Dynamic perfusion blood tube chip based on micro-fluidic core-shell fibers and preparation method of dynamic perfusion blood tube chip

Abstract

The invention discloses a dynamic perfusion blood tube chip based on micro-fluidic core-shell fibers and a preparation method thereof, and belongs to the technical field of biomedical materials.The preparation method comprises the steps that 1, hydrogel fibers with hollow inner cavities are prepared through the micro-fluidic technology; step 2, planting human coronary artery smooth muscle cells on the outer surface of the hydrogel fiber, and planting human umbilical vein endothelial cells in the cavity of the hydrogel fiber to realize three-dimensional co-culture of the two cells; and step 3, soaking the hydrogel fiber planted with the cells in a human coronary artery smooth muscle cell complete medium, connecting the hydrogel fiber with the human umbilical vein endothelial cell complete medium, and continuously pouring the human umbilical vein endothelial cell complete medium into an inner cavity of the hydrogel fiber planted with the cells to obtain the dynamic perfusion blood tube chip. The chip provided by the invention not only can realize three-dimensional co-culture of cells, but also realizes dynamic perfusion culture, and better simulates the structure and function of human blood vessels.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials and relates to a vascular chip, in particular to a dynamic perfusion vascular chip based on microfluidic core-shell fibers and a preparation method thereof. Background Art

[0002] As an emerging biomedical engineering method, organ chip technology has received widespread attention in the fields of disease research, drug screening and regenerative medicine in recent years. Compared with traditional two-dimensional cell culture and animal models, organ chip technology has unique advantages. Although two-dimensional cell culture is simple and easy, it lacks a three-dimensional microenvironment and cannot fully simulate the complex structure and function of in vivo tissues, resulting in low physiological relevance of research results. Although animal models can simulate the in vivo environment, there are species differences, and the research results are difficult to apply directly to humans. In addition, animal experiments are costly, time-consuming, and have ethical issues, which limit their widespread application.

[0003] As an important branch of organ chip technology, vascular chips can reconstruct the three-dimensional structure and dynamic perfusion environment of blood vessels at the microscale, providing a more physiologically relevant cell culture platform. Existing vascular chips usually use microfluidics technology to regulate the cell microenvironment by precisely controlling fluid flow. These chips show great potential in studying vascular biology, disease mechanisms, and drug screening. However, existing technologies still have shortcomings in how to construct blood vessels that are closer to the actual structure of the human body, such as cell co-culture problems, biomaterial cell compatibility, channel microstructure and other issues. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a dynamic perfusion vascular chip based on microfluidic core-shell fiber and a preparation method thereof, wherein the method uses microfluidic technology to prepare hydrogel fibers with a core-shell structure, plants human umbilical vein endothelial cells in the fiber lumen and plants human coronary artery smooth muscle cells on the outer surface, and then places the hydrogel fibers into a pre-designed chip, perfuse culture medium, and construct a vascular chip that can be dynamically perfused and cultured. The chip can not only realize three-dimensional co-culture of cells, but also realize dynamic perfusion culture, better simulate the structure and function of human blood vessels, and provide a new research method for studying the pathogenesis of human vascular diseases, drug screening, and personalized treatment.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing a dynamic perfusion blood vessel chip based on microfluidic core-shell fibers comprises the following steps:

[0007] Step 1: Prepare hydrogel fibers with hollow inner cavities using microfluidics technology; Step 2: Plant human coronary artery smooth muscle cells (HCASMCs) on the outer surface of the hydrogel fibers, and plant human umbilical vein endothelial cells (HUVECs) in the hydrogel fiber cavities to achieve three-dimensional co-culture of the two cells; Step 3: Soak the hydrogel fibers planted with cells in complete culture medium of human coronary artery smooth muscle cells, and connect them with complete culture medium of human umbilical vein endothelial cells, so that the complete culture medium of human umbilical vein endothelial cells is continuously infused into the inner cavity of the hydrogel fibers planted with cells, thereby obtaining a dynamic perfusion vascular chip.

