A microfluidic chip for in vitro lymphangiogenesis

Abstract

The application belongs to the technical field of micro-fluid organ chip and biomedical engineering, and particularly relates to a micro-fluid chip for lymphangiogenesis in vitro, which comprises a chip main body and a glass substrate fixedly connected with the chip main body, a gel culture channel and two culture liquid channels are arranged in parallel in the chip main body, and the two culture liquid channels are symmetrically arranged on the two sides of the gel culture channel; a plurality of trapezoidal micro columns are arranged between the gel culture channel and the culture liquid channels, and the wide bottom surface of the trapezoidal micro column faces the gel culture channel; a culture liquid inlet is arranged at one end of the culture liquid channel, and a culture liquid outlet is arranged at the other end of the culture liquid channel, and the culture liquid is driven to flow through the liquid level difference between the culture liquid inlet and the culture liquid outlet; and a cell inoculation port is arranged at the end of the gel culture channel. The application can not only stably construct a three-dimensional lymphatic vessel network, but also can maintain continuous interstitial flow without an external pump source, and is compatible with multi-factor pathological simulation and real-time observation.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic organ-on-a-chip and biomedical engineering technology, and particularly relates to a microfluidic chip for in vitro lymphatic vessel generation. Background Technology

[0002] With the increasing aging of the population, neurodegenerative diseases such as Alzheimer's disease have become a major public health problem. Recent studies have found that meningeal lymphatic vessels are a core pathway for clearing key metabolic waste products such as β-amyloid protein (Aβ) from the brain; their functional decline directly leads to the deposition of toxic proteins, exacerbating neuroinflammation. However, existing research methods have significant limitations: while animal models can simulate the in vivo environment, they suffer from strict ethical restrictions, large individual variability, difficulty in real-time observation of cell dynamics, and high experimental costs; traditional two-dimensional cell culture lacks three-dimensional extracellular matrix support, making it impossible to form physiologically functional tubular structures, let alone reproduce the fluid shear forces and mass transport gradients in vivo.

[0003] While existing microfluidic chip technology has seen improvements, it still has several shortcomings in culturing meningeal lymphatic vessels, a unique organ: First, the use of rectangular micropillars or walls to confine the gel makes it prone to overflow or collapse during injection, severely impacting experimental reproducibility. Second, it generally relies on external syringe pumps to drive the fluid, which is not only expensive but also fails to simulate the low-speed, continuous interstitial flow characteristic of meningeal tissue. Third, the culture space is mostly two-dimensional, limiting the construction of three-dimensional lymphatic networks. Fourth, it cannot integrate complex pathological microenvironment simulation functions such as inflammatory factor stimulation, immune cell migration, and drug intervention within a single chip. Furthermore, the optical permeability limitations of existing chips also hinder long-term live-cell imaging observation.

[0004] Therefore, a microfluidic chip for in vitro lymphatic vessel generation needs to be designed to solve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a microfluidic chip for in vitro lymphatic vessel generation to solve the above problems, so as to achieve the goal of both stably constructing a three-dimensional lymphatic network and maintaining continuous interstitial flow without external pump source, and being compatible with multi-factor pathological simulation and real-time observation.

[0006] To achieve the above objectives, the present invention provides the following solution: a microfluidic chip for in vitro lymphatic vessel generation, comprising a chip body and a glass substrate fixedly connected to the chip body; the chip body has parallelly arranged gel culture channels and two culture medium channels, the two culture medium channels being symmetrically arranged on both sides of the gel culture channels; a plurality of trapezoidal micropillars are disposed between the gel culture channels and the culture medium channels, the wide base of the trapezoidal micropillars facing the gel culture channels to utilize surface tension to restrict the extracellular matrix gel; one end of the culture medium channel has a culture medium inlet and the other end has a culture medium outlet, the culture medium flow is driven by the liquid level difference between the culture medium inlet and the culture medium outlet, and a continuous interstitial flow is formed in the gel region within the gel culture channel; a cell inoculation port is provided at the end of the gel culture channel.

