Biomimetic microfluidic chip simulating multi-organ interconnection and preparation method thereof

Abstract

The embodiment of the application discloses a kind of vein bionics microfluidic chip and its preparation and application method of simulating multi-organ interconnection.The chip includes a chip main body, and its microfluid channel system integrates at least three organ simulation units: a lung simulation chamber for culturing lung cells to simulate upstream organs that can produce systemic effects;An upstream colon cancer cell culture chamber for three-dimensional culture of colon cancer cells to simulate tumor primary lesions;And a downstream liver cell culture chamber for three-dimensional co-culture of liver cells and vascular endothelial cells to simulate the target organ of liver, and the three simulation chambers are communicated with each other by a vein bionics microfluid network, which simulates the complex blood circulation system of human body (including pulmonary circulation and systemic circulation), can realize the signal molecule transmission between organs, and simulate the invasion and metastasis process of colon cancer cells to the target organ of liver via the circulatory system after being affected by upstream lung signals.

Description

Technical Field

[0001] The embodiments of the present invention relate to the fields of biomedical engineering and microfluidics, and in particular to a leaf vein-inspired microfluidic chip that simulates multi-organ interconnection and its fabrication method. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors worldwide, with its high mortality rate primarily attributed to metastasis to distant organs, with the liver being the most prevalent target organ. Approximately 70% of colorectal cancer patients develop liver metastases, a key factor contributing to treatment failure and patient death. Colorectal cancer cells primarily metastasize via the circulatory system. Tumor cells detach and drain into the portal vein via the mesenteric veins, entering the liver via the bloodstream. There, they remain, infiltrate, and form new metastatic lesions within the complex vascular network of the hepatic sinusoids. However, tumor metastasis is not an isolated, localized event involving only the primary tumor and target organ, but rather a systemic process complexly regulated by the body's overall microenvironment. For example, epidemiological and basic research indicates that chronic inflammatory signals generated by distant organs such as the lungs can remotely influence the survival, invasion, and metastasis of colorectal cancer cells via the circulatory system. Therefore, a deeper understanding of the complete mechanisms of colorectal cancer liver metastasis requires not only studying its behavior within the portal vein and intrahepatic vascular network but also considering systemic regulatory factors contributed by other organs.

[0003] Currently, commonly used models for studying tumor metastasis mainly include traditional two-dimensional (2D) cell culture models, in vivo animal models, and emerging organ-on-a-chip models.

[0004] Two-dimensional cell culture models: Although simple to operate and inexpensive, they cannot simulate the three-dimensional (3D) cell growth environment, cell-cell interactions, and key physicochemical factors such as fluid shear forces in vivo, resulting in research results that are far from the real situation in vivo.

[0005] Animal models, such as nude mouse orthotopic transplantation or tail vein injection models, provide a systemic physiological environment and are currently the "gold standard" for research. However, animal models have inherent drawbacks, including high cost, long cycle time, complex operation, species differences, and difficulty in real-time, high-resolution dynamic process observation.

[0006] Microfluidic organ-on-a-chip models: As a cutting-edge in vitro research tool, they can precisely construct microenvironments simulating human organ function at the micrometer scale, enabling precise control of fluids, cells, biochemical factors, and other conditions. In recent years, various microfluidic chips for simulating tumor metastasis have been developed. These chips are typically manufactured using microfabrication techniques such as photolithography, soft etching, and 3D printing, and simulate blood vessels by designing microchannel networks. However, most existing tumor metastasis chips suffer from the following significant drawbacks:

[0007] First, the vascular network structure is overly simplified: To facilitate design and manufacturing, the vascular network within a chip is typically designed with simple geometric shapes, such as straight lines, Y-shaped branches, or grid-like structures. This simplified structure cannot realistically reproduce the complex hierarchical, multi-scale, asymmetric, and irregular topological features of in vivo vascular networks (such as the portal vein and its branches). Real vascular networks have a fractal structure that gradually narrows from the main vessels to the capillaries. This structure determines the complexity of hemodynamic characteristics (such as flow velocity distribution and shear force gradient), which are precisely the key factors affecting the interaction, retention, and extravasation of tumor cells with the vessel wall. Simplified geometric channels cannot accurately simulate these key physical processes.

[0008] Second, the system integration is insufficient, failing to simulate organ-organ crosstalk: Most existing models focus on simulating the binary relationship of "tumor primary site-target organ," neglecting the regulatory roles of other important organs at the system level. For example, they cannot simulate the complex, cross-organ cascade of reactions in which the lungs produce inflammatory factors after being stimulated by external factors (such as air pollutants), and how these factors act on colon cancer tissue through the simulated circulatory system, ultimately promoting its metastasis to the liver.

[0009] To address the issue of simplifying vascular networks, some researchers have proposed using readily available complex network structures in nature as templates, such as plant leaf veins, to construct biomimetic microfluidic channels. Plant leaf veins and animal vascular networks share a high degree of similarity in structure and function, both possessing efficient material transport capabilities and fractal network characteristics.

[0010] Against this backdrop, a prior Chinese invention patent, authorized publication number CN 102923639 B, discloses "A Precise Shaping Method for a Biomimetic Microfluidic System Based on Plant Leaf Veins." The main contribution of this patent lies in proposing a universal method for transforming the fragile structure of natural plant leaf veins into robust, durable, and reusable silicone rubber (PDMS) molds through a series of microfabrication processes. This patent (CN 102923639 B) successfully solves the problems of easy deformation, easy damage, and poor high-temperature resistance that exist when directly using natural leaf veins as molds, providing a feasible and universal manufacturing approach for accurately replicating the complex structure of natural leaf veins. However, existing technologies still suffer from problems such as distorted vascular network models, lack of specificity in biomimetic template selection, lack of complete microfluidic platform functional design, and lack of standardized application methodologies for simulating liver metastasis in colon cancer.

[0011] The inventors discovered that this patent and similar technologies merely provide a general method for manufacturing complex microchannels, and are not functional biomedical research tools in themselves. They do not address the deeper biological questions of how to utilize such complex channels to integrate multiple functional organ units to construct a microphysiological system capable of studying inter-organ signal crosstalk in systemic diseases such as cancer metastasis.

