A neurovascularized organoid chip for high-throughput drug screening

Abstract

This application discloses a neuromorphic organoid chip for high-throughput drug screening, belonging to the fields of microfluidic chips and drug screening technology. The chip includes a chip substrate and packaging module, a neuron culture module, an organoid culture module, a unidirectional trend connection module, a high-throughput drug delivery and distribution module, and a detection and sampling module. By integrating the neuron culture module, organoid culture module, unidirectional trend connection module with directional bias characteristics, and high-throughput drug delivery and distribution module on the same chip substrate, this invention achieves partitioned culture and directed connection of iPSC-induced neurons and three-dimensional tissues such as tumor organoids, supporting the construction of neuromorphic tumor models and high-throughput drug screening. This invention can guide neurites to preferentially extend towards the organoid side and inhibit the reverse migration of cells on the organoid side, offering advantages such as clear model boundaries, high throughput, and suitability for parallel drug testing and mechanism research.

Description

Technical Field

[0001] This invention relates to the fields of microfluidic chips, organoid chips, neural engineering, and drug screening technologies, and specifically to a neuralized organoid chip for high-throughput drug screening. Background Technology

[0002] Complex biological problems such as neurological diseases and neuro-tumor interactions often involve dynamic communication, directed migration, and differences in drug response between different types of cells and tissues. While existing two-dimensional cell culture systems are easy to operate, they struggle to simulate the spatial structure, cell polarity, and microenvironment gradients within the real body. Conventional organoid culture systems, although able to maintain certain three-dimensional structural features, typically lack controllable, stable, and reproducible interfacial connections with neural tissue, making it difficult to elucidate key mechanisms such as the formation of neuralized tissue, neurite invasion, or tumor microenvironment remodeling. Especially in drug screening, traditional culture systems suffer from limited throughput, large batch-to-batch variability, analytical difficulties, and uneven drug distribution, failing to meet the dual demands of mechanistic research and precise drug screening.

[0003] In recent years, microfluidic organoid-on-a-chip technology has provided a new approach for constructing complex tissue models. Microfluidic chips can achieve tissue culture conditions that more closely resemble the in vivo microenvironment through precise control of fluid shearing, material exchange, cell partitioning, and spatiotemporal gradient distribution. For neuron culture, it is necessary to obtain highly viable and directionally extendable neural networks within specific regions; for the culture of three-dimensional tissues such as tumor organoids, stable microstructures are needed to support cell aggregation, self-organization into spheres, and morphological maintenance during long-term culture. If these two aspects can be integrated into a single chip and a biased unidirectional migration pathway can be established, it is hoped that a neural tissue research platform with both structural controllability and functional analyzability can be constructed.

[0004] However, existing co-culture chips still have the following shortcomings: First, the neuronal and organoid regions are often connected only by simple straight channels, which cannot effectively restrict the reverse migration of large organoids or tumor cell clumps, resulting in chaotic co-culture boundaries and poor model reproducibility. Second, most chips have limited culture areas, making it difficult to meet the needs of high-throughput screening and to compare multiple drug concentrations, different combinations, or multiple biological replicates in parallel within the same chip. In addition, organoid culture often relies on matrix embedding such as Matrigel, which is complex and can affect the direct contact between cells, neural axons, or drug molecules, and is also not conducive to standardized screening.

[0005] Previous studies have shown that microporous arrays or hierarchically nested microwell structures can support tumor organoid formation under Matrigel-free or low-matrix conditions, improving organoid size consistency and array-based characterization capabilities. Simultaneously, Tesla valves with unidirectional flow resistance, tapered-expansion channels, or asymmetric microstructures can create directional bias without mechanical moving parts, providing geometric constraints for cell migration and tractor-directed extension. Combining these structures with neuronal-directed culture techniques allows for the construction of a high-throughput microfluidic chip with a "neuronal region-unidirectional connectivity region-organoid region," enabling preferential extension of neural synapses to the organoid side while inhibiting the migration of organoid-side cells or large tissues back to the neuronal side. This provides a new tool for studying neuromorphic tumors, neural invasion, drug regulation, and cell-cell interactions.

[0006] Therefore, there is an urgent need to develop a microfluidic chip platform that can support long-term co-culture of iPSC-induced neurons and organoids, and can achieve directional connectivity, high-throughput screening, and standardized analysis through microstructures and fluid structures, in order to make up for the shortcomings of existing technologies in tissue boundary control, parallel drug testing, and simulation of neural microenvironments. Summary of the Invention

[0007] In view of this, the present invention provides a neuralized organoid on-chip for high-throughput drug screening, which aims to solve the problems of poor tissue boundary control, low throughput, lack of directional connectivity and difficulty in standardizing parallel screening in the prior art.

