Device and method for promoting growth of endothelial glycocalyx using shear stress of blood flow

Abstract

Provided are a method for preparing a vascular chip, and a device and method for promoting the growth of endothelial glycocalyx on the basis of the vascular chip and microfluidic technology. The vascular chip has a bilayer structural model with a bionic design of human microvessels, and is prepared by processes such as photolithography and bonding. The device for promoting the growth of endothelial glycocalyx is constituted jointly by the vascular chip, a driving system, and a fluid shear stress generating system, wherein the driving system uses a microfluidic system to simulate the microenvironment within human blood vessels. The method for generating endothelial glycocalyx using fluid shear stress realizes the real-time observation of endothelial cells by means of endothelial cell seeding and dynamic perfusion culture.

Description

Apparatus and method for promoting endothelial glycocalyx growth using blood flow shear force TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to an apparatus and method for promoting endothelial glycocalyx growth. BACKGROUND

[0002] Vascular endothelial glycocalyx (VEG) is a protein-polysaccharide complex located on the luminal surface of vascular endothelial cells, with a thickness of 0.1-1.0 μm, synthesized and secreted by vascular endothelial cells, and located at the junction between circulating blood and blood vessel wall (Chen J, Gong D, Yi YH, et al. Role of vascular endothelial glycocalyx in the pathogenesis, diagnosis, and treatment of acute lung injury in sepsis [J]. Journal of the Chinese People's Liberation Army, 2021, 46(04): 398-403.). VEG is composed of various glycosaminoglycan side chains and glycoproteins, and its biological function and structure are relatively complex. VEG plays an important role in maintaining the stability of endothelial cell structure and function, preventing inflammatory cell adhesion, regulating blood cells and endothelial cells, maintaining the integrity of the vascular wall barrier function, regulating microcirculation blood flow, and inhibiting thrombus formation (Schmidt EP, Yang YM, Janssen wy, et al. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis [J]. Nat Med, 2012, 18(8): 1217-1223.). In many disease states, VEG will be damaged or shed, increasing the complexity of observation. And due to the lack of standardized and widely accepted VEG measurement methods, the comparison and verification of the observation results are limited. The main methods for promoting VEG growth at present include drug therapy (heparin and its derivatives), infusion of fresh frozen plasma, supplementation of glycosaminoglycans and glucocorticoids. Although there are many therapeutic drugs at present, there are some limitations. Many drugs not only have side effects, but also have high cost and are not easy to obtain in many resource-limited areas. Secondly, many theories and drugs for VEG repair are still in the laboratory stage and have not been fully verified.

[0003] In the normal blood system, vascular endothelial cells are affected by blood flow forces at all times. There are mainly three kinds of blood flow forces, namely hydrostatic pressure, circumferential tensile stress and fluid shear stress (ZHANG Y, ZENG Y, LIU JX, et al. Research progress of fluid shear stress regulating autophagy of vascular endothelial cells[J]. Biomedical Engineering Research, 2016, 35(01): 60-64.). Among them, fluid shear stress is the force generated by the friction between blood flow and the inner wall of the blood vessel, which is parallel to the long axis of the blood vessel, and is also the most important biomechanical stimulus for vascular endothelial cells, playing an important role in regulating endothelial cell function (QIN ZJ, CHEN SQ, WU YM, et al. Effects of material surface chemistry and fluid shear stress on human umbilical vein endothelial cells[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(16): 2516-2521.). VEG, as a highly complex and carbohydrate-rich plush structure on the apical surface of endothelial cells, helps the barrier properties of the vascular wall and is also involved in mechanical sensing and shear stress transduction to the endothelium. At present, high shear stress has been shown to maintain the integrity of VEG, but existing research has mainly focused on the static properties of VEG and its function in physiological conditions, and the research on its generation and integrity mechanism is still insufficient.