[0007] Preferably, the plurality of trapezoidal micropillars are uniformly arranged along the length of the gel culture channel, and a gap is provided between two adjacent trapezoidal micropillars.

[0008] Preferably, the width of the gel culture channel is 1.2 mm and the height is 200-300 μm; the width of the culture medium channel is 1.2 mm and the height is 200-300 μm.

[0009] Preferably, the liquid level difference between the culture medium inlet and the culture medium outlet is configured to maintain the average flow rate within the gel region in the range of 1-6 μm / s.

[0010] Preferably, the cell inoculation port is located at the top of the chip body, and the cell inoculation port is perpendicular to the gel culture channel.

[0011] Preferably, it also includes a drug inlet, which includes multiple independent sample dispensing holes that are connected to the culture medium channel.

[0012] Preferably, the thickness of the chip body is 5-8 mm, and the thickness of the glass substrate is 1 mm.

[0013] Preferably, the inner wall of the gel culture channel is surface modified and coated with at least one of fibronectin, type I collagen, laminin, and polylysine.

[0014] Preferably, the chip body has a size of 25mm × 25mm.

[0015] Preferably, the chip body is made of polydimethylsiloxane.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: 1. Establishing a stable three-dimensional meningeal lymphatic vessel culture environment: A three-channel structure combining a central gel culture channel with two side culture medium channels is adopted. The central region is filled with extracellular matrix materials such as fibrin gel, allowing meningeal lymphatic endothelial cells to be uniformly embedded in the three-dimensional matrix for growth. Compared with traditional two-dimensional culture methods, this structure can provide cells with a three-dimensional microenvironment that more closely resembles that of in vivo tissues, which is conducive to cell migration, polarization, budding, and lumen formation, thereby improving the efficiency of lymphatic network formation.

[0017] 2. Trapezoidal Micropillar Array Enhances Gel Stability: Traditional microfluidic chips typically use rectangular micropillars or vertical barriers to restrict the gel area, which can easily lead to gel leakage or culture medium contamination during dispensing. This invention employs a trapezoidal micropillar array structure, with micrometer-level gaps between adjacent micropillars. Under the combined effects of liquid surface tension and capillary action, this effectively restricts the position of the extracellular matrix gel, preventing it from entering the culture medium channels while ensuring free diffusion of the culture medium. This significantly improves the chip's sample loading success rate and experimental reproducibility.

[0018] 3. Stable interstitial flow without external syringe pump: Utilizing the level difference between the culture medium inlet and outlet, the culture medium is driven to flow slowly in the culture medium channels on both sides, forming a continuous and stable interstitial flow through the gel region. This flow method eliminates the need for complex equipment such as external syringe pumps and peristaltic pumps, reducing experimental costs and minimizing the impact of mechanical vibration and pulsed flow on cells, thus more closely resembling the physiological state of the meningeal lymphatic system in vivo.

[0019] 4. Applicable to a variety of experimental models: Not only is it suitable for meningeal lymphoendothelial cell culture, but it can also be used for VEGF-C induced lymphangiogenesis experiments, ANG-1 induced maturation experiments, HGF promotion experiments, TNF-α inflammatory stimulation experiments, Aβ protein damage experiments, PBMC immune cell migration experiments, and drug intervention experiments, thereby improving experimental efficiency.

[0020] 5. Facilitates real-time dynamic observation: The chip body is made of transparent polydimethylsiloxane material and permanently bonded to a high-transmittance glass substrate. It can be directly placed under an inverted fluorescence microscope, laser confocal microscope or live cell imaging system for continuous observation, realizing real-time recording of the migration, branching, lumen formation and immune cell migration processes of meningeal lymph endothelial cells.

