A microfluidic chip and method for brain organoid culture and multimodal monitoring

Abstract

The application provides a microfluidic chip and method for brain organoid culture and multi-modal monitoring, and belongs to the technical field of microfluidic chips and neural electrophysiology, which can partially solve the problem that the existing brain organoid research platform has single function and is difficult to realize synchronous acquisition of electrophysiological information and biochemical information. The microfluidic chip comprises a culture layer, an electrical coupling layer and an electrode layer which are sequentially stacked and connected to each other, wherein the culture layer is provided with a culture chamber for accommodating a brain organoid and a detection flow channel communicated with the culture chamber, and the spatial fixation of the brain organoid is realized through a limiting structure; the electrode layer is provided with a microelectrode array corresponding to the culture chamber, which is used for electric signal acquisition and electric stimulation; and the electrical coupling layer is used for establishing a stable electrical action path while isolating the fluid channel and the electrode circuit. The application is beneficial to long-term stable culture of brain organoids and neural function research.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip and neuroelectrophysiology technology, specifically relating to a microfluidic chip and method for brain organoid culture and multimodal monitoring. Background Technology

[0002] With the rapid development of neuroscience, bioengineering, and regenerative medicine, research into brain function and the mechanisms of nervous system diseases has deepened. Traditional two-dimensional cell culture models and animal models have, to some extent, promoted the development of neurobiology, but due to species differences or spatial limitations, they are difficult to realistically simulate the developmental process and complex neural network structure of the human brain. In recent years, brain organoids, as in vitro models obtained through three-dimensional culture and capable of partially reproducing the structural and functional characteristics of human brain tissue, have shown significant application value in fields such as neurodevelopmental research, analysis of the mechanisms of neurodegenerative diseases, and drug screening. Currently, the culture and functional research of brain organoids largely rely on static culture plates, spinnerets, or commercial microelectrode array culture systems. These culture methods can maintain the survival and development of organoids to a certain extent, but due to the relatively closed culture environment, the transport of nutrients and metabolites mainly depends on diffusion. Hypoxia and nutrient deficiency are prone to occur inside the organoids, especially in the core region, leading to central necrosis and limiting their long-term culture and functional maturation. In addition, existing studies on the assessment of the functional status of brain organoids mostly use single detection methods, such as relying solely on multi-electrode arrays for electrophysiological recording or detecting protein factors in the culture supernatant offline, making it difficult to achieve simultaneous acquisition of multi-dimensional information. Furthermore, while existing electrophysiological monitoring systems can record spontaneous or evoked electrical activity in brain organoids, their spatial resolution and long-term stability remain limited. Most systems only have signal acquisition capabilities, making it difficult to apply precise and controllable electrical stimulation to the organoids simultaneously with recording. On the other hand, the detection of neuroinflammatory factors, neurotrophic factors, or disease-related proteins typically requires periodic sampling and offline analysis, which is cumbersome, time-consuming, and fails to reflect the dynamic correlation between electrophysiological activity and biochemical changes.

[0003] Therefore, the existing technologies have the following shortcomings: First, the dynamic controllability of the brain organoid culture environment is insufficient, making it difficult to support long-term stable culture and functional maturation; second, electrophysiological monitoring and biochemical index detection are independent of each other, lacking the ability to perform multimodal simultaneous analysis; third, the existing platforms have low functional integration, making it difficult to achieve organoid culture, electrostimulation regulation, and in-situ detection of multiple indicators in the same system. To address these issues, a microfluidic chip and method for brain organoid culture and multimodal monitoring are proposed. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art, and to provide a microfluidic chip and method for brain organoid culture and multimodal monitoring.

[0005] The present invention provides a microfluidic chip for brain organoid culture and multimodal monitoring, comprising a culture layer, an electrical coupling layer and an electrode layer that are stacked and interconnected in sequence; The culture layer is provided with at least one culture chamber for accommodating brain organoids, a microfluidic channel communicating with the culture chamber, and a detection channel in fluid communication with the culture chamber; The culture chamber is connected to the microfluidic channel through a limiting structure, which is used to spatially fix the brain organoid while allowing the culture medium and brain organoid secretion products to pass through. The detection channel is used to guide the culture medium carrying the secreted products of the brain organoid to the detection area in order to obtain the biochemical information produced by the brain organoid during the culture process. The electrode layer is provided with a microelectrode array corresponding to the culture chamber. The microelectrode array is used to collect electrical signals from the brain organoid and / or to generate electrical stimulation to the brain organoid. The electrical coupling layer is disposed between the culture layer and the electrode layer, and forms an electrical interaction path between the microelectrode array and the culture chamber.

[0006] Furthermore, the limiting structure includes multiple slits, channels, or passages with feature dimensions smaller than the size of the brain organoid, to prevent the brain organoid from shifting under fluid action.

[0007] Specifically, the detection channel and the culture chamber maintain continuous fluid communication during the same culture process to guide the culture medium carrying the brain organoid secretion products to flow within the microfluidic chip.

