A three-dimensional tubular blood vessel organoid model chip and a construction method thereof

Abstract

The application discloses a three-dimensional tubular blood vessel organoid model chip and a construction method thereof, and belongs to the technical field of organ chip. The chip comprises an upper chip, a middle chip and a lower chip which are sequentially plasma bonded by polydimethylsiloxane (PDMS) material. The thickness of the upper chip is 5 mm, and three flow channel inlets and three flow channel outlets are arranged on the upper chip, and the pore diameters are all 4 mm. The thickness of the middle chip is 2 mm, and three flow channel inlets and three flow channel outlets which are coaxially communicated with the upper chip are arranged on the middle chip, and the pore diameters are all 1 mm. The thickness of the lower chip is 2 mm, and a microfluidic unit is arranged on the lower chip. The microfluidic unit comprises three parallel channels, a matrix channel is arranged in the middle, two perfusion channels are arranged on the two sides of the matrix channel, and the matrix channel and each perfusion channel are physically separated by a phase guide structure. The chip has the advantages of flexible structure design, low processing cost, good optical transparency, high simulation of the physiological microenvironment of blood vessels in the body, and the like, and provides a stable, reliable and visual in-vitro model platform for basic research of vascular biology.

Description

Technical Field

[0001] This invention belongs to the field of organ-on-a-chip technology, specifically relating to a three-dimensional tubular vascular organoid model chip and its construction method. Background Technology

[0002] Traditional in vitro vascular models typically culture endothelial cells on 2D surfaces such as plastic and glass, which still differ significantly from the structure and function of 3D blood vessels in vivo. Endothelial cells seeded on rigid substrates exhibit higher levels of contractile forces, leading to intercellular gap formation and impaired endothelial barrier. Furthermore, the shear stress generated by blood flow can enhance the expression of tight junction genes and proteins in blood vessels, while the culture medium above endothelial cells in traditional in vitro vascular models is usually static, failing to simulate the significant effects of in vivo blood flow on endothelial cell phenotype and function. Therefore, there is an urgent need to develop more physiologically relevant in vitro vascular models.

[0003] Emerging microfluidic models (also known as organ-on-chips) can simulate the three-dimensional structure and hemodynamics of blood vessels in vitro, offering advantages such as high controllability and non-invasive, high-resolution visualization. Currently, numerous microfluidic vascular models have been developed; however, most are designed from an engineering perspective, resulting in complex structures and requiring external devices such as infusion pumps, making operation cumbersome. In recent years, standardized microfluidic vascular models with simple operation have begun to achieve commercial scale. The OrganoPlate3-lane model stands out in terms of physiological relevance, compatibility, and ease of use. This model can construct blood vessels with good structure and barrier function, and allows for highly flexible design of co-culture modes. The outstanding advantages of the OrganoPlate3-lane model in vascular culture are: (1) constructing a 3D tubular structure; (2) allowing cells to directly contact the gel, simulating cell-extracellular matrix interaction; (3) applying blood flow perfusion to the blood vessels, which is driven by gravity and does not require additional tubing or pumps, and the perfusion can be applied simultaneously to all test units; (4) controllability; (5) high throughput; (6) compatibility with a variety of laboratory instruments; and (7) high-resolution visualization.

[0004] However, commercially available chips have significant limitations: they are expensive, rely on external procurement, and have fixed designs that cannot be flexibly adjusted. This restricts researchers from conducting large-scale customized in-depth exploration, becoming a bottleneck for the development of the field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a three-dimensional tubular vascular organoid model chip and its construction method, thereby solving the problems in the prior art.