[0004] Microfluidic technology is considered one of the important scientific and technological ones in the 21st century, which is a technology for manipulating fluid at micron or nanoscale. It can not only restore the blood flow environment, but also generate different intensities of fluid shear force according to the needs, and its application in biomedicine is becoming more and more widely (HUANG D. MAN JX. JIANG D, et al. Inertial microfluidics, recent advances [J]. Electrophoresis, 2020 41124. 2166-2187;IWANAGAT, MIURA N, BRAINARD B M, et al. A novel microchip flow chamber (total thrombus analysis system) to assess canine hemostasis [J] Front Vet Sci, 2020, 7: 307.) In the microfluidic channel, platelets can adhere to the bottom of the channel through the binding of surface receptors and corresponding protein ligands, while other blood cells will not adhere to the bottom (ZHANG T C, CHEN D. LIAO J, et al. Platelet aggregation on glass surface under physiological flow conditions [J]. J Med Biomech (Medical Biomechanics). 2022. 37(3): 425-432.) Therefore, constructing VEG model in vitro, making it intuitive and visual for research, can help us understand the generation mechanism of endothelial glycocalyx more deeply, and provide treatment ideas for diseases related to endothelial glycocalyx injury. SUMMARY

[0005] Based on the above technical deficiencies of the prior art, the present application provides a method for preparing a vascular chip, which comprises the following steps:

[0006] In a first aspect, the present application provides a method for preparing a vascular chip, which comprises the following steps:

[0007] S1. Template material preparation: the silicon wafer is first placed in acetone-isopropanol-ionized water for ultrasonic cleaning. After cleaning, the silicon surface is blown dry and baked to remove surface water molecules.

[0008] S2. Gluing: place the cleaned silicon wafer on the suction disc of the glue spreading machine, drop negative photoresist on the surface of the silicon wafer, and cover the silicon wafer with a layer of SU-8 photoresist.

[0009] S3. Pre-baking: the silicon wafer coated with photoresist is pre-baked until the photoresist is hardened, and then cooled.

[0010] S4. Exposure: after the photoresist is cured, the mask plate and the silicon wafer are put into an ultraviolet photoetching machine, so that the pattern on the mask plate is transferred to the SU-8 glue layer.

[0011] S5. Post-baking: the exposed silicon wafer is baked again, and then cooled.

[0012] S6. Development: the silicon wafer is immersed in a SU-8 special developing solution for development, and the SU-8 template is obtained after the development is completed.

[0013] S7. Pouring: the precursor polymer and the crosslinking agent are poured onto the SU-8 template, air bubbles are extracted, and then heated and cured, and then naturally cooled to obtain a patterned substrate.

[0014] S8. Substrate forming: the substrate is cut, and then the substrate is peeled off from the template to complete demolding.

[0015] S9. Substrate bonding: holes are punched on the demolded substrate, and the substrate and the glass substrate are surface treated, and then the activated surfaces of the two are contacted to form irreversible bonding at the interface of the two.

[0016] S10. Sterilization treatment: the prepared chip is sterilized, and the blood vessel chip is obtained.

[0017] Further, in step S1, the silicon wafer is selected as a 4-inch single-throw silicon wafer.

[0018] Further, in step S2, the negative photoresist drop is SU-82050 type negative photoresist.

[0019] Further, in step S2, the photoresist covered on the silicon wafer is obtained according to the corresponding relationship between the photoresist thickness and the rotating speed, and the spin coater is set to the corresponding rotating speed.

[0020] Further, in step S4, the mask plate is a mask plate with a pattern of a biomimetic human microvessel.

[0021] Further, in step S7, the precursor polymer is a material with high biocompatibility, good light transmittance, and surface modifiability.

[0022] Further, in step S7, the precursor polymer is selected from one or more of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC), and / or cyclic olefin polymer (COP).

[0023] Further, in step S9, the surface treatment of the PDMS substrate and the glass slide is selected as oxygen plasma.

[0024] Further, in step S10, the disinfection mode comprises any one of ultraviolet sterilization and high-pressure steam sterilization.

