Pneumatically-controlled cerebral arterial thrombosis dynamic micro-fluidic blood vessel chip integrated oxygen sensing system

Abstract

The invention discloses a pneumatically-controlled cerebral arterial thrombosis dynamic micro-fluidic blood vessel chip integrated oxygen sensing system. The system sequentially comprises a perfusion channel layer, a deformation layer, a glass oxygen barrier layer and a pore control layer from top to bottom, the perfusion channel layer is connected with an injection pump through an external silicone tube and is used for realizing dynamic culture of cells in the channel; the deformation layer is an elastic film and is deformed along with the increase of external air pressure, so that the channel forms a narrow area, and an oxygen probe is arranged in the elastic film and is used for oxygen monitoring; the air hole control layer is connected with a vacuum pump through an external silicone tube and applies air pressure to the deformation layer. The deformation layer is effectively controlled through an external vacuum pump, so that the stenosis of a channel is accurately controlled, different stenosis of blood vessels under physiological conditions is simulated, and changes of mechanical microenvironments such as fluid shear force, culture solution flow rate and flow and oxygen content chemical microenvironments are recorded; and a convenient and effective new platform is provided for studying the injury mechanism of ischemia reperfusion.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidic chip, and particularly relates to a pneumatic control ischemic stroke dynamic microfluidic blood vessel chip integrated oxygen sensing system. BACKGROUND

[0002] Acute ischemic stroke (AIS) is a leading cause of long-term disability and death worldwide, which is caused by sudden stenosis or occlusion of cerebral vessels, leading to focal brain dysfunction. Recanalization of occluded vessels through reperfusion therapy is the most effective treatment, including intravenous thrombolysis (IVT) and mechanical thrombectomy (MT). There are significant differences in blood flow dynamics during reperfusion between IVT and MT. IVT achieves recanalization by gradually dissolving thrombus within a specific time range (usually within 1 hour), although IVT is the preferred clinical treatment, its efficacy is limited by a narrow therapeutic window (within 4.5 hours) and low recanalization rate, especially in AIS patients caused by intracranial large vessel occlusion; while MT helps to quickly restore blood flow, and clinical studies have shown that IVT combined with MT is more helpful than single treatment in improving vascular inflammatory markers in AIS patients.

[0003] However, even after timely and successful reperfusion therapy, a significant number of acute stroke patients still suffer from disabling injuries. This is not only due to ischemia-induced irreversible damage, but also because sudden reperfusion can lead to more severe secondary inflammation and vascular damage, a condition known as ischemia-reperfusion injury (IRI). Studies have shown that hemodynamic changes downstream of the stenosis site after reperfusion can lead to a series of endothelial dysfunction, including inflammation, oxidative stress, decreased blood-brain barrier permeability, and energy metabolism dysfunction. Although animal experiments have shown that gradual restoration of blood flow is more effective than non-rapid restoration of blood flow in alleviating IRI and reducing reperfusion injury, MT (with or without IVT) has increasingly become the preferred option for clinical treatment of large vessel occlusive stroke. However, the mechanisms of secondary brain injury caused by different reperfusion therapies (IVT and MT) are still poorly understood, and the different effects of these methods on reperfusion outcomes deserve further study.

[0004] IRI pathological models are traditionally developed using animal models, 2D cell culture, and recently 3D microfluidic models. In animal models, the process of ischemia and reperfusion is simulated by performing middle cerebral artery occlusion (MCAO), which is consistent with MT, however, the MCAO model cannot fully reflect the characteristics of intravenous thrombolysis, in addition, animal models have limitations, including low throughput, high cost and ethical issues. Conventional 2D cell culture provides an alternative method with high throughput and cost-effectiveness, which usually adopts direct and indirect induction methods to simulate ischemic conditions, although these advantages, 2D models are difficult to accurately replicate the complex physiological functions of human tissues and organs.