[0021] 6. Simple manufacturing process and high repeatability: The chip body is manufactured using soft photolithography, which is a mature process with low processing costs, enabling mass production and making it suitable for research institutions and enterprises to promote and apply. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the overall invention; Figure 2 This is a schematic diagram of the bottom of the chip body of the present invention; Figure 3 for Figure 2 A magnified view of part A in the image.

[0024] The components include: 1. Gel culture channel; 2. Culture medium channel; 3. Cell inoculation port; 4. Culture medium inlet; 5. Culture medium outlet; 6. Chip body; 7. Glass substrate; and 8. Trapezoidal micropillars. Detailed Implementation

[0025] 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, and 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.

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] Reference Figures 1 to 3 As shown, this invention provides a microfluidic chip for in vitro lymphatic vessel generation, comprising a chip body 6 and a glass substrate 7 fixedly connected to the chip body 6. The chip body 6 has parallelly arranged gel culture channels 1 and two culture medium channels 2, symmetrically arranged on both sides of the gel culture channels 1. Multiple trapezoidal micropillars 8 are disposed between the gel culture channels 1 and the culture medium channels 2, with the wide base of the trapezoidal micropillars 8 facing the gel culture channels 1 to utilize surface tension to confine the extracellular matrix gel. One end of the culture medium channel 2 has a culture medium inlet 4, and the other end has a culture medium outlet 5. The culture medium flow is driven by the liquid level difference between the culture medium inlet 4 and the culture medium outlet 5, forming a continuous interstitial flow in the gel region within the gel culture channels 1. A cell inoculation port 3 is provided at the end of the gel culture channels 1.

[0028] The gel culture channel 1 is located in the central region of the chip body 6. It is mainly used to fill fibrin gel or other three-dimensional extracellular matrix materials and serves as a three-dimensional culture area for meningeal lymphoendothelial cells.

[0029] Two culture medium channels 2 are located on both sides of the gel culture channel 1 and are arranged parallel to the gel culture channel 1. They are used to transport culture medium, growth factors, inflammatory factors and drugs, and to establish a stable interstitial flow environment through the liquid level difference.

[0030] The scheme was further optimized by uniformly arranging multiple trapezoidal micropillars 8 along the length of the gel culture channel 1, with micron-sized gaps between adjacent trapezoidal micropillars 8 to allow for the diffusion of nutrients and biological factors.

[0031] Multiple trapezoidal micropillars 8 utilize liquid surface tension and capillary action to stably confine the extracellular matrix within the central culture region, while allowing free exchange of nutrients, growth factors, and metabolites in the culture medium through diffusion. The trapezoidal micropillars 8 employ a tapered structure, with the side closer to the gel culture region being wider than the side closer to the culture medium channel, thereby further enhancing gel fixation capacity and reducing the risk of gel leakage during sample loading.

[0032] Furthermore, the shape of the trapezoidal micropillar 8 is not limited to trapezoids; it can also be triangular or other shapes, as long as it is a microstructure that can produce a burst valve effect.

[0033] Further optimization of the scheme: the width of gel culture channel 1 is 1.2 mm and the height is 200-300 μm; the width of culture medium channel 2 is 1.2 mm and the height is 200-300 μm.

[0034] The scheme was further optimized so that the liquid level difference between the culture medium inlet 4 and the culture medium outlet 5 was configured to maintain the average flow rate in the gel region within the range of 1-6 μm / s.

[0035] In a further optimized design, the cell inoculation port 3 is located at the top of the chip body 6 and is perpendicular to the gel culture channel 1. It is used to inject gel mixed with meningeal lymph endothelial cells into the gel culture channel 1.

[0036] Further optimization of the scheme also includes a drug inlet, which includes multiple independent sample wells connected to culture medium channel 2, for adding vascular endothelial growth factor C, tumor necrosis factor-α, β-amyloid protein or peripheral blood mononuclear cell suspension respectively.

