Bionic blood brain barrier micro-fluidic chip device and application thereof

Abstract

The invention provides a bionic blood brain barrier micro-fluidic chip device and application thereof, and belongs to the technical field of micro-fluidic chips. According to the invention, by utilizing the micro-fluidic chip design of the modularized concentration gradient generation module and the blood brain barrier bionic module and the chip design of the unique snowflake-shaped concentration gradient generation module, the concentration gradient generation and the neuroinflammation model are combined on one platform, so that the drug screening can be efficiently carried out; and the dose-effect relationship and the synergistic effect of the medicine can be accurately evaluated.

Description

Technical Field

[0001] The present invention relates to the technical field of bionic chips, and in particular to a bionic blood-brain barrier microfluidic chip device and applications thereof. Background Art

[0002] The blood-brain barrier (BBB) ​​is a highly selective permeable barrier between the brain and the blood, composed of cerebral vascular endothelial cells, tight junctions, basement membranes, and supporting cells (such as astrocytes). Its main function is to protect the brain from potentially harmful substances while maintaining the stability of the microenvironment of the nervous system. The characteristics of the BBB make it a key challenge in drug development, as many drugs cannot effectively cross this barrier to treat central nervous system diseases.

[0003] In traditional studies, researchers use animal models or cell models to simulate the BBB. However, each of these models has its limitations. Animal models may not be able to fully simulate the characteristics of the human BBB, and animal experiments are usually long and costly, and usually require strict ethical review. Cell models, such as in vitro brain endothelial cell monolayer models, can provide a certain amount of information in drug screening experiments, but often cannot locally reproduce the complex structure and function of the BBB. Therefore, the development of a more accurate and reproducible BBB model is crucial for drug screening and neuroscience research.

[0004] Microfluidic chip technology has been widely used in biomedical research in recent years. This technology achieves highly biomimetic, high-throughput, low-loss, and low-cost experimental operations by precisely controlling fluids at the micro and nano scales. The advantages of microfluidic chips include efficient model establishment, low sample consumption, and high-sensitivity detection. Especially in drug screening and disease model construction, microfluidic chip technology can provide more physiologically relevant experimental conditions and improve the repeatability and reliability of experiments.

[0005] Modular design is an emerging trend in chip technology applications. Compared with traditional integrated chip design, modular design allows independent development, testing and combination of different functional modules, which has significant advantages:

[0006] First, modular design simplifies the chip design and manufacturing process, making it easier to create complex systems;

[0007] Secondly, it improves the flexibility of the experiment. Different modules can be combined and configured as needed to meet a variety of experimental requirements.

[0008] In addition, the modular design also lowers the technical threshold, making it easier for researchers in the field of life sciences to conduct experiments;

[0009] Finally, the modular design allows for separate collection of biological samples in different channels during an experiment, which is often difficult to achieve in a one-piece design.

[0010] In the drug development process, the drug's ability to cross the blood-brain barrier and its intervention effect on neuroinflammation are key factors. Traditional drug screening methods usually rely on in vitro cell models or animal models, but these methods are difficult to meet the needs of high throughput and high precision at the same time. Especially in the study of drug concentration and its synergistic effect, traditional methods often have limitations and cannot accurately evaluate the dose-effect relationship of the drug and the synergistic effect of the two. Summary of the invention

[0011] In order to solve the technical problems existing in the prior art, the present invention provides a novel solution, which utilizes the microfluidic chip design of the modular concentration gradient generation module and the blood-brain barrier bionic module, as well as the chip design of the unique snowflake-shaped concentration gradient generation module, to achieve the combination of concentration gradient generation and neuroinflammation model on one platform, so as to efficiently screen drugs and accurately evaluate the dose-effect relationship and synergistic effect of drugs.

[0012] The present invention provides a bionic blood-brain barrier microfluidic chip device, comprising: a concentration gradient generating module and a blood-brain barrier bionic module;

[0013] The concentration gradient generating module comprises a concentration gradient generating module chip and a pressure-sensitive adhesive; the concentration gradient generating module chip comprises a plurality of liquid inlets, a plurality of first liquid outlets and a snowflake-shaped microchannel, the snowflake-shaped microchannel comprises a plurality of annular microchannels and a plurality of S-shaped microchannels vertically intersecting the annular microchannels as a whole, the plurality of liquid inlets are located in the innermost annular microchannel and are respectively connected to one end of the plurality of S-shaped microchannels, the plurality of first liquid outlets are respectively connected to the other end of the plurality of S-shaped microchannels, and the drug can generate a concentration gradient of the drug by passing through the snowflake-shaped microchannel; the pressure-sensitive adhesive is connected to the concentration gradient generating module chip to seal the concentration gradient generating module chip; the pressure-sensitive adhesive is provided with a second liquid outlet corresponding to the first liquid outlet on one side close to the concentration gradient generating module chip;