[0005] The advent of tissue engineering technology provides a promising platform for constructing in vitro models closer to the dynamic microenvironment of blood vessels, which allows precise control of physical and chemical conditions, making it highly suitable for pathophysiological research, drug development and personalized medicine. Existing models rely on fixed geometry or simplified hypoxia / reoxygenation conditions and cannot fully simulate the complexity of clinical situations. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings of the prior art and provide a pneumatic control ischemic stroke dynamic microfluidic blood vessel chip integrated oxygen sensing system.

[0007] The purpose of the present application is achieved by the following technical solution: a pneumatic control ischemic stroke dynamic microfluidic blood vessel chip integrated oxygen sensing system, comprising a perfusion channel layer, a deformation layer, a glass oxygen barrier layer and a gas hole control layer, from top to bottom in turn perfusion channel layer, deformation layer, glass oxygen barrier layer and gas hole control layer.

[0008] The perfusion channel layer is connected to the injection pump through the external silicone tube, which is used to realize the dynamic culture of cells in the channel.

[0009] The deformation layer is an elastic film that deforms with increasing external air pressure, forming a narrow area in the channel, and the oxygen probe is arranged in the elastic film, which is used for oxygen monitoring.

[0010] The gas hole control layer is connected to the vacuum pump through the external silicone tube, which applies air pressure to the deformation layer.

[0011] Preferably, the perfusion channel layer, the deformation layer and the gas hole control layer are made of polydimethylsiloxane, and the contact surfaces of the adjacent two layers of the perfusion channel layer, the deformation layer, the glass oxygen barrier layer and the gas hole control layer are placed in the plasma treatment machine for cleaning treatment. The contact surface of the layer made of polydimethylsiloxane is treated for 120s, and the contact surface between the glass oxygen barrier layer is treated for 180s, and then transferred to a 60℃ oven for heating for 20 min, so that the four are tightly bonded to form a closed channel structure.

[0012] Preferably, the perfusion channel layer is a semi-open channel, which forms a closed microchannel after bonding with the deformation layer, and the microchannel is used for dynamic perfusion culture of cells, and the microchannel is provided with a liquid inlet and a liquid outlet.

[0013] Preferably, the gas hole control layer is provided with a gas hole, and the gas hole is connected to the vacuum pump through a steel needle and a silicone tube.

[0014] Preferably, the application steps of the system are as follows:

[0015] S1: surface pretreatment of the packaged chip model;

[0016] S2: static culture of cells in the chip;

[0017] S3: pathological simulation of cells in the chip and oxygen monitoring thereof.

[0018] Preferably, step S1 further comprises the following steps:

[0019] S11: placing the packaged chip model into an ultraclean sterile table and sterilizing under ultraviolet light for 4 h;

[0020] S12: washing the channel once with alcohol, washing the channel three times with sterile phosphate buffer solution, and adding a fibronectin solution with a concentration of 100 μg / mL to completely cover the channel;

[0021] S13: transferring to a 4°C refrigerator and standing for treatment for 12 h.

[0022] Preferably, step S2 further comprises the following steps:

[0023] S21: taking the model after surface treatment out of the refrigerator, rewarming in a 37°C incubator for 30-60 min, and after rewarming, transferring the model to an ultraclean workbench;

[0024] S22: using a pipette to suck out the fibronectin solution in the channel and using a vascular endothelial cell culture medium to rinse the channel;

[0025] S23: planting vascular endothelial cells in the microchannel at a density of 1×10 6 cell / mL;

[0026] S24: placing the microfluidic chip into a 37°C incubator containing 5% CO2 and incubating overnight to allow the cells to adhere and grow, and keeping the inlet and outlet covered with a set amount of vascular endothelial cell culture medium throughout the incubation.

