuromuscular junctions. The principle of the microelectrode array board 10 to electrically stimulate neurons through the microelectrodes 18 is based on the influence of the electric field on the neuronal membrane potential. When the microelectrode 18 generates current, it forms an electric field near the electrode, thereby changing the charge distribution on both sides of the cell membrane; this electric field causes the nerve cell to depolarize or hyperpolarize, thereby triggering an action potential; Specifically, the electrode plate 6, the microelectrode array plate 10 and the integrated circuit matrix 13 regulate the electrical stimulation parameters through an external electrical control device. The electrical stimulation parameters include current, voltage, pulse and current density, etc.: The current ranges from 0.1 to 10 microamperes; the voltage ranges from 0.05 to 10 volts, depending on the contact impedance between the microelectrode 18 and the nerve cells, which is usually in the kilo-ohm range; the pulse is a biphasic square wave, which includes a positive pulse and a negative pulse, which can better maintain the balance of the cells and reduce the electrochemical side effects caused by the monopolar pulse; the pulse width is controlled between tens and hundreds of microseconds (such as 100 to 500 microseconds), which is related to the activation time constant of the nerve cells; a shorter pulse time can reduce damage to the tissue while effectively activating the nerve cells; the pulse frequency ranges from 20 to 100 Hz, which helps trigger nerve cell activity or transmit information; current density is a key parameter for evaluating the intensity of electrical stimulation, which is controlled between 0 and 1 mA / cm². Too high a current density can damage nerve cells or other tissues around the microelectrode.

[0046] Step 5: Functional testing and analysis and evaluation; The bioelectric signals generated by nerve cells and muscle cells are monitored in real time through the microelectrode array board 10, and then the bioelectric signals are amplified, filtered, and processed through the integrated circuit matrix 13 and then output to an external computer for data processing, so as to complete the dynamic tracking, recording and data analysis of the cell reaction process and evaluate the function of the neuromuscular junction; An antibody that specifically binds to a motor neuron marker is selected to mark a pluripotency marker of a neuron, such as ChAT, SMA, β-tubulin III, etc. The pluripotency marker with fluorescence can be observed by a fluorescence microscope, so that physiological processes such as the connection and activity between the neuron and the muscle cell, the synapse formation and nerve conduction process of the neuron, the contraction and relaxation of the muscle cell, and the formation and structure of the neuromuscular junction can be dynamically observed; By integrating a tension sensor in the second culture chamber 2 (for example, integrated in a PDMS film, a PETE porous membrane or other flexible support materials), the muscle contraction force can be quantified and the abnormality of muscle tissue function can be observed; using electrical stimulation to perform a muscle contraction response test, the contraction force and responsiveness of muscle cells can be measured, and the impact of the disease on the muscle can be quantitatively evaluated or the functionality of the neuromuscular junction can be evaluated.

[0047] In combination with the above-mentioned usage method, the chip provided in this embodiment can be used to construct various neuromuscular disease models, study the physiological and pathological mechanisms of the disease, efficiently screen drugs in vitro, evaluate their efficacy and toxicity, and accelerate the process of new drug development.