[0014] The blood-brain barrier bionic module comprises an upper chip and a lower chip, the upper chip and the lower chip are separated by a porous film, the upper chip comprises a plurality of bionic blood vessel microchannels, each of the bionic blood vessel microchannels is provided with an independent bionic blood vessel microchannel inlet and a first bionic blood vessel microchannel outlet; the bionic blood vessel microchannel inlet corresponds to the first liquid outlet, the lower chip comprises a plurality of bionic brain microchannels, and the plurality of bionic blood vessel microchannels are arranged correspondingly to the plurality of bionic brain microchannels in a vertical direction, and each bionic brain microchannel is provided with an independent first bionic brain microchannel inlet and a first bionic brain microchannel outlet; a second bionic brain microchannel inlet and a second bionic brain microchannel outlet corresponding to the first bionic brain microchannel inlet and the first bionic brain microchannel outlet are provided on the porous film; a fifth bionic brain microchannel inlet and a fifth bionic brain microchannel outlet corresponding to the first bionic brain microchannel inlet and the first bionic brain microchannel outlet are also provided on the upper chip;

[0015] A third bionic brain microchannel inlet corresponding to the first bionic brain microchannel inlet of the lower chip is provided next to the first liquid outlet, and a fourth bionic brain microchannel inlet corresponding to the third bionic brain microchannel inlet is provided on the side of the pressure-sensitive adhesive close to the concentration gradient generating module chip; a second bionic blood vessel microchannel outlet and a third bionic brain microchannel outlet corresponding to the first bionic blood vessel microchannel outlet and the first bionic brain microchannel outlet are provided on the concentration gradient generating module chip; a third bionic blood vessel microchannel outlet and a fourth bionic brain microchannel outlet corresponding to the second bionic blood vessel microchannel outlet and the third bionic brain microchannel outlet are provided on the pressure-sensitive adhesive;

[0016] The concentration gradient generating module and the blood-brain barrier biomimetic module can be connected and separated by the pressure-sensitive adhesive.

[0017] Preferably, a concentration gradient of the drug is generated in the snowflake-shaped microchannel by a serial dilution method.

[0018] Preferably, the plurality of bionic blood vessel microchannels and the plurality of bionic brain microchannels are radial.

[0019] Preferably, the bionic blood vessel microchannel and the bionic brain microchannel are distributed horizontally.

[0020] Preferably, the number of the liquid inlets is 4, the number of the first liquid outlets is 16, and the number of the S-shaped microchannels is 16.

[0021] Preferably, the number of the bionic blood vessel microchannels is 16 and the number of the bionic brain microchannels is 16.

[0022] Preferably, the annular microchannel has at least 2 circles.

[0023] Preferably, the liquid inlet and the first liquid outlet have the same diameter, and the width of the S-shaped microchannel is greater than its depth, and both are smaller than the diameters of the liquid inlet and the first liquid outlet.

[0024] Preferably, the diameters of the liquid inlet and the first liquid outlet are 0.9 mm, and the width and depth of the S-shaped microchannel are 0.25 mm and 0.15 mm, respectively.

[0025] Preferably, the bionic blood vessel microchannel and the bionic brain microchannel have the same shape.

[0026] Preferably, the width of the bionic blood vessel microchannel and the bionic brain microchannel is greater than their depth, and the length of the bionic brain microchannel is equal to the length of the bionic blood vessel microchannel.

[0027] Preferably, the length, width and height of the bionic blood vessel microchannel and the bionic brain microchannel are 5 mm, 1 mm and 0.15 mm respectively.

[0028] Preferably, the upper chip, the lower chip and the concentration gradient generating module are made of polydimethylsiloxane.

[0029] Preferably, the porous film is made of at least one material selected from the group consisting of polycarbonate, polyethylene terephthalate, and polydimethylsiloxane.

[0030] The present invention also provides an application of the above-mentioned bionic blood-brain barrier microfluidic chip device in the screening of combined drug dosage.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] 1. Modular design reduces operational difficulty and technical threshold. The independent modular design of the present invention allows different functional modules (such as concentration gradient generation module and blood-brain barrier bionic module) to be designed, prepared and used separately. Compared with the existing highly integrated chip design, this modular design greatly simplifies the experimental operation process and lowers the threshold for life science researchers without engineering background to apply this type of microfluidic chip. Researchers can flexibly adjust the experimental plan through simple module combination and splitting during the experiment, which improves the versatility and ease of operation of the chip.