[0027] Preferably, step S3 further comprises the following steps:

[0028] S31: connecting the outlet and inlet of the perfusion channel with a steel needle and a silicone tube respectively, using a syringe to suck up a vascular endothelial cell complete culture medium containing 15% fetal bovine serum, and then connecting the silicone tube, setting the inlet flow rate to 100 μL / h, and providing the cells in the channel with a continuous flow of culture medium;

[0029] S32: placing the chip on an inverted fluorescence microscope stage or in a 37°C incubator;

[0030] S33: simulating different conditions;

[0031] S34: Real-time observation of cell state and taking pictures, using a device connected to an inverted fluorescence microscope to process pictures, real-time calculation of free oxygen content in the channel, and alarm when oxygen content is lower than the set value;

[0032] S35: Dissolving the drug to be screened in the cell culture medium at a therapeutic concentration for perfusion culture in the simulation process, and observing the morphological and arrangement changes of the cells under the inverted fluorescence microscope during the culture process.

[0033] Preferably, in step S33, the simulation is carried out under the conditions that the pressure provided by the vacuum pump is 90 kPa, and the inlet flow rate of the culture medium perfusion is maintained at 100 μL / h, and different conditions include brain ischemia process, brain ischemia gradual reperfusion process, and instantaneous reperfusion process;

[0034] Simulation of brain ischemia process: open the vacuum pump to make the elastic membrane of the deformation layer swell rapidly to form 80% stenosis in the channel, simulating the state of brain ischemia;

[0035] Simulation of brain ischemia gradual reperfusion process: after 1 h of pressurization, the pressure is gradually reduced to 0 at a rate of 1.5 kPa / min within 1 h;

[0036] Simulation of instantaneous reperfusion process: after 1 h of pressurization, the air pressure is quickly reduced to 0, the channel returns to normal and continues to be perfused for 1 h.

[0037] The present application has the following advantages: the present application provides effective control for the deformation layer through an external vacuum pump, thereby accurately controlling the stenosis of the channel, simulating different stenosis of blood vessels under physiological conditions, and recording changes in mechanical microenvironment such as fluid shear force, culture solution flow rate, flow, and chemical microenvironment such as oxygen content, thereby providing a convenient and effective new platform for studying the injury mechanism of ischemia-reperfusion. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Structure diagram of the position relationship of the perfusion channel layer, the deformation layer, the glass oxygen isolation layer, and the air hole control layer;

[0039] Figure 2 Structure diagram of the change of the elastic membrane;

[0040] Figure 3 Structure diagram of pressure-stenosis rate;

[0041] Figure 4 Structure diagram of the standard curve of the corresponding relationship between the light intensity emitted by the aerobic membrane and the oxygen content in the channel;

[0042] In the figure, 1 is the perfusion channel layer, 2 is the deformation layer, 3 is the glass oxygen isolation layer, 4 is the air hole control layer, 5 is the injection pump, and 6 is the vacuum pump. DETAILED DESCRIPTION

[0043] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some but not all of the embodiments of the present application. Generally, the components of the embodiments of the present application described and shown in the drawings can be arranged and designed in various different configurations.

[0044] Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts fall within the scope of protection of the present application.

[0045] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0046] It should be noted that: similar reference numerals and letters represent similar items in the following drawings, therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0047] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship commonly understood by those skilled in the art, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the indicated device or element must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second" and the like are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0048] In the description of the present application, it should also be noted that, unless otherwise explicitly specified and limited, the terms "arrangement", "installation", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be the communication inside two elements. For those of ordinary skill in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0049] In the present embodiment, as shown in FIG. 1, the present application comprises a first device 1 and a second device 2. Figure 1 and Figure 2As shown, a pneumatic control ischemic stroke dynamic microfluidic blood vessel chip integrated oxygen sensing system includes a perfusion channel layer 1, a deformation layer 2, a glass oxygen barrier layer 3, and a gas hole control layer 4, from top to bottom in turn for perfusion channel layer 1, deformation layer 2, glass oxygen barrier layer 3 and gas hole control layer 4;