[0048] Furthermore, the step 2 specifically includes: Step 21, iPSCs culture preparation and cultivation; Required reagents: mTeSR1 medium or E8 medium (for undifferentiated culture of iPSCs), Matrigel three-dimensional scaffold (1:100 dilution, for coating culture plates), PBS buffer (calcium-free, magnesium-free, for washing), and Accutase enzyme (for cell digestion); Plating: Coat 6-well plates with Matrigel 3D scaffolds and incubate at 37°C for at least 1 hour; Cell seeding: iPSCs were seeded into 6-well plates coated with Matrigel 3D scaffolds using mTeSR1 or E8 medium, and the medium was changed daily. Cell culture: Keep cells in an undifferentiated state and culture to 70%-80% confluence; confirm that the cell morphology is typical of iPSCs and there is no sign of differentiation; Step 22, iPSCs differentiate into motor neurons; Neural Differentiation Medium includes: DMEM / F12 (with GlutaMAX); N2 supplement (1%); B27 supplement (1%, without vitamin A); non-essential amino acids (NEAA, 1%); 2-mercaptoethanol (0.1 mM); Differentiation reagents include: CHIR99021 (Wnt agonist, 3 μM): promotes neuroectoderm formation; Retinoic acid (RA, 0.1 μM): promotes the differentiation of motor neurons; Purmorphamine (SHH agonist, 1 μM): activates the Sonic Hedgehog pathway and promotes motor neuron differentiation; BDNF (brain-derived neurotrophic factor, 10 ng / mL): supports neuronal differentiation; Differentiation steps: Step 221, neural ectoderm induction: when iPSCs reach 70%-80% confluence, the culture medium is replaced with neural differentiation medium containing CHIR99021 (3 μM) and cultured for 5 days to form neural ectoderm-like cells; Step 222, neural precursor formation: continue culturing, add RA (0.1 μM) and Purmorphamine (1 μM) to the culture medium to promote the generation of neural precursor cells. This stage lasts for 7 days; Step 223, motor neuron differentiation: replace the culture medium with neural differentiation medium supplemented with BDNF (10 ng / mL) and continue culturing for 7 days; the motor neurons should express the neural cell marker TUJ1 and motor neuron markers such as Hb9 and ChAT; Step 224, expected results: Motor neurons should be generated within 14 days and express motor neuron-specific markers, such as Hb9 and ChAT.

[0049] Step 23, iPSCs differentiate into skeletal muscle cells; Mesoderm Induction and Myocyte Differentiation Medium includes: DMEM / F12 (with GlutaMAX); N2 supplement (1%); B27 supplement (1%, without vitamin A); non-essential amino acids (NEAA, 1%); 2-mercaptoethanol (0.1mM); Differentiation reagents include: CHIR99021 (3 μM): Promotes mesoderm formation; FGF2 (20 ng / mL): promotes the generation of somite precursor cells; IGF-1 (50 ng / mL): promotes myocyte differentiation and fusion; Horse serum (2%): a promoter of myocyte differentiation; Differentiation steps: Step 231, mesoderm induction: iPSCs were transferred to mesoderm induction medium, CHIR99021 (3 μM) was added, and cultured for 48 hours; Step 232, muscle precursor differentiation: the culture medium was replaced with a culture medium containing FGF2 (20 ng / mL), cultured for 5 days to form somite-like cells, and the expression of PAX3 and PAX7 was detected; Step 233, myocyte differentiation: replace the culture medium with a medium containing IGF-1 (50 ng / mL) and 2% horse serum, and continue culturing for 7 days. Myogenic precursor cells should fuse to form myotubes, and the expression of Myogenin and MyoD should be detected; Step 234, expected results: After 7-10 days, the skeletal muscle cells should form myotube structures and express mature muscle-specific markers such as MyoD, Myogenin and Actin.

[0050] By means of the above method, neural cells and skeletal muscle cells can be accurately differentiated and, after reaching a certain density, introduced into the first culture chamber 1 and the second culture chamber 2 respectively for subsequent co-culture.

[0051] Furthermore, the step 4 also includes promoting the growth of nerve and muscle cells and the formation of neuromuscular junctions by the following methods: Step 41, coating the inner wall surface of the first culture chamber 1 with collagen, laminin or poly-D-lysine to promote the attachment, differentiation and elongation of nerve cells; Step 42, introducing astrocytes, microglia and vascular endothelial cells into the first culture chamber 1 for co-culture, so as to simulate the real environment of nerve cells in the body; glial cells can significantly improve the survival rate, stability and synaptic activity of neurons, and enhance the interaction between cells and matrices; Step 43, using a micro-patterned three-dimensional scaffold (made of sodium alginate, gelatin, etc.) in the first culture chamber 1 to provide three-dimensional support for the nerve cells, which helps to form a more complex neural network; Step 44, adding chemical inducers or growth factors, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), to the first culture chamber 1 and the second culture chamber 2 can promote the differentiation of nerve cells and the formation of synaptic connections in the early differentiation stage; designing chemical gradients in each culture chamber to guide the growth direction of neurites is helpful to form a specific neural network structure and simulate the real growth pattern of nerve cells in the body.