[0033] 2. Possess flexible concentration gradient generation capabilities. The concentration gradient generation module of the present invention realizes the multiple dilution of drugs through the bifurcation design of snowflake-shaped microchannels, that is, S-shaped microchannels and annular microchannels, thereby generating accurate and continuous concentration gradients on the same platform. Compared with the manual dilution of drug concentration or single-concentration drug processing methods in the prior art, the present invention can process multiple concentration levels simultaneously in a single experiment, greatly improving the efficiency of drug screening, especially when it is necessary to study drug synergy and dose-effect relationships, providing higher flexibility and accuracy.

[0034] 3. Possessing higher blood-brain barrier bionic ability. The blood-brain barrier bionic module of the present invention adopts a two-layer design of an upper chip and a lower chip. The upper chip simulates the vascular end of the blood-brain barrier and cultures cerebral vascular endothelial cells, etc. The lower chip simulates the brain end and cultures astrocytes or neurons, etc. Compared with existing single-layer or simple cell models, the present invention separates the two-layer cell culture system by a porous film, which can better simulate the real blood-brain barrier structure and function. In addition, the state of continuous dynamic culture allows the cell culture medium to provide additional fluid shear stress, which is more in line with the physiological state of blood flow in the human brain. This bionic design improves the accuracy of drug intervention effect evaluation and contributes to the development of central nervous system drugs.

[0035] 4. High-throughput drug screening and data acquisition capabilities. The present invention not only realizes efficient concentration gradient generation and BBB biomimetic functions, but also allows for the separate collection and analysis of biological samples after intervention with different drug concentrations through the splitting after module combination. This flexible design allows the culture medium effluent, protein, nucleic acid and other samples in each channel to be processed separately, providing accurate data support for drug screening experiments. Compared with the multi-sample separation that is difficult to perform in the highly integrated one-piece chip in the prior art, the design of the present invention significantly improves the quality and reliability of experimental data. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of an embodiment of a concentration gradient generation module of a bionic blood-brain barrier microfluidic chip device of the present invention;

[0037] Figure 2 This is an exploded schematic diagram of an embodiment of a blood-brain barrier bionic module of the bionic blood-brain barrier microfluidic chip device of the present invention;

[0038] Figure 3 It is a schematic diagram of the concentration gradient generation effect of an embodiment of the concentration gradient generation module of the bionic blood-brain barrier microfluidic chip device of the present invention.

[0039] In the figure: 1. concentration gradient generating module chip, 1-1. liquid inlet, 1-2-1. first liquid outlet, 1-2-2. second liquid outlet, 1-3. S-shaped microchannel, 1-4. annular microchannel; 2. pressure-sensitive adhesive; 3. upper chip, 3-1. bionic vascular microchannel, 3-2. bionic vascular microchannel inlet, 3-3-1. first bionic vascular microchannel outlet, 3-3-2. second bionic vascular microchannel outlet, 3-3-3. third bionic vascular microchannel outlet; 4. porous film; 5. lower chip, 5 -1, bionic brain microchannel, 5-2-1, the first bionic brain microchannel entrance, 5-2-2, the second bionic brain microchannel entrance, 5-2-3, the third bionic brain microchannel entrance, 5-2-4, the fourth bionic brain microchannel entrance, 5-2-5, the fifth bionic brain microchannel entrance, 5-3-1, the first bionic brain microchannel exit, 5-3-2, the second bionic brain microchannel exit, 5-3-3, the third bionic brain microchannel exit, 5-3-4, the fourth bionic brain microchannel exit, 5-3-5, the fifth bionic brain microchannel exit. DETAILED DESCRIPTION

[0040] The specific implementation modes of the present invention are described in detail below in conjunction with the accompanying drawings.

[0041] The present invention provides a bionic blood-brain barrier microfluidic chip device, comprising: a concentration gradient generating module and a blood-brain barrier bionic module;

[0042] The concentration gradient generating module comprises a concentration gradient generating module chip 1 and a pressure-sensitive adhesive 2; the concentration gradient generating module chip 1 comprises a plurality of liquid inlets 1-1, a plurality of first liquid outlets 1-2-1 and snowflake-shaped microchannels, the snowflake-shaped microchannels comprise a plurality of annular microchannels 1-4 and a plurality of S-shaped microchannels 1-3 which are vertically intersected with the annular microchannels 1-4 as a whole, the plurality of liquid inlets 1-1 are located in the innermost annular microchannel 1-4 and are respectively connected to one end of the plurality of S-shaped microchannels 1-3, the plurality of first liquid outlets 1-2-1 are respectively connected to the other end of the plurality of S-shaped microchannels 1-3, and the drug can generate a concentration gradient of the drug through the snowflake-shaped microchannel; the pressure-sensitive adhesive 2 is connected to the concentration gradient generating module chip 1 to seal the concentration gradient generating module chip 1; the pressure-sensitive adhesive 2 is provided with a second liquid outlet 1-2-2 corresponding to the first liquid outlet 1-2-1 on one side close to the concentration gradient generating module chip 1;

