3D visual bionic intestine-blood vessel-nerve interface organ simulation chip

Abstract

The invention discloses a 3D (three-dimensional) visual bionic intestine-blood vessel-nerve interface organ simulation chip, which is characterized in that a micro-fluidic chip is used as a platform to construct an intestinal tract-blood vessel-brain system which simulates biological transformation of perfluorinated compounds in intestinal tracts under near physiological conditions and biologically transports the perfluorinated compounds through blood vessels; a neuronal cell culture chamber, a vascular endothelial cell culture chamber and an intestinal tract cell culture chamber are respectively arranged on the micro-fluidic chip, the culture chambers are communicated through micro-columns, and the vascular endothelial cell culture chamber serves as a barrier in the middle; perfluorinated compounds enter corresponding cell culture cavities for culture through reagent injection channels of different cell culture channels, generated metabolites are discharged through the channels, and multi-organ interaction and toxicity induced by the perfluorinated compounds are analyzed. The chip can simulate an intestinal barrier structure, a blood vessel and a neural network structure, can highly simulate biological transport and transformation of a perfluorinated compound between intestines and brains, and effectively reduces cost and time.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to a 3D visualized bionic intestinal-vascular-nerve interface organ simulation chip. Background Art

[0002] More than 12,000 per- and polyfluoroalkyl substances (PFAS) have been developed and produced worldwide and are widely used in products such as food packaging bags, non-stick pans, and water-based film-forming foams. However, due to their high stability, persistence, toxicity, and long-distance transport ability, PFAS have triggered new ecological and environmental crises. Humans are exposed to PFAS through various pathways, including ingestion of contaminated food and water, and inhalation of contaminated dust and air. A growing number of studies have shown that PFAS exposure causes a series of adverse effects on human health, including vascular toxicity, metabolic dysfunction, endocrine disruption, and developmental toxicity. Therefore, studying the bioaccumulation of PFAS in multiple organs and their interactions is of great significance for assessing the toxicity of PFAS and its impact on life and health.

[0003] Currently, standard methods for toxicological analysis of compounds are primarily based on animal experiments. However, limitations such as long cycles, high costs, and large sample volumes make animal experiments challenging for the current large number of compound toxicological analyses. Therefore, there is an urgent need to develop in vitro models that require less reagent and sample, have shorter experimental cycles, more accurate dose responses, and are easier to observe. To this end, the US Food and Drug Administration (FDA) has proposed phasing out animal testing by 2035, emphasizing the development of more alternative, humanized in vitro models as a key direction for future development.

[0004] Microfluidic chips, often referred to as lab-on-a-chips or cell chips, range in size from micrometers to millimeters, similar in size to cells and their substructures. They can simulate the microenvironment of cells in the body and provide growth conditions closer to those in vivo. By reducing sample size and reagent costs, and providing researchers with precise control and predictability over the spatiotemporal dynamics of cellular microenvironments and fluids, they have the potential to replace traditional experiments. Currently, researchers have developed gut chips and brain chips to study the health effects of environmental pollutants, but most of them are based on a single organ and cannot accurately simulate the biological transport of exogenous environmental pollutants in the body. Summary of the Invention

[0005] The purpose of the present invention is to provide a 3D visualized biomimetic intestinal-vascular-neural interface organ simulation chip, which can simulate the intestinal barrier structure, blood vessels and neural network structure, and can highly simulate the biological transport and transformation of perfluorinated compounds between the intestine and the brain, effectively reducing costs and time.

[0006] The purpose of the present invention is achieved through the following technical solutions: A 3D visualized biomimetic intestinal-vascular-neural interface organ simulation chip, which uses a microfluidic chip as a platform to construct an intestinal-vascular-brain system that simulates the biotransformation of perfluorinated compounds in the intestine and their biotransport through blood vessels under near-physiological conditions. The microfluidic chip is provided with a neuronal cell culture chamber, a vascular endothelial cell culture chamber, and an intestinal cell culture chamber, wherein: The culture chambers are connected by microcolumns, and the vascular endothelial cell culture chamber acts as a barrier in the middle, connecting the neuronal cell culture chamber and the intestinal cell culture chamber on both sides and confining them in their respective channels; Perfluorinated compounds are injected into the corresponding cell culture chamber through the reagent injection channels of different cell culture channels for culture. The metabolites produced are discharged through the channels to analyze the multi-organ interactions and toxicity induced by perfluorinated compounds.