[0050] The perfusion channel layer 1 is connected with the injection pump 5 through the external silicone tube, and is used for realizing dynamic culture of cells in the channel;

[0051] The deformation layer 2 is an elastic film, and generates deformation with the increase of external air pressure, so that the channel forms a narrow area, and the elastic film is provided with an oxygen probe, and the oxygen probe is used for oxygen monitoring;

[0052] The gas hole control layer 4 is connected with the vacuum pump 6 through the external silicone tube, and applies air pressure to the deformation layer 2. The deformation layer 2 is provided with effective control through the external vacuum pump 6, so that the narrowness of the channel is accurately controlled, different narrowness of the blood vessel under physiological conditions is simulated, and the changes of the mechanical microenvironment such as fluid shear force, culture solution flow rate, flow, and the chemical microenvironment such as oxygen content are recorded, so that a convenient and effective new platform is provided for studying the injury mechanism of ischemia reperfusion. In the embodiment, the injection pump is provided with a syringe, and the liquid flow rate is regulated and controlled through the injection pump 5.

[0053] Further, the perfusion channel layer 1, the deformation layer 2 and the air hole control layer 4 are all made of polydimethylsiloxane, the contact surfaces between the adjacent two layers of the perfusion channel layer 1, the deformation layer 2, the glass oxygen barrier layer 3 and the air hole control layer 4 are placed in a plasma treatment machine for cleaning treatment, the contact surface between the layers made of polydimethylsiloxane is treated for 120 s, the contact surface between the layers and the glass oxygen barrier layer 3 is treated for 180 s, and then the four layers are transferred to a 60℃ oven for heating for 20 min, so that the four layers are tightly bonded to form a closed channel structure. Specifically, the channel size is length L = 2.5 cm, width W = 500 μm and height H = 120 μm. Further, the perfusion channel layer 1 is a semi-open channel, which forms a closed microchannel after being bonded with the deformation layer 2, the microchannel is used for dynamic perfusion culture of cells, the microchannel is provided with a liquid inlet and a liquid outlet, and the steel needle and the silica gel tube are connected with the syringe and the injection pump to realize continuous perfusion. Specifically, a standard soft etching method is used, a silicon wafer and SU-8 photoresist are used as raw materials, a 1500 r / min uniform coating speed is selected, a positive mold with a height of 120 μm is prepared, polydimethylsiloxane is cast and inverted, a channel layer and an air hole layer are cut out in a suitable size, a 1 mm puncher is used to punch holes at the liquid inlet and liquid outlet positions of the channel layer and the air hole positions of the air hole layer respectively, and the perfusion channel layer 1 and the air hole control layer 4 are obtained; 4 g of polydimethylsiloxane prepolymer and a curing agent are weighed according to a mass ratio of 10:1 and mixed uniformly in a disposable plastic cup; 10 mg of platinum (II) octaethylporphyrin (Pt (II) octaethylporphyrin, PtOEP) is dissolved in 10 ml of tetrahydrofuran, and the undissolved particles are filtered out using a screen, and the solution is poured into the polydimethylsiloxane and mixed uniformly; the mixture is placed in a vacuum drying machine for 4 h to volatilize the tetrahydrofuran; the extracted mixture is placed on the surface of a clean plastic petri dish cover, placed in a uniform coating machine, set to a rotation speed of 800 rmp / min and spin coating for 30 s; after spin coating, the mixture is transferred to a 60℃ oven for heating for 1-2 h to completely cure the polydimethylsiloxane, and then cooled to room temperature to obtain the deformation layer 2.

[0054] In the embodiment, the air hole control layer 4 is provided with an air hole, and the air hole is connected with the vacuum pump 6 through a steel needle and a silica gel tube. Specifically, the air hole is connected with the vacuum pump 6 through a steel needle and a silica gel tube to pressurize the deformation layer 2, and the main function of the glass oxygen barrier layer 3 is to prevent the air-permeable air hole control layer 4 from affecting the oxygen content monitoring of the deformation layer 2.