[0008] To optimize the above technical solutions, the specific measures taken also include:

[0009] Furthermore, in step one, the preparation method of the hydrogel fiber is: using a hydrogel precursor solution as an external phase and a supporting liquid as an internal phase, respectively injecting them into the external phase channel and the internal phase channel of a microfluidic fiber generating device, and solidifying the hydrogel precursor solution at the outlets of the external phase channel and the internal phase channel to obtain a hydrogel fiber with a hollow inner cavity; the microfluidic fiber generating device includes a coaxially assembled external phase capillary and an internal phase capillary, the internal phase capillary is the internal phase channel, and the channel between the internal phase capillary and the external phase capillary is the external phase channel; the diameter of the external phase capillary is 800 to 1000 μm, and the diameter of the internal phase capillary is 300 to 500 μm; accordingly, the outer diameter of the obtained hydrogel fiber is 750 to 950 μm, and the inner diameter is 350 to 550 μm.

[0010] Furthermore, in step one, the hydrogel precursor in the hydrogel precursor solution includes methacryloyl gelatin (GelMa) and polyethylene glycol diacrylate (PEGDA); the concentration of methacryloyl gelatin is 5-15 m / v%, preferably 10 m / v%, and the concentration of polyethylene glycol diacrylate is 1-4 v / v%, preferably 2.5 v / v%; the supporting liquid is polyvinyl alcohol (PVA) solution, and the concentration is 5-15 m / v%, preferably 10 m / v%; the flow rate of the external phase channel is 20-40 mL / h, and the flow rate of the internal phase channel is 10-30 mL / h.

[0011] Furthermore, in step one, the curing method is ultraviolet light curing, and the hydrogel precursor solution contains a photoinitiator; the photoinitiator is 2,4,6-trimethylbenzoyl lithium phosphite (LAP) with a concentration of 0.1 to 0.3 m / v%, preferably 0.2 m / v%.

[0012] Furthermore, in step 2, when the growth density of human coronary artery smooth muscle cells on the outer surface of the hydrogel fiber reaches 70-80%, human umbilical vein endothelial cells are implanted in the cavity of the hydrogel fiber.

[0013] Furthermore, in step 2, before the cells are implanted, the hydrogel fibers are sterilized; the sterilization method is: immersing the hydrogel fibers in a phosphate buffered saline solution (PBS) and irradiating them with ultraviolet light for 4 to 6 hours.

[0014] Furthermore, in step 2, the cell density of the human coronary artery smooth muscle cell suspension used for planting on the outer surface of the hydrogel fiber is 1×10 7 / mL; the method for planting human umbilical vein endothelial cells in the lumen of the hydrogel fiber is to inject a human umbilical vein endothelial cell suspension into the lumen of the hydrogel fiber; the cell density of the human umbilical vein endothelial cell suspension is 8×10 6 / mL.

[0015] Further, the specific method of step three is: fix the hydrogel fiber planted with cells on the chip; the chip has a fixing groove, and the groove walls at both ends of the fixing groove are provided with fixing holes, and the fixing holes are inserted with connectors; the hydrogel fiber is placed in the fixing groove, and the two ends are connected to two connectors respectively; the fixing groove contains complete culture medium of human coronary artery smooth muscle cells, and the hydrogel fiber is soaked; a connector is connected to complete culture medium of human umbilical vein endothelial cells through a PE tube, and the complete culture medium of human umbilical vein endothelial cells is pumped into the inner cavity of the hydrogel fiber. The chip is made of a customized template using polydimethylsiloxane (PDMS); the size of the middle fixing groove is 25mm long, 16mm wide, and 6mm high, and the left and right sides of the fixing groove are punched by a 1mm puncher, and then a 10μL spotting gun head is inserted at the punching point as a connector. The inner diameter of the PE tube is 0.6mm and the outer diameter is 0.9mm.

[0016] Furthermore, in step three, the flow rate of the complete culture medium of human umbilical vein endothelial cells infusing into the hydrogel fiber is 1 to 2 ml / h.

[0017] The present invention also provides a dynamic perfusion blood vessel chip based on microfluidic core-shell fibers prepared by the above preparation method.

[0018] The beneficial effects of the present invention are:

[0019] 1. The present invention relies on microfluidic technology and adopts a microfluidic fiber generation device to prepare hydrogel fibers with a shell-core structure (the core is the inner cavity), which has the advantages of simple operation, controllable inner and outer diameters, low cost, and easy replication.