[0006] The objective of this invention can be achieved through the following technical solutions: A method for constructing a three-dimensional tubular vascular organoid model chip includes the following steps: S1, a negative photoresist is coated on a silicon wafer, and a positive mold with a microfluidic network pattern is formed after exposure and development using a mask pattern; the microfluidic network pattern includes: protrusions corresponding to three parallel channels, with the middle protrusion corresponding to the matrix channel and the two sides corresponding to the infusion channels, and grooves for forming phase guide structures are provided between adjacent protrusions. S2, the polydimethylsiloxane PDMS prepolymer is cast and cured in the positive mold and the flat-bottomed container respectively; a lower PDMS chip with matrix channels, infusion channels and phase guide structure on the surface is obtained by peeling it off from the positive mold; a PDMS blank without microchannels is obtained by peeling it off from the flat-bottomed container, and after drilling, an upper PDMS chip and a middle PDMS chip are formed respectively; the bonding surfaces of the upper PDMS chip, the middle PDMS chip and the lower PDMS chip are treated with air plasma, and then aligned, bonded and cured in sequence; S3, inoculate extracellular matrix (ECM) gel into the matrix channel in the middle of the lower PDMS chip, and let it stand until the ECM gel polymerizes; S4, Inoculate the bottom of the inlet of the perfusion channel with an endothelial cell suspension, and tilt the culture dish containing the chip to allow the endothelial cells in the perfusion channel to settle and adhere to the side wall of the ECM gel. S5. After adding culture medium to the perfusion channel, the chip is placed horizontally on a shaker, and reciprocating fluid perfusion culture is performed by periodically switching the tilt angle of the shaker.

[0007] Furthermore, the process of obtaining the mask pattern includes: A white light interferometer was used to perform a three-dimensional scan on a chip prototype containing the target microfluidic structure to obtain three-dimensional point cloud data including channel width, depth, and the height and width of the phase guide structure. The obtained 3D point cloud data is imported into 3D modeling software for data repair and generates two 2D mask images for photolithography. The first mask image contains three channels and two phase guides, and its height is the phase guide height when used. The second mask contains only three channels, and its height is the chip channel height when used.

[0008] Further, in S2: a 4mm diameter punch is used to punch holes in the 5mm thick PDMS blank after peeling to form the upper PDMS chip; a 1mm diameter punch is used to punch holes in the 2mm thick PDMS blank after peeling to form the middle PDMS chip.

[0009] Furthermore, the ECM gel in S3 was prepared by mixing 1M HEPES, 37g / L NaHCO3 solution and rat tail collagen in a volume ratio of 1:1:8.

[0010] Furthermore, between S3 and S4, a channel modification step is included: the perfusion channel is coated with human fibronectin at a concentration of 0.1 mg / ml, placed at 37°C for 2 hours, and then removed and washed.

[0011] Furthermore, the tilt angle of the shaker is 7°, and the angle switching time interval is set to 8 minutes.

[0012] A three-dimensional tubular vascular organoid model chip was prepared using the above-described construction method.

[0013] The aforementioned three-dimensional tubular vascular organoid model chip includes: an upper chip, a middle chip, and a lower chip, wherein: The upper chip is 5mm thick and has 3 flow channel inlets and 3 flow channel outlets, each with a diameter of 4mm. The thickness of the middle layer chip is 2mm, and it is provided with 3 flow channel inlets and 3 flow channel outlets that are coaxially connected to the upper layer chip, and the aperture of each channel is 1mm. The lower chip is 2 mm thick and has a microfluidic unit on it. The microfluidic unit includes three parallel channels, with a matrix channel in the middle and two infusion channels on either side of the matrix channel. The matrix channel and each infusion channel are physically separated by a phase guide structure.

[0014] The matrix channel width in the aforementioned lower-layer chip is 350µm, and the infusion channels on both sides are 300µm wide and 200µm deep.

[0015] The aforementioned matrix channel is filled with cross-linked polymerized extracellular matrix (ECM) gel, and the physical boundary of the ECM gel is confined at the phase guide structure. The inner wall of the perfusion channel is coated with a layer of human fibronectin, and an endothelial cell layer formed by tilting sedimentation is attached at the physical interface adjacent to the ECM gel.