[0008] Specifically, a protein detection component for contacting the culture medium is provided in the detection area corresponding to the detection channel, and the protein detection component is configured to perform in-situ detection of the target protein within the microfluidic chip.

[0009] Preferably, each microelectrode unit in the microelectrode array is configured to switch between an electrical signal acquisition state and an electrical stimulation state for electrophysiological recording and electrical stimulation modulation of the brain organoid.

[0010] Specifically, the culture layer is provided with multiple independent culture chambers, each of which is provided with an independent microelectrode array and detection channel to realize the parallel culture and functional monitoring of multiple brain organoids.

[0011] Another aspect of the present invention provides a microfluidic method for brain organoid culture and multimodal monitoring, the method being implemented using the aforementioned microfluidic chip for brain organoid culture and multimodal monitoring, and comprising the following steps: S1: The brain organoid and the three-dimensional support matrix are placed into the culture chamber to form a fixed culture structure; S2: Supply culture medium to the culture chamber through the microfluidic channel, and maintain the spatial position of the brain organoid in the culture chamber through the limiting structure; S3: The microelectrode array is used to collect the electrophysiological signals generated by the brain organoid, and the outflowing culture medium is guided to the detection area corresponding to the detection channel to obtain the target protein information secreted by the brain organoid.

[0012] Furthermore, in step S3, during the acquisition of the electrophysiological signals, electrical stimulation with preset parameters is applied to the brain organoid through the microelectrode array to regulate or induce its neural activity state.

[0013] Furthermore, the target protein includes biomarkers associated with neurological functional status, neurological injury, or neurological disease.

[0014] Furthermore, the microfluidic method performs time correlation analysis on the acquired electrophysiological signal data and the detection results of the target protein to assess the functional changes of the brain organoid under stimuli or pathological conditions.

[0015] The beneficial effects of this invention are as follows: This invention, by setting up a culture chamber structure with physical restraint, effectively immobilizes brain organoids while allowing the exchange of culture medium and secreted products, improving the problems of displacement and restricted nutrient exchange in traditional culture methods, and facilitating the long-term stable culture of brain organoids. This invention integrates a microelectrode array, electrical coupling structure, and detection channel into the same microfluidic chip, enabling the synchronous acquisition of electrophysiological signals and target protein information generated by the same brain organoid without interrupting culture, avoiding information fragmentation caused by multiple separate detection methods in existing technologies. The microelectrode array of this invention can switch between recording and stimulation states, realizing real-time monitoring and controllable intervention of neural activity in brain organoids, suitable for studying neural network development, abnormal discharges, and stimulus response processes. This invention sets up multiple independent culture units on the same chip, each configured with corresponding electrodes and detection structures, allowing for the simultaneous culture and monitoring of multiple brain organoids, improving experimental efficiency and the reproducibility of results. Attached Figure Description

[0016] Figure 1This is a schematic diagram of the overall exploded structure of a microfluidic chip for brain organoid culture and multimodal monitoring according to a specific embodiment of the present invention. Figure 2 This is a schematic diagram of the culture layer structure of a microfluidic chip for brain organoid culture and multimodal monitoring, according to a specific embodiment of the present invention. Figure 3 This is a schematic diagram of the electrical coupling layer of a microfluidic chip for brain organoid culture and multimodal monitoring, according to a specific embodiment of the present invention. Figure 4 This is a schematic diagram of the electrode layer structure of a microfluidic chip for brain organoid culture and multimodal monitoring according to a specific embodiment of the present invention. Figure 5 This is a partial enlarged view of the culture area composed of three layers of a microfluidic chip for brain organoid culture and multimodal monitoring, according to a specific embodiment of the present invention. Figure 6 This is a flowchart illustrating the steps of a microfluidic method for brain organoid culture and multimodal monitoring, according to a specific embodiment of the present invention.

[0017] The structure consists of: a 100-layer culture layer; a 200-layer electrical coupling layer; and a 300-layer electrode layer. 101 First inlet; 102 Second inlet; 103 First outlet; 104 Second outlet; 105 Detection area; 106 Culture chamber; 107 Limiting structure; 108 Detection flow channel; 109 Microfluidic channel; 201 Electrical connection hole; 301 Microelectrode array; 302 Signal interface area; 303 Microelectrode unit. Detailed Implementation

[0018] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] like Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown, a microfluidic chip for brain organoid culture and multimodal monitoring provided by a specific embodiment of the present invention includes a culture layer 100, an electrical coupling layer 200 and an electrode layer 300 that are stacked and interconnected in sequence. The culture layer 100 includes at least one culture chamber 106 for accommodating brain organoids, a microfluidic channel 109 communicating with the culture chamber 106, and a detection channel 108 in fluid communication with the culture chamber 106. The culture chamber 106 is connected to the microfluidic channel 109 via a limiting structure 107, which spatially fixes the brain organoid while allowing the culture medium and brain organoid secretions to pass through. The detection channel 108 guides the culture medium carrying the brain organoid secretions to the detection area 105 to obtain biochemical information generated by the brain organoid during culture. The electrode layer 300 is provided with a microelectrode array 301 that is vertically arranged in relation to the culture chamber 106. The microelectrode array 301 is used to collect electrical signals from brain organoids and / or to generate electrical stimulation to brain organoids. An electrical coupling layer 200 is disposed between the culture layer 100 and the electrode layer 300 to form a stable electrical action path between the microelectrode array 301 and the culture chamber 106 while isolating the fluid channel and the electrode circuit.

