A three-chamber microfluidic chip device for in vitro real-time monitoring of neuro-tumor-immune microenvironment and application

Abstract

The application discloses a three-cavity microfluidic chip device and application for nerve-tumor-immune microenvironment in vitro real-time monitoring. The chip device adopts a three-layer structure: the bottom layer integrates microelectrode and conductive polymer to realize nerve electrophysiological regulation; the middle layer is a nanopore membrane, realizing axon penetration and cell physical partition; the top layer is provided with a tumor organoid culture pool integrating lymphatic circulation flow channel, simulating immune cell dynamic chemotaxis. The application can realize three-dimensional penetration and domination of nerve axon to 3D tumor organoid, synchronously complete electric signal stimulation, nerve transmitter sampling and cell behavior real-time monitoring, effectively solve the defects of traditional co-culture model, such as dimension loss, insufficient functional isolation, signal coupling distortion and inability to dynamically monitor, and is suitable for nerve-tumor interaction mechanism research and neurogenic antitumor drug screening, and has the advantages of high-fidelity anatomical simulation, precise electrophysiological regulation and real-time non-destructive dynamic monitoring.

Description

Technical Field

[0001] This application relates to a three-chamber microfluidic chip device and its application for real-time in vitro monitoring of the neural-tumor-immune microenvironment, belonging to the fields of biomedical engineering and microfluidic chip technology. Background Technology

[0002] In the field of tumor microenvironment research, the interaction between the nervous and immune systems (i.e., neuroimmune regulation) has become a key entry point for understanding tumor progression and cancer-related psychological disorders. However, existing in vitro research methods have significant limitations in simulating this complex interactive system. Current mainstream techniques mainly rely on traditional Transwell dishes or two-dimensional (2D) plate co-culture systems. These methods typically involve simply mixing and seeding tumor cells, neurons, and immune cells on the same plane, resulting in a lack of necessary physical partitioning between cells and making it difficult to simulate the unique three-dimensional spatial hierarchy of in vivo tissues. In this environment, neurons cannot form directional axonal growth, and tumor cells often grow in a monolayer adherent manner. This differs significantly from the 3D mass structure of solid tumors in vivo and the three-dimensional innervation of nerve fibers, limiting the realistic observation of the translation process of nerve endings entering the tumor tissue.

[0003] Furthermore, existing technologies lack dynamism and precision in signal modulation, making it difficult to meet the research needs of complex mechanisms. Traditional co-culture protocols mostly employ static culture modes, lacking dynamic modules that simulate in vivo circulatory systems (such as lymphatic and blood circulation), resulting in a significant simplification of the chemotaxis and invasion processes of immune cells towards tumors. More importantly, existing devices generally lack integrated electrophysiological modulation and real-time microenvironment monitoring methods. Researchers typically rely on endpoint detection methods (such as immunofluorescence staining after the experiment) to extrapolate results, failing to capture the instantaneous dynamics of neurotransmitter release by neurons upon electrical stimulation, or to achieve in-situ, continuous sampling of molecular concentration gradients in the microenvironment. This "black box" experimental design obscures the critical time points for the translation of neural signals into immunosuppressive signals, resulting in a lack of high-fidelity experimental data to support the development of intervention strategies targeting systemic pathological processes such as the "liver-brain axis."

[0004] Therefore, developing a microfluidic chip device that enables three-dimensional co-culture of nerve-tumor-immune systems, dynamic electrophysiological regulation, and in-situ real-time monitoring has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] The object of the present invention is to overcome the limitations of existing co-culture models in terms of lack of dimension, insufficient functional isolation, distorted signal coupling, and inability to perform dynamic monitoring; the present application aims to provide a real-time monitoring device for tumor-immune microenvironment with three-dimensional innervation function of nerve axons, which is mainly used for constructing a highly bionic tumor-nerve-immune (TNI) triple co-culture system to simulate multicellular interactions in complex organ microenvironment under ex vivo conditions.