[0052] Step 45, electrical stimulation is performed in the third culture chamber 3 to promote nerve cell development, synapse formation and optimization of electrophysiological characteristics, while monitoring the electrophysiological activities of the cells in real time.

[0053] Furthermore, the pluripotency markers in step 5 also include: Acetylcholine receptors (AChR) were fluorescently labeled with α-BTX (α-Bungarotoxin) to detect neuromuscular junction formation; Synaptophysin, a synaptic vesicle protein present in the presynaptic region of motor neurons, is used for fluorescent labeling to confirm the formation of neuromuscular junctions; TUJ1, MAP2, and MyoD were stained for the detection of motor neuron and skeletal muscle cell-specific markers.

[0054] Embodiment 2: This embodiment provides a microfluidic device using the neuromuscular multi-coupled organoid chip as described in the first embodiment, including an integrated circuit matrix, a microenvironment detection sensor, and a dynamic fluid transmission unit; The microenvironment detection sensor comprises a first culture chamber detection sensor, a second culture chamber detection sensor and a third culture chamber detection sensor; A first culture chamber detection sensor is disposed between the culture layer and the circuit layer at the first culture chamber, and is used to detect microenvironmental data of the first culture chamber; the microenvironmental data includes fluid flow rate, chemical gradient, temperature and dissolved oxygen level; A second culture chamber detection sensor is disposed between the culture layer and the circuit layer at the second culture chamber and is used to detect microenvironment data of the second culture chamber; A third culture chamber detection sensor is disposed between the culture layer and the circuit layer in the third culture chamber and is used to detect microenvironment data of the third culture chamber; The dynamic fluid transmission part includes a plurality of micro pumps and valves, which are respectively arranged at the liquid inlet and the liquid outlet, and are used to input nutrient solution and / or medicine into the microfluidic channel in a program-controlled manner, and to output metabolic waste of cells; this can simulate the blood circulation in the body, and provide continuous or time-divided nutrient solution and / or medicine to nerve cells and muscle cells, respectively, to ensure that each organoid grows and develops under the most suitable conditions; through the flow control of micro pumps and valves, precise fluid flow rate and gradient environment (such as gradient of oxygen, nutrient and growth factor concentration) can be achieved in the microfluidic channel; The microfluidic module, the microenvironment detection sensor and the dynamic fluid transmission unit are electrically connected to regulate the microenvironment in the chip.

[0055] The integrated circuit matrix is ​​electrically connected to the microfluidic module and the microelectrode array board, and is used to control the chip to perform data acquisition, electrical stimulation, and microfluidic management.

[0056] Furthermore, a tension sensor may be provided in the PETE porous membrane below the second culture chamber to monitor muscle contraction force, thereby observing abnormalities in muscle tissue function; an electrophysiological sensor may be provided in the PETE porous membrane below the first culture chamber to monitor the conduction of nerve signals; an optical sensor may be integrated in the PETE porous membrane below any culture chamber to monitor oxygen and / or pH in the culture chamber.

[0057] Furthermore, an oxygen control device may be added to each culture chamber to ensure that the oxygen concentration is always kept within an appropriate range, especially when the density of nerve cells is high, to help prevent hypoxia.

[0058] Furthermore, micro sensors can be added to each culture chamber to monitor temperature, pH and dissolved oxygen levels in real time to ensure the stability of the microenvironment; too high or too low temperature will affect the activity of nerve cells and synapse formation, and pH fluctuations will also affect the culture effect.