[0043] The blood-brain barrier bionic module comprises an upper chip 3 and a lower chip 5, wherein the upper chip 3 and the lower chip 5 are separated by a porous film 4, wherein the upper chip 3 comprises a plurality of bionic blood vessel microchannels 3-1, each of which is provided with an independent bionic blood vessel microchannel inlet 3-2 and a first bionic blood vessel microchannel outlet 3-3-1; wherein the bionic blood vessel microchannel inlet 3-2 corresponds to the first liquid outlet 1-2-1, and the lower chip 5 comprises a plurality of bionic brain microchannels 5-1, and the plurality of bionic blood vessel microchannels 3-1 are vertically aligned with the plurality of bionic brain microchannels 5-1. It should be arranged that each bionic brain microchannel 5-1 is provided with an independent first bionic brain microchannel inlet 5-2-1 and a first bionic brain microchannel outlet 5-3-1; the porous film 4 is provided with a second bionic brain microchannel inlet 5-2-2 and a second bionic brain microchannel outlet 5-3-2 corresponding to the first bionic brain microchannel inlet 5-2-1 and the first bionic brain microchannel outlet 5-3-1; the upper chip is also provided with a fifth bionic brain microchannel inlet 5-2-5 and a fifth bionic brain microchannel outlet 5-3-5 corresponding to the first bionic brain microchannel inlet 5-2-1 and the first bionic brain microchannel outlet 5-3-1;

[0044] A third bionic brain microchannel inlet 5-2-3 corresponding to the first bionic brain microchannel inlet 5-2-1 of the lower chip 5 is provided next to the first liquid outlet 1-2-1, and a fourth bionic brain microchannel inlet 5-2-4 corresponding to the third bionic brain microchannel inlet 5-2-3 is provided on the side of the pressure-sensitive adhesive 2 close to the concentration gradient generating module chip 1; a second bionic blood vessel microchannel outlet 3-3-2 and a third bionic brain microchannel outlet 5-3-3 corresponding to the first bionic blood vessel microchannel outlet 3-3-1 and the first bionic brain microchannel outlet 5-3-1 are provided on the concentration gradient generating module chip 1; a third bionic blood vessel microchannel outlet 3-3-3 and a fourth bionic brain microchannel outlet 5-3-4 corresponding to the second bionic blood vessel microchannel outlet 3-3-2 and the third bionic brain microchannel outlet 5-3-3 are provided on the pressure-sensitive adhesive 2;

[0045] The concentration gradient generating module and the blood-brain barrier biomimetic module can be connected and separated by the pressure-sensitive adhesive 2 .

[0046] According to a specific embodiment of the present invention, a concentration gradient of the drug is generated in the snowflake-shaped microchannel by a serial dilution method.

[0047] According to a specific embodiment of the present invention, the plurality of bionic blood vessel microchannels 3-1 and the plurality of bionic brain microchannels 5-15-1 are radial.

[0048] According to a specific embodiment of the present invention, the bionic blood vessel microchannel and the bionic brain microchannel 5 - 1 are distributed horizontally.

[0049] According to a specific embodiment of the present invention, the number of the liquid inlets 1-1 is 4, the number of the first liquid outlets 1-2-1 is 16, and the number of the S-shaped microchannels 1-3 is 16.

[0050] According to a specific embodiment of the present invention, the number of the bionic blood vessel microchannels is 16, and the number of the bionic brain microchannels 5-1 is 16.

[0051] According to a specific embodiment of the present invention, the annular microchannel 1-4 has at least 2 circles.

[0052] According to a specific embodiment of the present invention, the diameters of the liquid inlet 1-1 and the first liquid outlet 1-2-1 are the same, and the width of the S-shaped microchannel 1-3 is greater than the depth, and both are smaller than the diameters of the liquid inlet 1-1 and the first liquid outlet 1-2-1.

[0053] According to a specific embodiment of the present invention, the diameters of the liquid inlet 1-1 and the first liquid outlet 1-2-1 are 0.9 mm, and the width and depth of the S-shaped microchannel 1-3 are 0.25 mm and 0.15 mm, respectively.

[0054] According to a specific embodiment of the present invention, the bionic blood vessel microchannel 3-1 and the bionic brain microchannel 5-1 have the same shape.

[0055] According to a specific embodiment of the present invention, the width of the bionic blood vessel microchannel 3-1 and the bionic brain microchannel 5-1 is greater than the depth, and the length of the bionic brain microchannel 5-1 is equal to the length of the bionic blood vessel microchannel 3-1.

[0056] According to a specific embodiment of the present invention, the length, width and height of the bionic blood vessel microchannel 3-1 and the bionic brain microchannel 5-1 are 5 mm, 1 mm and 0.15 mm respectively.