[0006] In order to achieve the above object, the present application adopts the following technical solution:

[0007] The present application provides a three-chamber microfluidic chip device for real-time in vitro monitoring of neuro-tumor-immune microenvironment, comprising a top tumor-immune interaction layer, a middle axon guidance and signal screening interface layer, and a bottom electrophysiology integrated substrate layer which are arranged sequentially from top to bottom; the bottom electrophysiology integrated substrate layer is provided with a micropore array, a conductive polymer layer and a microelectrode array; the middle axon guidance and signal screening interface layer is provided with a nanoporous membrane, the membrane surface is coated with a nerve induction factor, and is provided with multi-point sampling channels and an induction factor injection flow channel; the top tumor-immune interaction layer is provided with a communicated tumor organoid open culture pool and a microfluidic flow channel provided with a simulated lymphatic circulation loop, the microfluidic flow channel of the simulated lymphatic circulation loop is provided with an immune cell introduction inlet and a flow control micro-pump access point, and the tumor organoid open culture pool is provided with a flow field control valve.

[0008] The three-layer structure physically forms a vertical coupling structure, that is, the three-layer structure is sequentially arranged along the same vertical axis direction, so that the vertical innervation of axons can be simulated with high fidelity.

[0009] The tumor organoid open culture pool and the microfluidic flow channel provided with the simulated lymphatic circulation loop as a whole form a "convex" shape or a similar structure (due to the simulated lymphatic circulation loop, the microfluidic flow channel as a whole is an arc-shaped channel).

[0010] Further, the conductive polymer is PEDOT:PSS.

[0011] Further, the nerve induction factor is nerve growth factor (NGF) and / or netrin-1 (Netrin-1).

[0012] Further, the pore diameter of the nanoporous membrane is 0.5-1 μm.

[0013] The present application also provides a method of using the above chip device, comprising the following steps: 1) constructing neurospheres on the bottom layer and monitoring the maturity; 2) placing tumor organoids on the nanoporous membrane of the middle layer, and inducing axons to penetrate vertically for innervation; 3) introducing immune cells into the top layer and starting circulation simulation; 4) performing electrical stimulation intervention and conducting real-time sampling monitoring.

[0014] This application also provides the application of the above-mentioned chip device in i) neuro-tumor interaction research, ii) immune killing monitoring, iii) screening of neurogenic antitumor drugs, and iv) neurotransmitter signal translation analysis.

[0015] This application provides a three-chamber microfluidic chip device for real-time in vitro monitoring of the neural-tumor-immune microenvironment, aiming to solve the technical problem that the complex process of transcoding tumor microenvironment signals to the central nervous system is difficult to reproduce in vitro. Its specific functional principle and the technical problem it solves are as follows: Anatomical Reconstruction of Physical Channels: Liver cancer is not only a local metabolic disease, but its remodeled microenvironment also activates the vagal nerve endings distributed in the liver through physical contact, forming a "command center" for transmitting signals to the central nervous system. Existing technologies are mostly horizontally laid out, which cannot simulate the anatomical features of axons extending vertically deep into the tissue and controlling organs. However, the three-chamber microfluidic chip of this application simulates real physical channels through a vertical three-dimensional structure.

[0016] Dynamic simulation of signal translation: The function of the vagus nerve is not only conduction, but also the translation of chemical disturbances in the tumor microenvironment into specific neuroelectric signals. This application utilizes conductive hydrogel (PEDOT:PSS) and microelectrode arrays as the underlying interface to establish a controllable "electrochemical signal translation" simulation system. By simulating nerve impulses through active electrical stimulation, the system's immediate regulation of immune behavior is studied.

[0017] Coupling of Microcirculation and Immune Chemotaxis: Clinical observations have shown a high degree of coupling between depressive symptoms and tumor progression. This application utilizes the principles of shear force and flow field control in fluid mechanics to design a simulated lymphatic circulation loop, aiming to recreate the complex dynamic process of immune cells infiltrating into tumor organoids under the guidance of neural signals in a dynamic fluid environment.

[0018] Compared with the prior art, this application has the following beneficial effects: This application, through innovative physical structure and bioelectric interface design, has significant advantages over traditional co-culture models, providing a core tool for challenging conventional wisdom and clarifying how neural signals are transmitted. Specifically, it offers the following advantages: High-fidelity anatomical simulation (vertical control): Breaking through the limitations of traditional horizontal chips, it realizes "penetrating" three-dimensional control of axons over 3D tumor organoids, greatly improving the structural realism of liver cancer microenvironment remodeling research.