[0037] Each sample well is connected to the corresponding culture medium channel 2, allowing the addition of meningeal lymphoendothelial cells, peripheral blood mononuclear cells, Aβ protein, VEGF-C, ANG-1, HGF, TNF-α, and other drugs or biological factors according to experimental needs, enabling rapid switching between different experimental conditions.

[0038] Further optimization of the scheme: the thickness of the chip body 6 is 5-8mm, the thickness of the glass substrate 7 is 1mm, and the chip body 6 and the glass substrate 7 are bonded together after oxygen plasma treatment to form a closed microfluidic culture system.

[0039] To further optimize the process, the inner wall of gel culture channel 1 is surface modified and coated with at least one of fibronectin, type I collagen, laminin, and polylysine.

[0040] The design was further optimized so that the chip body 6 has a size of 25mm×25mm, which is suitable for the observation stage of standard inverted fluorescence microscopes and laser confocal microscopes.

[0041] Further optimization of the design: the chip body 6 is made of polydimethylsiloxane.

[0042] Example 1 I. Overall Chip Structure This embodiment provides a three-dimensional microfluidic chip for in vitro meningeal lymphatic vessel formation and culture.

[0043] The overall dimensions of the chip body 6 are preferably 25mm × 25mm. The chip body 6 is made of transparent polydimethylsiloxane (PDMS) with a thickness of 5–8mm. Its lower surface is permanently bonded to a transparent glass substrate 7 with a thickness of approximately 1mm to form a closed culture system. PDMS material has good biocompatibility, oxygen permeability, and optical transparency, which can meet the requirements for long-term culture of meningeal lymphoendothelial cells and facilitate real-time observation under fluorescence microscopy and confocal microscopy.

[0044] Before bonding the chip body 6 to the glass substrate 7, it needs to undergo hydrophilic treatment.

[0045] The chip body 6 is composed of a culture area, a sample loading area, and a fluid control area. The culture area is located in the center of the chip, the sample loading area is distributed around the chip, and the fluid control area is connected to the inlet and outlet of the culture medium.

[0046] The entire chip is formed in one step using soft lithography, ensuring that the dimensions of each growth channel remain consistent and that the fluid distribution is uniform.

[0047] A gel culture channel 1 is set in the center, and culture medium channels 2 are set on both sides.

[0048] Gel culture channel 1 is mainly used to fill three-dimensional extracellular matrix materials. Preferably, one or more of the following are used as the culture medium: Fibrin Matrix, Matrigel, Collagen I, and GelMA.

[0049] In this embodiment, fibrin gel with a concentration of 6-10 mg / mL is preferably used as a three-dimensional culture scaffold.

[0050] Meningeal lymphoendothelial cells are uniformly mixed in the gel to form a stable three-dimensional culture system.

[0051] The gel culture channel 1 is preferably 1.2 mm wide and about 300 μm high. This size ensures that the cells have sufficient space to grow while maintaining rapid diffusion of nutrients in the culture medium.

[0052] The width of the culture medium channel 2 is preferably 1.2 mm, and the height is approximately 300 μm. The culture medium channel 2 is connected to the culture medium inlet 4 and the culture medium outlet 5, respectively, and a continuous and stable flow is established through the liquid level difference.

[0053] Nutrients, growth factors, and oxygen in the culture medium on both sides can diffuse into the central culture area through the gel, thereby maintaining the long-term stable culture of meningeal lymphoendothelial cells.

[0054] An array of trapezoidal micropillars 8 is disposed between the gel culture channel 1 and the culture medium channel 2. The trapezoidal micropillars 8 are uniformly arranged along the length of the gel culture channel 1. Several micrometer-level gaps are formed between two adjacent trapezoidal micropillars 8. The trapezoidal micropillars 8 are trapezoidal in shape, with their wider base facing the gel culture area and their narrower base facing the culture medium channel. This structure can make full use of the surface tension of the liquid to stably fix the fibrin gel in the central culture area, avoiding lateral flow or entry into the culture medium channel during gel dispensing.