[0012] In summary, existing technologies either suffer from overly simplified vascular models or, while solving the fabrication of complex channels, lack systematic design, functional integration, and application methodologies for specific pathological models (especially cancer metastasis models involving multi-organ interconnections). Therefore, there is an urgent need to develop a novel multi-organ integrated microfluidic chip that not only leverages the complex structural advantages of natural leaf veins but also integrates upstream signaling organs (such as the lungs), primary tumor foci (such as colon cancer), and downstream target organs (such as the liver). Furthermore, a standardized experimental methodology should be established to construct a more precise and efficient biomimetic platform in vitro capable of studying the impact of inter-organ signal crosstalk on the tumor metastasis cascade response. Summary of the Invention

[0013] This invention provides a leaf vein-inspired biofluidic chip simulating multi-organ interconnection and its fabrication method, aiming to construct an integrated in vitro platform capable of studying how signals from remote organs (such as lung inflammation) regulate the metastasis of downstream tumors (such as colon cancer) to target organs (such as the liver) through the circulatory system. This invention integrates lung simulation units, colon cancer primary lesion simulation units, and liver target organ simulation units, and uses a highly biomimetic leaf vein network to simulate the complex human circulatory system, thereby providing a more precise, efficient, and low-cost biomimetic platform for deeply revealing the systemic regulatory mechanisms of cancer metastasis and developing novel treatment strategies.

[0014] In a first aspect, Embodiment 1 of the present invention provides a leaf vein-inspired microfluidic chip simulating multi-organ interconnection, comprising:

[0015] The chip body is composed of a polymer layer with a microstructure bonded to a substrate layer. The microfluidic system is structurally divided into a pulmonary circulation zone and an integral circulation zone. The microfluidic system includes:

[0016] At least one lung simulation chamber, located within the pulmonary circulation zone, is a hollow chamber with a first sample loading port for inoculating and three-dimensionally culturing colon cancer cells to simulate the primary tumor site;

[0017] At least one upstream colon cancer cell culture chamber, located within the systemic circulation zone, is a hollow chamber with a first sample loading port for inoculating and three-dimensionally culturing colon cancer cells to simulate the primary tumor site;

[0018] At least one downstream hepatocyte culture chamber, located within the systemic circulation zone, is a hollow chamber with a second sample loading port for three-dimensional co-culture of hepatocytes and vascular endothelial cells to simulate the liver target organ microenvironment;

[0019] The leaf vein-inspired microfluidic network, made based on the structure of natural plant leaf veins, constitutes the fluid pathways in the pulmonary circulation zone and systemic circulation zone, and connects the organ simulation chambers in their respective zones; as well as inlet and outlet ports, which are used to connect external pipelines and fluid pumps to construct a perfusion system that simulates human blood circulation.

[0020] As one embodiment, the inlet includes: a pulmonary circulation inlet (7) and a systemic circulation inlet (9); the outlet includes: a pulmonary circulation outlet (8) and a systemic circulation outlet (10). When the chip is working, the pulmonary circulation outlet and the systemic circulation inlet are connected through an external pipeline, so that the fluid carrying signal molecules flowing out of the lung simulation chamber can enter the systemic circulation zone. The fluid first flows through and surrounds the upstream colon cancer cell culture chamber in the systemic circulation zone. The tumor cells that invade from the culture chamber enter the flow channel of the leaf vein biomimetic microfluidic network. The fluid carrying tumor cells and upstream signal molecules continues to flow in the leaf vein biomimetic microfluidic network and finally reaches the downstream hepatocyte culture chamber to simulate the process of tumor cell migration under the signal regulation of the upstream organ and adhesion and extravasation at the target organ. The fluid finally flows out from the systemic circulation outlet.

[0021] As one embodiment, the lung simulation chamber (3), the upstream colon cancer cell culture chamber (4), the downstream hepatocyte culture chamber (5), and the leaf vein biomimetic microfluidic network (6) are all integrally formed on the bottom surface of the polymer layer.

[0022] As one embodiment, the polymer layer (1) is a PDMS layer, and the substrate layer (2) is a PMMA or glass layer.

[0023] Secondly, embodiments of the present invention also provide a method for fabricating a chip according to the first aspect, comprising:

[0024] Acquisition and digitization of leaf vein template: Fresh target leaves are selected, placed in an aqueous solution of sodium carbonate and sodium hydroxide, heated, rinsed, and the remaining leaf tissue is peeled off. After air drying, a flat leaf vein skeleton is obtained. A high-resolution grayscale image of the leaf vein skeleton is obtained by scanning. The grayscale image is binarized and vectorized to obtain the leaf vein template file of the target leaf. The leaf vein template file contains a leaf vein network.

[0025] Preparation of the master mold: Based on the leaf vein template file, the layout of the pulmonary circulation area and the systemic circulation area is designed, and a lung simulation chamber, an upstream colon cancer cell culture chamber, and a downstream hepatocyte culture chamber are added in the corresponding areas to obtain the target layout file. A high-precision photomask is fabricated based on the target layout file. A layer of photoresist is spin-coated on a silicon substrate and pre-baked to obtain a photoresist layer. The photoresist layer is exposed, post-baked, and developed to obtain the silicon-based master mold.

[0026] PDMS chip molding and assembly: The PDMS prepolymer and curing agent are mixed and placed in a vacuum dryer for degassing. The degassed mixture is slowly poured onto the silicon-based master mold treated with trimethylchlorosilane vapor and cured in an oven. After curing, the PDMS layer is peeled off from the silicon-based master mold. Pulmonary circulation inlet, pulmonary circulation outlet, systemic circulation inlet, systemic circulation outlet, and sample loading ports for each culture chamber are fabricated at the target positions on the PDMS layer. The PDMS layer and PMMA substrate layer are placed together in a plasma cleaner for treatment. After removal, the treated surfaces of the PDMS layer and the PMMA substrate layer are aligned and bonded together, and then heated in an oven to enhance the bonding strength.