[0025] In a second aspect, the present application provides a device for promoting endothelial glycocalyx growth, which is composed of a blood vessel chip, a driving system and a fluid shear stress generating system.

[0026] Further, the blood vessel chip is obtained by the preparation method of the first aspect of the present application, and is a double-layer structure model designed according to the human microvessel, wherein the upper layer is made of a material with high biocompatibility and good light transmission, and the lower layer is a glass substrate. The blood vessel chip has a rectangular shape with a length of 2 cm.

[0027] Further, the material of the upper layer of the blood vessel chip is selected from one or more of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC) and / or cyclic olefin polymer (COP).

[0028] Further, the upper layer of the blood vessel chip is provided with a microfluidic channel, which is designed according to the structure of human microarteriole-capillary-microvenule and is a cell perfusion channel and a cell culture area.

[0029] Further, the microfluidic channel is a wideband with a structure of wide-narrow-wide, which is a bionic human microvessel structure.

[0030] Preferably, the width of the microfluidic channel is 600um-300um-600um, and the observed part of the present application is the part with a width of 300um.

[0031] Further, the driving system is performed by using an Elveflow microfluidic system, which includes a flow controller, a control system, a liquid storage tube and a flow sensor.

[0032] Further, an external pressure source and / or a vacuum source are connected to the flow controller, the external pressure source and / or the vacuum source press the liquid in the liquid storage tube through air pressure, and the microfluidic flow in the channel is carried out through the flow sensor and the flow controller.

[0033] Further, the external pressure source and / or the vacuum source are connected to a pressure sensor, and the precise control of the air pressure achieves the precise control of the microfluidic flow in the microfluidic channel.

[0034] Preferably, the pressure source or the vacuum source can be an air compressor, a vacuum pump and a gas cylinder.

[0035] Further, the fluid shear stress generating system is mainly realized by liquid flow in the channel, and the liquid in the liquid storage pipe is pressed out by a driving system to make the fluid enter the microfluidic channel, based on the channel involved, the inlet is 2ul / min, the direction is unified, and the stable and pulse-free flow rate is adopted, so that the stable fluid shear stress is formed.

[0036] Further, in order to facilitate real-time observation, the blood vessel chip is placed in a living cell workstation, the living cell workstation is placed under a microscope, and real-time observation of cells is carried out.

[0037] Further, the fluid shear stress simulation device further comprises a metabolite collection pipe, and the culture medium and cell metabolites flowing through the chip and the cell metabolite outlet pipeline are collected through the metabolite collection pipe.

[0038] Further, the fluid shear stress generated in the channel can be calculated by the following formula:

[0039]

[0040] In the formula, Q is the channel flow (m / s); h is the channel height (m); and w is the channel width (m). 3

[0041] In a third aspect, the present application provides a method for generating endothelial glycocalyx by using fluid shear stress, which comprises the following steps:

[0042] S1. Preparing a blood vessel chip, the blood vessel chip is obtained by the preparation method of the first aspect of the present application and is sterilized;

[0043] S2. Infusing a bovine fibrinogen solution in the channel of the blood vessel chip for incubation;

[0044] S3. Using a buffer to rinse the channel in the blood vessel chip, inoculating human umbilical vein endothelial cells, and carrying out static culture;

[0045] S4. After the cells adhere, using a microfluidic system to apply stable fluid for continuous dynamic culture;

[0046] S5. After dynamic culture, fixing the cells and the endothelial glycocalyx in the channel;

[0047] S6. Using wheat germ agglutinin staining to carry out immunofluorescence staining of the endothelial glycocalyx, and observing and photographing under a fluorescence microscope.

[0048] Further, in step S1, the blood vessel chip is a double-layer structure model designed according to the shape of a biomimetic human microvessel, the upper layer is made of a material with high biocompatibility and good light transmission, and the lower layer is a glass substrate (Glass substrate). The shape of the blood vessel chip is rectangular, and the length is 2cm.