[0057] According to a specific embodiment of the present invention, the upper chip 3, the lower chip 5 and the concentration gradient generating module are made of polydimethylsiloxane.

[0058] According to a specific embodiment of the present invention, the porous film 4 is made of at least one material selected from the group consisting of polycarbonate, polyethylene terephthalate, and polydimethylsiloxane.

[0059] The present invention also provides an application of the above-mentioned bionic blood-brain barrier microfluidic chip device in the screening of combined drug dosage.

[0060] Example 1

[0061] like Figure 1-2 As shown, the present invention provides a bionic blood-brain barrier microfluidic chip device, comprising: a concentration gradient generating module and a blood-brain barrier bionic module;

[0062] The concentration gradient generating module comprises a concentration gradient generating module chip 1 and a pressure-sensitive adhesive 2; the concentration gradient generating module chip 1 comprises a plurality of liquid inlets 1-1, a plurality of first liquid outlets 1-2-1 and snowflake-shaped microchannels, the snowflake-shaped microchannels comprise a plurality of annular microchannels 1-4 and a plurality of S-shaped microchannels 1-3 which are vertically intersected with the annular microchannels 1-4 as a whole, the plurality of liquid inlets 1-1 are located in the innermost annular microchannel 1-4 and are respectively connected to one end of the plurality of S-shaped microchannels 1-3, the plurality of first liquid outlets 1-2-1 are respectively connected to the other end of the plurality of S-shaped microchannels 1-3, and the drug can generate a concentration gradient of the drug through the snowflake-shaped microchannel; the pressure-sensitive adhesive 2 is connected to the concentration gradient generating module chip 1 to seal the concentration gradient generating module chip 1; the pressure-sensitive adhesive 2 is provided with a second liquid outlet 1-2-2 corresponding to the first liquid outlet 1-2-1 on one side close to the concentration gradient generating module chip 1;

[0063] The blood-brain barrier bionic module comprises an upper chip 3 and a lower chip 5, wherein the upper chip 3 and the lower chip 5 are separated by a porous film 4, wherein the upper chip 3 comprises a plurality of bionic blood vessel microchannels 3-1, each of which is provided with an independent bionic blood vessel microchannel inlet 3-2 and a first bionic blood vessel microchannel outlet 3-3-1; wherein the bionic blood vessel microchannel inlet 3-2 corresponds to the first liquid outlet 1-2-1, and the lower chip 5 comprises a plurality of bionic brain microchannels 5-1, and the plurality of bionic blood vessel microchannels 3-1 are vertically aligned with the plurality of bionic brain microchannels 5-1. It should be arranged that each bionic brain microchannel 5-1 is provided with an independent first bionic brain microchannel inlet 5-2-1 and a first bionic brain microchannel outlet 5-3-1; the porous film 4 is provided with a second bionic brain microchannel inlet 5-2-2 and a second bionic brain microchannel outlet 5-3-2 corresponding to the first bionic brain microchannel inlet 5-2-1 and the first bionic brain microchannel outlet 5-3-1; the upper chip is also provided with a fifth bionic brain microchannel inlet 5-2-5 and a fifth bionic brain microchannel outlet 5-3-5 corresponding to the first bionic brain microchannel inlet 5-2-1 and the first bionic brain microchannel outlet 5-3-1;

[0064] A third bionic brain microchannel inlet 5-2-3 corresponding to the first bionic brain microchannel inlet 5-2-1 of the lower chip 5 is provided next to the first liquid outlet 1-2-1, and a fourth bionic brain microchannel inlet 5-2-4 corresponding to the third bionic brain microchannel inlet 5-2-3 is provided on the side of the pressure-sensitive adhesive 2 close to the concentration gradient generating module chip 1; a second bionic blood vessel microchannel outlet 3-3-2 and a third bionic brain microchannel outlet 5-3-3 corresponding to the first bionic blood vessel microchannel outlet 3-3-1 and the first bionic brain microchannel outlet 5-3-1 are provided on the concentration gradient generating module chip 1; a third bionic blood vessel microchannel outlet 3-3-3 and a fourth bionic brain microchannel outlet 5-3-4 corresponding to the second bionic blood vessel microchannel outlet 3-3-2 and the third bionic brain microchannel outlet 5-3-3 are provided on the pressure-sensitive adhesive 2;

[0065] The concentration gradient generating module and the blood-brain barrier biomimetic module can be connected and separated by the pressure-sensitive adhesive 2 .