[0027] As one embodiment, the acquisition and digitization of the leaf vein template includes: selecting fresh photinia leaves, placing them in an aqueous solution of 7% (w / v) sodium carbonate and 10% (w / v) sodium hydroxide, heating to boiling and maintaining for 20 minutes, removing them and rinsing them repeatedly with distilled water, gently peeling off the remaining leaf tissue with a soft brush, air-drying to obtain a flat leaf vein skeleton, and scanning to obtain a grayscale image of the leaf vein skeleton with a resolution of 1200 dpi.

[0028] As one embodiment, the preparation of the master mold includes: adding circular chambers of a predetermined diameter on both sides of the leaf vein network, which serve as a lung simulation chamber, an upstream colon cancer cell culture chamber, and a downstream hepatocyte culture chamber, respectively; spin-coating a layer of SU-8 2050 photoresist on a 4-inch silicon wafer at 500 rpm for 10 seconds and at 2000 rpm for 30 seconds; pre-baking includes baking at 65°C for 10 minutes and baking at 95°C for 30 minutes to obtain a photoresist layer with a thickness of approximately 110 μm; exposing the layer to ultraviolet light using a SUSSMJB4 lithography machine at an exposure dose of 350 mJ / cm²; post-baking includes baking at 65°C for 5 minutes and baking at 95°C for 15 minutes; and then developing the layer in SU-8 developer for approximately 10 minutes to obtain the silicon-based master mold.

[0029] In one embodiment, during the molding and assembly of the PDMS chip, the PDMS prepolymer and curing agent are mixed at a mass ratio of 10:1 and placed in a vacuum dryer for 30 minutes to degas. The degassed mixture is then slowly poured onto the silicon-based master mold treated with trimethylchlorosilane vapor and cured in an 80°C oven for 1.5 hours. After curing, the PDMS layer is peeled off from the silicon-based master mold to obtain the polymer layer. Pulmonary circulation inlets, pulmonary circulation outlets, systemic circulation inlets, systemic circulation outlets, and sample loading ports for each culture chamber are fabricated at the target locations on the PDMS layer. The PDMS layer and the PMMA substrate layer are placed together in a plasma cleaner and treated for 45 seconds at a vacuum of 700 mTorr and high power. After removal, the treated surfaces of the two layers are immediately aligned and bonded together, and then heated in an 80°C oven for 10 minutes to enhance the bonding strength. Finally, the chip is secured with screws and nuts.

[0030] Thirdly, embodiments of the present invention provide a method for establishing a multi-organ interconnected tumor metastasis model, which establishes a cell model based on the microfluidic chip described in the first aspect;

[0031] The method includes:

[0032] Chip pretreatment: The microfluidic chip is rinsed with ethanol and deionized water, sterilized by autoclaving, and bovine fibrinogen solution is injected into the microfluidic system for incubation in an incubator;

[0033] Three-dimensional culture of multiple organ cells: A cell suspension containing lung cells, colon cancer cells, hepatocytes, and vascular endothelial cells is prepared. The cell suspension is mixed with a fibrin hydrogel precursor solution. The hydrogel mixture containing the lung cells is injected into the lung simulation chamber through a corresponding sample dispensing port. The hydrogel mixture containing the colon cancer cells is injected into the upstream colon cancer cell culture chamber through the first sample dispensing port. The hydrogel mixture containing the hepatocytes and vascular endothelial cells is injected into the downstream hepatocyte culture chamber through the second sample dispensing port. The microfluidic chip is placed in an incubator for static incubation to allow the hydrogel to solidify and form a three-dimensional tissue.

[0034] Dynamic perfusion and transfer simulation:

[0035] a. Establish a circulation path: Seal all sample application ports with sterile pins, connect the peristaltic pump to the inlet and outlet of the chip through tubing, and construct a complete circulation path that simulates the connection between pulmonary circulation and systemic circulation;

[0036] b. Set process parameters: Perform continuous dynamic culture at a constant flow rate, and introduce stimulants into the pulmonary circulation zone at specific time points to induce the generation of signaling molecules in the lung simulation chamber;

[0037] Observation and analysis of the transfer process:

[0038] a. Real-time imaging: Using an inverted confocal fluorescence microscope, live cell imaging of the chip is performed daily; by observing the colon cancer cells pre-labeled with fluorescent proteins, the entire process of their invasion from the upstream colon cancer cell culture chamber after being affected by upstream lung signals, entering the leaf vein biomimetic microfluidic network, and appearing, adhering and proliferating in the liver tissue microenvironment of the downstream hepatocyte culture chamber is tracked.

[0039] b. Data analysis: Quantitative analysis of the acquired fluorescence images.

[0040] As one embodiment, in the chip pretreatment, the microfluidic chip is rinsed three times with 75% ethanol and deionized water for 5 minutes each time, and then autoclaved; 1.5 mg / mL bovine fibrinogen solution is injected into the microfluidic system of the chip, and incubated in a 37°C incubator for 1 hour.

[0041] In the three-dimensional culture of multiple organ cells, the cells used included human alveolar epithelial cells (HPAEpic), colon cancer cells (HT-29), hepatocytes (HepG2), and vascular endothelial cells (HUVEC). The final concentration of the fibrin hydrogel precursor solution was 3 mg / mL fibrinogen and 6 U / mL thrombin. After mixing the cell suspension and the fibrin hydrogel precursor solution at a 1:1 volume ratio, the cells were seeded as follows: a. HPAEpic and HUVEC were mixed at a 1:7 ratio, resulting in a total cell concentration of 8 × 10⁻⁶ cells / mL. 7 4 µL of the hydrogel mixture was injected into the lung simulation chamber through the corresponding sample dispensing port, with a concentration of cells / mL.

[0042] b. The concentration is 2.5 × 10⁻⁶ 6 A hydrogel mixture of HT-29 cells / mL was injected into the upstream colon cancer cell culture chamber through the first sample loading port, at a rate of 2 µL per chamber.

[0043] c. Mix HepG2 and HUVEC at a ratio of 7:1 to obtain a total cell concentration of 7.5 × 10⁻⁶. 6 4 µL of the hydrogel mixture was injected into the downstream hepatocyte culture chamber through the second sample dispensing port at cells / mL.