[0049] ​Further, the upper layer of the blood vessel chip is selected from one or more of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC) and / or cyclic olefin polymer (COP).

[0050] Further, the upper layer of the blood vessel chip is provided with a microfluidic channel, which is designed according to the arteriole-capillary-venule structure of the human vascular network, and is a cell perfusion channel and a cell culture area.

[0051] Further, the microfluidic channel is a wide band of coarse-fine-coarse, which is a biomimetic human microvessel structure.

[0052] Preferably, the microfluidic channel is a wide band of 600um-300um-600um, and the observation of the present application is the part with a width of 300um.

[0053] Further, in step S2, the concentration of bovine fibrinogen is 5mg / ml, and incubation is carried out at 4℃ overnight.

[0054] Further, in step S3, the concentration of human umbilical vein endothelial cells is 5×10 6 CFU / ml. Beneficial effects

[0055] 1. The blood vessel chip provided by the present application has many advantages as a new type of in vitro model.

[0056] (a) The blood vessel chip integrated with a perfusion system (continuous perfusion, bidirectional perfusion, etc.) is superior to ordinary static models, and the shear force, physical and chemical gradient formed by fluid flow in the vascular system can be more close to the dynamic environment in vivo;

[0057] (b) By replicating different in vivo processes, single-factor variables can be controlled in vitro, which facilitates people to better understand and study the influence of different pathological factors on vascular endothelial function. For example, by changing the structure of the blood vessels, the changes of cells in different regions and different blood flow states and the influence on the function of vascular endothelium can be observed;

[0058] (c) The blood vessel chip allows real-time monitoring of cell behavior and drug response, which plays an important role in rapid evaluation of drug effect and toxicity test.

[0059] 2. The device for promoting endothelial glycocalyx growth provided by the present application has the following advantages:

[0060] (a) The chip is placed in a live cell workstation, which facilitates real-time observation of changes in cell morphology and skeleton-related changes during dynamic culture environment;

[0061] (b) The microfluidic system can precisely control the flow, such as cell culture, drug screening, etc.

[0062] (c) The system can achieve stable flow without pulse, provide controlled shear stress, and control the experiment through computer software, making the experiment process more convenient and accurate.

[0063] 3. By constructing endothelial glycocalyx with the present application, various biological experiments can be carried out:

[0064] (a) Simulate the vascular environment: The microfluidic chip can simulate the microenvironment in the blood vessel, including blood flow dynamics and the interaction between cells and extracellular matrix;

[0065] (b) Research in high glucose environment: Through the microfluidic system, endothelial cells can be cultured in a high glucose environment to study the effect of high glucose on endothelial glycocalyx, such as the study of changes in glycocalyx and its constituent glycans in vascular endothelial cells under high glucose conditions;

[0066] (c) Drug screening and toxicity testing: The microfluidic system can be used to screen compounds with preventive and therapeutic activity for diseases related to endothelial glycocalyx, while conducting drug toxicity testing;

[0067] (d) Disease model construction: Microfluidic technology can be used to construct disease models of endothelial glycocalyx dysfunction, such as diabetic vasculopathy, to further study the pathological mechanisms and potential treatment options;

[0068] (e) Endothelial cell function research: The microfluidic system can be used to study the function of endothelial cells under different physiological and pathological conditions, including vascular permeability, leukocyte adhesion, and anticoagulant function, etc.

[0069] (f) Multi-omics analysis: Combined with microfluidic chip technology, multi-omics analysis such as transcriptome, proteome and metabolome can be carried out to further understand the role of endothelial glycocalyx in diseases;

[0070] (g) Research on the protective effect of endothelial glycocalyx: The microfluidic system can be used to study the function of endothelial glycocalyx in protecting vascular endothelium, regulating vascular permeability and nitric oxide release induced by blood flow shear stress, etc.