[0008] This invention provides a neuralized organoid microarray for high-throughput drug screening, comprising: The chip substrate and packaging module include a chip substrate unit and a capping packaging unit; the chip substrate unit is used to carry a neuron culture module, an organoid culture module, a unidirectional trend connection module, and a high-throughput drug delivery and distribution module; the capping packaging unit is disposed above the chip substrate unit and sealed together with the chip substrate unit to form a closed or semi-closed microfluidic network. The neuron culture module includes a cell seeding unit, an adherent culture unit, and a directional growth unit; the adherent culture unit is used to form a planar or shallow cavity structure suitable for neuronal cell adhesion and long-term culture; the directional growth unit is disposed on the side of the adherent culture unit near the unidirectional trend connection module, and is used to guide neuronal processes to extend along a predetermined direction. An organoid culture module is disposed on the chip substrate unit and spaced apart from the neuron culture module; the organoid culture module includes an organoid seeding unit and a microwell array unit; the microwell array unit is composed of multiple microwells arranged in an array, used for confined culture of three-dimensional organoids; A unidirectional trend connection module, disposed between the neuron culture module and the organoid culture module, is used to establish a directionally biased material exchange and neural extension pathway between the two modules. This allows signals from the neuron culture module to be preferentially transmitted to the organoid culture module, while reducing the probability of cells or tissues from the organoid culture module migrating backwards to the neuron culture module. The unidirectional trend connection module includes at least one connection channel, which is fluidly connected to both the neuron culture module and the organoid culture module. A directional bias unit is disposed within the connection channel. This unit prioritizes the extension of neural processes from the neuron culture module to the organoid culture module and inhibits the backward migration of cells or tissues from the organoid culture module to the neuron culture module. A high-throughput drug delivery and distribution module, including a common injection unit and a flow equalization distribution unit, is disposed on the chip substrate unit; the common injection unit includes at least one common injection port for introducing drug solution into the chip; the flow equalization distribution unit is composed of a branched flow equalization network or a resistance-matching channel for distributing input liquid in parallel to the neuron culture module and / or the organoid culture module; The detection and sampling module includes an imaging observation unit, which reserves a transparent window or a low autofluorescence observation area in the corresponding region of the chip for real-time observation of the cell or tissue state in the neuron culture module, the unidirectional trend connection module and the organoid culture module.

[0009] Compared with the prior art, the present invention has the following beneficial effects: First, this invention spatially separates the neuronal culture area and the organoid culture area and couples them through unidirectional trend connection channels. While maintaining the culture stability of each type of tissue, it enables neurites to preferentially extend towards the organoid, reducing the risk of reverse contamination of the neuronal area by organoid-side cells and significantly improving model boundary clarity and experimental reproducibility. Second, this invention employs a hierarchical microwell array structure to support the array formation and culture of organoids under Matrigel-free or low-matrix conditions, resulting in more uniform organoid size and more controllable position, facilitating real-time imaging, automated analysis, and parallel comparisons under multiple conditions. Third, this invention integrates a high-throughput distribution and gradient drug delivery network, enabling parallel execution of multiple drug, multiple concentration, and multiple replicate experiments on the same chip or in the same batch of chips, significantly reducing reagent consumption and labor costs, and improving screening efficiency. Furthermore, this chip can be used to study neuromorphic tumor formation, neural invasion, neuro-tumor interaction, drug resistance, and combination therapy mechanisms, and can also be extended to brain organoids, peripheral nerve organoids, and other neuromorphic tissue systems, demonstrating good versatility and scalability. Because the chip is compatible with microscopic imaging, electrical activity recording, and endpoint molecular analysis, this invention is suitable not only as a platform for drug efficacy evaluation but also as a tool for basic mechanism research. Attached Figure Description

[0010] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0011] Figure 1 This is a module relationship diagram of a neuralized organoid chip for high-throughput drug screening according to an embodiment of the present invention; Figure 2 This is a layout diagram of the neuron culture module, organoid culture module, and unidirectional trend connection module in an embodiment of the present invention. Figure 3 This is a flowchart illustrating the use of a neuralized organoid chip for high-throughput drug screening according to an embodiment of the present invention. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] like Figures 1-2 As shown, this embodiment provides a neuromorphic organoid chip for high-throughput drug screening. The chip integrates a neuron culture module, an organoid culture module, a unidirectional trend connection module, and a high-throughput drug delivery and distribution module on the same substrate. It can be used to study the interaction mechanism between iPSC-induced neurons and three-dimensional tissues such as tumor organoids, construct neuromorphic tumor models, and conduct high-throughput screening of anti-tumor drugs, neuromodulatory drugs, and combination therapy strategies.

[0014] The system of this invention consists of six core modules: a chip substrate and packaging module, a neuron culture module, an organoid culture module, a unidirectional trend connection module, a high-throughput drug delivery and distribution module, and a detection and sampling module. These six modules work together organically through a spatial partitioning structure, a microfluidic transport network, directional migration channels, and a detection feedback interface to achieve partitioned culture, directional interaction, high-throughput parallel drug delivery, and joint detection of multiple indicators between neurons and organoids.