[0020] Second, the hydrogel fiber prepared by the present invention is constructed using a composite hydrogel system with good biocompatibility, so its surface and interior have large pores, which are conducive to the attachment of cells and the exchange of substances between cells and culture medium, and have good biocompatibility, and cells are easy to grow and reproduce. Specifically, the hydrogel precursor used in the present invention includes methacrylated gelatin (GelMa) and polyethylene glycol diacrylate (PEGDA), wherein the naturally derived GelMa has good biocompatibility, is conducive to the adhesion and proliferation of cells, and can be quickly and conveniently cured by ultraviolet light, and the shell-core fiber is generated online in real time; at the same time, the addition of the PEGDA component can adjust the mechanical properties of the fiber, on the one hand, the fiber can better maintain its shell-core structure, on the other hand, by adjusting the concentration of PEGDA, the shell-core fiber equivalent to the mechanical properties of the real blood vessel wall in vivo can be obtained, and the mechanical properties of blood vessels in vivo can be more realistically simulated. In general, the composite of the two hydrogel components of GelMa and PEGDA makes the hydrogel fiber have both biocompatibility and mechanical properties, thereby more realistically reproducing the structure and physiological function of blood vessels in vivo in vitro. In addition, the two types of cells grow in different locations without interfering with each other, forming a co-culture state, which better mimics the vascular structure in the human body.

[0021] 3. The vascular chip prepared by the present invention introduces a micropump system to control the flow of culture medium, simulating the flow of blood in blood vessels, thereby realizing the study of the effects of shear force on cell growth. The vascular chip can be used to construct various human vascular disease models, study their pathogenesis, drug screening and personalized treatment, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the preparation process of a dynamic perfusion vascular chip based on microfluidic core-shell fiber, wherein a is a schematic diagram of the structure of a microfluidic fiber generation device, b is a schematic diagram of the core-shell structure of the hydrogel fiber, and c is a schematic diagram of the dynamic perfusion vascular chip;

[0023] Figure 2 are physical pictures of a microfluidic fiber generating device and a hydrogel fiber, wherein a is a physical picture of the microfluidic fiber generating device, and b is a physical picture of the generated hydrogel fiber;

[0024] Figure 3 is the characterization result of the hydrogel fiber, wherein a is the observation picture of the hydrogel fiber under an optical microscope, b is the observation picture of the hydrogel fiber loaded with red nanoparticles under a fluorescence microscope, c is the cross-sectional picture of the hydrogel fiber under a scanning electron microscope, and d is the outer surface picture of the hydrogel fiber under a scanning electron microscope;

[0025] Figure 4These are microscopic images of human coronary artery smooth muscle cells and human umbilical vein endothelial cells cultured on hydrogel fibers for 24 h, 48 h, and 72 h, respectively;

[0026] Figure 5 It is an immunofluorescence image of human coronary artery smooth muscle cells and human umbilical vein endothelial cells cultured on hydrogel fibers, among which human coronary artery smooth muscle cells specifically express α-SMA, human umbilical vein endothelial cells specifically express CD31, and all cell nuclei are stained with DAPI;

[0027] Figure 6 This is a physical picture of a dynamic perfusion vascular chip based on microfluidic core-shell fibers, where a is an overall physical picture of the entire device, b is a physical picture of the chip, and c is a physical picture of the dynamic perfusion vascular chip after perfusion of culture medium. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments.

[0029] This embodiment provides a dynamic perfusion blood vessel chip based on microfluidic core-shell fibers, and the preparation method includes the following steps:

[0030] Step 1: Prepare hydrogel fibers with hollow cavities using microfluidics technology.

[0031] (1) Preparation of microfluidic fiber generation device

[0032] Select a long capillary glass tube with an inner diameter of 800 μm and an outer diameter of 1000 μm, use a diamond knife to cut out a tube segment of about 3.5 cm long, and use fine sandpaper to carefully polish one end of the tube segment to make it smooth and smooth to ensure the smooth progress of subsequent operations. This tube segment is the external phase capillary; select a long capillary glass tube with an inner diameter of 300 μm and an outer diameter of 450 μm, also use a diamond knife to cut out a tube segment of about 7 cm long, and use fine sandpaper to carefully polish one end of the tube segment to make it smooth and smooth to ensure the smooth progress of subsequent operations. This tube segment is the internal phase capillary. Soak the prepared external phase capillary and internal phase capillary together in a 75% alcohol solution and clean them in an ultrasonic instrument for 30 minutes to ensure that the inside and outside of the pipe are thoroughly cleaned. After cleaning, blow the pipe dry with high-pressure nitrogen to ensure that the inner wall is completely dry to avoid any residual liquid affecting the experimental results. After completing the above steps, a capillary channel with a clean inner wall can be obtained, ready for subsequent experimental operations.