[0055] Compared with traditional rectangular micropillars, trapezoidal micropillars 8 can further improve gel retention and reduce damage to the culture area caused by factors such as gel shrinkage and cell traction during experiments.

[0056] In addition, the trapezoidal micropillar array maintains a continuous diffusion pathway, allowing VEGF-C, ANG-1, HGF, TNF-α and other soluble biological factors in the culture medium to enter the central gel region through diffusion, forming a stable concentration gradient and providing continuous stimulation to meningeal lymphoendothelial cells.

[0057] The chip body 6 has multiple independent circular sample loading ports on its top, each of which is connected to the gel culture channel 1 and the culture medium channel 2. Each culture medium channel 2 has a culture medium inlet 4 and a culture medium outlet 5 at both ends, and the gel culture channel 1 has an independent gel sample loading port to facilitate operations such as extracellular matrix injection, cell seeding, and culture medium replacement.

[0058] The diameter of the sample dispensing well is preferably 2–5 mm, and can be adjusted appropriately according to experimental needs. A liquid reservoir of a certain depth is formed around the sample dispensing well to increase the storage capacity of the culture medium and reduce liquid level changes caused by evaporation during the culture process.

[0059] The chip body 6 adopts an open-type liquid level difference drive method, which does not rely on an external peristaltic pump, syringe pump, or pressure control system. During the experiment, only the liquid level of the culture medium inlet 4 needs to be added to be higher than that of the culture medium outlet 5 to form a stable pressure difference between the culture medium channels 2, thereby driving the culture medium to flow slowly. This flow method has the advantages of simple structure, stable operation, low cost, and convenient maintenance.

[0060] The flow of liquid causes the pressure difference to gradually decrease. Therefore, the culture medium inlet 4 needs to be designed with a storage tank, and a sufficient amount of culture medium needs to be added to the storage tank to slow down the rate at which the pressure difference decreases.

[0061] Because the culture medium diffuses through the central gel region, a stable and uniform interstitial flow is formed within the gel. This interstitial flow continuously provides nutrients to the meningeal lymphoendothelial cells while promptly removing cellular metabolic waste, and creates a concentration gradient of biological factors, which is beneficial for cell migration and lymphatic vessel budding.

[0062] By adjusting the height difference between the culture medium inlet 4 and the culture medium outlet 5, a flow rate of approximately 1–6 μm / s can be formed in the central gel region to simulate the tissue fluid flow environment under normal physiological and inflammatory conditions of meningeal tissue, respectively.

[0063] The chip body 6 is manufactured using soft photolithography.

[0064] First, the chip structure is drawn according to the CAD design drawings, and the chip photolithography mask is obtained by using high-precision mask fabrication technology.

[0065] Subsequently, SU-8 negative photoresist is spin-coated onto the clean silicon wafer surface. The spin-coating thickness is determined based on the height of the growth channels, preferably controlled at around 300 μm. After spin-coating, pre-baking, UV exposure, post-baking, and development are performed sequentially to form a microchannel master mold on the silicon wafer surface.

[0066] Polydimethylsiloxane (PDMS) prepolymer and curing agent were thoroughly mixed at a mass ratio of 10:1, and air bubbles generated during the mixing process were removed in a vacuum degassing device. The PDMS mixture was then slowly poured onto the surface of the SU-8 master mold and cured at 70°C for about 2 hours. After curing, the PDMS layer was peeled off.

[0067] Using a special punch, culture medium inlet 4, culture medium outlet 5, and sample addition hole are punched at the corresponding positions in the PDMS layer.

[0068] Subsequently, the chip body 6 and the glass substrate 7 were treated with oxygen plasma to form active hydroxyl groups on their surfaces. The treated chip body 6 immediately covered the surface of the glass substrate 7, and permanent bonding was completed at room temperature to form a closed microfluidic chip.