[0015] Specifically, a neural organoid on-chip for high-throughput drug screening includes: a chip substrate and a packaging module, including a chip substrate unit and a capping packaging unit; The chip substrate unit is used to carry the neuron culture module, organoid culture module, unidirectional trend connection module, and high-throughput drug delivery and distribution module; the capping unit is disposed above the chip substrate unit and sealed together with the chip substrate unit to form a closed or semi-closed microfluidic network.

[0016] The chip substrate and encapsulation module are used to construct the overall chip support platform, form a closed or semi-closed microfluidic culture environment, and provide stable structural support and biocompatible interfaces for each functional area. The chip substrate unit is prepared using glass, silicon wafers, cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), photocurable resins, SU-8, or other biocompatible materials. It is used to support neuron culture modules, organoid culture modules, unidirectional trend connection modules, and high-throughput drug delivery and distribution modules. This substrate unit can be designed as a rigid, semi-flexible, or flexible structure as needed to balance the requirements of microscopic imaging, processing precision, liquid sealing, and mass production. The capping encapsulation unit is located above the chip substrate and is bonded to the substrate through plasma bonding, thermocompression bonding, UV curing, adhesive bonding, or other sealing methods to form a closed or partially open microfluidic network. This unit is used to limit culture medium evaporation, maintain a sterile environment, stabilize the flow field within the channels, and provide the structural basis for inlet / outlet interfaces, sampling ports, and observation windows.

[0017] In one alternative implementation, the chip substrate and packaging module further include a liquid inlet / outlet interface unit; The inlet and outlet interface units are arranged at the edge or top of the chip and are respectively connected to the neuron culture module, organoid culture module, unidirectional trend connection module and high-throughput drug delivery and distribution module. They are used to introduce cell suspension, culture medium, drug solution, washing solution and functional probe, and to discharge waste liquid or sampling liquid. The inlet / outlet interface unit adopts a standard Luer interface, capillary interface or needle puncture interface.

[0018] The inlet / outlet interface unit is located at the edge or top of the chip and connects to the neuron culture module, organoid culture module, unidirectional trend connection module, and high-throughput drug delivery and dispensing module, respectively. It is used to introduce cell suspension, culture medium, drug solution, washing solution, and functional probes, and to discharge waste or sampling fluid. This unit can use a standard Luer interface, capillary interface, or needle puncture interface to adapt to external pumps, reservoirs, or automated liquid workstations.

[0019] The neuron culture module includes a cell seeding unit, an adherent culture unit, and a directional growth unit. The adherent culture unit is used to form a planar or shallow cavity structure suitable for neuronal cell adhesion and long-term culture. The directional growth unit is located on the side of the adherent culture unit near the unidirectional trend connection module and is used to guide neuronal processes to extend in a predetermined direction.

[0020] The neuron culture module is used to achieve directional attachment, differentiation, maturation, neurite extension, and neural network construction of neurons on one side of the chip, and serves as the signaling endpoint for the neuralized microenvironment. The cell seeding unit receives iPSC-induced neurons, neural progenitor cell differentiation systems, primary neurons, or other cells capable of forming neural networks, and controls the initial cell distribution density and seeding location through local confinement structures, ensuring stable attachment to the predetermined culture area. The adherent culture unit forms planar or shallow cavity structures suitable for neuronal adhesion and long-term culture. The surface can be further modified with poly-L-lysine, laminin, Matrigel-alternating matrix, or other functional modifications to improve neuronal adhesion rate, survival rate, and network formation ability. This unit supports long-term neuronal maintenance and neural network maturation. The directional growth unit includes micropatterned adhesion strips, axonal guiding microgrooves, confinement channels, or other microstructures to guide neuronal cell body distribution and neurite extension along predetermined directions, thereby improving the controllability and reproducibility of the neural network structure and enhancing its projection ability towards the unidirectional trend connection module or organoid culture module.

[0021] In one optional implementation, the neuron culture module further includes an independent perfusion unit; the independent perfusion unit is separately connected to the adherent culture unit and is used to supply culture medium, differentiation-inducing factors, drugs or dyes to the neuron culture module side to achieve local microenvironment regulation.

[0022] Specifically, the independent perfusion unit is separately connected to the neuron culture module, used to independently supply culture medium, differentiation-inducing factors, drugs, or dyes to the neuron side, and to achieve local microenvironment regulation. This unit can reduce the direct interference of the culture conditions on the organoid culture module side to the neuron culture module side, maintaining the relative independence of the culture conditions in the two regions.

[0023] In one optional embodiment, the cavity height of the adherent culture unit is 50-300 μm; the surface of the adherent culture unit is modified with a poly-L-lysine layer, a laminin layer, a Matrigel substitute matrix layer, or a composite functionalized coating thereof; the directional growth unit includes micropatterned adhesion strips, axonal guiding microgrooves, or confined channels for guiding the distribution of neuronal cells and the extension of neurites along a predetermined direction.