[0020] Specifically, the culture layer 100 is made of a biocompatible polymer material and has a first inlet 101 and a second inlet 102. The first inlet 101 is used to continuously introduce culture medium, and the second inlet 102 is used to inject a mixture of brain organoids and three-dimensional support matrix. The culture layer 100 also has a first outlet 103 and a second outlet 104. The first outlet 103 is used for gas-liquid exchange, and the second outlet 104 is used for culture medium to flow out and enter the detection channel 108.

[0021] Furthermore, the culture chamber 106 has a microscale cavity structure, which is suitable for the three-dimensional culture of single or small numbers of brain organoids, and its periphery is connected to the microfluidic channel through the limiting structure 107.

[0022] Furthermore, such as Figure 5 As shown, the limiting structure 107 includes multiple slits, channels or passages with feature sizes smaller than the size of brain organoids. On the one hand, it can prevent brain organoids from shifting under perfusion conditions, and on the other hand, it allows the free diffusion and exchange of nutrients, metabolites and brain organoid secretion factors, thereby maintaining a stable microenvironment.

[0023] Based on the above basic implementation method, the detection channel 108 and the culture chamber 106 maintain a continuous fluid communication relationship during the same culture process, which is used to guide the culture medium carrying brain organoid secretion products to flow inside the microfluidic chip and finally enter the detection area 105.

[0024] Furthermore, the detection area 105 is located at the edge of the culture layer 100, which is suitable for arranging biochemical detection components to achieve in-situ detection of target proteins in the culture medium.

[0025] In one specific embodiment, the electrical coupling layer 200 is made of an insulating material and has a plurality of electrical communication holes 201 disposed thereon. The electrical communication holes 201 are aligned vertically with the culture chamber 106 and the microelectrode array 301, so that a low-impedance and stable electrical coupling path is formed between the brain organoid and the microelectrode array 301, while avoiding direct contact between the culture medium and the electrode circuit.

[0026] In this embodiment, the electrode layer 300 includes a microelectrode array 301 and a signal interface region 302, wherein the microelectrode array 301 is composed of a plurality of microelectrode units 303, and each microelectrode unit 303 can be connected to an external electrophysiological recording device or stimulation device.

[0027] Furthermore, each microelectrode unit 303 in the microelectrode array 301 is configured to switch between an electrical signal acquisition state and an electrical stimulation state to achieve electrophysiological recording and electrical stimulation regulation of brain organoids; the culture layer 100 is provided with multiple independent culture chambers 106, each culture chamber 106 is respectively provided with an independent microelectrode array 301 and a detection channel 108, thereby realizing parallel culture and multimodal monitoring of multiple brain organoids.

[0028] In one specific implementation, such as Figure 6 As shown, the present invention also provides a microfluidic method for brain organoid culture and multimodal monitoring. The method is implemented using the aforementioned microfluidic chip for brain organoid culture and multimodal monitoring, and includes the following steps: S1: Brain organoids and three-dimensional support matrix are placed together in culture chamber 106 to form a fixed culture structure; S2: The culture medium is supplied to the culture chamber 106 through the microfluidic channel 109, and the spatial position of the brain organoid in the culture chamber 106 is maintained by the limiting structure 107. S3: Use the microelectrode array 301 to collect the electrophysiological signals generated by brain organoids, and guide the outflowing culture medium to the detection area 105 corresponding to the detection channel 108 to obtain the target protein information secreted by brain organoids.

[0029] Specifically, the culture medium is continuously perfused during the culture process, so that the brain organoids are always in a stable nutritional and metabolic environment.

[0030] Furthermore, electrophysiological signals include spontaneous discharge signals from brain organoids or electrical activity signals induced under stimuli.

[0031] In another specific embodiment, in step S3, during the acquisition of electrophysiological signals, electrical stimulation with preset parameters is applied to brain organoids through a microelectrode array to regulate or induce their neural activity state; the target protein includes biomarkers related to neural function state, neural damage, or neural disease.

[0032] In this embodiment, the electrical stimulation can be low-frequency stimulation to promote neural differentiation and network maturation of brain organoids; or high-frequency stimulation to induce abnormal neural activities such as epileptiform discharges.

[0033] Specifically, by simultaneously monitoring electrophysiological signals and the release of target proteins, in vitro models related to diseases such as epilepsy or nerve damage can be constructed.