[0019] Real-time, non-destructive dynamic monitoring: The integration of in-situ microsampling channels and transparent imaging windows allows researchers to simultaneously acquire neurophysiological data, neurotransmitter gradients, and immune killing dynamics without damaging the cellular microenvironment, enabling real-time monitoring of the entire process.

[0020] Precise electrophysiological modulation interface: Through the underlying integrated microelectrode array, it is possible to accurately simulate neural signals of different frequencies (such as sympathetic / parasympathetic nerve excitation), providing a digital intervention method for analyzing the specific functions of the "liver-brain axis" physical channel.

[0021] Systematic evaluation of drug screening: The device provides a complete "tumor-neurology-immune" interactive closed loop, which can not only evaluate the efficacy of traditional chemical drugs, but also screen novel intervention drugs that target the vagus nerve signal transduction process, providing a more practical drug screening platform for improving liver cancer-related depression. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the microfluidic triple culture device.

[0023] Figure 2 A schematic diagram of the experimental process for constructing and monitoring a tumor-immune microenvironment chip with neural innervation. Detailed Implementation

[0024] To make the technical solution of this application clearer and easier to understand, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0025] Unless otherwise specified, the experimental or testing methods described in the following examples are conventional methods; the reagents and materials described are obtained from conventional commercial sources unless otherwise specified.

[0026] Example 1: Structure and Application of a Three-Cavity Microfluidic Chip Device 1. Structural Design of a Three-Cavity Microfluidic Chip Device The device is a three-layer vertically coupled biochip architecture (arranged sequentially along the same vertical axis), such as... Figure 1 As shown, the specific structural components are as follows: Bottom layer: Electrophysiological integrated base layer. It features a microporous array to induce neurons to form 3D neurospheres, which are placed on a conductive polymer (PEDOT:PSS, full name poly3,4-ethylenedioxythiophene: polystyrene sulfonate, an aqueous solution of a polymer with conductivity, optical transparency and solution processing properties). A microelectrode array (MEA) is attached under the conductive polymer layer, giving the device the ability to perform real-time electrical stimulation and electrical signal acquisition of neural networks.

[0027] Middle layer: axon guidance and signal screening interface layer. The core component is a nanopore membrane with a pore diameter ranging from 0.5 μm to 1.0 μm. This pore diameter allows axons to penetrate but retains the cell bodies of neurons and tumor cells. The purpose is to utilize the physical size exclusion effect of the pores to only allow vertical penetration and growth of nerve axons, while retaining neuronal cell bodies in the bottom layer, achieving accurate anatomical partitioning. The membrane is coated with neurotransmitters (such as nerve growth factor [NGF], netrin-1 [Netrin-1]) to induce vertical upward growth of axons; the nanopore membrane is provided with an inducer injection channel and multi-point sampling channels. The inducer injection channel is used for injecting inducers, and the multi-point sampling channels allow a micro-syringe to collect micro-volume liquid samples from the membrane surface at different time points and positions for analyzing the local concentrations of neurotransmitters and cytokines.

[0028] Top layer: tumor-immune interaction layer, which is provided with an open culture pool for tumor organoids, used to accommodate and culture 3D tumor organoids, and integrates a set of microfluidic channels simulating the lymphatic circulation loop. Its structural features are as follows: through topological design, the open culture pool for tumor organoids and the microfluidic channels simulating the lymphatic circulation loop integrally form a "convex" shape or similar structure, and then a microfluidic pump access point / port arranged on the channel (the access point is used for connecting a micro-circulation pump to support dynamic recruitment and infiltration of immune cells in a hydrodynamic environment) realizes continuous dynamic circulation of the culture medium; this enables shear force to be formed at the confluence of the channels and the central culture pool, and microfluidic technology is used to precisely control the fluid shear force to simulate the low-speed lymphatic flow field environment in vivo. An immune cell introduction inlet is arranged on the channel for injecting immune cells, so as to support dynamic introduction of activated immune cells. An optical observation window is constructed with a highly transparent material, and combined with an auxiliary micro-sampling channel, real-time in-situ monitoring of immune cell chemotaxis, recruitment and molecular secretion processes is realized. In addition, a flow field control valve is arranged on the culture pool, and the flow rate is adjusted through the flow field control valve to simulate the low-speed shear force environment of interstitial fluid, prompting T cells to spontaneously aggregate on the surface of tumor organoids during circulation.