[0024] Specifically, in the neuron culture module, the adherent culture unit can employ a shallow culture cavity, an array of culture islands, or a long, narrow cavity with microgrooves. The cavity height is preferably 50-300 μm to balance nutrient exchange and microscopic imaging clarity. The bottom can be treated with polylysine, laminin, Matrigel dilution coating, peptide modification, or dopamine interface activation to enhance the adhesion, differentiation, and neurite extension capabilities of iPSC-induced neurons. Parallel microgrooves, radial guide ridges, or locally patterned adhesions can be placed on the side of the neuron culture module near the unidirectional trend connection module to direct neurites towards the connection channel, enhancing cross-regional connection efficiency. The neuron culture module has separate inlets, outlets, and buffer cavities for perfusion, medium exchange, and drug treatment of neuron-specific culture medium, avoiding interference with the culture requirements of the organoid culture module.

[0025] The organoid culture module is set on the chip substrate unit and spaced apart from the neuron culture module; the organoid culture module includes an organoid seeding unit and a microwell array unit; the microwell array unit is composed of multiple microwells arranged in an array, used for confined culture of three-dimensional organoids.

[0026] The organoid culture module is used on the other side of the chip to achieve high-throughput formation, stable confinement, parallel culture, and screening of organoids or three-dimensional cell clusters, and serves as a receptor or response end for neural interactions and drug evaluation. The organoid seeding unit receives cell suspensions related to tumor organoids, brain organoids, patient-derived organoids, or other three-dimensional cell clusters, and uses local sedimentation, confined aggregation, or low-shear perfusion to allow cells to enter pre-defined microstructures to form three-dimensional tissues. The microwell array unit, located inside the organoid culture module, includes micropore arrays, microwell arrays, or hierarchically nested microwell array structures. This structure enables automatic cell aggregation, homogenization into clusters, and spatially confined culture through recessed cavities of different sizes, resulting in organoids with good consistency in size, morphology, and distribution.

[0027] In one optional implementation, the organoid culture module further includes a confined culture unit and a parallel screening unit; the confined culture unit is used to maintain the organoid within a predetermined microwell during perfusion culture or fluid exchange by means of microwell aspect ratio design, peripheral confining edges, low shear flow field control or local support structure; the parallel screening unit consists of multiple repeatedly set organoid culture microunits, used to realize simultaneous control experiments with multiple drugs, different doses or different treatment conditions.

[0028] Specifically, the confined culture unit is used to maintain organoids within predetermined microwells during perfusion culture or medium exchange, reducing the probability of tissue drift, fusion, or loss. This unit can improve organoid culture stability through microwell aspect ratio design, peripheral confining edges, low-shear flow field control, or local support structures. The microwells preferably employ a layered structure with a larger upper opening and a smaller lower confinement to facilitate cell sedimentation and aggregation, organoid fixation, and long-term spheroidization culture. This region can support Matrigel-free culture, low-concentration hydrogel-assisted culture, or surface-confined culture. After organoid formation, it can be partially exposed to the culture medium to allow direct contact with drugs, neural processes, immune cells, or detection probes. The microwell diameter is preferably 50-1000 μm, and the depth is preferably 50-1200 μm; microwell arrays of different scales can be set on the same chip to culture organoids of different sizes or at different maturity stages. The bottom of the microwell can be equipped with micropores, mesh supports, or flexible limiting shoulders to prevent organoid drift; simultaneously, organoid recovery and subsequent molecular analysis can be achieved through backflushing, local aspiration, or a removable cap. The parallel screening unit consists of multiple repeatedly configured organoid culture microunits, which can be linked with the equalization distribution unit to achieve simultaneous control experiments with multiple drugs, different doses, or different treatment conditions, thereby meeting the needs of high-throughput screening and statistical analysis.

[0029] A unidirectional trend connection module, positioned between the neuron culture module and the organoid culture module, establishes a directionally biased pathway for material exchange and neural extension between them. This prioritizes the transmission of signals from the neuron culture module to the organoid culture module while reducing the probability of reverse migration of cells or tissues from the organoid culture module to the neuron culture module. The unidirectional trend connection module includes at least one connection channel, which is fluidly connected to both the neuron culture module and the organoid culture module. A directional bias unit is located within the connection channel. This unit prioritizes the extension of neural processes from the neuron culture module to the organoid culture module and inhibits the reverse migration of cells or tissues from the organoid culture module to the neuron culture module.