[0071] (h) Changes in endothelial glycocalyx under pathological conditions: Microfluidic technology can help study the changes in endothelial glycocalyx under pathological conditions, such as sepsis, and its impact on disease progression. BRIEF DESCRIPTION OF DRAWINGS

[0072] Figure 1 is the design idea of the vascular chip.

[0073] Figure 2 is a schematic diagram of the upper structure of the vascular chip. Note: width (a) = 600 μm; (b) = 300 μm; (c) = 600 μm.

[0074] Figure 3 is a schematic diagram of the fabrication of a blood vessel chip.

[0075] Figure 4 is a photograph of a SU-8 template.

[0076] Figure 5 is a photograph of a molded PDMS substrate.

[0077] Figure 6 is a photograph of a molded PDMS substrate after punching.

[0078] Figure 7 is a photograph of a blood vessel chip.

[0079] Figure 8 is a device for inducing endothelial glycocalyx generation using fluid shear stress.

[0080] Figure 9 is a method for endothelial glycocalyx growth.

[0081] Figure 10 is the effect of different fluid shear stress on endothelial glycocalyx under confocal microscope.

[0082] Figure 11 is the effect of different fluid shear stress on endothelial glycocalyx under conventional optical microscope. DETAILED DESCRIPTION

[0083] The specific embodiments of the present application will be further described with the following non-limiting examples. It is to be understood that the examples are only used to more fully describe and further illustrate my application. Accordingly, the examples should not be construed as limiting the scope of the present application, but merely as being illustrative. Further, the technical features involved in the following examples can be combined with each other as long as there is no conflict. Any modification, equivalent replacement, improvement, and the like made within the spirit and principle of the present application shall be included in the scope of the present application.

[0084] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available unless otherwise specified.

[0085] Glycosaminoglycans: A class of linear acidic polysaccharides with rich functions, which are combined with core proteins to form proteoglycans through covalent bonds with serine residues and exist in cell membranes and extracellular matrix. They can affect biological processes such as embryonic development, enzyme activity, extracellular matrix assembly, ligand-receptor binding, and cell signaling by regulating the functions of different proteins. The interaction of glycosaminoglycans and proteins is involved in diseases including cardiovascular disease, cancer, infectious disease, neurodegenerative disease, inflammatory response, and wound healing.

[0086] Elveflow microfluidic system: It is a system designed by Elveflow, France, for liquid processing in cell experiments, can realize perfusion of various culture media, can realize stable culture medium perfusion and replacement between several solutions, can control shear stress in a large flow range, realizes automation of cell culture microfluidic process, and is used for cell culture of chip laboratory, flow cell and perfusion chamber, and is used to create continuous flow in cell culture process and detect flow.

[0087] Photoresist: Photoresist refers to an etch-resistant film material whose solubility changes through the irradiation or radiation of an exposure source such as ultraviolet light, electron beam, X-ray, etc.

[0088] Example 1 Preparation of a blood vessel chip

[0089] The blood vessel chip in the application is designed by imitating human microvessels, as shown in FIGS. 1-3. The specific preparation process is as follows: 1. Preparation of a template

[0090] Template material preparation: As shown in FIG. 3A, a 4-inch single-throw silicon wafer is selected as the template material, and the silicon wafer is ultrasonically cleaned in acetone-isopropyl alcohol-ionized water in sequence. After cleaning, the silicon surface is blown dry with a nitrogen gun, and is baked on a hot plate at 180℃ for more than 30min to remove surface water molecules.

[0091] Gluing: As shown in FIG. 3B, place the cleaned silicon wafer on the chuck of the spin coater, and use a dropper to drop SU-82050 type negative photoresist on the surface of the silicon wafer. According to the corresponding relationship between the thickness of the photoresist and the rotation speed, set the spin coater to the corresponding rotation speed, so that the silicon wafer is uniformly covered with a layer of photoresist.

[0092] Soft bake: Place the SU-8 coated silicon wafer on a hot plate for soft bake, set the baking temperature to 85℃, bake until the photoresist hardens, then take the silicon wafer off the hot plate and cool it naturally at room temperature.