[0069] After bonding is completed, the chip is placed at 70°C for 30 minutes to further heat it to improve the bonding strength and prevent leakage during long-term cultivation.

[0070] Before the formal experiment, the chip was placed in a UV sterilization chamber for 30 minutes, thoroughly cleaned with 75% ethanol, and then rinsed with sterile PBS to ensure the chip was sterile.

[0071] Subsequently, fibronectin or type I collagen is pre-coated in the culture channel to enhance the adhesion between meningeal lymphoendothelial cells and the substrate material.

[0072] During formal culture, fibrin gel was first prepared, and meningeal lymphoendothelial cells were uniformly mixed into the gel according to the experimental design.

[0073] Cell density is 1×10 6 ~5×10 6 cells / mL.

[0074] Using a micropipette, slowly inject approximately 30 μL of cell-gel mixture into gel culture channel 1 to completely fill the entire central culture area.

[0075] Due to the surface tension constraint generated by the trapezoidal micropillar array, the gel can remain stably in the central culture region without entering the culture medium channels on both sides.

[0076] After the gel is applied, place the chip in a 37°C incubator and let it stand for 20-30 minutes to allow the fibrin gel to fully cross-link and solidify.

[0077] After the gel solidifies, add complete culture medium to the left and right culture channels 2 respectively, adding about 20 μL of culture medium to each side.

[0078] During the culture process, the culture medium is changed every 24 hours.

[0079] The culture medium can be added according to the experimental design to establish different experimental models by adding VEGF-C, ANG-1, HGF, TNF-α, Aβ protein, drug candidate molecules, or combinations thereof.

[0080] As the culture time is extended, meningeal lymphoendothelial cells gradually migrate, proliferate, and connect with each other, forming a continuous three-dimensional lymphatic network in the gel.

[0081] To simulate the inflammatory microenvironment of the meninges in neurodegenerative diseases such as Alzheimer's, Aβ protein and TNF-α can be added to the culture medium. The Aβ concentration is controlled at 25–100 ng / mL, and the TNF-α concentration at 10–20 ng / mL. Inflammatory factors in the culture medium can slowly diffuse along culture channel 2 to the central gel region, forming a stable concentration gradient under the combined action of interstitial flow. Long-term exposure of meningeal lymphoendothelial cells to this inflammatory microenvironment can alter their cell migration rate, lumen formation ability, network coverage, and cell viability.

[0082] Researchers can use confocal microscopy to continuously observe the process of meningeal lymphatic vessel formation and combine it with immunofluorescence staining to analyze changes in the expression of lymphatic endothelial marker proteins such as LYVE-1, PROX-1, VEGFR-3 and Podoplanin, thereby evaluating the influence of the inflammatory microenvironment on meningeal lymphogenesis.

[0083] It can also be used to simulate the recruitment process of meningeal immune cells. In the experiment, a stable meningeal lymphatic network is first formed in the central gel region. Then, a suspension of peripheral blood mononuclear cells (PBMCs) is added to one side of the culture medium channel 2, creating a continuous interstitial flow through the liquid level difference. Under the influence of chemokines, PBMCs gradually migrate through the micropillar array and towards the central lymphatic region. After 24 hours of culture, the number of PBMCs that have migrated to the central region can be counted using fluorescence microscopy. Combined with chemokine receptor blocking experiments such as CCR7 and CXCR4, the recruitment capacity of immune cells under different inflammatory conditions can be analyzed. This experiment can further evaluate the role of the meningeal lymphatic system in the regulation of neuroinflammation and the transport of immune cells.

[0084] Example 2: Microfluidic Chip for Cultivating Meningeal Lymphatic Vessels Based on Surface Modification Based on Example 1, in order to further improve the adhesion ability of meningeal lymphoendothelial cells (HLECs) on the surface of the chip body 6 microchannels, this example performs biofunctionalization treatment on the inner surface of the chip.