[0016] The beneficial effects of this invention are: 1. This invention uses a white light interferometer to perform non-contact 3D scanning of a chip prototype containing target microchannels (phaseguides) to obtain high-resolution point cloud data. After repair, a mask is generated, and an SU-8 positive mold is fabricated on a silicon wafer using ultraviolet lithography. This mold is then used to fabricate the PDMS chip. This solution, based on proprietary processing technology, eliminates the dependence on external procurement for traditional commercial organ-on-a-chip (such as OrganoPlate), significantly reducing production costs. Simultaneously, it overcomes the bottleneck of fixed and inflexible designs in commercial chips, enabling fully customizable design parameters and providing an economical and efficient underlying technical tool for large-scale customized in-depth exploration.

[0017] 2. This invention employs a plasma-bonded assembly of a 5mm thick (4mm pore size) upper chip, a 2mm thick (1mm pore size) middle chip, and a lower chip containing microchannels, forming a culture medium storage pool between the upper and middle layers. During use, culture medium is added, and the chip is placed on a shaker with a 7° tilt angle and an 8-minute switching interval for cultivation. This liquid level difference structure, utilizing specific height and pore size, combined with the periodic tilting of the shaker, allows the liquid to flow back and forth within the bottom microchannels entirely by gravity. This avoids the need for complex and cumbersome tubing devices such as external syringe pumps required in traditional microfluidic models, and successfully overcomes the limitation of traditional 2D static culture in simulating blood flow effects. It provides continuous physiological fluid shear force for blood vessel growth, enhancing the expression of tight junction genes and proteins.

[0018] 3. This invention employs a design where the lower microfluidic unit features a central matrix channel and two side-by-side perfusion channels, separated by two Phaseguides. During endothelial cell inoculation, the culture dish containing the chip is placed on its side (at a 75° angle to the horizontal), allowing cells in the upper perfusion channel to settle for 25 minutes. The Phaseguide (2.2 mm in length, 100 μm in width, and 60 μm in height) acts as a physical barrier, effectively limiting the ECM gel and allowing subsequently inoculated cells to directly contact the gel, highly mimicking cell-extracellular matrix interactions in vivo. Furthermore, the specific 75° tilt angle forces endothelial cells to settle precisely under gravity and attach to the lateral ECM gel wall, thus avoiding the formation of intercellular gaps and barrier damage caused by inoculation on a rigid substrate, successfully constructing a three-dimensional tubular vascular structure with complete physiological barrier function.

[0019] 4. The upper, middle and lower layers of this invention are all cast from polydimethylsiloxane (PDMS) material and then plasma bonded and cured. Utilizing the natural optical transparency and lack of autofluorescence interference of PDMS material, the chip is compatible with high-resolution visualization detection tools (such as live cell imaging instruments). Researchers can directly observe the morphology, density and angiogenesis process of blood vessels for several consecutive days without damaging the culture environment through bright field and HOECHST fluorescence staining. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the channels in the organoid chip of the present invention; Figure 2 These are bottom and side views of the organoid chip seeding of cells according to the present invention, and channel scan images of the organoid chip; Figure 3 This is a photograph of the mask used in this invention; Figure 4 This invention comprises a top view of the upper, middle, and lower layer chips, as well as a side view and a top view of the three-layer combined chip; Figure 5 This is the morphology of the chip after the gel has been inoculated in the central channel of the chip of this invention; Figure 6 These are bright-field images after HUVECs cells have been seeded; Figure 7 This is a bottom view of the lower chip of the present invention, which is a bright-field and fluorescence image of live cell imaging after HOECHST staining of blood vessels. Figure 8 The images show fluorescence images of hoechst and F-actin stained after blood vessel fixation on day 8. Figure 9 This is a schematic diagram of the pathway structure corresponding to one of the channels in the organoid chip of this invention.