[0033] (2) Assembly of microfluidic fiber generation device

[0034] First, select a glass slide as the base platform. Figure 1As shown in a, one end of the external phase capillary is firmly fixed to one side of the glass slide using AB quick-drying glue to ensure its stability and positioning accuracy. Subsequently, one end of the internal phase capillary is inserted into the end of the external phase capillary and fixed with AB quick-drying glue to ensure that the ends of the two tubes are flush and present a clear "concentric circle" structure in the cross section. This step is critical to ensure the precise flow of fluid within the device. After the pipeline connection is completed, install a needle at the end connection of the two tubes and seal it with AB quick-drying glue. As shown in Figure 2 As shown in middle a, a simple microfluidic fiber generation device was successfully assembled.

[0035] (3) Preparation of hydrogel precursor solution and supporting fluid

[0036] The formula of the hydrogel precursor solution is 10% (m / v) methacrylated gelatin (GelMa) + 2.5% (v / v) polyethylene glycol diacrylate (PEGDA) + 0.2% (m / v) 2,4,6-trimethylbenzoyl lithium phosphite (photoinitiator LAP). The specific preparation steps are as follows: add the above ingredients to an appropriate amount of deionized water in sequence, stir and mix them thoroughly with an oscillating mixer, and place them in a 37°C water bath and heat until they are completely dissolved.

[0037] The supporting liquid formula is 10% (m / v) polyvinyl alcohol (PVA). The specific preparation steps are as follows: PVA is added to deionized water, stirred at a moderate speed using a magnetic stirrer, and heated in a 40° C. water bath overnight until it is completely dissolved.

[0038] (4) Preparation of hydrogel fibers

[0039] First, the microfluidic fiber generation device is protected from light. Then, deionized water is introduced into the spotting needle connecting the two capillaries through a polyethylene (PE) tube to exhaust the air inside. At this time, the flow rate of deionized water is set to 10mL / h. Subsequently, the hydrogel precursor solution is passed into the external phase capillary through a PE tube, and the flow rate is set to 30ml / h. At the same time, a polyvinyl alcohol (PVA) aqueous solution is passed into the internal phase capillary, and the flow rate is set to 20mL / h. After the two solutions are injected through the corresponding pipes, the fluid outflow at the outlet is observed. When the fluid at the outlet is stable and flows out continuously, it means that the system has reached a stable state. Finally, after the fluid flows out stably, the outlet is immediately irradiated with ultraviolet light (UV). After the hydrogel precursor solution is completely exhausted and the curing is completed, the hydrogel fiber is taken out and rinsed with PBS, and then transferred to a clean container. Figure 1 Figure b is a schematic diagram of the structure of the hydrogel fiber, where the core structure refers to the hollow inner cavity of the hydrogel fiber. Figure 2 Figure b is a physical picture of the prepared hydrogel fiber.

[0040] (5) Characterization of hydrogel fibers

[0041] First, the prepared hydrogel fibers were immersed in PBS and observed under an optical microscope to record their structure and measure their inner and outer diameters. Subsequently, the hydrogel fibers were treated with an ethanol gradient dehydration method to gradually remove the water therein, and then subjected to supercritical drying to completely dehydrate the hydrogel fibers to obtain dry fibers with stable structures. The dried hydrogel fibers were observed under a scanning electron microscope (SEM) to record their microstructural characteristics.

[0042] In order to better characterize the hollow structure of the hydrogel fiber, 1% (v / v) red polystyrene fluorescent nanoparticles (excitation wavelength 580nm, emission wavelength 605nm) were added to the hydrogel precursor solution to prepare fluorescently labeled hydrogel fibers, which were then fully washed with PBS to remove unbound nanoparticles and other impurities. The washed hydrogel fibers were cut into small segments with a cutting knife to expose their cross sections. Finally, these hydrogel fiber segments were placed under a confocal microscope for observation.