[0085] Because PDMS material is naturally hydrophobic, cell adhesion efficiency is low when cells directly contact the PDMS surface, leading to problems such as cell detachment, lumen breakage, and network instability during long-term culture. Therefore, after chip fabrication, the inner wall of the microchannels is modified to be hydrophilic.

[0086] First, the PDMS chip was treated with oxygen plasma for 60–120 s to form a large number of hydroxyl groups on the PDMS surface, thereby improving its hydrophilicity. Then, a 50 μg / mL Fibronectin solution was injected into each culture channel of the chip, and the chip was incubated at 37°C for 1 h to allow Fibronectin to be uniformly adsorbed onto the PDMS surface. After incubation, unbound proteins were washed with sterile PBS, and cells were immediately seeded.

[0087] Alternatively, type I collagen (Collagen I), laminin, gelatin, or poly-L-Lysine can be used to coat the culture channels to further improve cell adhesion properties.

[0088] After surface modification, meningeal lymphoendothelial cells formed a continuous cell layer after 24 hours of culture, with a significantly increased cell spreading area and tighter intercellular connections. The lymphatic network formed after 7 days of culture showed significantly better continuity than that of untreated PDMS chips, effectively reducing cell detachment during culture and improving long-term culture stability.

[0089] During the culture period, observations using laser confocal microscopy revealed that in the surface-modified chip, meningeal lymphoendothelial cells were able to form a regular arrangement along the direction of interstitial flow and gradually establish a continuous tubular structure. The average number of branches, average branch length, and network coverage area were all higher than those in the unmodified chip.

[0090] Therefore, this embodiment further improves the stability of the chip under long-term dynamic culture conditions, making it more suitable for meningeal lymphatic vessel formation and drug screening experiments.

[0091] Example 3: Microfluidic chip for simulating inflammatory microenvironment and recruiting immune cells Based on the chip structure described in Example 1, this example further adds an immune cell migration experiment module.

[0092] First, a mature meningeal lymphatic network was established within gel culture channel 1. During the culture process, 100 ng / mL VEGF-C and 20 ng / mL ANG-1 were continuously added to the culture medium, and the cells were cultured for 7–10 days to allow the meningeal lymphatic endothelial cells to form a stable three-dimensional tubular structure.

[0093] Subsequently, inflammatory culture medium containing TNF-α and Aβ proteins was added to the left culture medium channel 2, and normal culture medium was added to the right culture medium channel 2. By forming a stable concentration gradient between the two culture mediums, a continuous inflammatory microenvironment was established in the central gel region.

[0094] Furthermore, peripheral blood mononuclear cells (PBMCs) were added to the left culture medium channel, with a cell concentration preferably of 1×10⁻⁶. 6 cells / mL. By adjusting the liquid level difference, a continuous interstitial flow of approximately 4 μm / s is formed within the chip, causing PBMCs to migrate towards the meningeal lymphatic region under the combined action of chemokines and fluid shear forces.

[0095] After 24 hours of culture, the number of PBMCs entering the central gel region and adhering to the surface of lymphatic vessels was counted using fluorescence microscopy. PBMCs were then further treated with CCR7 or CXCR4 neutralizing antibodies to evaluate the effects of different chemotactic signaling pathways on the migration ability of immune cells.

[0096] Experimental results show that the number of PBMCs migrating significantly increases under inflammatory stimulation conditions, while the number of PBMCs migrating significantly decreases after blocking CCR7 or CXCR4. This indicates that the chip can stably simulate the immune cell recruitment process under meningeal inflammation and can be used for the study of the meningeal immune microenvironment.

[0097] Example 4: Microfluidic culture platform for drug screening This embodiment utilizes a microfluidic chip to establish a meningeal lymphatic vessel drug evaluation platform.