[0044] The chip was placed in a 37°C incubator and incubated overnight to allow the hydrogel to solidify and form a three-dimensional tissue.

[0045] In the set process parameters, cell culture medium preheated to 37°C is injected into the chip at a constant flow rate of 60 µL / min. The chip is placed in a culture environment of 37°C and 5% CO2, and its dynamic culture process is divided into two stages:

[0046] a. Stabilization period: First, a continuous dynamic culture is carried out for 2 days to allow the simulated tissues of each organ to reach a stable state;

[0047] b. Stimulation and observation period: Starting from day 3, a specific stimulant (such as LPS) was added to the culture medium in the pulmonary circulation zone, while the systemic circulation zone was kept in the regular culture medium. The culture was continued for 3-5 days to simulate the promoting effect of pulmonary signals on liver metastasis of colorectal cancer.

[0048] The cell culture medium was 50% EGM + 50% DMEM.

[0049] Compared with the prior art, the technical solution provided by the embodiments of the present invention has at least the following positive effects:

[0050] 1. Highly systematic and biomimetic: For the first time, three key organs—lung, colon cancer, and liver—are integrated onto a single chip. By simulating the circulatory system through a biomimetic leaf vein network, the model can study the regulatory mechanism of systemic signals generated by upstream organs on downstream tumor metastasis, greatly improving the physiological relevance of the model.

[0051] 2. Unity of structure and function: The leaf vein biomimetic microfluidic network not only simulates complex blood vessels in structure, but also becomes a key pathway for connecting multiple organs, transmitting signal molecules and transporting tumor cells, realizing the integrated design of "primary lesion-circulatory system-target organ-regulatory organ".

[0052] 3. Dynamic Visualization and Multidimensional Analysis: The transparent chip design combined with fluorescent labeling technology enables real-time, dynamic, and high-resolution live-cell imaging of the entire metastasis process of tumor cells in a multi-organ interconnected environment. Furthermore, samples can be easily collected from each organ compartment after the experiment for in-depth analysis at the gene and protein levels.

[0053] 4. High reproducibility of the method: This invention provides a standardized experimental procedure for chip fabrication and multi-organ co-culture, ensuring the reliability and reproducibility of the research results and making it possible for high-throughput drug screening and research on complex disease mechanisms. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 This is a schematic diagram of the structure of the leaf vein biomimetic microfluidic chip that simulates multi-organ interconnection provided in Embodiment 1 of the present invention.

[0056] Figure 2 This is a reference schematic diagram of a leaf vein-inspired microfluidic chip that simulates multi-organ interconnection, provided in an embodiment of the present invention.

[0057] Figure labels: 1. Pulmonary circulation zone; 2. Systemic circulation zone; 3. Lung simulation chamber; 4. Upstream colon cancer cell culture chamber; 5. Downstream hepatocyte culture chamber; 6. Leaf vein biomimetic microfluidic network; 7. Pulmonary circulation inlet; 8. Pulmonary circulation outlet; 9. Systemic circulation inlet; 10. Systemic circulation outlet. Detailed Implementation

[0058] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, the accompanying drawings show only the parts relevant to the present invention, and not all of the structures.

[0059] The existing technology has the following main problems:

[0060] Distortion of Vascular Network Models: Traditional microfluidic chips, in order to simplify design and manufacturing, often employ linear, grid-like, or simple Y-shaped branching geometries to simulate the microchannel networks within them. This overly simplified structure differs significantly from real vascular networks in vivo, especially complex networks like the portal vein system, which exhibit multi-level branching, asymmetry, and fractal characteristics. This results in the chip's inability to accurately reproduce the complex hemodynamic environment in vivo (such as flow velocity distribution and shear force gradients), thus affecting the fidelity of simulations of key behaviors of tumor cells within blood vessels, such as migration, adhesion, and residence, and significantly reducing the physiological relevance of research results.

[0061] The selection of biomimetic templates lacks specificity: Although existing technologies (such as Chinese patent CN 102923639 B) have proposed methods for using plant leaf veins as templates to create complex microchannels, this technology is a general forming process. Its purpose is to accurately replicate the physical structure of any plant leaf vein, without addressing how to select and match leaf veins according to specific biological simulation needs. To simulate the metastasis of colon cancer to the liver via the portal vein, a biomimetic network that is highly similar to the portal vein system in terms of topology, branching angles, and diameter grading ratios is needed. However, existing technologies do not provide guidance or solutions for this, resulting in biomimetic techniques remaining at the level of "formal resemblance" rather than "spiritual resemblance."

[0062] Lack of system integration and functional design: Existing leaf vein biomimetic fabrication technology aims to obtain an open microfluidic channel structure. However, a functional tumor metastasis research platform is more than just a biomimetic channel. It needs to be a complete, integrated, and operable microfluidic system, including a cell culture area simulating the primary tumor site, a cell co-culture area simulating the target organ (liver) microenvironment, inlet and outlet channels and connecting channels for precise control of fluid and substance exchange, and a cell culture area simulating the regulatory organ (such as the lung). Current technology does not disclose such an integrated chip design capable of simulating multiple key links in the tumor metastasis chain.

[0063] Lack of standardized application methodologies: Even with biomimetic microfluidic structures or chips, how to effectively and reproducibly simulate the complex pathophysiological process of lung-mediated liver metastasis in colon cancer remains an unsolved problem. Existing technologies mainly focus on the "manufacturing" level, lacking a complete set of "application" methodologies, including how to co-culture different types of cells, how to set the perfusion conditions that best simulate portal vein blood flow in vivo, how to induce tumor invasion and entry into circulation, and how to conduct dynamic observation and quantitative analysis.