[0029] 2. A method for simulating multicellular interactions in complex organ microenvironments in vitro based on a three-chamber microfluidic chip device The experimental procedure is as shown in Figure 2 , specifically comprising the following steps: First stage: neural substrate construction and functional maturation Substrate coating: a layer of laminin (Laminin) or poly-L-lysine (PLL) is coated on the surface of the microelectrode array (MEA) at the bottom layer of the chip to enhance cell adhesion.

[0030] Neurosphere induction: primary neurons or iPSC-derived neural progenitor cells are seeded in the micropore array of the bottom layer.

[0031] Electrophysiological monitoring: Using the underlying integrated conductive polymer (PEDOT:PSS) interface, the maturation state of the neurosphere is non-destructively monitored through electrochemical impedance spectroscopy (EIS) until stable spontaneous discharge activity is recorded.

[0032] Phase Two: 3D Tumor Organoid Localization and Axonal Vertical Innervation Organoid seeding: Precisely place tumor organoids, which have been cultured and matured in vitro and have a diameter of about 300 μm-500 μm, directly above the middle nanoporous membrane (0.8 μm pore size).

[0033] Chemotaxis induction: A matrix gel containing a gradient of nerve growth factor (NGF) is injected into the inducing factor injection channel, and the axons of the underlying neurosphere are guided vertically upward through the nanoporous membrane by chemotaxis.

[0034] Dominance confirmation: The morphological characteristics of axon insertion into the tumor organoid are observed through a real-time imaging system to establish physical connection.

[0035] Phase 3: Introduction of immune cells and activation of the circulatory system Cell activation: Cells are activated in vitro using CD3 / CD28 antibodies and then fluorescently labeled (e.g., CellTracker Blue).

[0036] Dynamic perfusion: Activated T cells are introduced from the immune cell inlet into the open culture pool of tumor organoids at the top layer, and a micro-circulation pump is started.

[0037] Circulation simulation: The flow rate is adjusted by controlling the valve through the flow field to simulate the low-speed shear force environment of the interstitial fluid, which promotes the spontaneous aggregation of T cells to the surface of tumor organoids during circulation.

[0038] Phase 4: Neuromodulation Intervention and Multidimensional Data Monitoring Resting-state observation: Recording the baseline infiltration depth and killing efficiency of T cells in tumor organoids without external stimulation.

[0039] Stimulation-state simulation: Applying electrical stimulation of a specific pattern (such as high-frequency pulses) to the underlying neural array to simulate the excitation state of the nervous system.

[0040] Signal acquisition: The concentration of neurotransmitters (such as norepinephrine) at the interface was extracted simultaneously through multi-point sampling channels, and the transient changes in T cell infiltration behavior were observed by combining confocal imaging to analyze the regulatory role of neural signals on the formation of immune synapses.

[0041] Example 2: Functional verification of a three-cavity microfluidic chip device (axon penetration and electrical signal translation efficiency) This embodiment aims to demonstrate that the device can achieve vertical innervation of nerve axons and successfully translate electrical stimulation into chemical signal changes.

[0042] 1. Experimental Setup Bottom layer: Primary rat hippocampal neurons were inoculated and cultured for 7 days to form a mature network.

[0043] Middle layer: coated with 50 μg / mL Netrin-1 inducing factor.

[0044] Monitoring point: Extract mid-layer liquid samples every 6 hours using sampling microchannels.

[0045] 2. Observational data Axonal penetration rate: Observed by high-magnification confocal microscopy, at 48 hours, approximately 85±5% of the micropores showed nerve axons penetrating and entering the top chamber.

[0046] Quantitative neurotransmitter release: Biphasic electrical stimulation with a frequency of 20 Hz and a pulse width of 1 ms was applied to the subcutaneous layer.

[0047] Results: The microsampling channel detected that the norepinephrine (NE) concentration rapidly increased from baseline (0.2 nM) to 4.5 nM within 5 minutes after stimulation, demonstrating that the device has a highly efficient electrochemical signal translation capability.