[0098] First, meningeal lymphoendothelial cells were cultured in gel culture channel 1 for 7 days to form a continuous lymphatic network. Then, different drugs were added to the left and right culture channels according to experimental needs, including: VEGF-C; ANG-1; HGF; VEGF-C + ANG-1 combined group; VEGF-C + ANG-1 + HGF combined group; inflammatory stimulation group (TNF-α + Aβ); and candidate drug intervention group. During culture, the culture medium was changed every 24 hours, and observation was performed continuously for 14 days.

[0099] After the experiment, lymphatic vessel formation was evaluated by Calcein-AM live cell staining, LYVE-1 immunofluorescence staining, and PROX-1 immunofluorescence staining, and the following indicators were statistically analyzed: lymphatic vessel coverage area; average branch length; number of branch points; average vessel diameter; network connectivity; cell viability; and cell migration speed.

[0100] The results show that this chip can stably support long-term culture of meningeal lymphatic vessels and can sensitively reflect the promoting or inhibiting effects of different drugs on lymphatic vessel formation, and can be used as a drug screening platform for meningeal lymphatic vessels.

[0101] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0102] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A microfluidic chip for in vitro lymphatic vessel generation, characterized in that, The device includes a chip body (6) and a glass substrate (7) fixedly connected to the chip body (6). The chip body (6) has parallel gel culture channels (1) and two culture medium channels (2) inside. The two culture medium channels (2) are symmetrically arranged on both sides of the gel culture channels (1). Multiple trapezoidal micropillars (8) are arranged between the gel culture channels (1) and the culture medium channels (2). The wide bottom surface of the trapezoidal micropillars (8) faces the gel culture channels (1) to use surface tension to restrict the extracellular matrix gel. One end of the culture medium channel (2) has a culture medium inlet (4) and the other end has a culture medium outlet (5). The culture medium flows through the liquid level difference between the culture medium inlet (4) and the culture medium outlet (5) and forms a continuous interstitial flow in the gel region of the gel culture channel (1). The end of the gel culture channel (1) has a cell inoculation port (3).

2. A microfluidic chip for in vitro lymphatic vessel generation according to claim 1, characterized in that, Multiple trapezoidal micropillars (8) are uniformly arranged along the length of the gel culture channel (1), and a gap is provided between two adjacent trapezoidal micropillars (8).

3. A microfluidic chip for in vitro lymphatic vessel generation according to claim 1, characterized in that, The gel culture channel (1) has a width of 1.2 mm and a height of 200-300 μm; the culture medium channel (2) has a width of 1.2 mm and a height of 200-300 μm.

4. A microfluidic chip for in vitro lymphatic vessel generation according to claim 1, characterized in that, The liquid level difference between the culture medium inlet (4) and the culture medium outlet (5) is configured to maintain the average flow rate within the gel region in the range of 1-6 μm / s.

5. A microfluidic chip for in vitro lymphatic vessel generation according to claim 1, characterized in that, The cell inoculation port (3) is located on the top of the chip body (6) and is perpendicular to the gel culture channel (1).

6. A microfluidic chip for in vitro lymphatic vessel generation according to claim 1, characterized in that, It also includes a drug inlet, which includes multiple independent sample inlets connected to the culture medium channel (2).

7. A microfluidic chip for in vitro lymphatic vessel generation according to claim 1, characterized in that, The thickness of the chip body (6) is 5-8 mm, and the thickness of the glass substrate (7) is 1 mm.

8. A microfluidic chip for in vitro lymphatic vessel generation according to claim 1, characterized in that, The inner wall of the gel culture channel (1) is surface modified and coated with at least one of fibronectin, type I collagen, laminin, and polylysine.

9. A microfluidic chip for in vitro lymphatic vessel generation according to claim 1, characterized in that, The chip body (6) has a size of 25mm×25mm.

10. A microfluidic chip for in vitro lymphatic vessel generation according to claim 1, characterized in that, The chip body (6) is made of polydimethylsiloxane.

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