[0055] Further, the application steps of the system are as follows:

[0056] S1: surface pretreatment is performed on the packaged chip model; specifically, step S1 further includes the following steps:

[0057] S11: the packaged chip model is placed in an ultraclean sterile table and ultraviolet sterilized for 4 h;

[0058] S12: Rinse the channel once with alcohol and three times with sterile phosphate buffer, and add 100 μg / mL fibronectin solution to completely cover the channel;

[0059] S13: Transfer to a 4°C refrigerator and let it stand for 12 hours.

[0060] S2: Static culture of cells in the chip; specifically, step S2 further includes the following steps:

[0061] S21: Take the surface-treated model out of the refrigerator and rewarm it in a 37°C incubator for 30-60 minutes. After rewarming, transfer the model to a clean bench.

[0062] S22: Use a pipette to aspirate the fibronectin solution in the channel and rinse the channel with vascular endothelial cell culture medium;

[0063] S23: 1×10 6 The density of cells / mL was seeded in the microchannel;

[0064] S24: Place the microfluidic chip in an incubator at 37°C and 5% CO2 for overnight incubation to allow the cells to adhere to the wall and grow. During the entire incubation process, keep the liquid inlet and outlet covered with a set amount of vascular endothelial cell culture medium.

[0065] S3: Pathological simulation of cells in the chip is performed and oxygen monitoring is performed on the cells. Specifically, step S3 further includes the following steps:

[0066] S31: Connect the outlet and inlet of the perfusion channel with a steel needle and silicone tubing, respectively. Use a syringe to draw in complete endothelial cell culture medium containing 15% fetal bovine serum, then connect it to the silicone tubing. Set the inlet flow rate to 100 μL / h to provide a continuous flow of culture medium to the cells in the channel.

[0067] S32: Place the chip on the stage of an inverted fluorescence microscope or in a 37°C incubator;

[0068] S33: Simulating different conditions; further, in step S33, the simulation is performed under the condition that the pressure of the vacuum pump (6) is 90 kPa and the inlet flow rate of the culture medium perfusion is maintained at 100 μL / h. The different conditions include cerebral ischemia process, cerebral ischemia gradual reperfusion process and instantaneous reperfusion process;

[0069] Simulating cerebral ischemia process: Turn on the vacuum pump (6) to make the elastic film of the deformation layer (2) expand rapidly, forming 80% stenosis in the channel, simulating the cerebral ischemia state, such as Figure 3 shown.

[0070] Simulate the process of cerebral ischemia and gradual reperfusion: after 1 h of pressure, the pressure is gradually reduced to 0 within 1 h at a rate of 1.5 kPa / min;

[0071] Simulate the process of transient reperfusion: after 1 h of pressure, the air pressure is quickly reduced to 0, the channel returns to normal and continues to perfuse for 1 h.

[0072] S34: Real-time observation of cell state and photography, picture processing using equipment connected to the inverted fluorescence microscope, real-time calculation of free oxygen content in the channel, and alarm when the oxygen content is lower than the set value; Specifically, the determination of the standard curve: to calculate the oxygen content index of the "blood" in the ischemia and hypoxia microfluidic chip during real-time culture, the standard curve of the emission light of the oxygen film and the oxygen content in the channel needs to be fitted in advance. Different oxygen content gradient standard solutions are added to the channel, the channel is placed under the inverted fluorescence microscope, and the fixed green fluorescence excitation is used to save the emission light images of the channel with different oxygen content gradients. The corresponding relationship between the emission light of the oxygen film and the oxygen content in the channel is established, and the red pixel value of the channel is calculated using an algorithm, which is linearly fitted with the known oxygen content to calculate the standard curve. Whether the continuous use will affect the fluorescence emission process of PtOEP is tested, and the same chip is subjected to a cycle experiment from low to high oxygen content and from high to low oxygen content. The results are shown in Figure 4 2 The higher standard curve value is used as the formula for calculating the oxygen content in this experiment:

[0073] ;

[0074] Among them is the emission light intensity, the unit is pixel value, is the oxygen content, the unit is %, which represents the dissolved oxygen percentage in the solution.