[0034] In another specific embodiment, the microfluidic method performs time correlation analysis on the acquired electrophysiological signal data and the detection results of the target protein to assess the functional changes of the brain organoid under stimuli or pathological conditions.

[0035] Furthermore, time correlation analysis includes correlating the trends of electrophysiological signal changes with the results of target protein accumulation detection to analyze the relationship between changes in neural activity and biochemical responses.

[0036] Furthermore, this association analysis can be used to assess the effects of electrical stimulation interventions or the impact of pathological factors on brain organoid function.

[0037] Example 1: Brain organoid maturation culture and epileptiform electrical activity monitoring In one specific implementation, this embodiment adopts the following... Figures 1 to 5 The microfluidic chip implementation shown here, used for brain organoid culture and multimodal monitoring, illustrates the process of brain organoid maturation culture, epileptiform electrical activity induction, electrophysiological signal acquisition, and detection of target proteins related to nerve injury within the same microfluidic chip. In this embodiment, the electrical stimulation and electrical signal acquisition processes are achieved through the coordinated operation of the culture layer 100, the electrical coupling layer 200, and the electrode layer 300.

[0038] In this embodiment, the microfluidic chip includes a culture layer 100, an electrical coupling layer 200, and an electrode layer 300, which are stacked and interconnected in sequence. The culture layer 100 is used to form a three-dimensional culture space for the brain organoid and a flow path for secreted products. The electrical coupling layer 200 is used to establish an electrical interaction path between the culture layer 100 and the electrode layer 300. The electrode layer 300 is used to acquire electrical signals and / or perform electrical stimulation on the brain organoid.

[0039] The fabrication and assembly of chips includes the following steps: First, a culture layer 100 is prepared. The culture layer 100 is made of a biocompatible polymer material and includes a first inlet 101, a second inlet 102, a first outlet 103, a second outlet 104, a detection area 105, a culture chamber 106, a limiting structure 107, a detection channel 108, and a microfluidic channel 109. Specifically, the first inlet 101 is used to introduce the culture medium, the second inlet 102 is used to introduce the mixture of brain organoids and the three-dimensional support matrix, the first outlet 103 is used for gas-liquid exchange, and the second outlet 104 is used to guide the culture medium to the detection channel 108. The culture chamber 106 is used to contain the brain organoids. The limiting structure 107 is disposed between the culture chamber 106 and the microfluidic channel 109. The characteristic dimensions of the slits, pores, or channels of the limiting structure 107 are smaller than the size of the brain organoids, thereby allowing the culture medium, nutrients, metabolites, and secretions from the brain organoids to pass through while preventing the brain organoids from migrating out of the culture chamber 106 with the fluid flow.

[0040] Furthermore, the detection channel 108 maintains fluid communication with the culture chamber 106, and the detection channel 108 guides the culture medium carrying brain organoid secretion products to the detection area 105. A protein detection component is installed in the detection area 105. The protein detection component adopts an immunochromatographic detection device targeting neuron-specific enolase as a specific form, and is used to perform in-situ detection of target proteins related to nerve damage in the culture medium.

[0041] Next, an electrical coupling layer 200 is prepared. The electrical coupling layer 200 is disposed between the culture layer 100 and the electrode layer 300, and multiple electrical communication holes 201 are provided on the electrical coupling layer 200. The electrical communication holes 201 are vertically aligned with the culture chamber 106 and the microelectrode array 301. Conductive hydrogel, conductive dielectric, or metallized structures are disposed within the electrical communication holes 201 to form a stable electrical interaction path between the microelectrode array 301 and the brain organoid within the culture chamber 106, while simultaneously reducing the risk of interference caused by direct contact between the culture medium and the electrode circuitry.

[0042] Electrode layer 300 is fabricated again. Electrode layer 300 includes microelectrode array 301, signal interface region 302, and multiple microelectrode units 303. Microelectrode array 301 is positioned corresponding to culture chamber 106, and each microelectrode unit 303 can be connected to external electrophysiological recording equipment and / or electrostimulation equipment through signal interface region 302. Each microelectrode unit 303 can switch between electrical signal acquisition state and electrical stimulation state, thereby enabling electrophysiological recording and electrostimulation modulation of brain organoids during the same culture process.

[0043] During chip assembly, the culture layer 100, electrical coupling layer 200, and electrode layer 300 are sequentially aligned and bonded, ensuring that the culture chamber 106, electrical communication hole 201, and microelectrode array 301 correspond vertically to each other. After assembly, buffer solution or culture medium is injected into the microfluidic channel 109 to check the channel sealing and the liquid exchange state within the culture chamber 106; simultaneously, electrode impedance detection or electrical signal testing confirms whether the electrical interaction path between the microelectrode array 301 and the electrical coupling layer 200 is connected.