[0030] The unidirectional trend connection module establishes a directional biased pathway for material exchange and neurite extension between the neuron culture module and the organoid culture module. This prioritizes signal transmission from the neuron culture module to the organoid culture module, while reducing the probability of reverse migration of cells or tissues from the organoid culture module to the neuron culture module. The connection channel, located between the neuron and organoid culture modules, consists of several parallel, series, or grid-arranged microchannels used to establish local microenvironment coupling between the two regions. The channel size can be optimized based on neurite extension requirements and organoid cell size characteristics. The directional bias unit, located within the connection channel, includes Tesla valve units, asymmetric expansion / contraction cavities, micropillar arrays, unidirectional ratchet structures, slit-expansion combinations, or combinations thereof. This unit alters the flow resistance distribution, migration path geometry, and local spatial constraints to ensure a preferential trend of fluid exchange, soluble factor diffusion, and neurite extension from the neuron culture module to the organoid culture module. By utilizing geometric asymmetry to generate different pressure drops when fluid flows in the forward and reverse directions, and combining this with differences in channel width, height, bending radius, and micropillar arrangement, directional selectivity is achieved at the level of cell movement and tissue migration. Relatively slender and flexible neural processes can extend through narrow channels toward the organoid culture module, while larger organoid cell clusters or tumor cell clusters find it more difficult to reverse their passage back to the neuron culture module. The width of the connecting channel is preferably 3-50 μm, the height is preferably 3-30 μm, and the length is preferably 50-5000 μm; segmented widths and gradient cross-sections can also be incorporated within the connecting channel to further regulate migration selectivity.

[0031] In one optional implementation, the unidirectional trend connection module further includes a neural projection guidance unit and a reverse migration inhibition unit; the neural projection guidance unit provides a preferred path for the cross-regional extension of axons or synapse-like structures through a narrow confinement structure, a gradually widened channel, or a surface adhesion regulation layer; the reverse migration inhibition unit increases the difficulty for cells or tissues on the organoid culture module side to migrate to the neuron culture module side through geometric sieving, asymmetric blockade, local width restriction, or flow field bias.

[0032] The neural projection guidance unit provides preferential pathways for the cross-regional extension of axons or synapse-like structures through narrow confinement structures, gradually widened channels, or surface adhesion regulation layers. This enables neurons to form directional connections towards the organoid culture module, simulating projection, invasion, regulation, or pathological coupling processes between neuralized tissues. The reverse migration inhibition unit increases the difficulty for cells, large granular tissue masses, or high-density cell clusters from the organoid culture module to migrate towards the neuron culture module. Through geometric sieving, asymmetric blockade, local width restriction, or flow field bias, it reduces the probability of reverse cross-regional migration without significantly affecting the passage of neurites. The unidirectional trend connection module can also be loaded with ECM proteins, chemokines, anti-adhesion coatings, or degradable barrier layers to enhance directional neurite elongation, restrict the crossing of non-target cells, and simulate the boundaries of neuralized tissues.

[0033] The high-throughput drug delivery and distribution module includes a common injection unit and a flow equalization distribution unit, which are disposed on the chip substrate unit. The common injection unit includes at least one common injection port for introducing the drug solution into the chip. The flow equalization distribution unit is composed of a branch flow equalization network or a resistance matching channel for distributing the input liquid in parallel to the neuron culture module and / or the organoid culture module.

[0034] Specifically, the high-throughput drug delivery and distribution module is used to distribute candidate drugs, probes, stimulating factors, or processing solutions to the neuron culture module and / or organoid culture module in a preset manner, enabling parallel execution of multiple experiments on the same chip and controlled comparison under controlled conditions. The common injection unit includes at least one common injection port for introducing candidate drugs, culture replenishment medium, fluorescent probes, immunoassay reagents, or processing solutions into the chip, and is connected to the subsequent flow equalization network and branch channels. The flow equalization and distribution unit, composed of a branch flow equalization network, resistance-matching channels, or a dendritic flow distribution structure, is used to distribute the input liquid to multiple parallel screening units at a relatively uniform flow rate, reducing experimental bias caused by inconsistent flow rates between different parallel screening units.

[0035] In one optional embodiment, the high-throughput drug delivery and distribution module further includes a concentration gradient generation unit and a rinsing and switching unit; the concentration gradient generation unit mixes different concentrations of drugs or factors in a preset ratio and outputs them to multiple culture sites through a serpentine mixing channel, a bifurcation confluence structure or a laminar flow diffusion mixing network; the rinsing and switching unit includes a bypass rinsing channel, a valve-controlled branch or a buffer inlet, used to achieve channel cleaning, condition switching and residual liquid removal before and after drug delivery.

[0036] Specifically, the concentration gradient generation unit uses a serpentine mixing channel, a bifurcation confluence structure, a laminar diffusion mixing network, or other microfluidic gradient generation structures to mix different concentrations of drugs or factors in a preset ratio and output them to multiple culture sites for dose-dependent testing and efficacy window screening. The rinsing and switching unit includes bypass rinsing channels, valve-controlled branches, or buffer inlets to clean channels, switch conditions, and remove residual liquid before and after drug administration, thereby reducing cross-contamination and improving the reusability of multi-round screening experiments.

[0037] In one optional implementation, the concentration gradient generating unit is a Christmas tree-shaped gradient network or a serpentine mixing channel network, used to mix at least two drug solutions in a preset ratio to form multiple drug output terminals with different concentrations, each drug output terminal being fluidly connected to a neuron culture module or organoid culture module of a parallel screening unit.