[0093] Exposure: As shown in FIG. 3C, after the photoresist is cured, place the mask plate with the microvessel pattern and the silicon wafer into the ultraviolet photoetching machine. The mask plate and the silicon wafer SU-8 glue layer surface will maintain a small gap, and then use ultraviolet light to irradiate the glue to cause a chemical reaction, transferring the pattern on the mask plate to the SU-8 glue layer.

[0094] Post exposure bake: Place the exposed silicon wafer on a hot plate again at a temperature of 95℃ for baking for 30min, and then cool it at room temperature.

[0095] Development: As shown in Figure 3D, the silicon wafer is immersed in SU-8 developer (MicroChem's SU-8 developer) for development, and the SU-8 template obtained after development is shown in Figure 4.

[0096] 2. PDMS pouring, as shown in Figure 3E

[0097] The two solutions of PDMS precursor polymer and crosslinking agent are poured into a cup at a mass ratio of 10:1, and a glass rod is used to stir them uniformly. The uniformly mixed solution is poured onto the SU-8 template and placed in a vacuum chamber. Air bubbles are extracted by a vacuum pump, and the solution is heated and cured in an 80°C oven for 1 h, and then naturally cooled to obtain a PDMS substrate with patterns.

[0098] 3. PDMS substrate forming, as shown in Figure 3F

[0099] The PDMS substrate is cut with a blade to retain only the patterned area, and then the PDMS is peeled off from the template to complete demolding, as shown in Figure 5.

[0100] 4. PDMS substrate bonding, as shown in Figure 3G

[0101] Holes are punched in the demolded substrate, and the demolded PDMS substrate and the glass substrate are surface treated by oxygen plasma to activate the surfaces. Then the activated surfaces of the two are brought into contact to form irreversible bonding at the interface between them, as shown in Figure 6. At this time, a closed channel is formed between the glass substrate and the chip, which is the cell culture area, as shown in the red area of Figure 7.

[0102] 5. Sterilization and disinfection treatment

[0103] The prepared chip is placed on an ultraclean bench and subjected to ultraviolet disinfection for 1 h to obtain the blood vessel chip of the present application. The blood vessel chip has a rectangular shape, a length of 2 cm, a width of 600 μm / 300 μm, and a height of 150 μm. The observation part of the present application has a length of 1 cm, a width of 300 μm, and a height of 150 μm.

[0104] Example 2 Construction of a fluid shear force simulation device

[0105] 1. Composition of the fluid shear force simulation device

[0106] The fluid shear force simulation device is composed of a blood vessel chip, a driving system, and a fluid shear force generating system.

[0107] The upper layer of the blood vessel chip is provided with a microfluidic channel, which is designed according to the structure of human microarteriole-capillary-microvenule and is a cell perfusion channel and a cell culture area.

[0108] The drive system uses the Elveflow microfluidic system, which includes a flow controller, a control system, a liquid reservoir, and a flow sensor. An external pressure source and / or vacuum source is connected to the flow controller. The external pressure source and / or vacuum source uses air pressure to force the liquid out of the liquid reservoir. The flow sensor and flow controller control the microfluidic flow rate within the channel.

[0109] Furthermore, an external pressure source and / or vacuum source connected to an external pressure sensor enables precise control of the microfluidic flow rate within the microfluidic channel through precise control of air pressure.

[0110] The fluid shear force generation system is mainly achieved through liquid flow within the channels. A drive system forces liquid out of the reservoir tube and into the microfluidic channels. Based on the channels involved, an inlet flow rate of 2 μl / min is used, with a uniform direction and a stable, pulse-free flow rate to generate stable fluid shear force. After the cells have adhered to the channel walls for 48 hours, the chip is connected to an "L"-shaped needle and tubing, which are then connected to the drive system. Fluid is continuously applied for 24 hours in a stable, pulse-free mode.