[0075] Algorithm for mapping emission light intensity to oxygen content: place the chip with vascular endothelial cells under the green excitation light of the fluorescence microscope, observe the image information of the narrow downstream we need, obtain the original emission light image taken by the microscope, and detect the average value of the red pixels in the narrow downstream of the channel after brightness, exposure, and background removal uniformization treatment. The specific steps are as follows:

[0076] A1: Background removal: to remove background pixels and exclude the influence of background interference on data, use image subtraction algorithm in matlab, select background image as the minuend and channel image as the subtrahend:

[0077]

[0078] ​Wherein, imgChannel is the original image, imgBackground is the background image around the narrow downstream channel;

[0079] To carry out subtraction processing, imgBackground is enlarged to the size of the original image imgChannel, and the average image pixel is obtained , is the normalized processing image after the channel image is subtracted from the background image;

[0080] A2: Differentiated expansion: to make the image brightness change trend more obvious, the image is processed by exposure, and the exposure adjustment formula is:

[0081]

[0082] Wherein, k is the exposure adjustment coefficient, k selected for all pictures is the same, and , which is equivalent to the same processing for all images, and will not affect the change trend of the image itself;

[0083] A3: Select the image downstream of the narrow channel, and perform arithmetic average on the RGB pixel value of each pixel point to obtain the average red pixel component:

[0084] .

[0085] S35: The drug to be screened is dissolved in the cell culture medium at a therapeutic concentration, and is used for perfusion culture in the simulation process. The morphology and arrangement changes of the cells are observed under an inverted fluorescence microscope during the culture process.

[0086] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A pneumatically controlled ischemic stroke dynamic microfluidic vascular chip integrated oxygen sensing system, characterized by: It comprises a perfusion channel layer (1), a deformation layer (2), a glass oxygen barrier layer (3) and a pore control layer (4), which are, from top to bottom, the perfusion channel layer (1), the deformation layer (2), the glass oxygen barrier layer (3) and the pore control layer (4); The perfusion channel layer (1) is connected to the injection pump (5) via an external silicone tube, and is used to achieve dynamic cell culture in the channel; The deformable layer (2) is an elastic membrane, and deforms as the external air pressure increases, so that the channel forms a narrow area, and an oxygen probe is provided in the elastic membrane, and the oxygen probe is used for oxygen monitoring; The pore control layer (4) is connected to a vacuum pump (6) via an external silicone tube to apply air pressure to the deformation layer (2).

2. The pneumatically controlled ischemic stroke dynamic microfluidic vascular chip integrated oxygen sensing system according to claim 1 is characterized by: The perfusion channel layer (1), the deformation layer (2) and the pore control layer (4) are all made of polydimethylsiloxane. The contact surfaces of the two adjacent layers of the perfusion channel layer (1), the deformation layer (2), the glass oxygen barrier layer (3) and the pore control layer (4) are placed upward in a plasma treatment machine for cleaning. The layer contact surface made of polydimethylsiloxane is treated for 120 seconds, and the contact surface between the layer and the glass oxygen barrier layer (3) is treated for 180 seconds. The layer is then transferred to a 60°C oven and heated for 20 minutes to ensure that the four layers are tightly fitted to form a closed channel structure.

3. The pneumatically controlled ischemic stroke dynamic microfluidic vascular chip integrated oxygen sensing system according to claim 2 is characterized by: The perfusion channel layer (1) is a semi-open channel, and forms a closed microchannel when bonded with the deformation layer (2). The microchannel is used for dynamic perfusion culture of cells, and a liquid inlet and a liquid outlet are provided on the microchannel.