[0066] The bionic blood-brain barrier microfluidic chip device of the present invention can be used to study the intervention effect of different drug components on neuroinflammation when they are used in combination at different concentrations. Its application method is as follows: the upper chip 3 and the lower chip 5 of the blood-brain barrier bionic module can be pre-cultured with cerebral vascular endothelial cells and astrocytes respectively to construct the blood-brain barrier, and then lipopolysaccharide, Aβ protein, 6-hydroxydopamine or proinflammatory cytokines are injected into the brain end channel of the blood-brain barrier (i.e., the bionic brain microchannel 5-1 of the lower chip 5) to establish a neuroinflammation model, and finally the concentration gradient generation module is assembled with the blood-brain barrier bionic module to complete the intervention of different drug components in combination at different concentrations on the neuroinflammation model, and the intervention effect of the drug on neuroinflammation is evaluated by detecting neuroinflammation biomarkers before and after the intervention.

[0067] Example 2

[0068] The concentration gradient generating module chip 1 comprises a plurality of liquid inlets 1-1, a plurality of first liquid outlets 1-2-1 and snowflake-shaped microchannels, the snowflake-shaped microchannels comprising two annular microchannels 1-4 and 16 S-shaped microchannels 1-3 which are vertically intersected with the annular microchannels 1-4 as a whole, the four liquid inlets 1-1 are located in the innermost annular microchannel 1-4 and are respectively connected to one end of the S-shaped microchannel 1-3, the 16 first liquid outlets 1-2-1 are respectively connected to the other ends of the plurality of S-shaped microchannels 1-3, the diameters of the liquid inlet 1-1 and the first liquid outlet 1-2-1 are 0.9 mm, the width and depth of the S-shaped microchannel 1-3 are 0.25 mm and 0.15 mm respectively, and the drug can generate a concentration gradient of the drug through the snowflake-shaped microchannel; the pressure-sensitive adhesive 2 is connected to the concentration gradient generating module chip 1 to seal the concentration gradient generating module chip 1; the pressure-sensitive adhesive 2 is provided with a second liquid outlet 1-2-2 corresponding to the first liquid outlet 1-2-1 on one side close to the concentration gradient generating module chip 1;

[0069] The blood-brain barrier bionic module includes an upper chip 3 and a lower chip 5, and the upper chip 3 and the lower chip 5 are separated by a porous film 4. The porous film 4 is used to physically separate the upper chip and the lower chip and provide attachment points for cells seeded on the upper and lower sides of the film. In addition, the porous film 4 is an important guarantee for the growth of different cell partitions in the chip and the signal conduction between cells. The upper chip 3 includes 16 bionic vascular microchannels 3-1, each of which is provided with an independent bionic vascular microchannel inlet 3-2 and a first bionic vascular microchannel outlet 3-3-1; the bionic vascular microchannel inlet 3-2 corresponds to the first liquid outlet 1-2-1, and the required cells (such as cerebral vascular endothelial cells, cerebral vascular pericytes, etc.) can be pre-cultured in the bionic vascular microchannel 3-1, and when the blood-brain barrier model is completed, it can be used to establish a neuroinflammation model. The lower chip 5 includes 16 bionic brain microchannels 5-1, and the bionic blood vessel microchannel 3-1 is arranged corresponding to the bionic brain microchannel 5-1 in the vertical direction. The length, width and height of the bionic blood vessel microchannel 3-1 and the bionic brain microchannel 5-1 are 5 mm, 1 mm and 0.15 mm respectively. Each bionic brain microchannel 5-1 is provided with an independent first bionic brain microchannel inlet 5-2-1 and a first bionic brain microchannel outlet 5-3-1. The required cells (such as astrocytes, microglia, neurons, etc.) can be pre-cultured in the bionic brain microchannel 5-1. When the blood-brain barrier model is completed, the neuroinflammation model can be established. Different modeling drugs (such as lipopolysaccharide, Aβ protein, etc.) can be introduced into the bionic brain microchannel 5-1 according to different research needs to construct different disease models. The porous film 4 is provided with a second bionic brain microchannel inlet 5-2-2 and a second bionic brain microchannel outlet 5-3-2 corresponding to the first bionic brain microchannel inlet 5-2-1 and the first bionic brain microchannel outlet 5-3-1;

[0070] A third bionic brain microchannel inlet 5-2-3 corresponding to the first bionic brain microchannel inlet 5-2-1 of the lower chip 5 is provided next to the first liquid outlet 1-2-1, and a fourth bionic brain microchannel inlet 5-2-4 corresponding to the third bionic brain microchannel inlet 5-2-3 is provided on the side of the pressure-sensitive adhesive 2 close to the concentration gradient generating module chip 1; a second bionic blood vessel microchannel outlet 3-3-2 and a third bionic brain microchannel outlet 5-3-3 corresponding to the first bionic blood vessel microchannel outlet 3-3-1 and the first bionic brain microchannel outlet 5-3-1 are provided on the concentration gradient generating module chip 1; a third bionic blood vessel microchannel outlet 3-3-3 and a fourth bionic brain microchannel outlet 5-3-4 corresponding to the second bionic blood vessel microchannel outlet 3-3-2 and the third bionic brain microchannel outlet 5-3-3 are provided on the pressure-sensitive adhesive 2;

[0071] The concentration gradient generating module and the blood-brain barrier biomimetic module can be connected and separated by the pressure-sensitive adhesive 2 .