[0111] Preferably, the pressure or vacuum source can be an air compressor, a vacuum pump, or a gas cylinder.

[0112] Furthermore, to facilitate real-time observation, the vascular chip was placed inside a live-cell workstation, which was then placed under a microscope for real-time cell observation.

[0113] Furthermore, the fluid shear force simulation device also includes a metabolite collection tube, through which the culture medium and cell metabolites flowing through the chip are collected.

[0114] To further improve the efficiency of fluid shear force-induced endothelial calyx formation, the following optimization measures can be taken:

[0115] (a) Optimize the parameters for generating fluid shear force to produce a more suitable shear force for endothelial cell growth.

[0116] (b) During the culture process, specific growth factors can be added to promote the formation of endothelial calyx.

[0117] 2. The fluid wall shear force generated in the channel can be calculated using the following formula:

[0118]

[0119] In the formula, Q is the channel flow rate (m³ / s). 3 / s); h is the channel height (m); w is the channel width (m).

[0120] 3. Fluid shear stress simulation device operation process (Figure 8)

[0121] (a) Connect an external pressure and / or vacuum source to the flow controller, and connect a pressure sensor to the external pressure and / or vacuum source;

[0122] (b) Control the pressure of the external pressure and / or vacuum source and the flow rate of the microfluidic channel through the control system, and adjust the pressure through the flow sensor to obtain the required flow rate in the setting;

[0123] (c) Place the blood vessel chip in the live cell workstation and connect it to the microfluidic system through the hose and "L" shaped needle;

[0124] (d) According to the air pressure controlled by the control system, press the liquid in the liquid storage tube out through the external pressure and / or vacuum source, and inject the precisely controlled flow rate into the blood vessel chip stably.

[0125] In order to facilitate real-time observation, place the live cell workstation under the microscope for real-time observation of cells, and collect the medium and cell metabolites flowing through the chip through the pipeline and the collection tube.

[0126] Example 3 Method for generating endothelial glycocalyx using fluid shear stress (Figure 9)

[0127] (a) Use normal saline to prepare bovine fibrin solution, and prepare the concentration to be 5 mg / ml;

[0128] (b) Infuse the bovine fibrinogen solution into the chip channel for incubation, and incubate overnight in a 4°C refrigerator;

[0129] (c) Use PBS buffer to rinse the channel in the chip, and after rinsing 3 times, inoculate human umbilical vein endothelial cells, with a cell inoculation density of 5 x 10 6 / ml, and statically culture for 48 h until the cells adhere;

[0130] (d) After the cells adhere, use the microfluidic system to apply stable fluid, and continuously and dynamically culture for 24 h;

[0131] (e) After dynamic culture, fix the cells and endothelial glycocalyx in the channel with paraformaldehyde;

[0132] (f) Use wheat germ agglutinin staining to perform immunofluorescence staining of endothelial glycocalyx, and observe the cell morphology under a fluorescence microscope and take pictures.

[0133] Example 4 Effect of different fluid shear stresses on endothelial glycocalyx

[0134] After static culture for 48 h, the cells were intervened with flow rates of 2 μl / min, 1 μl / min, 0.5 μl / min and 0 μl / min to simulate different blood flow environments in human body. The effects of different fluid shear stress on the generation of endothelial glycocalyx were explored by comparing the fluid shear stress stimulation after the flow rate was reduced from 2 μl / min.

[0135] The results show that the vascular endothelial cells can grow into relatively dense villous structures. Under the confocal microscope, it can be observed that the growth of villous structure gradually decreases with the gradual reduction of flow rate; when the flow rate is 0 μl / min, i.e. static culture, it is difficult to observe the peripheral villous structure, as shown in Fig. 10.

[0136] Under the ordinary microscope, it can be observed that when the flow rate is 2 μl / min, the cells gradually elongate, and the cell arrangement is relatively orderly, mainly parallel to the fluid direction; with the gradual reduction of flow rate, the cell arrangement direction has no obvious regularity, and the cells change from spindle-shaped to oval-shaped, as shown in Fig. 11.