[0007] It can be seen from the technical solution provided by the above-mentioned present invention that the above-mentioned chip can simulate the intestinal barrier structure, blood vessels and neural network structure, and can highly simulate the biological transport and transformation of perfluorinated compounds between the intestine and the brain, effectively reducing costs and time. The evaluation results will be more objective and true, and can be applied to the in vitro toxicological evaluation of various environmental pollutants and drugs, providing new technical support for the health risk assessment of agricultural environmental pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0009] Figure 1 A schematic diagram of the structure of a 3D visualized bionic intestinal-vascular-nerve interface organ simulation chip provided by an embodiment of the present invention; Figure 2 This is a structural diagram of the intestinal-vascular-brain system of the microfluidic chip according to an embodiment of the present invention; Figure 3 This is a graph showing the CCK-8 results of the perfluorinated compounds described in the examples of the present invention on intestinal tract, vascular endothelial cells and nerve cells. DETAILED DESCRIPTION

[0010] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments, and do not constitute a limitation of the present invention. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0011] like Figure 1 The figure shows a schematic diagram of the structure of a 3D visualized biomimetic intestinal-vascular-neural interface organ simulation chip provided by an embodiment of the present invention. The simulation chip uses a microfluidic chip as a platform to construct an intestinal-vascular-brain system that simulates the biotransformation of perfluorinated compounds in the intestine and their biotransport through the blood vessels under near-physiological conditions. The microfluidic chip is respectively provided with a neuronal cell culture chamber 2, a vascular endothelial cell culture chamber 3, and an intestinal cell culture chamber 4, wherein: The culture chambers are connected by microcolumns, and the vascular endothelial cell culture chamber 3 acts as a barrier in the middle, connecting the neuronal cell culture chamber 2 and the intestinal cell culture chamber 4 on both sides and confining them in their respective channels; This setup allows vascular microtubule sprouts to migrate and grow toward the intestinal or neuronal channels and grow on one side of the extracellular matrix (ECM) hydrogel, interacting only with vascular endothelial cells; e.g. Figure 2 Figure 2 shows the structure of the microfluidic chip gut-blood vessel-brain system according to an embodiment of the present invention; A is a bright field image of the vascular lumen, B is a bright field image of the neural network, and C is a bright field image of the intestine; Perfluorinated compounds are injected into the corresponding cell culture chamber through the reagent injection channels of different cell culture channels for culture. The metabolites produced are discharged through the channels to analyze the multi-organ interactions and toxicity induced by perfluorinated compounds.

[0012] In a specific implementation, the step of allowing the perfluorinated compound to enter the corresponding cell culture chamber through reagent injection channels of different cell culture channels for culture includes: The resuspended human colon cancer cell (HCT116) suspension was added to intestinal cell culture chamber 4, and then the simulation chip was placed on its side in a 37°C incubator for 30 min; The resuspended human umbilical vein endothelial cell (HUVEC) suspension was added to the vascular endothelial cell culture chamber 3, and then the simulation chip was placed flat in a 37°C incubator for 30 min; The resuspended mouse hippocampal neuronal cell (HT22) suspension was added to the neuronal cell culture chamber 2, and then the simulation chip was placed flat in a 37°C incubator overnight; After the system is stabilized, the perfluorochemical stock solution is dissolved in the culture medium, and then injected into the intestinal cell culture chamber 4, and then placed in a 37°C incubator for 24 hours.

[0013] In the specific implementation, the multi-organ interactions and toxicity induced by perfluorinated compounds were analyzed. The specific process is as follows: Prepare a culture medium containing 1 mg / mL dextran solution (FITC-Dextran 70kDa). Add 5 μL of preheated culture medium containing 1 mg / mL dextran solution to intestinal cell culture chamber 4. Replace the medium in endothelial cell culture chamber 3 as usual and incubate in a 37°C incubator for 24 h. After incubation, collect the culture medium from endothelial cell culture chamber 3 and measure the fluorescence intensity of the culture medium to assess the permeability of the intestinal epithelial barrier. Set up three replicates for each group to eliminate errors caused by experimental operation and measurement. After the simulation chip was running normally, 50 ppm of perfluorinated compounds were added to the intestinal cell culture chamber 4 and cultured for 24 hours. Then, cell activity, the length, branch points, and number of vascular sprouts and axons in the endothelial cell culture chamber 3 and the neuronal cell culture chamber 2 were measured to characterize the degree of vascular damage. Vascular endothelial cadherin (VE-cadherin) and β3-tubulin are two key proteins that maintain tight junctions between vascular endothelial cells and neurons. Their expression can reflect structural integrity and normal function. Therefore, we used immunohistochemistry to detect the expression and localization of these two proteins.