[0043] Figure 3 Figure a is an observation of the hydrogel fiber under an optical microscope, b is an observation of the hydrogel fiber loaded with red nanoparticles under a fluorescence microscope, c is a cross-sectional view of the hydrogel fiber under a scanning electron microscope, and d is an external surface view of the hydrogel fiber under a scanning electron microscope. It can be clearly seen from these images that the prepared hydrogel fiber has structural characteristics similar to blood vessels, and there are a large number of loose reticular structures on the surface of the hydrogel fiber.

[0044] (6) Morphology control of hydrogel fibers

[0045] First, the external phase flow rate was fixed at 30mL / h, and the internal phase flow rate was set to 10mL / h, 15mL / h, 20mL / h, 25mL / h and 30mL / h respectively. Under this condition, the inner and outer diameters and core-shell ratios of the obtained hydrogel fibers were compared and analyzed. The experimental results show that with the increase of the external phase flow rate, the core-shell ratio of the hydrogel fiber tends to gradually decrease. Subsequently, the fixed internal phase flow rate was adjusted to 25mL / h, and the external phase flow rate was set to 20mL / h, 25mL / h, 30mL / h, 35mL / h and 40mL / h respectively. In this group of experiments, the inner and outer diameters and core-shell ratios of the hydrogel fibers were also compared and analyzed. The results showed that with the increase of the external phase flow rate, the core-shell ratio gradually increased. It can be seen from these two groups of experiments that by adjusting the internal and external phase flow rates, the inner and outer diameters and core-shell ratios of the hydrogel fibers can be effectively regulated, thereby achieving precise control of the morphology of the hydrogel fibers.

[0046] Step 2: Plant human coronary artery smooth muscle cells (HCASMCs) on the outer surface of the hydrogel fiber, and plant human umbilical vein endothelial cells (HUVECs) in the lumen of the hydrogel fiber to achieve three-dimensional co-culture of the two cells.

[0047] (1) Cell culture

[0048] Human umbilical vein endothelial cells (HUVECs) were cultured in ECM medium containing endothelial cell growth factor, 10% fetal bovine serum and 1% double antibodies (penicillin and streptomycin). Similarly, human coronary artery smooth muscle cells (HCASMCs) were cultured in complete coronary artery smooth muscle cell medium containing growth factors, insulin, 10% fetal bovine serum and 1% double antibodies (penicillin and streptomycin). Both cells were placed in a carbon dioxide incubator (37°C, 5% CO2, saturated humidity), and the culture medium was changed every other day. The cell culture cycle is about 2-3 days, until the cells grow to 80-90% confluence, and then 0.25% trypsin is used for digestion and passage.

[0049] (2) Human coronary artery smooth muscle cells were implanted on the outer surface of the hydrogel fibers

[0050] First, the prepared hydrogel fibers were cut into segments of about 4 cm and their two ends were sealed. Subsequently, the hydrogel fibers were sterilized under ultraviolet light for 4 to 6 hours. After sterilization, the hydrogel fibers were soaked in sterile culture medium for 24 hours. Then, the soaked hydrogel fibers were transferred to the well plate and 2 mL of a cell concentration of 1×10 7 / mL human coronary artery smooth muscle cell suspension. Then, the well plate was placed in an incubator for 12 hours. After the culture was completed, the hydrogel fiber was taken out, transferred to a new well plate, and supplemented with sufficient culture medium. Figure 4 The growth of human coronary artery smooth muscle cells on the outer surface of the hydrogel fiber after culturing for 24, 48, and 72 hours, respectively, was shown. The results showed that the cells grew well on the outer surface of the hydrogel fiber.

[0051] (3) Implantation of human umbilical vein endothelial cells in the lumen of hydrogel fibers

[0052] The cells were cultured in an incubator for 2 to 3 days until the growth density of human coronary artery smooth muscle cells on the surface of the hydrogel fibers reached about 70 to 80%. The hydrogel fibers were then taken out of the incubator and the hydrogel portions at both ends of the hydrogel fibers were cut off with a sterile blade. Next, a 10 μL spotting pipette tip was used to sample the cells at a concentration of about 8×10 6 / mL human umbilical vein endothelial cell suspension was injected into the fiber lumen, and then the treated hydrogel fiber was placed in the incubator again for further culture. Figure 4The growth of human umbilical vein endothelial cells after being cultured on the lumen of hydrogel fibers for 24, 48, and 72 hours, respectively, was shown. The results also showed that the cells grew well in the lumen of hydrogel fibers.