[0059] Embodiment three: like Figure 5 As shown, this embodiment provides an application method of the neuromuscular multi-coupled organoid chip as described in Embodiment 1, comprising: Step a1, obtaining skin cells or blood cells from patients with amyotrophic lateral sclerosis (ALS), reprogramming to obtain induced pluripotent stem cells with the mutated gene characteristics of the disease, and differentiating them into motor nerve cells and muscle cells; Step a2, introducing the motor nerve cells into the first culture chamber; introducing the muscle cells into the second culture chamber; co-culturing into motor neuron organoids and muscle organoids, forming neuromuscular junctions in the third culture chamber through microfluidic channels, and obtaining an amyotrophic lateral sclerosis disease model; Step a3, observing abnormal phenomena of motor neurons, muscle contraction and synaptic function in the disease model, and recording electrophysiological data through a microelectrode array plate; Step a4, using the disease model to perform drug screening, testing the improvement effects of antioxidant drugs, neurotrophic factors, etc. on motor nerve cell survival and muscle function.

[0060] In this way, cells from ALS patients can be used to prepare ALS disease models in order to study the effects of related gene mutations on the development and function of motor neurons and screen potential drugs.

[0061] The specific experimental process is as follows: Experimental materials / equipment preparation: Cell sources include: Skin fibroblasts or blood monocytes from ALS patients; iPSC reprogramming kit (such as Sendai virus or mRNA).

[0062] Culture materials include: Matrigel 3D scaffold (or other ECM); collagen, laminin, or poly-D-lysine; DMEM medium; Neuronal Differentiation Medium; Mesoderm induction medium; Growth factors: such as BDNF, GDNF, IGF-1, FGF2, etc.

[0063] The training equipment includes: The device in Example 2 (comprising a plurality of neuromuscular multi-coupled organoid microfluidic chips); 6-well plate or 24-well plate; CO2 incubator (37°C, 5% CO2); Inverted microscope; Cryopreservation medium (10% DMSO + 90% FBS or other cryopreservation medium).

[0064] Analysis tools include: Fluorescence microscopy (for detecting organoid marker expression); RT-qPCR equipment (for gene expression analysis); Confocal microscopy (for structural analysis).

[0065] The experimental process includes: Stage 1, Differentiation of Motor Neurons and Muscle Cells: Obtain a skin biopsy or peripheral blood sample from an ALS patient, and differentiate motor neurons and muscle cells according to the method in step 2; Phase 2: Neuromuscular multi-coupled organoid microfluidic chip culture: The Matrigel three-dimensional scaffold was spread on the bottom of the first culture chamber of the neuromuscular multi-coupled organoid microfluidic chip and solidified at 37°C for 10 minutes; Each culture chamber of the chip is coated with a matrix such as collagen, laminin or poly-D-lysine to promote cell attachment and growth; Motor neuron differentiation medium (containing growth factors such as BDNF and GDNF) is injected into the chip through the dynamic fluid transfer unit to ensure its uniform distribution and prevent bubbles from entering the microfluidic channel to avoid affecting the attachment effect of the matrix; Motor nerve cells were planted in the first culture chamber of the chip; muscle cells were planted in the second culture chamber of the chip; the corresponding cell suspension injection speed was: 0.5-1 μl / s; the cell density was maintained at 10 6 cells / mL to ensure uniform distribution; Neuromuscular co-culture was performed by the method of step 3; the flow rate of the culture medium was controlled at 0.1 μL / min; the temperature in the chip was kept constant at 37°C; the pH in the chip was controlled within the range of 7.2-7.4; Perform electrical stimulation and chemical induction by the method in step 4; and input chemical stimulants such as acetylcholine into the chip through the dynamic fluid transmission part to simulate in vivo signal transmission; Phase 3: Functional testing and disease phenotype analysis of neuromuscular multi-coupled organoid microfluidic chips: Input a marker (such as a calcium ion indicator) into the chip through a dynamic fluid transfer unit, and observe the chip using a fluorescence microscope; dynamically adjust the fluid flow rate and culture medium composition according to the observation results to optimize the functional detection conditions; The neural activity in the third culture chamber was detected by a microelectrode array plate to evaluate the electrophysiological function of motor neurons; Phase 4, drug screening and phenotypic testing: Divide multiple chips into multiple experimental groups, pump different concentrations of drug candidates (such as ALS therapeutic drugs such as Riluzole) through the dynamic fluid transmission part, and set a blank control group; The third culture chamber was electrically stimulated through an integrated circuit matrix: the pulse frequency was 20 Hz, the pulse width was 200 microseconds, and the voltage was 0.05-0.1 volts; the electrophysiological activity of nerve cells was synchronously detected through a microelectrode array plate, and the data acquisition frequency was 1 kHz, so as to observe the effects of drugs on nerve conduction function; 1 µM acetylcholine was pumped through the dynamic fluid transfer unit for 30 seconds to observe the contraction response of muscle cells, thereby detecting cell viability and evaluating drug toxicity; changes in the expression of motor neuron markers were detected by fluorescence microscopy.