[0044] During the brain organoid loading process, the brain organoid is mixed with a three-dimensional support matrix and introduced into the culture chamber 106 through the second inlet 102, so that the brain organoid is located in the culture chamber 106 and confined to the culture area corresponding to the microelectrode array 301 by the limiting structure 107. Subsequently, culture medium is continuously supplied through the first inlet 101. The culture medium enters the culture chamber 106 through the microfluidic channel 109, exchanges nutrients, metabolites and secretions with the brain organoid, and then flows to the detection channel 108 and the detection area 105 through the second outlet 104.

[0045] During the brain organoid maturation culture stage, low-frequency biphasic square wave pulses are applied to the brain organoids within the culture chamber 106 via a microelectrode array 301 to promote the maturation of the brain organoid neural network. As a specific implementation, regular low-frequency electrical stimulation is applied starting from the 7th day of culture. The pulse width of the low-frequency electrical stimulation is 2 ms, the frequency is 1 Hz, and the intensity is 0.2 mA, with stimulation lasting for 2 hours daily. This stimulation is alternately output by multiple microelectrode units 303 located in the central region of the culture chamber 106. During stimulation, the microelectrode array 301 synchronously acquires local field potential signals from the brain organoids to monitor their response to the low-frequency electrical stimulation.

[0046] During the developmental monitoring phase, starting from day 10 of culture, spontaneous electrical activity signals from brain organoids were periodically collected using a microelectrode array 301. As a specific implementation, continuous spontaneous field potential recordings were performed weekly, with a sampling rate set to 20 kHz and a bandwidth filtering range of 0.1 Hz to 3000 Hz. Acquisition indicators included one or more of the following: spontaneous discharge frequency, oscillatory wave occurrence, and network burst activity, to assess the establishment process of brain organoid electrophysiological function.

[0047] After the brain organoids reach a predetermined maturation state, baseline electrophysiological recording is performed. The microfluidic chip is placed in a constant temperature environment, the electrode layer 300 is connected to a multi-channel electrophysiological recording system, and the spontaneous field potential activity of the brain organoids is continuously recorded using the microelectrode array 301 as baseline data for subsequent stimulus response analysis.

[0048] During the epileptiform electrical activity induction phase, high-frequency electrical stimulation is applied to the brain organoid via a microelectrode array 301 to induce abnormal neural activity. In one specific implementation, the high-frequency electrical stimulation is a biphasic square wave pulse with a pulse width of 1 ms, a frequency of 50 Hz, an intensity of 0.5 mA, and a duration of 10 s, synchronously output by multiple microelectrode units 303 located in the central region of the culture chamber 106. After the high-frequency electrical stimulation ends, the microelectrode array 301 switches from the electrical stimulation state to the electrical signal acquisition state to continuously acquire the electrophysiological response signals following the stimulation.

[0049] During long-term electrophysiological monitoring, the field potential signal after stimulation is continuously or intermittently acquired using a microelectrode array 301. As a specific implementation, continuous monitoring is performed for 48 hours after high-frequency electrical stimulation, and electrophysiological data is saved at predetermined intervals. This electrophysiological data is used to extract one or more of the following indicators: cluster discharge frequency, spike discharge rate, network synchronicity index, duration of epileptiform discharges, and amplitude of epileptiform discharges.

[0050] During the protein detection stage, the culture medium flowing out of the culture chamber 106 carries brain organoid secretion products into the detection channel 108 and contacts the protein detection component in the detection area 105. In this embodiment, the protein detection component is used to detect neuron-specific enolase, which is a target protein associated with neuronal injury. The detection results are obtained by colorimetry, specifically by qualitatively or semi-quantitatively interpreting the neuron-specific enolase based on the presence or absence of color development of the detection line in the protein detection component, the color intensity, or the correspondence with a preset colorimetric standard.

[0051] Furthermore, a time correlation analysis was performed on the electrophysiological signal data after high-frequency electrical stimulation and the neuron-specific enolase colorimetric interpretation results obtained from the detection area 105. This time correlation analysis included mapping the spike discharge rate, network synchronicity index, epileptiform discharge duration or amplitude to the color change or colorimetric grade of the detection line in the protein detection component, in order to assess the relationship between changes in neural activity in brain organoids under epileptiform electrical activity and changes in neuron-specific enolase release.

[0052] It should be noted that this embodiment is used to illustrate that the microfluidic chip of this application can support brain organoid maturation culture, low-frequency electrical stimulation to promote maturation, high-frequency electrical stimulation to induce abnormal electrical activity, electrophysiological signal acquisition, and target protein detection within the same chip. The above-mentioned electrical stimulation parameters, electrical signal acquisition parameters, and detection indicators are specific implementation methods and do not imply that the results of related disease models are necessarily directly generated by the chip structure itself, nor do they constitute a limitation on the scope of protection of this invention.