[0048] Example 2: Simulation of the "liver-brain axis" mechanism (the effect of neural regulation on immune killing) This embodiment simulates how vagal nerve signals inhibit the anti-tumor activity of T cells in the context of liver cancer-related depression.

[0049] 1. Experimental Groups Control group (Resting): The neural basal layer is in a resting state.

[0050] Stress-Simulated group: High-frequency discharge (50Hz) simulating "psychological stress" is applied to the neural basal layer.

[0051] Intervention group (Antagonist): In addition to the stimulation group, β-receptor blockers were perfused into the top layer.

[0052] 2. Quantification of experimental results

[0053] 3. Data Analysis Conclusions Experimental data show that pathological signals released from nerve axons (induced by electrical stimulation) significantly inhibited the infiltration ability of T cells into tumor organoids (reducing it by approximately 70%) and their cytotoxic activity. This inhibitory effect can be effectively reversed by blocking neurotransmitter receptors during translation.

[0054] In summary, the chip device provided in this application can be applied to: 1) Research on neuro-tumor interaction mechanisms: This is used to observe in real time how nerve axons penetrate physical barriers and exert targeted control over tumor organoids in 3D space, revealing the direct regulatory role of neurotransmitters on tumor growth, invasion and metabolic remodeling.

[0055] 2) Dynamic monitoring of immune killing efficiency: Using a simulated lymphatic circulation system, observe the dynamic process of immune cells (such as T cells and NK cells) chemotaxis, infiltration and cytotoxicity of tumor tissue under different neural electrical signal interference.

[0056] 3) Screening of neurogenic antitumor drugs: As an innovative drug screening platform, this device can evaluate how drugs targeting neural receptors or neurotransmitter signals can enhance the clinical efficacy of immunotherapy (such as PD-1 / L1 inhibitors) by modulating the "neuro-immune axis".

[0057] 4) Systematic signal translation analysis: Through the built-in microsampling channel, the concentration gradients of neurotransmitters (such as norepinephrine and acetylcholine) and cytokines in the microenvironment are extracted and quantitatively analyzed in real time to resolve the molecular logic of peripheral nerve signal translation into tumor immune response.

[0058] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from this application, and these improvements and additions should also be considered within the scope of protection of this application.

Claims

1. A three-chamber microfluidic chip device for real-time in vitro monitoring of the neural-tumor-immune microenvironment, characterized in that, The system comprises, from top to bottom, a top tumor-immune interaction layer, a middle axon guidance and signal screening interface layer, and a bottom electrophysiological integrated base layer. The bottom electrophysiological integrated base layer consists of, from top to bottom, a microporous array, a conductive polymer layer, and a microelectrode array. The middle axon guidance and signal screening interface layer has a nanoporous membrane coated with neural inducing factors and includes multi-point sampling channels and inducing factor injection channels. The top tumor-immune interaction layer has a connected tumor organoid open culture pool and a microfluidic channel simulating a lymphatic circulation loop. The microfluidic channel simulating a lymphatic circulation loop has an immune cell introduction inlet and a flow-controlled micropump access point. The tumor organoid open culture pool has a flow field control valve.

2. The chip device according to claim 1, characterized in that, The conductive polymer is PEDOT:PSS.

3. The chip device according to claim 1, characterized in that, The neural inducing factors are nerve growth factor (NGF) and / or neural axon guidance factor 1 (Netrin-1).

4. The chip device according to claim 1, characterized in that, The pore size of the nanoporous membrane is 0.5-1 μm.

5. A method of using the chip device according to any one of claims 1-4, characterized in that, Includes the following steps: 1) Construct neurospheres at the bottom layer and monitor their maturity; 2) Place tumor organoids on the middle layer nanoporous membrane to induce vertical axonal penetration and innervation; 3) Introduce immune cells at the top layer and initiate cyclic simulation; 4) Intervene with electrical stimulation and monitor in real time.

6. The application of the chip device according to any one of claims 1-4 in i) neuro-tumor interaction research, ii) immune killing monitoring, iii) screening of neurogenic antitumor drugs, and iv) neurotransmitter signal translation analysis.

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