[0014] In addition, reactive oxygen species (ROS), an important signaling molecule for the progression of inflammation, were further measured using a 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe. The following steps were performed: 10 μM phosphate buffered saline (PBS) was prepared, and the chip was flushed with phosphate buffered saline (PBS) for 5 minutes using a flow pump 24 hours after administration. Fluorescence solution was then flowed for 30 minutes, and observation was performed using a fluorescence microscope in the dark. At the same time, a negative control group was set up, and three parallel experiments were set up in each group to eliminate the errors caused by experimental operations and measurements.

[0015] like Figure 3 The graph shows the CCK-8 results of the perfluorinated compounds described in the examples of the present invention on the intestinal tract, vascular endothelial cells and nerve cells. Figure 3 It can be seen that with the increase of PFAS concentration, the cell survival rate of HT22 cells when the concentration is higher than 200 ppm, HUVEC cells when the concentration is higher than 100 ppm, and HCT116 cells when the concentration is higher than 50 ppm all decreases to below 50%.

[0016] In addition, the preparation process of the microfluidic chip is as follows: Spin coating: First, drop negative photoresist (model SU8-2050) on the silicon wafer and spin coat at 800 rpm for 60 s; Pre-baking: Then heat the silicon wafer at 65°C for 3 minutes and at 95°C for 7 minutes, and cool it naturally to room temperature; repeat this operation 3 times, and extend the pre-baking time after each spin-off; Exposure, used to initiate cross-linking of the photoresist: Specifically, the mask is placed on the silicon wafer and pressed with glass, then transferred to the exposure stage as a whole for 3 minutes (1 minute per exposure, 3 separate exposures). The mask, consisting of a substrate and a light-shielding film, is a pattern transfer tool or master in the microelectronics manufacturing process, used to transfer the pattern to the silicon wafer. Post-baking is used to ensure that the cross-linking reaction is complete. Specifically, after exposure, the mask and silicon wafer are separated, and the silicon wafer is placed on a hot plate and heated at 65°C for 3 minutes and then at 95°C for 20 minutes, and then cooled naturally to room temperature. Development is used to remove uncrosslinked photoresist: Specifically, the silicon wafer is thoroughly washed with developer to remove uncrosslinked photoresist, and the surface of the silicon wafer is cleaned with isopropyl alcohol to obtain a chip template; the chip template is then placed in a 65°C oven and baked for 30 minutes to evaporate the residual isopropyl alcohol; Silanization treatment: Specifically, the chip template after development treatment is placed in a vacuum desiccator, 10 μL of silanization reagent is added, and the vacuum pump is turned on to form a negative pressure in the vacuum desiccator. The chip template is silanized in the atmosphere of the silanization reagent to complete the production of the chip positive mold; Glue pouring: The polydimethylsiloxane (PDMS) prepolymer and initiator are thoroughly stirred and mixed, then poured onto the prepared chip positive mold. After vacuum degassing for 20 minutes to remove bubbles, the mixture is placed in a 65°C oven for heating and curing for more than 2 hours. The PDMS substrate (about 5mm thick) is peeled off, and the chip is cut and punched. The main component of the initiator is a silicon-containing crosslinking agent. Bonding: Place the cut PDMS substrate and chip together in a plasma cleaning machine for plasma cleaning, then quickly put the two together, press gently, and place the bonded chip in a 65°C oven for 20 minutes to strengthen the bonding, and finally obtain a microfluidic chip.

[0017] The material of the microfluidic chip is a light-permeable and air-permeable polydimethylsiloxane (PDMS) polymer; the mass ratio of the polydimethylsiloxane (PDMS) monomer to the initiator is 10:1.

[0018] In specific implementation, such as Figure 1 As shown, the vascular endothelial cell culture chamber 3 is provided with an array microcolumn, and the length and width of the array microcolumn are 0.5*0.15 mm; The length and width of a single culture chamber are 18.5*1 mm; each culture chamber is provided with a liquid inlet and outlet 1, which plays a role of ventilation and serves as an air vent.