[0053] Example 2: Alzheimer's Disease-Related Pathological Simulation and Multimodal Detection In another specific implementation, this embodiment adopts the following... Figures 1 to 5 The microfluidic chip shown is used for brain organoid culture and multimodal monitoring to simulate and monitor functional changes in brain organoids under Alzheimer's disease-related pathological conditions in vitro. This embodiment is used to illustrate one application of the microfluidic chip in in vitro research on neurodegenerative diseases and does not imply that related pathological changes are necessarily directly generated by the chip structure itself.

[0054] In this embodiment, the microfluidic chip includes a culture layer 100, an electrical coupling layer 200, and an electrode layer 300 that are stacked and interconnected in sequence. The culture layer 100 is provided with a culture chamber 106, a limiting structure 107, a microfluidic channel 109, a detection channel 108, and a detection area 105; the electrical coupling layer 200 is provided with electrical communication holes 201 corresponding to the culture chamber 106 and the microelectrode array 301; the electrode layer 300 is provided with a microelectrode array 301, a signal interface area 302, and multiple microelectrode units 303.

[0055] During chip fabrication, the culture layer 100 is made of a biocompatible polymer material, and a limiting structure 107 is formed around the culture chamber 106. The slits, channels, or passages of the limiting structure 107 have characteristic dimensions smaller than the size of the brain organoid, which are used to restrict the brain organoid from migrating out of the culture chamber 106 with the fluid flow under perfusion conditions, while allowing the culture medium, nutrients, metabolites, and secretions to pass through. The detection channel 108 is in fluid communication with the culture chamber 106 and extends to the detection area 105. A protein detection component is disposed in the detection area 105. The protein detection component can be a dual immunochromatographic detection element, an antibody-functionalized detection membrane, or other structures suitable for the detection of target proteins, for detecting Alzheimer's disease-related biomarkers in the culture medium.

[0056] An electrical coupling layer 200 is disposed between the culture layer 100 and the electrode layer 300, and multiple electrical connection holes 201 are formed thereon. The electrical connection holes 201 are vertically aligned with the culture chamber 106 and the microelectrode array 301. Conductive hydrogel, conductive medium, or metallized structures are disposed within the electrical connection holes 201 to form an electrical action path between the microelectrode array 301 and the culture chamber 106. Multiple microelectrode units 303 in the electrode layer 300 are connected to the signal interface region 302 to achieve electrophysiological signal acquisition and / or electrical stimulation.

[0057] During chip assembly, the culture layer 100, electrical coupling layer 200, and electrode layer 300 are sequentially aligned and bonded, ensuring that the culture chamber 106, electrical communication via 201, and microelectrode array 301 correspond vertically to each other. After assembly, the fluid connectivity between the microfluidic channel 109, culture chamber 106, detection channel 108, and detection area 105 is checked by perfusing culture medium, and the electrical interaction path between the microelectrode array 301 and the culture chamber 106 is confirmed by electrical signal testing.

[0058] In practical applications, the brain organoids are mixed with a three-dimensional support matrix and introduced into the culture chamber 106 through the second inlet 102, so that the brain organoids are located in the culture chamber 106 and a fixed culture structure is formed by the three-dimensional support matrix. Subsequently, culture medium is continuously supplied to the culture chamber 106 through the first inlet 101. The culture medium enters the culture chamber 106 through the microfluidic channel 109, exchanges nutrients, metabolites and secretions with the brain organoids, and then flows to the detection channel 108 and the detection area 105.

[0059] While employing the same microfluidic chip structure, the core difference between this embodiment and Embodiment 1 lies in the electrical signal scheme, specifically in the electrical stimulation frequency, stimulation intensity, and subsequent electrical signal acquisition method. Embodiment 1 uses a 1Hz, 0.2mA low-frequency biphasic square wave pulse for maturation stimulation and a 50Hz, 0.5mA high-frequency electrical stimulation to induce epileptiform electrical activity; this embodiment, however, uses a 0.5Hz, 100mV to 200mV low-frequency, low-amplitude biphasic balanced pulse for maturation stimulation and performs long-term electrical signal detection and collection during Alzheimer's disease-related pathological simulations.

[0060] In one specific implementation, the brain organoids are selected from early-stage brain organoids differentiated from human induced pluripotent stem cells, which have weak spontaneous electrical activity and immature neural networks. After the brain organoids are loaded into the culture chamber 106, they are first perfused with culture medium at a constant low flow rate to allow the brain organoids to adapt to the on-chip culture environment.

[0061] During the electrical stimulation maturation phase, low-frequency, low-amplitude biphasic balanced pulses are applied to the brain organoid via a microelectrode array 301. In one specific implementation, the low-frequency, low-amplitude biphasic balanced pulses have a frequency of 0.5 Hz and an amplitude of 100 mV to 200 mV, applied for 2 hours daily, with a 10-hour interval before re-application. This electrical stimulation process is output through multiple microelectrode units 303 to simulate the rhythmic electrical activity environment of early neural networks. During stimulation, the microelectrode array 301 collects spontaneous electrophysiological activity signals from the brain organoid at fixed times daily, with each collection session lasting 30 minutes, to obtain electrical activity data during the transition from an immature to a mature state.