[0064] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an in vitro research tool and its method of use that can more realistically and accurately simulate the process of liver metastasis in colorectal cancer. To this end, this invention proposes the following technical improvements:

[0065] Addressing the lack of specificity and purposefulness in existing biological applications, such as the core of patent CN 102923639 B, which focuses on the "forming method" itself and aims to "precisely replicate" the veins of any plant with "hard leaf texture and clear veins," representing a general microfluidic manufacturing technology, this patent does not address how to screen and select plant veins based on specific biological simulation needs. One of the innovations of this invention lies in its approach: instead of arbitrarily selecting veins, it purposefully selects specific plant veins (such as leaves from specific banyan or maple varieties) that are highly similar to the portal vein network in terms of topological structure, branching angles, and diameter grading ratios through analysis of the anatomical and hemodynamic characteristics of the portal vein system. This ensures the structural similarity and functional relevance of the biomimetic model.

[0066] The current technology lacks a complete and functional chip system design. For example, patent CN 102923639 B only discloses how to manufacture a single-layer, open microfluidic structure, without providing a complete, closed microfluidic chip system for specific biological research. A functional tumor metastasis chip, in addition to a biomimetic vascular network, integrates other functional units. For example, the chip designed in this invention not only includes a leaf vein channel network mimicking the portal vein, but also integrates a lung simulation chamber for simulating the upstream signaling organ, an upstream tumor cell culture chamber for culturing colon cancer cell clusters (the "primary lesion"), a downstream hepatocyte culture chamber for co-culturing hepatocytes (the "target organ"), and independent inlets and outlets for introducing different fluids (such as culture media, drugs, and immune cells). These functional units are organically connected through microchannels to form an integrated system capable of simulating the entire process of "lung regulation - tumor cell shedding - entry into circulation - targeted colonization".

[0067] Addressing the lack of methodologies in existing technologies for simulating specific pathological processes, such as the CN 102923639 B patent which focuses on "preparing complex leaf vein microfluidic structures," its application prospects are general and do not disclose any specific biological or medical applications. This invention not only provides a physical "chip," but more importantly, it provides a complete "color cancer liver metastasis simulation method." This method includes: how to culture lung cells, colorectal cancer cells, and hepatocytes separately in specific regions of the chip; how to set the flow rate and shear force of the biomimetic blood flow to simulate the physiological conditions of the portal vein; how to induce tumor cells to invade and enter the biomimetic vascular network; and how to use this system for real-time microscopic observation to quantitatively analyze the migration, adhesion, survival, and extravasation behavior of tumor cells in the complex vascular network. Therefore, this model can be used for the screening and evaluation of anti-metastatic drugs.

[0068] like Figure 1 As shown, this embodiment of the invention provides a leaf vein-inspired biofluidic chip that simulates multi-organ interconnection. The microfluidic chip of this embodiment is composed of a polymer layer with microstructures and a substrate layer bonded together. The polymer layer and the substrate layer form a closed microfluidic system, which is structurally divided into a pulmonary circulation zone 1 and an integrated circulation zone 2. The microfluidic system includes: at least one lung simulation chamber 3, at least one upstream colon cancer cell culture chamber 4, at least one downstream hepatocyte culture chamber 5, a leaf vein-inspired microfluidic network 6, an inlet, and an outlet.

[0069] The lung simulation chamber 3 is located within the pulmonary circulation zone 1 and has a sample loading port for inoculating and culturing lung cells to simulate the upstream signaling organ.

[0070] The upstream colon cancer cell culture chamber 4 is located within the systemic circulation zone 2. It is a hollow chamber with a first sample loading port for inoculating and three-dimensionally culturing colon cancer cells to simulate the primary tumor lesion.

[0071] Downstream hepatocyte culture chamber 5, located within systemic circulation zone 2, is a hollow chamber with a second sample loading port, used for three-dimensional co-culture of hepatocytes and vascular endothelial cells to simulate the liver target organ microenvironment.

[0072] The leaf vein biomimetic microfluidic network 6 is made based on the natural plant leaf vein structure. The leaf vein biomimetic microfluidic network 6 constitutes the fluid pathways in the pulmonary circulation zone (1) and the systemic circulation zone (2) respectively, and connects the organ simulation chambers in their respective zones.

[0073] The inlet and outlet are used to connect external pipelines and a fluid pump to construct a perfusion system simulating human blood circulation. The inlet includes a pulmonary circulation inlet 7 and a systemic circulation inlet 9, and the outlet includes a pulmonary circulation outlet 8 and a systemic circulation outlet 10. The physical structure of the chip in this embodiment is as follows: Figure 2 As shown.

[0074] When the chip is in operation, the pulmonary circulation outlet 8 and the systemic circulation inlet 9 are connected via external tubing, allowing the fluid carrying signal molecules flowing from the lung simulation chamber 3 to enter the systemic circulation zone 2. Within the systemic circulation zone 2, the fluid first flows through and surrounds the upstream colon cancer cell culture chamber 4. Tumor cells invading from the upstream colon cancer cell culture chamber 4 enter the channels of the leaf vein-inspired microfluidic network 6. The fluid carrying tumor cells and upstream signal molecules continues to flow within the leaf vein-inspired microfluidic network 6, ultimately reaching the downstream hepatocyte culture chamber 5, simulating the migration of tumor cells under the signal regulation of the upstream organ and their adhesion and extravasation at the target organ. The fluid finally flows out from the systemic circulation outlet 10. The pulmonary circulation inlet 7 is used to inject culture medium into the chip, and the systemic circulation outlet 10 is used to discharge waste liquid or collect samples, thus forming a fluid circulation system.

[0075] The lung simulation chamber 3, the upstream colon cancer cell culture chamber 4, the downstream hepatocyte culture chamber 5, and the leaf vein-inspired microfluidic network 6 are all integrally molded on the bottom surface of the polymer layer. The polymer layer is a PDMS layer, and the base layer is a PMMA or glass layer.

[0076] The microfluidic chip in this invention organically connects the lung simulation chamber, the upstream colon cancer cell culture chamber, and the downstream hepatocyte culture chamber through a multi-level branching microfluidic network (i.e., leaf vein biomimetic microfluidic network) with a complex topological structure derived from natural plant leaf veins. This forms an integrated design of "lung-primary lesion-biomimetic vascular network-target organ", which can connect multiple organs, transmit signal molecules, and simulate the regulatory mechanism of systemic signals generated by upstream organs on downstream tumor metastasis, greatly improving the physiological relevance of the model.