[0038] Specifically, the chip can contain 2, 4, 8, 16, 24, 48, or more parallel screening units. Each unit includes a neuron culture module, a connective culture module, and an organoid culture module to meet the requirements of drug screening and statistical reproducibility. A tree-like shunt, serpentine mixing channel, or Christmas tree-shaped gradient network is configured upstream to form different drug concentrations, combination ratios, or time-series processing schemes. Each unit can achieve independent liquid supply via valves, pluggable interfaces, external peristaltic pumps, syringe pumps, or gravity-driven systems, or it can achieve low-cost, high-throughput operation through a common main channel and bypass flow-limiting structures. This module is compatible with multiple endpoint readings, including cell viability analysis, neurite length quantification, organoid volume and morphology analysis, drug penetration evaluation, cytotoxicity, immune killing, and co-culture interface fluorescence tracing.

[0039] The detection and sampling module includes an imaging observation unit. The imaging observation unit reserves a transparent window or a low autofluorescence observation area in the corresponding area of ​​the chip for real-time observation of the cell or tissue state in the neuron culture module, the unidirectional trend connection module, and the organoid culture module.

[0040] Specifically, the detection and sampling module is used for real-time observation, quantitative detection, and sample extraction of the neuron-organoid interaction process within the chip, thereby supporting mechanism research and drug efficacy evaluation. The imaging observation unit reserves a transparent window or low autofluorescence observation area in the corresponding region of the chip for bright-field, fluorescence, confocal, or live-cell imaging, and records in real time the extension of neurites, changes in organoid morphology, cell migration, network connection establishment, and dynamic responses after drug treatment.

[0041] In one optional implementation, the detection and sampling module further includes a sampling interface unit and an electrical / sensing integrated unit; the sampling interface unit is connected to a local culture chamber or channel for extracting supernatant, metabolic fluid, secretory factor samples or local processing fluid; the electrical / sensing integrated unit reserves microelectrode interfaces, impedance detection sites, metabolic sensor sites or other functional integration areas within the chip for monitoring neural network electrical activity, cell activity, metabolic level, barrier state or changes in the local microenvironment.

[0042] The sampling interface unit is connected to a local culture chamber or channel for extracting supernatant, metabolic fluid, secreted factor samples, or local processing solutions for subsequent molecular detection, biochemical analysis, or omics studies. The electrical / sensing integration unit can reserve microelectrode interfaces, impedance detection sites, metabolic sensor sites, or other functional integration areas within the chip to monitor neural network electrical activity, cell activity, metabolic levels, barrier status, or changes in the local microenvironment, thereby providing multi-dimensional evaluation indicators for drug screening.

[0043] In one optional embodiment, the chip substrate unit is made of a biocompatible material; the biocompatible material includes, but is not limited to, glass, silicon wafer, cyclic olefin polymer, cyclic olefin copolymer, polydimethylsiloxane, polymethyl methacrylate, photocurable resin or composite materials thereof; the capping unit is bonded to the chip substrate unit by a sealing method; the sealing method includes, but is not limited to, plasma bonding, thermoforming bonding, UV curing or adhesive bonding; the neuron culture module, organoid culture module and unidirectional trend connection module are formed on the chip substrate unit by soft lithography, injection molding, thermoforming, laser processing, micromilling, 3D printing or mold preparation processes.

[0044] Specifically, the main chip material can be PDMS, COC, COP, PMMA, photocurable resin, SU-8, glass, or their composite materials. Microstructure fabrication can employ soft lithography, injection molding, thermoforming, laser processing, micromilling, digital light processing (DLP) 3D printing, two-photon printing, or mold fabrication processes. For hierarchical microwell structures and complex Tesla valve structures, high-precision 3D printing is preferred for creating a master mold, which is then replicated to form mass-produced chips. The chip surface can be selectively modified according to the needs of different areas to achieve differentiated functions such as promoting neuronal adhesion, organoid culture, and preventing backflow in connective tissue.

[0045] The chip substrate and packaging module constitute the physical carrier and microfluidic environment foundation of the entire device; the neuron culture module and organoid culture module are responsible for neural network construction and three-dimensional tissue culture, respectively; the unidirectional trend connection module establishes directionally biased interaction pathways between two regions; the high-throughput drug delivery and dispensing module enables parallel processing of multiple conditions; and the detection and sampling module is responsible for dynamic observation and quantitative analysis. All modules form a collaborative system through structural partitioning, fluid pathways, directional connections, and signal acquisition interfaces, ultimately realizing a high-throughput microfluidic chip platform for research on neural tissue interaction mechanisms and drug screening.

[0046] like Figure 3 As shown, the specific usage method of the chip is as follows: Chip pretreatment: The chips undergo sterilization, degassing, and surface modification. Neuron culture modules are grafted with adhesion-promoting molecules, while organoid culture modules undergo low-adsorption treatment or localized hydrogel modification according to culture requirements.

[0047] Neuron seeding: iPSC-induced neurons or their precursor cells are seeded into a neuron culture module and cultured under static or low-speed perfusion conditions to allow the cells to attach, differentiate, and gradually form a neural network.