[0019] The cell culture chamber of the simulated chip is made of Matrigel. Specifically, the cell culture chamber provides a scaffold system similar to that in the body, creating a growth environment similar to that in the body, promoting cell proliferation and differentiation, and exhibiting similar in vivo tissue structure and functional characteristics.

[0020] The above-mentioned simulation chip can be used to simulate the spatial arrangement of intestinal cells related to intestinal barrier function, the spatial arrangement of cells related to vascular function in the portal vein system, and the spatial arrangement of nerve cells related to the neural network.

[0021] It should be noted that the contents not described in detail in the embodiments of the present invention belong to the prior art known to those skilled in the art.

[0022] In summary, the simulation chip described in the embodiment of the present invention is demand-oriented, with the goal of reducing the need for animal experiments, achieving more accurate toxicity assessments, and promoting personalized medicine. It has developed microfluidic chip technology, and proposed a neuron-blood vessel-intestine model using gel three-dimensional culture technology. The simulation chip can highly simulate the biological transport and transformation of perfluorinated compounds between the intestine and the brain, effectively reducing costs and time. The evaluation results will be more objective and realistic, and can be applied to the in vitro toxicological evaluation of various environmental pollutants and drugs, providing new technical support for the health risk assessment of agricultural environmental pollutants.

[0023] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims. The information disclosed in the background technology section of this article is only intended to deepen the understanding of the overall background technology of the present invention, and should not be regarded as an admission or any form of implication that the information constitutes prior art already known to those skilled in the art.

Claims

1. A 3D visualized biomimetic intestinal-vascular-nerve interface organ simulation chip, characterized in that: The simulation chip uses a microfluidic chip as a platform to construct an intestinal-vascular-brain system that simulates the biotransformation of perfluorinated compounds in the intestine and their biotransport through blood vessels under near-physiological conditions. The microfluidic chip is respectively provided with a neuronal cell culture chamber, a vascular endothelial cell culture chamber, and an intestinal cell culture chamber, wherein: The culture chambers are connected by microcolumns, and the vascular endothelial cell culture chamber acts as a barrier in the middle, connecting the neuronal cell culture chamber and the intestinal cell culture chamber on both sides and confining them in their respective channels; Perfluorinated compounds are injected into the corresponding cell culture chamber through the reagent injection channels of different cell culture channels for culture. The metabolites produced are discharged through the channels to analyze the multi-organ interactions and toxicity induced by perfluorinated compounds.

2. The 3D visualized bionic intestinal-vascular-nerve interface organ simulation chip according to claim 1, characterized in that: The step of allowing the perfluorochemical compound to enter the corresponding cell culture chamber through the reagent injection channels of different cell culture channels for culture specifically includes: The resuspended human colon cancer cell HCT116 suspension was added to the intestinal cell culture chamber, and then the simulation chip was placed on its side in a 37°C incubator for 30 min; The resuspended human umbilical vein endothelial cell (HUVEC) suspension was added into the vascular endothelial cell culture chamber, and then the simulation chip was placed flat in a 37°C incubator for 30 min; The resuspended mouse hippocampal neuronal cell HT22 suspension was added to the neuronal cell culture chamber, and then the simulation chip was placed flat in a 37°C incubator overnight; After the system is stabilized, the perfluorochemical stock solution is dissolved in the culture medium, then injected into the intestinal cell culture chamber, and then placed in a 37°C incubator for 24 hours.

3. The 3D visualized bionic intestinal-vascular-nerve interface organ simulation chip according to claim 2, characterized in that: Analyze the multi-organ interactions and toxicity induced by perfluorinated compounds. The specific process is as follows: Prepare a culture medium containing 1 mg / mL dextran solution. Add 5 μL of preheated culture medium containing 1 mg / mL dextran solution to the intestinal cell culture chamber. Replace the medium in the vascular endothelial cell culture chamber as usual, and incubate in a 37°C incubator for 24 hours. After incubation, collect the culture medium from the vascular endothelial cell culture chamber and measure the fluorescence intensity of the culture medium to assess the permeability of the intestinal epithelial barrier. Set up three replicates for each group to eliminate the influence of experimental operation and measurement errors. After the chip was simulated to operate normally, 50 ppm of perfluorinated compounds were added to the intestinal cell culture chamber and cultured for 24 hours. Then, cell activity, vascular sprout and axon length, branch points, and mesh number were measured in the endothelial cell culture chamber and the neuronal cell culture chamber to characterize the degree of vascular damage. Reactive oxygen species (ROS), as an important signaling molecule for the progression of inflammation, were further measured using a 2',7'-dichlorofluorescein diacetate probe, where: Prepare 10 μM phosphate buffered saline (PBS). First, use a flow pump to rinse the chip with phosphate buffered saline (PBS) for 5 minutes after 24 hours of drug administration. Then, flow fluorescent solution for 30 minutes and observe with a fluorescence microscope in the dark. At the same time, a negative control group was set up, and three parallel experiments were set up in each group to eliminate the errors caused by experimental operations and measurements.