[0077] Embodiment 2 of the present invention provides a method for fabricating a leaf vein-inspired microfluidic chip that simulates multi-organ interconnection, comprising:

[0078] (1) Acquisition and digitization of leaf vein templates: Fresh photinia leaves were selected and placed in an aqueous solution of 7% (w / v) sodium carbonate and 10% (w / v) sodium hydroxide, heated to boiling and maintained for 20 minutes. After removal, the leaves were repeatedly rinsed with distilled water, and the remaining leaf tissue was gently peeled off with a soft brush. After air drying, a smooth leaf vein skeleton was obtained. Grayscale images of the leaf vein skeleton were acquired at a resolution of 1200 dpi using an HP ScanJet Pro N4600 fnw1 scanner. The images were binarized and vectorized to obtain the leaf vein template file of the target leaf. The leaf vein template file contains the leaf vein network.

[0079] (2) Preparation of the master mold: The leaf vein template file was edited, and the layout of the pulmonary circulation zone 1 and systemic circulation zone 2 was designed according to the leaf vein template file. The lung simulation chamber 3, the upstream colon cancer cell culture chamber 4, and the downstream hepatocyte culture chamber 5 were added to the corresponding areas to obtain the target layout file. A high-precision chromium plate photomask was prepared according to the target layout file. A layer of SU-8 2050 photoresist was spin-coated on a 4-inch silicon wafer. By controlling the spin-coating speed (500 rpm for 10s, 2000 rpm for 30s) and the pre-baking process (65℃ for 10min, 95℃ for 30min), a photoresist layer with a thickness of about 110μm was obtained. Ultraviolet light exposure was performed using a SUSS MJB4 lithography machine with an exposure dose of 350 mJ / cm². After post-baking (65℃ for 5min, 95℃ for 15min), it was developed in SU-8 developer for about 10 minutes to obtain the silicon-based master mold.

[0080] (3) PDMS Chip Molding and Assembly: The Sylgard 184 PDMS prepolymer and curing agent were thoroughly mixed at a mass ratio of 10:1 and degassed in a vacuum dryer for 30 minutes. The degassed mixture was then slowly poured onto a master mold that had been treated with trimethylchlorosilane vapor for 30 minutes and cured in an oven at 80°C for 1.5 hours. After curing, the PDMS layer was carefully peeled off from the master mold. A Harris Uni-Core™ 0.6mm punch was used to... Figure 1 As shown, pulmonary circulation inlet 7, pulmonary circulation outlet 8, systemic circulation inlet 9, systemic circulation outlet 10, and sample loading ports for each culture chamber were fabricated at the target locations on the PDMS layer. The PDMS layer and the cleaned PMMA substrate layer 2 were placed together in a plasma cleaner (PDC-002, Harrick Plasma) and treated for 45 seconds at a vacuum of 700 mTorr and high power. Immediately after removal, the treated surfaces of both layers were aligned and bonded together, and then heated in an 80°C oven for 10 minutes to enhance bonding strength. Finally, the chip was secured with screws and nuts.

[0081] The acquisition and digitization of leaf vein templates may include: selecting fresh photinia leaves, placing them in an aqueous solution of 7% (w / v) sodium carbonate and 10% (w / v) sodium hydroxide, heating to boiling and maintaining for 20 minutes, removing them and rinsing them repeatedly with distilled water, gently peeling off the remaining leaf tissue with a soft brush, air-drying to obtain a flat leaf vein skeleton, and scanning to obtain a grayscale image of the leaf vein skeleton with a resolution of 1200 dpi.

[0082] The preparation of the master mold may include: adding circular chambers of a predetermined diameter on both sides of the leaf vein network, which serve as the lung simulation chamber 3, the upstream colon cancer cell culture chamber 4, and the downstream hepatocyte culture chamber 5, respectively; spin-coating a layer of SU-8 2050 photoresist on a 4-inch silicon wafer at 500 rpm for 10 seconds and at 2000 rpm for 30 seconds; pre-baking including baking at 65°C for 10 minutes and baking at 95°C for 30 minutes to obtain a photoresist layer with a thickness of approximately 110 μm; exposing the layer to ultraviolet light using a SUSS MJB4 lithography machine at an exposure dose of 350 mJ / cm²; post-baking including baking at 65°C for 5 minutes and baking at 95°C for 15 minutes; and then developing the layer in SU-8 developer for approximately 10 minutes to obtain the silicon-based master mold.

[0083] In the PDMS chip molding and assembly process, PDMS prepolymer and curing agent are mixed at a mass ratio of 10:1 and placed in a vacuum dryer for 30 minutes to degas. The degassed mixture is then slowly poured onto the silicon-based master mold treated with trimethylchlorosilane vapor and cured in an 80°C oven for 1.5 hours. After curing, the PDMS layer is peeled off from the silicon-based master mold to obtain the polymer layer. Pulmonary circulation inlet 7, pulmonary circulation outlet 8, systemic circulation inlet 9, systemic circulation outlet 10, and sample loading ports for each culture chamber are fabricated at the target locations on the PDMS layer. The PDMS layer and PMMA substrate layer are placed together in a plasma cleaner and treated for 45 seconds at a vacuum of 700 mTorr and high power. After removal, the treated surfaces of both layers are immediately aligned and bonded together, and then heated in an 80°C oven for 10 minutes to enhance bonding strength. Finally, the chip is secured with screws and nuts.

[0084] Embodiment 3 of the present invention provides a method for establishing a multi-organ interconnected tumor metastasis model, comprising the following steps:

[0085] Chip pretreatment: The microfluidic chip is rinsed with ethanol and deionized water, sterilized by autoclaving, and bovine fibrinogen solution is injected into the microfluidic system for incubation in an incubator.

[0086] Three-dimensional culture of multiple organ cells: Prepare a cell suspension containing lung cells, colon cancer cells, hepatocytes, and vascular endothelial cells; mix the cell suspension with fibrin hydrogel precursor solution; inject the hydrogel mixture containing lung cells into the lung simulation chamber 3 through the corresponding sample dispensing port; inject the hydrogel mixture containing colon cancer cells into the upstream colon cancer cell culture chamber 4 through the first sample dispensing port; inject the hydrogel mixture containing hepatocytes and vascular endothelial cells into the downstream hepatocyte culture chamber 5 through the second sample dispensing port; place the microfluidic chip in an incubator for static incubation to allow the hydrogel to solidify and form a three-dimensional tissue.