[0053] (4) Cell immunofluorescence staining

[0054] First, the hydrogel fibers seeded with two types of cells were taken out, and the surface and inner cavity of the hydrogel fibers were gently cleaned with PBS. Next, the cells were fixed with 4% paraformaldehyde solution for 30 minutes. Subsequently, 0.5% Triton X-100 solution was used for treatment for 30 minutes to increase the permeability of the cell membrane. After the treatment, 5% bovine serum albumin solution was used for blocking for 30 minutes. Next, the hydrogel fibers were placed in a solution containing the primary antibody and incubated overnight at 4°C. The next day, the hydrogel fibers were transferred to a solution containing the secondary antibody and incubated at room temperature for 2 hours. Afterwards, DAPI staining solution was added for staining for 10 minutes. Finally, observation was performed under a fluorescence microscope, and the image was recorded, such as Figure 5 According to the experimental results, both cells specifically expressed their corresponding proteins, confirming that human umbilical vein endothelial cells and human coronary artery smooth muscle cells can be co-cultured in the same hydrogel fiber.

[0055] Step 3: Immerse the cell-seeded hydrogel fiber in a complete culture medium of human coronary artery smooth muscle cells, and connect it with a complete culture medium of human umbilical vein endothelial cells, so that the complete culture medium of human umbilical vein endothelial cells is continuously infused into the inner cavity of the cell-seeded hydrogel fiber to obtain a dynamic perfusion vascular chip.

[0056] (1) Design and production of chip structure

[0057] According to the pre-designed CAD model, an acrylic mold is first prepared. Then, polydimethylsiloxane (PDMS) material is used for molding to obtain a PDMS chip. Figure 1 As shown in c, the chip has a fixed groove. A 1mm puncher is used to accurately punch holes on the left and right sides of the fixed groove, and a 10μL spotting gun tip is inserted into the fixed hole. To ensure the cleanliness and sterility of the device, the PDMS chip and the inserted spotting gun tip are thoroughly disinfected with a 75% alcohol solution. After disinfection, the device is placed under ultraviolet light for 4 hours for further sterilization. After sterilization, the device is transferred to the clean bench for subsequent operations.

[0058] (2) Construction of Vascular Chip

[0059] The hydrogel fiber planted with two types of cells was transferred to the surface of the chip, and the tips of the spotting guns on the left and right sides of the chip were used to accurately clamp the two ends of the hydrogel fiber to ensure that the fiber was firmly fixed and did not shift on the chip. Subsequently, the complete culture medium of human coronary artery smooth muscle cells was poured into the fixing groove to soak the hydrogel fiber; at the same time, one side of the spotting gun tip was connected to the complete culture medium of human umbilical vein endothelial cells through a PE tube, and the complete culture medium of human umbilical vein endothelial cells was pumped into the inner cavity of the hydrogel fiber through the micropump system, and the pumping flow rate was 1-2mL / 24h, thereby completing the construction of the dynamic perfusion vascular chip. Figure 6 Figures a and b show actual pictures of the dynamic perfusion vascular chip based on microfluidic core-shell fibers, and figure c shows the dynamic perfusion state of the culture medium in the chip, further proving the successful construction of the vascular chip.

[0060] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the reagents, materials and operating procedures used herein are reagents, materials and conventional procedures widely used in the corresponding fields.

[0061] It should be noted that the terms such as "upper", "lower", "left", "right", "front", "back", etc. cited in the invention are only for the convenience of description and are not used to limit the scope of implementation of the present invention. Changes or adjustments in their relative relationships should be regarded as the scope of implementation of the present invention without substantially changing the technical content.

[0062] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a dynamic perfusion blood vessel chip based on microfluidic core-shell fiber, characterized in that: The following steps are involved: Step 1: Prepare hydrogel fibers with hollow cavities using microfluidics technology; Step 2: planting human coronary artery smooth muscle cells on the outer surface of the hydrogel fiber and planting human umbilical vein endothelial cells in the cavity of the hydrogel fiber; Step 3: Immerse the cell-seeded hydrogel fiber in a complete culture medium of human coronary artery smooth muscle cells, and connect it with a complete culture medium of human umbilical vein endothelial cells, so that the complete culture medium of human umbilical vein endothelial cells is continuously infused into the inner cavity of the cell-seeded hydrogel fiber to obtain a dynamic perfusion vascular chip.