[0048] Organoid construction: Tumor cells, patient-derived cell clusters, tumor tissue digests, or other organoid initiating cells are seeded into the organoid microwell array region, and organoids are formed through gravity sedimentation, self-aggregation, and perfusion culture.

[0049] Establishing cross-regional connections: During continued culture, neural processes extend along guide structures and unidirectional trend connection channels toward the organoid, forming contact with cells on the surface or inside the organoid.

[0050] Drug processing: The high-throughput drug delivery and distribution module inputs candidate drugs, combination drug regimens, immune cells or stimulating factors to other modules, which then act in parallel within a set time.

[0051] Results detection: Bright field, fluorescence, live cell imaging, electrophysiological recording, calcium imaging, endpoint staining or molecular analysis were used to evaluate the degree of neurotransmission, invasive behavior, drug inhibition effect and toxicity.

[0052] Sample recovery: After the experiment, neurons, connective tissue samples or organoids can be selectively recovered for subsequent single-cell sequencing, protein detection, metabolic analysis or pathological sections.

[0053] A preferred embodiment of the present invention is as follows: A high-throughput microfluidic chip with eight parallel screening units was constructed using a glass substrate and a PDMS capping layer. Each unit has a neuron culture module on the left and an organoid culture module on the right, and they are connected in the middle by 8-20 parallel unidirectional trend connection channels.

[0054] The neuron culture module is set up with an adherent culture cavity with a width of 1-5 mm and a cavity height of 100-200 μm. The bottom of the cavity is modified with poly-D-lysine and laminin. A parallel microgroove with a width of 5-20 μm is set on the side near the unidirectional trend connection module to guide iPSC to induce neuronal axon extension.

[0055] The organoid culture module is equipped with multiple hierarchical nested microwells. The upper diameter of each microwell is 300-800 μm, the lower limiting diameter is 100-400 μm, and the depth is 300-1000 μm. These microwells are used to culture tumor organoids derived from patients.

[0056] The central unidirectional trend connection module uses a combination of Tesla valve and slit channel. The narrowest part of the channel is 5-15μm wide and 5-10μm high, which allows neural protrusions to pass through and inhibits the reverse migration of large organoid cell clusters.

[0057] A tree-like concentration gradient network is set up upstream of the chip, which can simultaneously generate multiple drug concentrations and deliver them to each parallel unit.

[0058] In this procedure, mature neural networks are first cultured in the neuronal region, and then tumor cell clusters are seeded into the organoid culture module to form organoids. These organoids are then continuously co-cultured and treated with candidate drugs. Live-cell imaging is used to observe the extension of neurites into organoids, changes in organoid volume, and cell death. Immunofluorescence is combined with the detection of synaptic markers, neural markers, and tumor proliferation / apoptosis markers to evaluate the neurotissue interaction and the effectiveness of drug intervention.

[0059] This implementation method has a clear structure, is easy to operate, and has a high throughput, enabling stable realization of neuron-organoid co-culture, directional connection, and parallel drug screening.

[0060] Although embodiments of the invention have been described in conjunction with the accompanying drawings, those skilled in the art can make various modifications and variations without departing from the spirit and scope of the invention, and such modifications and variations all fall within the scope defined by the appended claims.

Claims

1. A neuralized organoid on-chip for high-throughput drug screening, characterized in that, include: The chip substrate and packaging module include a chip substrate unit and a capping packaging unit; the chip substrate unit is used to carry a neuron culture module, an organoid culture module, a unidirectional trend connection module, and a high-throughput drug delivery and distribution module; the capping packaging unit is disposed above the chip substrate unit and sealed together with the chip substrate unit to form a closed or semi-closed microfluidic network. The neuron culture module includes a cell seeding unit, an adherent culture unit, and a directional growth unit; the adherent culture unit is used to form a planar or shallow cavity structure suitable for neuronal cell adhesion and long-term culture; the directional growth unit is disposed on the side of the adherent culture unit near the unidirectional trend connection module, and is used to guide neuronal processes to extend along a predetermined direction. An organoid culture module is disposed on the chip substrate unit and spaced apart from the neuron culture module; the organoid culture module includes an organoid seeding unit and a microwell array unit; the microwell array unit is composed of multiple microwells arranged in an array, used for confined culture of three-dimensional organoids; A unidirectional trend connection module, disposed between the neuron culture module and the organoid culture module, is used to establish a directionally biased material exchange and neural extension pathway between the two modules. This allows signals from the neuron culture module to be preferentially transmitted to the organoid culture module, while reducing the probability of cells or tissues from the organoid culture module migrating backwards to the neuron culture module. The unidirectional trend connection module includes at least one connection channel, which is fluidly connected to both the neuron culture module and the organoid culture module. A directional bias unit is disposed within the connection channel. This unit prioritizes the extension of neural processes from the neuron culture module to the organoid culture module and inhibits the backward migration of cells or tissues from the organoid culture module to the neuron culture module. A high-throughput drug delivery and distribution module, including a common injection unit and a flow equalization distribution unit, is disposed on the chip substrate unit; the common injection unit includes at least one common injection port for introducing drug solution into the chip; the flow equalization distribution unit is composed of a branched flow equalization network or a resistance-matching channel for distributing input liquid in parallel to the neuron culture module and / or the organoid culture module; The detection and sampling module includes an imaging observation unit, which reserves a transparent window or a low autofluorescence observation area in the corresponding region of the chip for real-time observation of the cell or tissue state in the neuron culture module, the unidirectional trend connection module and the organoid culture module.