[0022] In the diagram: 1-Upper infusion channel inlet; 2-Upper infusion channel outlet; 3-Gel channel inlet; 4-Gel channel outlet; 5-Lower infusion channel inlet; 6-Lower infusion channel outlet. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1 The channel layout of the three-dimensional tubular vascular organoid model chip is as follows: Figure 1 As shown, the pathway structure corresponding to one of the channels (any one of the upper perfusion channel, gel channel, or lower perfusion channel) is as follows: Figure 9 As shown; In this embodiment, the three-dimensional tubular vascular organoid model chip includes, from top to bottom, a stacked and bonded upper chip, a middle chip, and a lower chip. The upper chip is 5mm thick and has 3 flow channel inlets and 3 flow channel outlets, each with a diameter of 4mm. The thickness of the middle layer chip is 2mm, and it is provided with 3 flow channel inlets and 3 flow channel outlets that are coaxially connected to the upper layer chip, and the aperture of each channel is 1mm. The lower chip is 2 mm thick and has a microfluidic unit on it. The microfluidic unit includes three parallel channels, with a matrix channel in the middle and two infusion channels on either side of the matrix channel. The matrix channel and each infusion channel are physically separated by a phase guide structure.

[0025] The matrix channel in the lower chip has a width of 350µm, and the infusion channels on both sides have a width of 300µm and a depth of 200µm.

[0026] The phase guide structure has a length of 2.2 mm, a width of 100 μm, and a height of 60 μm.

[0027] Example 2 The method for constructing the three-dimensional tubular vascular organoid model chip in Example 1 includes the following steps: S1. Manufacturing of microfluidic device molds A Keyence white light interferometer was used to perform non-contact 3D scanning on a chip prototype containing the target microfluidic structure, acquiring high-resolution 3D point cloud data including channel width, depth, and precise height and width of the phase guide. The obtained 3D point cloud data was imported into 3D modeling software for data repair, and based on this reverse data, a 2D mask pattern suitable for photolithography (such as...) was generated. Figure 3(As shown in the image) Place the glass slide with photoresist flat on a 65°C hot plate and bake for 10 minutes, then place it flat on a 95°C hot plate and bake for another 10 minutes to evaporate the solvent from the photoresist. After removing the optical glass and letting it cool in the dark, measure the thickness using a side-mounted instrument before exposure. Measure the thickness at three different locations and take the average value. Calculate the film thickness based on the thickness before spin-coating. Place the mask on the corresponding optical glass and turn on the exposure machine to expose it, causing a chemical reaction in the photoresist and changing the properties of the photoresist in the photosensitive areas. Place the exposed silicon wafer flat on a 65°C hot plate and bake for 3 minutes, then place it flat on a 95°C hot plate and bake for 10 minutes. After baking, remove the silicon wafer and let it cool, awaiting development. After post-baking, wash away the photoresist outside the channels with developer, gently blow the surface with an air gun, and then rinse the wafer with isopropanol. If white substances are present, further development is required; if no white substances are present, rinse thoroughly with isopropanol and dry with an air gun. Spray the finished silicon wafer surface with an alcohol spray bottle, then dry it with an air gun to remove residual developer and acetone. Observe the mold under a microscope to check for any damage.

[0028] S2, chip manufacturing steps and PDMS layer assembly Cast polydimethylsiloxane (PDMS) prepolymer (PDMS substrate: curing agent = 10:1 (w / w)) and degas in a vacuum chamber for 2 hours. Cur the PDMS overnight in a ventilated oven at 80°C. Peel the PDMS off the silicon wafer. Drill holes 1-6 (e.g., ...). Figure 1 (As shown). For the holes in the upper layer, use a 4 mm diameter punch; for the holes in the middle layer, use a 1 mm diameter punch. Clean the PDMS board by repeatedly sticking and peeling off tape. Then treat the PDMS board with air plasma for 40 seconds (40 mW, 50 cm³). First, place the cleaned lower two layers of chips under light and visually inspect the corresponding positions of the six holes. Slowly align them from left to right and then attach them. After confirming alignment, pinch and fix them by hand again to ensure a firm bond without air bubbles. Then place them in an 80°C oven for curing. After curing, the lower two layers of chips and the upper layer of chips are plasma cleaned again, then aligned and attached using the same method, and placed in the oven for curing. The three-layer chip assembly is now complete, and the effect is as shown. Figure 4 As shown.