2. The method for preparing a dynamic perfusion blood vessel chip based on microfluidic core-shell fiber according to claim 1, characterized in that: In step 1, the preparation method of the hydrogel fiber is as follows: using a hydrogel precursor solution as an external phase and a supporting liquid as an internal phase, respectively injecting the hydrogel precursor solution into an external phase channel and an internal phase channel of a microfluidic fiber generation device, and solidifying the hydrogel precursor solution at the outlets of the external phase channel and the internal phase channel to obtain a hydrogel fiber with a hollow inner cavity; The microfluidic fiber generation device comprises an external phase capillary and an internal phase capillary which are coaxially assembled, the internal phase capillary is an internal phase channel, and the channel between the internal phase capillary and the external phase capillary is an external phase channel; The diameter of the outer capillary is 800-1000 μm, and the diameter of the inner capillary is 300-500 μm.

3. The method for preparing a dynamic perfusion blood vessel chip based on microfluidic core-shell fiber according to claim 2, characterized in that: In step 1, the hydrogel precursor in the hydrogel precursor solution includes methacrylated gelatin and polyethylene glycol diacrylate; the concentration of methacrylated gelatin is 5 to 15 m / v%, and the concentration of polyethylene glycol diacrylate is 1 to 4 v / v%; The supporting liquid is a polyvinyl alcohol solution with a concentration of 5 to 15 m / v%; The flow rate of the external phase channel is 20-40 mL / h, and the flow rate of the internal phase channel is 10-30 mL / h.

4. The method for preparing a dynamic perfusion blood vessel chip based on microfluidic core-shell fiber according to claim 2, characterized in that: In step 1, the curing method is ultraviolet light curing, and the hydrogel precursor solution contains a photoinitiator; The photoinitiator is 2,4,6-trimethylbenzoyl lithium phosphite, and the concentration is 0.1-0.3 m / v%.

5. The method for preparing a dynamic perfusion blood vessel chip based on microfluidic core-shell fiber according to claim 1, characterized in that: In step 2, when the growth density of human coronary artery smooth muscle cells on the outer surface of the hydrogel fiber reaches 70-80%, human umbilical vein endothelial cells are then implanted in the cavity of the hydrogel fiber.

6. The method for preparing a dynamic perfusion blood vessel chip based on microfluidic core-shell fiber according to claim 1, characterized in that: In step 2, the hydrogel fibers are sterilized before the cells are implanted; The sterilization method is as follows: immersing the hydrogel fiber in a phosphate buffered saline solution and irradiating it with ultraviolet light for 4 to 6 hours.

7. The method for preparing a dynamic perfusion blood vessel chip based on microfluidic core-shell fiber according to claim 1, characterized in that: In step 2, the cell density of the human coronary artery smooth muscle cell suspension used for planting on the outer surface of the hydrogel fiber is 1×10 7 / mL; The method for implanting human umbilical vein endothelial cells in the lumen of hydrogel fibers is to inject a human umbilical vein endothelial cell suspension into the lumen of the hydrogel fibers; the cell density of the human umbilical vein endothelial cell suspension is 8×10 6 / mL.

8. The method for preparing a dynamic perfusion blood vessel chip based on microfluidic core-shell fiber according to claim 1, characterized in that: The specific method of step three is: fixing the hydrogel fiber planted with cells on the chip; The chip has a fixing groove, and fixing holes are opened on the groove walls at both ends of the fixing groove, and connectors are inserted in the fixing holes; the hydrogel fiber is placed in the fixing groove, and the two ends are respectively connected to the two connectors; The fixed tank is filled with complete culture medium of human coronary artery smooth muscle cells, which soaks the hydrogel fiber; a connector is connected to complete culture medium of human umbilical vein endothelial cells through a PE tube, and the complete culture medium of human umbilical vein endothelial cells is pumped into the inner cavity of the hydrogel fiber.

9. The method for preparing a dynamic perfusion blood vessel chip based on microfluidic core-shell fiber according to claim 1, characterized in that: In step 3, the flow rate of the complete culture medium of human umbilical vein endothelial cells into the hydrogel fiber is 1-2 ml / h.

10. A dynamic perfusion blood vessel chip based on microfluidic core-shell fiber prepared by the preparation method according to any one of claims 1 to 9.

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