[0066] Embodiment 4: like Figure 6 As shown, this embodiment provides an application method of the neuromuscular multi-coupled organoid chip as described in Embodiment 1, comprising: Step b1, obtaining induced pluripotent stem cells from patients with spinal muscular atrophy, and differentiating motor nerve cells and muscle cells, wherein the induced pluripotent stem cells carry an SMN1 gene mutation; Step b2, introducing the motor nerve cells into the first culture chamber; introducing the muscle cells into the second culture chamber; co-culturing into motor neuron organoids and muscle organoids, forming neuromuscular junctions in the third culture chamber through microfluidic channels, and obtaining a spinal muscular atrophy disease model; Step b3, using electrophysiological recording and molecular markers to detect motor neuron signal transduction, including synaptic protein levels and neuronal connectivity levels, to evaluate the structure and function of neuronal synapses; Step b4, testing compounds or genes that enhance SMN protein expression in the disease model to evaluate the effects of the compounds or genes on improving the interaction between neurons and muscles or delaying the progression of neuronal degeneration.

[0067] Embodiment five: like Figure 7 As shown, this embodiment provides an application method of the neuromuscular multi-coupled organoid chip as described in Embodiment 1, comprising: Step c1, obtaining induced pluripotent stem cells with dystrophin gene mutation from patients with Duchenne muscular dystrophy, and differentiating motor nerve cells and muscle cells; Step c2, introducing the motor nerve cells into the first culture chamber; introducing the muscle cells into the second culture chamber; co-culturing into motor neuron organoids and muscle organoids, forming neuromuscular junctions in the third culture chamber through microfluidic channels, and obtaining a Duchenne muscular dystrophy disease model; Step c3, setting a tension sensor in the second culture chamber to measure muscle contraction force; Step c4, conducting muscle contraction response test through electrophysiological stimulation; Step c5, testing gene therapy, expression effect of exogenous dystrophin protein and drug response through the disease model.

[0068] Embodiment six: As shown in FIG8 , this embodiment provides an application method of the neuromuscular multi-coupled organoid chip as described in Embodiment 1, comprising: Step d1, obtaining induced pluripotent stem cells from a healthy body and differentiating them into motor neurons and muscle cells; Step d2, introducing the motor nerve cells into the first culture chamber; introducing the muscle cells into the second culture chamber; co-culturing into motor neuron organoids and muscle organoids, and forming neuromuscular junctions in the third culture chamber through microfluidic channels; Step d3, adding specific antibodies into the third culture chamber to simulate the attack of autologous antibodies on the neuromuscular junction to obtain a myasthenia gravis disease model; Step d4, observing the effect of the antibody on muscle contraction, and recording abnormal data of the response of muscle organoids to neuronal signals, for evaluating the expression level of acetylcholine receptors at synapses; Step d5, testing antibody inhibitors, immunosuppressive drugs or neurotransmitter enhancing drugs through the disease model, and recording signal transduction or muscle function data.