4. The 3D visualized bionic intestinal-vascular-nerve interface organ simulation chip according to claim 1, characterized in that: The preparation process of the microfluidic chip is as follows: Spin coating: First, drop the negative photoresist onto the silicon wafer and spin coat at 800 rpm for 60 s. Pre-baking: Then heat the silicon wafer at 65°C for 3 minutes and at 95°C for 7 minutes, and cool it naturally to room temperature; repeat this operation 3 times, and extend the pre-baking time after each spin-off; Exposure, used to initiate cross-linking of the photoresist: Specifically, the mask is placed on the silicon wafer, pressed with glass, and then transferred to the exposure stage for 3 minutes. The mask, consisting of a substrate and a light-shielding film, is a pattern transfer tool or master in the microelectronics manufacturing process, used to transfer the pattern to the silicon wafer. Post-baking is used to ensure that the cross-linking reaction is complete. Specifically, after exposure, the mask and silicon wafer are separated, and the silicon wafer is placed on a hot plate and heated at 65°C for 3 minutes and then at 95°C for 20 minutes, and then cooled naturally to room temperature. Development is used to remove uncrosslinked photoresist: Specifically, the silicon wafer is thoroughly washed with developer to remove uncrosslinked photoresist, and the surface of the silicon wafer is cleaned with isopropyl alcohol to obtain a chip template; the chip template is then placed in a 65°C oven and baked for 30 minutes to evaporate the residual isopropyl alcohol; Silanization treatment: Specifically, the chip template after development treatment is placed in a vacuum desiccator, 10 μL of silanization reagent is added, and the vacuum pump is turned on to form a negative pressure in the vacuum desiccator. The chip template is silanized in the atmosphere of the silanization reagent to complete the production of the chip positive mold; Glue pouring: The polydimethylsiloxane (PDMS) prepolymer and initiator are thoroughly stirred and mixed, and then poured onto the prepared chip positive mold. After vacuum degassing for 20 minutes to remove bubbles, the mixture is placed in a 65°C oven for heating and curing for more than 2 hours. The PDMS substrate is peeled off, and the chip is cut and punched. The initiator is a silicon-containing crosslinking agent. Bonding: Place the cut PDMS substrate and chip together in a plasma cleaning machine for plasma cleaning, then quickly put the two together and press them gently. Place the bonded chip in a 65°C oven for 20 minutes to strengthen the bonding, and finally obtain a microfluidic chip.

5. The 3D visualized bionic intestinal-vascular-nerve interface organ simulation chip according to claim 1, characterized in that: The vascular endothelial cell culture chamber is provided with an array of microcolumns, and the length and width of the array microcolumns are 0.5*0.15 mm; The length and width of a single culture chamber are 18.5*1 mm; each culture chamber is provided with a liquid inlet and outlet, which plays a role of ventilation and serves as an air vent.

6. The 3D visualized bionic intestinal-vascular-nerve interface organ simulation chip according to claim 4, characterized in that: The material of the microfluidic chip is a light-permeable and breathable polydimethylsiloxane (PDMS) polymer; The ratio of polydimethylsiloxane (PDMS) monomer to initiator is 10:

1.

7. The 3D visualized bionic intestinal-vascular-nerve interface organ simulation chip according to claim 1, characterized in that: The material used for the cell culture chamber of the simulation chip is matrix glue.

8. The 3D visualized bionic intestinal-vascular-nerve interface organ simulation chip according to claim 1, characterized in that: The simulation chip can be used to simulate the spatial arrangement of intestinal cells related to intestinal barrier function, the spatial arrangement of cells related to vascular function in the portal vein system, and the spatial arrangement of nerve cells related to the neural network.

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