[0062] During the maturity monitoring phase, the collected electrophysiological signals were used to extract one or more indicators, including average peak firing rate, network burst activity, and functional connectivity matrix complexity. The temporal changes of these indicators were used to assess the neural network maturation process of brain organoids under low-frequency, low-amplitude electrical stimulation. When network burst activity stabilized or functional connectivity indicators reached predetermined conditions, the electrical stimulation program was stopped, and normal culture medium was continued to be perfused to record baseline electrophysiological activity and biochemical background information prior to pathological simulation.

[0063] During the pathological simulation phase, pathological inducers related to Alzheimer's disease are added to the culture medium to create neurodegenerative pathological simulation conditions. The pathological inducers enter the culture chamber 106 with the culture medium and act on the brain organoids. The secretory products generated during the culture process enter the detection channel 108 and the detection area 105 with the outflowing culture medium.

[0064] During pathological simulation, the microelectrode array 301 acquires electrophysiological signals from brain organoids using long-term monitoring. In one specific implementation, the sampling rate of the electrode layer 300 is set to 30 kHz. The electrophysiological data is used for spike signal detection, spike sorting, average spike firing rate calculation, network burst activity statistics, and functional connectivity analysis.

[0065] During the biochemical detection stage, the culture medium flowing out of the culture chamber 106 enters the detection channel 108 and contacts the protein detection component in the detection area 105. In this embodiment, the protein detection component is used to detect at least one of interleukin-6 and phosphorylated Tau protein. Interleukin-6 is used to characterize neuroinflammatory-related responses, and phosphorylated Tau protein is used to characterize Alzheimer's disease-related pathological responses. The detection results are obtained by colorimetry, specifically by qualitative or semi-quantitative interpretation of the target protein based on the presence or absence of color development of the detection line in the protein detection component, the color intensity, or the correspondence with a preset colorimetric standard.

[0066] Furthermore, a time-correlation analysis was performed on the long-term electrophysiological signal data acquired by the microelectrode array 301 during the pathological simulation and the detection results of interleukin-6 and / or phosphorylated Tau protein obtained from the detection region 105. This time-correlation analysis included mapping the average peak firing rate, network burst activity rate, functional connectivity indicators, or synchronicity indicators to the target protein detection results to assess the relationship between changes in brain organoid neural network function and biochemical responses under Alzheimer's disease-related pathological conditions.

[0067] Therefore, this embodiment demonstrates that the microfluidic chip of this application, through the synergistic cooperation of the culture layer 100, the electrical coupling layer 200, and the electrode layer 300, achieves electrostimulation maturation culture of brain organoids, Alzheimer's disease-related pathological simulation, long-term electrophysiological signal acquisition, network function index analysis, and target protein detection within the same chip. Compared with the aforementioned epileptiform electrical activity monitoring embodiment, the electrical signal scheme of this embodiment focuses on long-term maturation promotion at low frequency and low amplitude, long-term electrical signal detection and collection during chronic pathological disturbances, and detection of neuroinflammation and Tau-related proteins, thus demonstrating the multimodal monitoring capability of the microfluidic chip of this application suitable for in vitro model research of different neurological diseases.

[0068] In summary, the embodiments disclosed herein have at least the following technical effects: This invention provides a culture chamber 106 with physical restraint function within the culture layer 100, and uses a restraint structure 107 to fix brain organoids within the culture area, preventing displacement or loss of organoids under fluid perfusion conditions without relying on an external force field. Simultaneously, the restraint structure 107 allows free diffusion and exchange of culture medium, nutrients, and metabolites, effectively alleviating the problems of insufficient nutrition and easy central necrosis in the core region of organoids under traditional static culture conditions, thereby supporting the long-term stable culture and functional maturation of brain organoids. This invention integrates a microelectrode array 301, an electrical coupling layer 200, and a detection channel 108 into a single microfluidic chip, enabling the synchronous acquisition of electrophysiological signals and target protein information generated during the culture of the same brain organoid without interrupting the culture process. Compared to existing technologies where electrophysiological and protein detection must be performed step-by-step and offline, this invention avoids information loss due to sample transfer and time lag, significantly improving the ability to analyze the dynamic relationship between neural activity and biochemical responses. In the microelectrode array 301 of this invention, each microelectrode unit 303 can switch between an electrical signal acquisition state and an electrical stimulation state, allowing the same electrode to be used for both high spatiotemporal resolution electrophysiological recording and the application of programmable electrical stimulation intervention. This structural design enables electrical stimulation modulation to be implemented simultaneously with monitoring neural activity in brain organoids, which is beneficial for studying the effects of electrical stimulation on neural network development, plasticity changes, and abnormal discharge behavior. This invention incorporates a protein detection component within the detection channel 108 that directly contacts the culture medium, enabling in-situ detection of the target protein while the culture medium flows within the chip, eliminating the need for frequent sampling or offline analysis. This approach reduces human error and minimizes interference with the culture system, making it particularly suitable for simultaneous monitoring of long-term electrophysiological recordings and the cumulative release of protein factors. This invention enables parallel culture and multimodal monitoring of multiple brain organoids by setting up multiple independent culture chambers 106 in the culture layer 100, and configuring an independent microelectrode array 301 and detection channel 108 in each culture chamber. This structure not only improves experimental throughput, but also facilitates inter-group comparison and statistical analysis under the same experimental conditions, enhancing the reliability and reproducibility of experimental results. Based on the aforementioned advantages of structural and functional integration, this invention enables the cultivation of brain organoids, electrical stimulation intervention, electrophysiological monitoring, and detection of disease-related proteins within a single platform. It is applicable to the construction of in vitro models and mechanistic studies of various neurological diseases, including epilepsy and Alzheimer's disease. Furthermore, by performing time-correlation analysis on electrophysiological signals and biochemical indicators, reliable data support can be provided for evaluating the efficacy of physical stimulation or other interventions.