[0072] Among them, the concentration gradient generation module and the blood-brain barrier bionic module are quickly assembled by pressure-sensitive adhesive 2. In the assembled state, the 16 first liquid outlets 1-3-1 of the concentration gradient generation module completely cover the bionic blood vessel microchannel inlet 3-2 of the upper chip 3; in the drug intervention experiment stage, the cell culture medium containing different drugs is introduced clockwise into the 4 liquid inlets 1-1 of the concentration gradient generation module, and the drugs are diluted in multiples through the S-shaped microchannel 1-3 and the annular microchannel 1-4 to form concentration gradients of different drugs. In this way, different concentrations of different drugs can be combined on the same chip to implement intervention experiments on neuroinflammatory disease models. The design of the array channel is to increase the test throughput. The more array channels are set, the more concentration gradients are formed, and the finer the concentration gradients are divided, which is conducive to the development of concentration-dependent experiments.

[0073] The upper chip 3 of the blood-brain barrier bionic module is provided with a bionic vascular microchannel 3-1 array, which can be used to culture cell types at the vascular end of the blood-brain barrier, such as cerebral vascular endothelial cells and cerebral vascular pericytes, during the cell culture stage; during the drug intervention experiment stage, the concentration gradient generating module is assembled with the blood-brain barrier bionic module, and the bionic vascular microchannel inlet 3-2 of the upper chip 3 corresponds to the first liquid outlet 1-2-1 of the concentration gradient generating module chip 1, so that the liquid in the concentration gradient generating module directly flows into the bionic vascular microchannel 3-1; after the drug intervention experiment is completed, the upper chip 3 and the concentration gradient generating module are separated, and at the end of the experiment, biological samples (such as RNA, protein, etc.) in different channels of the bionic vascular microchannel 3-1 array are collected separately.

[0074] Similarly, the lower chip 5 is provided with an array of bionic brain microchannels 5-1 corresponding to the upper chip 3, which can culture cell types at the brain end of the blood-brain barrier, such as astrocytes, microglia, and neurons, during the cell culture stage. After the blood-brain barrier is constructed, a neuroinflammation model is established by continuously perfusing modeling substances, such as lipopolysaccharide, Aβ protein, 6-hydroxydopamine or proinflammatory cytokines, into the bionic brain microchannels 5-1. The mutually independent bionic brain microchannels 5-1 are respectively provided with a first bionic brain microchannel inlet 5-2 and a first bionic brain microchannel outlet 5-3, which also facilitates the separate collection of biological samples (such as RNA, protein, etc.) in different channels of the channel array at the end of the experiment.

[0075] In the present invention, the porous film 4 serves to separate and communicate the upper chip 3 and the lower chip 5. During the cell inoculation process, the film physically separates the upper and lower regions, so that the cells cultured on the upper and lower sides of the film only appear in a fixed region, avoiding migration through the film. In addition, during the entire experimental process, the cells in the microchannels of the upper chip 3 and the lower chip 5 can conduct real-time intercellular material exchange and signal conduction through the film micropores, thereby helping to establish a more complex bionic physiological microenvironment.