Claims

1. A method for preparing a blood vessel chip, the method comprising the following steps: S1. Template material preparation: a silicon wafer is sequentially placed in acetone-isopropyl alcohol-ionized water and ultrasonically cleaned, after the cleaning is completed, the silicon surface is blown dry and baked to remove surface water molecules; S2. Gluing: the cleaned silicon wafer is placed on a glue spreading machine suction disc, negative photoresist is dropped on the surface of the silicon wafer, and the silicon wafer is covered with a layer of photoresist; S3. Pre-baking: the silicon wafer coated with photoresist is pre-baked until the photoresist becomes hard, and then cooled; S4. Exposure: after the photoresist is cured, the mask plate and the silicon wafer are placed in a UV photoetching machine, and the pattern on the mask plate is transferred to the SU-8 resist layer; S5. Post-baking: the silicon wafer after exposure is baked again, and then cooled; S6. Development: the silicon wafer is immersed in SU-8 special developing solution for development, and the SU-8 template is obtained after the development is completed; S7. Pouring: a mixed solution of precursor polymer and crosslinking agent is poured onto the SU-8 template, air bubbles are extracted and heated to solidify, and then naturally cooled to obtain a substrate with patterns; S8. Substrate forming: the substrate is cut, and then the substrate is peeled off from the template to complete demolding; S9. Substrate bonding: holes are punched on the demolded substrate, and the substrate and the glass substrate are surface treated, then the activated surfaces of the two are contacted to form irreversible bonding at the interface of the two; S10. Sterilization and disinfection treatment: the prepared chip is sterilized, and the blood vessel chip of the application is obtained.

2. The preparation method according to claim 1, wherein the mask plate in step S4 is a mask plate with a pattern of a biomimetic human microvessel.

3. The preparation method according to any one of claims 1 or 2, wherein the precursor polymer in step S7 is selected from materials with high biocompatibility and good light transmission.

4. The preparation method according to any one of claims 1-3, wherein the precursor polymer in step S7 is selected from one or more of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC) and / or cyclic olefin polymer (COP).

5. A device for promoting the growth of endothelial glycocalyx, the device being composed of a blood vessel chip, a driving system and a fluid shear stress generating system.

6. The device according to claim 5, wherein a microfluidic channel is arranged on the upper layer of the blood vessel chip, the channel is designed according to the structure of human microarteriole-capillary-microvenule, and is a cell perfusion channel and a cell culture area.

7. The device according to any one of claims 5 or 6, wherein the driving system is implemented by using an Elveflow microfluidic system, which comprises a flow controller, a control system, a liquid storage tube and a flow sensor.

8. The device according to any one of claims 5-7, wherein the fluid shear stress generating system is mainly realized by liquid flow in the channel, liquid in the liquid storage tube is pressed out by the driving system, and then the fluid enters the microfluidic channel, based on the channel involved, the flow rate at the inlet is 2 μl / min, the direction is unified, and a stable and pulseless flow rate is adopted, so as to form a stable fluid shear stress.

9. The apparatus according to any one of claims 5-8, wherein the fluid shear force can be calculated using the following formula: where Q is the channel flow rate (m 3 / s); h is the channel height (m); and w is the channel width (m).

10. A method for generating endothelial glycocalyx using fluid shear stress, the method comprising the steps of: S1. Preparing a vascular chip and sterilizing; S2. Infusing a bovine fibrinogen solution into the vascular chip channel for incubation; S3. Using a buffer to rinse the channel in the vascular chip, inoculating human umbilical vein endothelial cells, and static culture; S4. After the cells adhere, using a microfluidic system to apply stable fluid for continuous dynamic culture; S5. After dynamic culture, fixing the cells and endothelial glycocalyx in the channel; S6. Immunofluorescence staining of endothelial glycocalyx using wheat germ agglutinin staining, fluorescence microscopy observation and photography.

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