4. The pneumatically controlled ischemic stroke dynamic microfluidic vascular chip integrated oxygen sensing system according to claim 3 is characterized by: The pore control layer (4) is provided with pores, and the pores are connected to the vacuum pump (6) via a steel needle and a silicone tube.

5. The pneumatically controlled ischemic stroke dynamic microfluidic vascular chip integrated oxygen sensing system according to claim 1 is characterized by: The application steps of the system are as follows: S1: Surface pretreatment of the packaged chip model; S2: Static culture of cells in the chip; S3: Pathological simulation of cells in the chip and oxygen monitoring.

6. The pneumatically controlled ischemic stroke dynamic microfluidic vascular chip integrated oxygen sensing system according to claim 5 is characterized by: The step S1 further includes the following steps: S11: Place the packaged chip model in an ultra-clean sterilization table and sterilize with UV for 4 hours; S12: Rinse the channel once with alcohol and three times with sterile phosphate buffer, and add 100 μg / mL fibronectin solution to completely cover the channel; S13: Transfer to a 4°C refrigerator and let it stand for 12 hours.

7. The pneumatically controlled ischemic stroke dynamic microfluidic vascular chip integrated oxygen sensing system according to claim 6, characterized in that: The step S2 further includes the following steps: S21: Take the surface-treated model out of the refrigerator and rewarm it in a 37°C incubator for 30-60 minutes. After rewarming, transfer the model to a clean bench. S22: Use a pipette to aspirate the fibronectin solution in the channel and rinse the channel with vascular endothelial cell culture medium; S23: 1×10 6 The density of cells / mL was seeded in the microchannel; S24: Place the microfluidic chip in an incubator at 37°C and 5% CO2 for overnight incubation to allow the cells to adhere to the wall and grow. During the entire incubation process, keep the liquid inlet and outlet covered with a set amount of vascular endothelial cell culture medium.

8. The pneumatically controlled ischemic stroke dynamic microfluidic vascular chip integrated oxygen sensing system according to claim 7 is characterized by: The step S3 further includes the following steps: S31: Connect the outlet and inlet of the perfusion channel with a steel needle and silicone tubing, respectively. Use a syringe to draw in complete endothelial cell culture medium containing 15% fetal bovine serum, then connect it to the silicone tubing. Set the inlet flow rate to 100 μL / h to provide a continuous flow of culture medium to the cells in the channel. S32: Place the chip on the stage of an inverted fluorescence microscope or in a 37°C incubator; S33: Simulate different conditions; S34: Observe the cell status in real time and take photos. Use a device connected to an inverted fluorescence microscope to process the images and calculate the free oxygen content in the channel in real time. When the oxygen content falls below the set value, an alarm is issued. S35: The drug to be screened is dissolved in the cell culture medium at a therapeutic concentration and used for perfusion culture during the simulation process. During the culture process, the morphology and arrangement changes of the cells are observed under an inverted fluorescence microscope.

9. The pneumatically controlled ischemic stroke dynamic microfluidic vascular chip integrated oxygen sensing system according to claim 8, characterized in that: In step S33, the simulation is performed under the condition that the pressure of the vacuum pump (6) is 90 kPa and the inlet flow rate of the culture medium perfusion is maintained at 100 μL / h. Different conditions include cerebral ischemia process, cerebral ischemia gradual reperfusion process and instantaneous reperfusion process; Simulating the cerebral ischemia process: turning on the vacuum pump (6) causes the elastic film of the deformation layer (2) to expand rapidly, forming an 80% stenosis in the channel, simulating the cerebral ischemia state; Simulate the gradual reperfusion process of cerebral ischemia: after pressurization for 1 hour, gradually reduce the pressure to 0 at a rate of 1.5 kPa / min within 1 hour; Simulate the transient reperfusion process: after pressurization for 1 hour, the air pressure is quickly reduced to 0, the channel returns to normal and perfusion continues for 1 hour.

Images