[0069] In other embodiments, more disease models can be constructed with reference to the above-mentioned disease models, so as to provide a reliable and convenient platform for studying neuromuscular physiological and pathological mechanisms, screening drugs, and other aspects.

[0070] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A neuromuscular multi-coupled organoid microfluidic chip, characterized in that: include: In the culture layer, a first culture chamber, a second culture chamber and a third culture chamber are arranged; The first culture chamber is used to culture neural cells; The second culture chamber is used to culture muscle cells; A microfluidic channel for connecting the first culture chamber and the second culture chamber is provided between the two chambers; a liquid inlet and a liquid outlet are provided at both ends of the microfluidic channel; the liquid inlet is provided at one end close to the first culture chamber; The third culture chamber is arranged on the microfluidic channel and is used to culture the neuromuscular junction formed by the axons of nerve cells and muscle cells; A microfluidic module is arranged in the center of the third culture chamber; an electrode plate is inlaid on the bottom surface of the microfluidic module; a microelectrode array plate is arranged in the circuit layer below the electrode plate; a gap between the electrode plate and the microelectrode array plate constitutes a discharge space for electrically stimulating and detecting electrophysiological functions of cells entering the discharge space; a plurality of vertical channels are opened on the side wall of the microfluidic module for connecting the third culture chamber with the discharge space; Corresponding sensing points are evenly distributed around the microelectrodes of the microelectrode array plate, and the microelectrodes and the sensing points are connected by a one-to-one corresponding first wire for transmitting electrical signals from the microelectrodes; an integrated circuit matrix and a second wire connected to the integrated circuit matrix are evenly distributed around the sensing points for controlling the chip and processing the electrical signals.

2. The chip according to claim 1, characterized in that: The microfluidic channel comprises a plurality of axial separation zones, and a plurality of permeation holes are arranged on the separation zones.

3. The chip according to claim 1, characterized in that: The chip substrate includes polydimethylsiloxane.

4. The chip according to claim 1, characterized in that: The number of the first culture chambers is 1; the number of the second culture chambers is 2; the number of the liquid inlets is 2; the number of the liquid outlets is 1; the first culture chamber and the liquid outlet are arranged at both ends of the microfluidic channel; the second culture chamber and the liquid inlet are symmetrically arranged on both sides of the microfluidic channel.

5. The chip according to claim 1, characterized in that: The material of the microelectrode is TiN.

6. A neuromuscular multi-coupled organoid microfluidic device using the chip as described in any one of claims 1 to 5, characterized in that: It includes an integrated circuit matrix, a microenvironment detection sensor, and a dynamic fluid transmission unit; The microenvironment detection sensor comprises a first culture chamber detection sensor, a second culture chamber detection sensor and a third culture chamber detection sensor; The first culture chamber detection sensor is disposed between the culture layer and the circuit layer at the first culture chamber, and is used to detect microenvironmental data of the first culture chamber; the microenvironmental data includes fluid flow rate, chemical gradient, temperature and dissolved oxygen level; The second culture chamber detection sensor is disposed between the culture layer and the circuit layer at the second culture chamber, and is used to detect microenvironment data of the second culture chamber; The third culture chamber detection sensor is arranged between the culture layer and the circuit layer in the third culture chamber, and is used to detect the microenvironment data of the third culture chamber; The dynamic fluid transmission part includes a plurality of micro pumps and valves, which are respectively arranged at the liquid inlet and the liquid outlet, and are used to programmatically input nutrient solution and / or medicine into the microfluidic channel and output metabolic waste of cells; The microfluidic module is electrically connected to the microenvironment detection sensor and the dynamic fluid transmission unit, and is used to regulate the microenvironment in the chip; The integrated circuit matrix is ​​electrically connected to the microfluidic module and the microelectrode array board.