[0087] Dynamic perfusion and transfer simulation:

[0088] a. Establish a circulation path: Seal all sample application ports with sterile pins, connect the peristaltic pump to the inlet and outlet ports of the chip through tubing, and construct a complete circulation path that simulates the connection between pulmonary circulation and systemic circulation.

[0089] b. Set process parameters: Perform continuous dynamic culture at a constant flow rate, and introduce stimulants into the pulmonary circulation zone 1 at specific time points to induce the generation of signaling molecules in the lung simulation chamber 3.

[0090] Observation and analysis of the transfer process:

[0091] Real-time imaging: Using an inverted confocal fluorescence microscope, live-cell imaging of the chip is performed daily; by observing colon cancer cells pre-labeled with fluorescent proteins, the entire process of their invasion from the upstream colon cancer cell culture chamber 4 after being affected by upstream lung signals, entering the leaf vein biomimetic microfluidic network 6, and appearing, adhering and proliferating in the liver tissue microenvironment of the downstream hepatocyte culture chamber 5 is tracked.

[0092] Data analysis: Quantitative analysis of the acquired fluorescence images.

[0093] In the chip pretreatment, the microfluidic chip can be rinsed three times with 75% ethanol and deionized water for 5 minutes each time, and then autoclaved. 1.5 mg / mL bovine fibrinogen solution is injected into the microfluidic system of the chip and incubated in a 37°C incubator for 1 hour.

[0094] In the three-dimensional culture of multiple organ cells, lung cells were represented by human alveolar epithelial cells (HPAEpic), and colon cancer cells were represented by HT-29 cells at a concentration of 2.5 × 10⁻⁶. 6 cells / mL; hepatocytes were HepG2, vascular endothelial cells were HUVEC, HepG2 and HUVEC were mixed at a ratio of 7:1, and the total cell concentration was 7.5 × 10⁻⁶ cells / mL; 6 The final concentration of the fibrin hydrogel precursor solution was 3 mg / mL fibrinogen and 6 U / mL thrombin. The cell suspension and the fibrin hydrogel precursor solution were mixed at a 1:1 volume ratio. The hydrogel mixture containing HT-29 cells was injected into the upstream colon cancer cell culture chamber 3 through the first sample feeding port, 2 µL per chamber. The hydrogel mixture containing HepG2 / HUVEC cells was injected into the downstream hepatocyte culture chamber 4 through the second sample feeding port, 4 µL per chamber. The chip was placed in a 37°C incubator and incubated overnight to allow the hydrogel to solidify and form a three-dimensional tissue.

[0095] In setting the process parameters, dynamic culture is carried out at a constant flow rate of 60 µL / min; a routine culture of 2 days can be performed first to stabilize the organs and tissues, and then a specific stimulant is added to the culture medium in the pulmonary circulation zone 1, and culture is continued for 3-5 days to simulate the promoting effect of pulmonary signals on liver metastasis of colon cancer; the cell culture medium is: 50% EGM + 50% DMEM.

[0096] Specifically, (1) Cell preparation and culture: Lung cells could be human alveolar epithelial cells (HPAEpic), human colon cancer cells (HT-29) transfected with lentivirus expressing mCherry red fluorescent protein; human liver cancer cells (HepG2); and human umbilical vein endothelial cells (HUVEC). HT-29 and HepG2 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin; HUVEC was cultured in EGM-2 medium. All cells were placed in a cell culture incubator at 37°C and 5% CO2.

[0097] (2) Chip pretreatment and cell seeding: The assembled chip was disinfected and moistened by perfusing with 75% ethanol and sterile PBS for 15 minutes each. Then, it was perfused with 1.5 mg / mL bovine fibrinogen solution and incubated at 37°C for 1 hour. Excess liquid was aspirated. Preparation of cell-hydrogel mixture: HT-29 cells were resuspended in DMEM to a concentration of 5 × 10⁻⁶ cells / mL. 6 The concentration of cells / mL was rapidly mixed with an equal volume of a mixture of 6 mg / mL fibrinogen and 12 U / mL thrombin (operated on ice), and 2 µL of this mixture was injected into the upstream colon cancer cell culture chamber 4 through the first injection port. Similarly, HepG2 and HUVEC were mixed at a 7:1 ratio and resuspended in DMEM to a total concentration of 1.5 × 10⁻⁶ cells / mL. 7 The cell / mL sample was mixed with an equal volume of hydrogel precursor solution, and 4 µL of the mixture was injected into the downstream hepatocyte culture chamber 5 through the second sample loading port. The chip was then placed in an incubator and allowed to stand for 1 hour until the hydrogel was completely cured.

[0098] (3) Dynamic perfusion culture: Seal all sample loading ports with a sterile steel needle. Connect the peristaltic pump (Langer Instruments, BT100-2J) to the inlet through a silicone tube with an inner diameter of 0.5 mm. Set the flow rate to 60 µL / min and perfuse the cell culture medium (50% EGM-2 + 50% DMEM) preheated to 37°C into the chip for continuous dynamic culture for 5 days.

[0099] (4) Observation of the metastasis process: Starting from day 2 of culture, the chip was scanned and imaged daily using a Zeiss LSM 900 confocal microscope. Using a 20x objective lens, green and red fluorescence channels (for observing HT-29 cells) and bright field channels were acquired respectively. On day 5 of culture, red fluorescent HT-29 cells were observed to have migrated from the upstream colon cancer cell culture chamber 4 and appeared in the downstream hepatocyte culture chamber 5, forming close contact with the liver tissue therein, indicating the successful establishment of the metastasis model.

[0100] Compared with the prior art, the technical solution provided by the embodiments of the present invention has at least the following positive effects:

[0101] High biomimicry: By using natural leaf veins as a template, the leaf vein biomimetic microfluidic network of the present invention can realistically reproduce the complex multi-level bifurcation structure of blood vessels in the body (such as the portal vein), and can provide a hemodynamic microenvironment that is closer to the physiological state.