[0069] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A microfluidic chip for brain organoid culture and multimodal monitoring, characterized in that, It includes a culture layer, an electrical coupling layer, and an electrode layer that are stacked and interconnected in sequence; The culture layer is provided with at least one culture chamber for accommodating brain organoids, a microfluidic channel communicating with the culture chamber, and a detection channel in fluid communication with the culture chamber; The culture chamber is connected to the microfluidic channel through a limiting structure, which is used to spatially fix the brain organoid while allowing the culture medium and brain organoid secretion products to pass through. The detection channel is used to guide the culture medium carrying the secreted products of the brain organoid to the detection area in order to obtain the biochemical information produced by the brain organoid during the culture process. The electrode layer is provided with a microelectrode array corresponding to the culture chamber. The microelectrode array is used to collect electrical signals from the brain organoid and / or to generate electrical stimulation to the brain organoid. The electrical coupling layer is disposed between the culture layer and the electrode layer, and forms an electrical interaction path between the microelectrode array and the culture chamber.

2. The microfluidic chip for brain organoid culture and multimodal monitoring according to claim 1, characterized in that, The limiting structure includes multiple slits, channels, or passages with feature dimensions smaller than the size of the brain organoid, to prevent the brain organoid from shifting under fluid action.

3. The microfluidic chip for brain organoid culture and multimodal monitoring according to claim 1, characterized in that, The detection channel and the culture chamber maintain continuous fluid communication during the same culture process to guide the flow of the culture medium carrying the secretory products of the brain organoids within the microfluidic chip.

4. The microfluidic chip for brain organoid culture and multimodal monitoring according to claim 3, characterized in that, A protein detection component for contacting the culture medium is provided in the detection area corresponding to the detection channel. The protein detection component is configured to perform in-situ detection of the target protein within the microfluidic chip.

5. The microfluidic chip for brain organoid culture and multimodal monitoring according to claim 1, characterized in that, Each microelectrode unit in the microelectrode array is configured to switch between an electrical signal acquisition state and an electrical stimulation state for electrophysiological recording and electrical stimulation modulation of the brain organoid.

6. The microfluidic chip for brain organoid culture and multimodal monitoring according to any one of claims 1 to 5, characterized in that, The culture layer is provided with multiple independent culture chambers, each of which is provided with an independent microelectrode array and detection channel to realize the parallel culture and functional monitoring of multiple brain organoids.

7. A microfluidic method for brain organoid culture and multimodal monitoring, characterized in that, The method is implemented using a microfluidic chip for brain organoid culture and multimodal monitoring according to any one of claims 1 to 6, and includes the following steps: S1: The brain organoid and the three-dimensional support matrix are placed into the culture chamber to form a fixed culture structure; S2: Supply culture medium to the culture chamber through the microfluidic channel, and maintain the spatial position of the brain organoid in the culture chamber through the limiting structure; S3: The microelectrode array is used to collect the electrophysiological signals generated by the brain organoid, and the outflowing culture medium is guided to the detection area corresponding to the detection channel to obtain the target protein information secreted by the brain organoid.

8. The microfluidic method for brain organoid culture and multimodal monitoring according to claim 7, characterized in that, In step S3, during the acquisition of the electrophysiological signals, electrical stimulation with preset parameters is applied to the brain organoid through the microelectrode array to regulate or induce its neural activity state.

9. The microfluidic method for brain organoid culture and multimodal monitoring according to claim 7, characterized in that, The target proteins include biomarkers associated with neurological functional status, neurological injury, or neurological disease.

10. The microfluidic method for brain organoid culture and multimodal monitoring according to claim 7, characterized in that, The microfluidic method performs time correlation analysis on the collected electrophysiological signal data and the detection results of the target protein to assess the functional changes of the brain organoids under stimulation or pathological conditions.

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