7. An application method of the chip according to any one of claims 1 to 5, characterized in that: include: Step a1, obtaining skin cells or blood cells from patients with amyotrophic lateral sclerosis, obtaining induced pluripotent stem cells with disease mutation gene characteristics through reprogramming, and differentiating them into motor nerve cells and muscle cells; Step a2, introducing the motor nerve cells into the first culture chamber; introducing the muscle cells into the second culture chamber; co-culturing into motor neuron organoids and muscle organoids, forming neuromuscular junctions in the third culture chamber through microfluidic channels, and obtaining an amyotrophic lateral sclerosis disease model; Step a3, observing abnormal phenomena of motor neurons, muscle contraction and synaptic function in the disease model, and recording electrophysiological data through a microelectrode array plate; Step a4, using the disease model to perform drug screening, testing the improvement effects of antioxidant drugs and neurotrophic factors on motor neuron survival and muscle function.

8. An application method of the chip according to any one of claims 1 to 5, characterized in that: include: Step b1, obtaining induced pluripotent stem cells from patients with spinal muscular atrophy, and differentiating motor nerve cells and muscle cells, wherein the induced pluripotent stem cells carry an SMN1 gene mutation; Step b2, introducing the motor neurons into the first culture chamber; The muscle cells are introduced into the second culture chamber; co-cultured into motor neuron organoids and muscle organoids, and a neuromuscular junction is formed in the third culture chamber through a microfluidic channel to obtain a spinal muscular atrophy disease model; Step b3, using electrophysiological recording and molecular markers to detect motor neuron signal transduction, including synaptic protein levels and neuronal connectivity levels, to evaluate the structure and function of neuronal synapses; Step b4, testing compounds or genes that enhance SMN protein expression in the disease model to evaluate the effects of the compounds or genes on improving the interaction between neurons and muscles or delaying the process of neuronal degeneration.

9. An application method of the chip as claimed in any one of claims 1 to 5, characterized in that: include: Step c1, obtaining induced pluripotent stem cells with dystrophin gene mutation from patients with Duchenne muscular dystrophy, and differentiating motor nerve cells and muscle cells; Step c2, introducing the motor nerve cells into the first culture chamber; introducing the muscle cells into the second culture chamber; co-culturing into motor neuron organoids and muscle organoids, forming neuromuscular junctions in the third culture chamber through microfluidic channels, and obtaining a Duchenne muscular dystrophy disease model; Step c3, setting a tension sensor in the second culture chamber to measure muscle contraction force; Step c4, conducting muscle contraction response test through electrophysiological stimulation; Step c5, testing gene therapy, expression effect of exogenous dystrophin protein and drug response through the disease model.

10. An application method of the chip according to any one of claims 1 to 5, characterized in that: include: Step d1, obtaining induced pluripotent stem cells from a healthy body and differentiating them into motor neurons and muscle cells; Step d2, introducing the motor neurons into a first culture chamber; The muscle cells are introduced into a second culture chamber; co-cultured into motor neuron organoids and muscle organoids, and a neuromuscular junction is formed in a third culture chamber through a microfluidic channel; Step d3, adding specific antibodies into the third culture chamber to simulate the attack of autologous antibodies on the neuromuscular junction to obtain a myasthenia gravis disease model; Step d4, observing the effect of the antibody on muscle contraction, and recording abnormal data of the response of muscle organoids to neuronal signals, for evaluating the expression level of acetylcholine receptors at synapses; Step d5, testing antibody inhibitors, immunosuppressive drugs or neurotransmitter enhancing drugs through the disease model, and recording signal transduction or muscle function data.

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