2. The neural organoid chip according to claim 1, characterized in that, The chip substrate and packaging module also include a liquid inlet / outlet interface unit; The inlet and outlet interface units are arranged on the edge or top of the chip and are respectively connected to the neuron culture module, organoid culture module, unidirectional trend connection module and high-throughput drug delivery and distribution module. They are used to introduce cell suspension, culture medium, drug solution, washing solution and functional probe, and to discharge waste liquid or sampling liquid. The inlet / outlet interface unit adopts a standard Luer interface, capillary interface, or needle puncture interface.

3. The neural organoid chip according to claim 1, characterized in that, The neuron culture module also includes an independent perfusion unit; The independent perfusion unit is separately connected to the adherent culture unit and is used to supply culture medium, differentiation induction factors, drugs or dyes to the neuron culture module to achieve local microenvironment regulation.

4. The neural organoid chip according to claim 1, characterized in that, The cavity height of the adherent culture unit is 50-300 μm; The surface of the adherent culture unit is modified with a polylysine layer, a laminin layer, a Matrigel substitute matrix layer, or a composite functionalized coating thereof. The directional growth unit includes micropatterned adhesion strips, axonal guiding microgrooves, or confined channels, used to guide the distribution of neuronal cells and the extension of neurites along a predetermined direction.

5. The neural organoid chip according to claim 1, characterized in that, The organoid culture module further includes a confined culture unit and a parallel screening unit; The confined culture unit is designed with a microwell depth-to-width ratio, peripheral confining edges, low-shear flow field control, or local support structure to maintain the organoid within the predetermined microwell during perfusion culture or fluid exchange. The parallel screening unit consists of multiple repeatedly configured organoid culture microunits, used to achieve simultaneous control experiments with multiple drugs, different doses, or different treatment conditions.

6. The neural organoid chip according to claim 1, characterized in that, The unidirectional trend connection module also includes a neural projection guidance unit and a reverse migration inhibition unit; The neural projection guidance unit provides a preferred path for the cross-regional extension of axons or synapse-like structures through a narrow confinement structure, a gradually widened channel, or a surface adhesion regulation layer; the reverse migration inhibition unit increases the difficulty of migration of cells or tissues from the organoid culture module to the neuron culture module through geometric sieving, asymmetric blockade, local width restriction, or flow field bias.

7. The neural organoid chip according to claim 1, characterized in that, The high-throughput drug delivery and distribution module also includes a concentration gradient generation unit and a flushing and switching unit; The concentration gradient generating unit mixes drugs or factors of different concentrations in a preset ratio and outputs them to multiple culture sites through a serpentine mixing channel, a bifurcation confluence structure, or a laminar flow diffusion mixing network. The flushing and switching unit includes a bypass flushing channel, a valve-controlled branch, or a buffer inlet, used to achieve channel cleaning, condition switching, and residual liquid removal before and after drug administration.

8. The neural organoid chip according to claim 7, characterized in that, The concentration gradient generating unit is a Christmas tree-shaped gradient network or a serpentine mixing channel network, used to mix at least two drug solutions in a preset ratio to form multiple drug output terminals with different concentrations. Each drug output terminal is fluidly connected to the neuron culture module or organoid culture module of one of the parallel screening units.

9. The neural organoid chip according to claim 1, characterized in that, The detection and sampling module further includes a sampling interface unit and an electrical / sensing integrated unit; the sampling interface unit is connected to a local culture chamber or channel and is used to extract supernatant, metabolic fluid, secretory factor samples or local processing fluid; the electrical / sensing integrated unit reserves microelectrode interfaces, impedance detection sites, metabolic sensor sites or other functional integration areas within the chip for monitoring neural network electrical activity, cell activity, metabolic level, barrier state or changes in the local microenvironment.

10. The neural organoid chip according to claim 1, characterized in that, The chip substrate unit is made of a biocompatible material; the biocompatible material includes, but is not limited to, glass, silicon wafer, cyclic olefin polymer, cyclic olefin copolymer, polydimethylsiloxane, polymethyl methacrylate, photocurable resin or composite materials thereof; The capping unit is bonded to the chip substrate unit by a sealing method; the sealing method includes, but is not limited to, plasma bonding, thermosetting bonding, UV curing or adhesive bonding. The neuron culture module, the organoid culture module, and the unidirectional trend connection module are formed on the chip substrate unit using soft lithography, injection molding, hot pressing, laser processing, micromilling, 3D printing, or mold preparation processes.

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