[0076] The upper chip 3, the lower chip 5, the porous film 4 and the concentration gradient generating module can be composited with different materials. The upper chip 3, the lower chip 5 and the concentration gradient generating module are made of polydimethylsiloxane (PDMS); the porous film can be made of at least one material such as polycarbonate (PC), polyethylene terephthalate (PET), PDMS, etc.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. A bionic blood-brain barrier microfluidic chip device, characterized in that: include: Concentration gradient generation module and blood-brain barrier bionic module; The concentration gradient generating module comprises a concentration gradient generating module chip and a pressure-sensitive adhesive; the concentration gradient generating module chip comprises a plurality of liquid inlets, a plurality of first liquid outlets and a snowflake-shaped microchannel, the snowflake-shaped microchannel comprises a plurality of annular microchannels and a plurality of S-shaped microchannels vertically intersecting the annular microchannels as a whole, the plurality of liquid inlets are located in the innermost annular microchannel and are respectively connected to one end of the plurality of S-shaped microchannels, the plurality of first liquid outlets are respectively connected to the other end of the plurality of S-shaped microchannels, and the drug can generate a concentration gradient of the drug by passing through the snowflake-shaped microchannel; the pressure-sensitive adhesive is connected to the concentration gradient generating module chip to seal the concentration gradient generating module chip; the pressure-sensitive adhesive is provided with a second liquid outlet corresponding to the first liquid outlet on one side close to the concentration gradient generating module chip; The blood-brain barrier bionic module comprises an upper chip and a lower chip, the upper chip and the lower chip are separated by a porous film, the upper chip comprises a plurality of bionic blood vessel microchannels, each of the bionic blood vessel microchannels is provided with an independent bionic blood vessel microchannel inlet and a first bionic blood vessel microchannel outlet; the bionic blood vessel microchannel inlet corresponds to the first liquid outlet, the lower chip comprises a plurality of bionic brain microchannels, and the plurality of bionic blood vessel microchannels are arranged correspondingly to the plurality of bionic brain microchannels in a vertical direction, and each bionic brain microchannel is provided with an independent first bionic brain microchannel inlet and a first bionic brain microchannel outlet; a second bionic brain microchannel inlet and a second bionic brain microchannel outlet corresponding to the first bionic brain microchannel inlet and the first bionic brain microchannel outlet are provided on the porous film; a fifth bionic brain microchannel inlet and a fifth bionic brain microchannel outlet corresponding to the first bionic brain microchannel inlet and the first bionic brain microchannel outlet are also provided on the upper chip; A third bionic brain microchannel inlet corresponding to the first bionic brain microchannel inlet of the lower chip is provided next to the first liquid outlet, and a fourth bionic brain microchannel inlet corresponding to the third bionic brain microchannel inlet is provided on the side of the pressure-sensitive adhesive close to the concentration gradient generating module chip; a second bionic blood vessel microchannel outlet and a third bionic brain microchannel outlet corresponding to the first bionic blood vessel microchannel outlet and the first bionic brain microchannel outlet are provided on the concentration gradient generating module chip; a third bionic blood vessel microchannel outlet and a fourth bionic brain microchannel outlet corresponding to the second bionic blood vessel microchannel outlet and the third bionic brain microchannel outlet are provided on the pressure-sensitive adhesive; The concentration gradient generating module and the blood-brain barrier bionic module can be connected and separated by the pressure-sensitive adhesive.

2. The bionic blood-brain barrier microfluidic chip device according to claim 1, characterized in that: The concentration gradient of the drug is generated in the snowflake-shaped microchannel by a serial dilution method.

3. The bionic blood-brain barrier microfluidic chip device according to claim 1, characterized in that: The plurality of bionic blood vessel microchannels and the plurality of bionic brain microchannels are all radial.

4. The bionic blood-brain barrier microfluidic chip device according to claim 1, characterized in that: The bionic blood vessel microchannel and the bionic brain microchannel are distributed horizontally.

5. The bionic blood-brain barrier microfluidic chip device according to claim 1, characterized in that: The number of the liquid inlets is 4, the number of the first liquid outlets is 16, and the number of the S-shaped microchannels is 16.

6. The bionic blood-brain barrier microfluidic chip device according to claim 5, characterized in that: There are 16 bionic blood vessel microchannels and 16 bionic brain microchannels.

7. The bionic blood-brain barrier microfluidic chip device according to claim 1, characterized in that: The annular microchannel has at least 2 circles.

8. The bionic blood-brain barrier microfluidic chip device according to claim 1, characterized in that: The liquid inlet and the first liquid outlet have the same diameter, and the width of the S-shaped microchannel is greater than its depth, and both are smaller than the diameters of the liquid inlet and the first liquid outlet.

9. The bionic blood-brain barrier microfluidic chip device according to claim 8, characterized in that: The diameters of the liquid inlet and the first liquid outlet are 0.9 mm, and the width and depth of the S-shaped microchannel are 0.25 mm and 0.15 mm, respectively.

10. The bionic blood-brain barrier microfluidic chip device according to claim 1, characterized in that: The bionic blood vessel microchannel has the same shape as the bionic brain microchannel.

11. The bionic blood-brain barrier microfluidic chip device according to claim 10, characterized in that: The width of the bionic blood vessel microchannel and the bionic brain microchannel is greater than the depth, and the length of the bionic brain microchannel is equal to the length of the bionic blood vessel microchannel.

12. The bionic blood-brain barrier microfluidic chip device according to claim 10, characterized in that: The length, width and height of the bionic blood vessel microchannel and the bionic brain microchannel are 5 mm, 1 mm and 0.15 mm respectively.

13. The bionic blood-brain barrier microfluidic chip device according to claim 1, characterized in that: The upper chip, the lower chip and the concentration gradient generating module are made of polydimethylsiloxane.

14. The bionic blood-brain barrier microfluidic chip device according to claim 1, characterized in that: The porous film is made of at least one material selected from the group consisting of polycarbonate, polyethylene terephthalate and polydimethylsiloxane.

15. Use of the bionic blood-brain barrier microfluidic chip device according to any one of claims 1 to 14 in screening of combined drug dosages.

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