[0102] System integration: This invention integrates four key functional units—the primary tumor site, the circulatory vascular network, the target organ, and the lung organ—onto a single chip, which can simulate the complete chain of "lung regulation-invasion-circulation-colonization" in tumor metastasis;

[0103] Dynamic visualization: The transparent design of the chip, combined with fluorescent labeling technology, enables real-time, dynamic, and high-resolution live-cell imaging of the entire process of tumor cell metastasis, providing a powerful visualization tool for revealing the metastasis mechanism;

[0104] High reproducibility of the method: This invention provides a standardized chip preparation and experimental operation procedure, including specific equipment parameters, reagent concentrations and operation times, which ensures the reliability and reproducibility of the research results and makes high-throughput drug screening possible.

[0105] Note that the above description is merely a preferred embodiment of the present invention and the technical principles employed. Those skilled in the art will understand that the present invention is not limited to the specific embodiments described herein, and various obvious changes, readjustments, and substitutions can be made without departing from the scope of protection of the present invention. Therefore, although the present invention has been described in detail through the above embodiments, the present invention is not limited to the above embodiments, and may include many other equivalent embodiments without departing from the concept of the present invention, the scope of which is determined by the scope of the appended claims.

Claims

1. A leaf vein-inspired microfluidic chip simulating multi-organ interconnection, characterized in that, include: It is composed of a polymer layer with a microstructure and a base layer bonded together. The polymer layer and the base layer bond together to form a closed microfluidic system. The microfluidic system is structurally divided into a pulmonary circulation zone (1) and an integral circulation zone (2). The microfluidic system includes: At least one lung simulation chamber (3), located within the pulmonary circulation zone (1), has a sample loading port for inoculating and culturing lung cells to simulate an upstream signaling organ; At least one upstream colon cancer cell culture chamber (4) is located within the systemic circulation zone (2), which is a hollow chamber with a first sample loading port for inoculating and three-dimensionally culturing colon cancer cells to simulate the primary tumor site; At least one downstream hepatocyte culture chamber (5) is located within the systemic circulation zone (2). It is a hollow chamber with a second sample loading port for three-dimensional co-culture of hepatocytes and vascular endothelial cells to simulate the liver target organ microenvironment. A leaf vein biomimetic microfluidic network (6), which is made according to the natural plant leaf vein structure, constitutes the fluid pathways in the pulmonary circulation zone (1) and systemic circulation zone (2) respectively, and connects the organ simulation chambers in their respective areas; as well as The inlet and outlet are used to connect external pipelines and fluid pumps to construct a perfusion system that simulates human blood circulation.

2. The chip according to claim 1, characterized in that, The inlet includes a pulmonary circulation inlet (7) and a systemic circulation inlet (9); the outlet includes a pulmonary circulation outlet (8) and a systemic circulation outlet (10). When the chip is working, the pulmonary circulation outlet (8) and the systemic circulation inlet (9) are connected through an external pipeline, so that the fluid carrying signal molecules flowing out of the lung simulation chamber (3) can enter the systemic circulation zone (2). The fluid first flows through and surrounds the upstream colon cancer cell culture chamber (4) in the systemic circulation zone (2). The tumor cells that invade from the upstream colon cancer cell culture chamber (4) enter the flow channel of the leaf vein biomimetic microfluidic network (6). The fluid carrying tumor cells and upstream signal molecules continues to flow in the leaf vein biomimetic microfluidic network (6) and finally reaches the downstream hepatocyte culture chamber (5) to simulate the process of tumor cell migration under the signal regulation of upstream organs and adhesion and extravasation at the target organ. The fluid finally flows out from the systemic circulation outlet (10).

3. The chip according to claim 1, characterized in that, The lung simulation chamber (3), the upstream colon cancer cell culture chamber (4), the downstream hepatocyte culture chamber (5), and the leaf vein biomimetic microfluidic network (6) are all integrally formed on the bottom surface of the polymer layer.

4. A method for establishing a multi-organ interconnected tumor metastasis model, characterized in that, Cell model establishment based on the microfluidic chip as described in any one of claims 1 to 3; The method includes: Chip pretreatment: The microfluidic chip is rinsed with ethanol and deionized water, sterilized by autoclaving, and bovine fibrinogen solution is injected into the microfluidic system for incubation in an incubator; Three-dimensional culture of multiple organ cells: Prepare a cell suspension containing lung cells, colon cancer cells, hepatocytes and vascular endothelial cells; mix the cell suspension with fibrin hydrogel precursor solution; inject the hydrogel mixture containing the lung cells into the lung simulation chamber (3) through the corresponding sample dispensing port. The hydrogel mixture containing the colon cancer cells is injected into the upstream colon cancer cell culture chamber (4) through the first sample loading port. The hydrogel mixture containing the hepatocytes and vascular endothelial cells is injected into the downstream hepatocyte culture chamber (5) through the second sample loading port. The microfluidic chip was placed in an incubator and incubated statically to allow the hydrogel to solidify and form a three-dimensional tissue. Dynamic perfusion and transfer simulation: a. Establish a circulation path: Seal all sample application ports with sterile pins, connect the peristaltic pump to the inlet and outlet of the chip through tubing, and construct a complete circulation path that simulates the connection between pulmonary circulation and systemic circulation; b. Set process parameters: Continuous dynamic culture is carried out at a constant flow rate, and stimulants can be introduced into the pulmonary circulation zone (1) at a specific time point to induce the lung simulation chamber (3) to generate signal molecules; Observation and analysis of the transfer process: Real-time imaging: The chip is imaged daily using an inverted confocal fluorescence microscope; by observing the colon cancer cells pre-labeled with fluorescent protein, the entire process of their invasion from the upstream colon cancer cell culture chamber (4) after being affected by upstream lung signals, entering the leaf vein biomimetic microfluidic network (6), and appearing, adhering and proliferating in the liver tissue microenvironment of the downstream hepatocyte culture chamber (5) is tracked. Data analysis: Quantitative analysis of the acquired fluorescence images.

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