[0029] S3, Day 0: After sterilization by UV irradiation for 1.5 hours, place in a sterile petri dish. Neutral Collagen I was inoculated into the intermediate ECM channel: Two 1.5 mL EP tubes were placed on ice. A gel was prepared by mixing 1 M HEPES, 37 g / L NaHCO3, and rat tail collagen Collagen I at a volume ratio of 1:1:8. 100 μL was prepared each time, and the procedure was performed on ice. Specific preparation steps are as follows: 1) Mix 10 μL of 1M HEPES with 10 μL of 37 g / L NaHCO3 until homogeneous; 2) Add 80 μL of rat tail collagen I, and quickly pipette up and down at least 20 times to mix thoroughly, avoiding air bubbles. 3) Place the glue on ice and use within 10 minutes.

[0030] The dispensing steps are as follows: 1) Hold the pipette perpendicular to the ECM channel inlet, with the tip directly above the bottom of the ECM channel inlet (well 3), and dispense the ECM gel; the gel volume is 1.4 μL. It is crucial to pay attention to the dispensing technique here, as the plasma-treated PDMS is hydrophilic, and even after several hours of UV irradiation, its surface hydrophobicity has not fully recovered. Therefore, the dispensing speed must be extremely slow. Once the gel is visually observed entering the 2.2 mm phase guide, control the pipette with your finger to dispense the gel at a slow (near-static) speed until it passes smoothly through the ECM. If the speed is too fast, the gel will not remain in the central channel and will overflow into the adjacent channels. A successful gel looks like... Figure 5 As shown.

[0031] 2) Place the chip in a cell culture incubator (37°C, 5% CO2) and let it stand for 15 minutes until the gel is completely polymerized.

[0032] 3) Remove the chip and add 50µLHBSS to the gel inlet / outlet (wells 3 and 4) to prevent the gel from drying out.

[0033] 4) Place the chip back into the incubator. Cell seeding can be performed 3 hours later or the next day. For primary HUVECs, seeding overnight yields better cell tube formation.

[0034] S4, fibronectin for channel modification The specific procedure is as follows: Coat the upper channel with 0.1 mg / ml human fibronectin, place on a shaker at 37°C for 2 hours, remove the coating, and wash twice with PBS.

[0035] S5. Day 1: HUVECs are seeded in the upper channel. Cells are collected. Primary HUVECs of passage 5 or less are selected. Once the cells reach 80% confluence, they are digested with trypsin. A schematic diagram of the process is shown below. Figure 2 As shown.

[0036] The specific operating steps are as follows: 1) Calculate the number of viable cells in the cell suspension, resuspend in complete endothelial cell culture medium to obtain a density of 1.0. 10 7 A single-cell suspension per mL.

[0037] 2) Discard the HBSS (well 3) at the entrance of the gel channel.

[0038] 3) Position the pipette tip directly above the bottom of the upper perfusion channel inlet (well 1) and inoculate with 2 µL of cell suspension. Note that the cell suspension needs to be resuspended periodically during inoculation to ensure uniform cell density and prevent sedimentation.

[0039] 4) Add 50µL of endothelial cell culture medium to the inlet of the upper perfusion channel (well 1). During the addition process, be careful to keep the pipette tip against the well wall and avoid direct contact between the pipette tip and the microfluidic channel to prevent disturbing the culture medium or forming air bubbles.

[0040] 5) Place the dish containing the chip on its side (at a 75° angle to the horizontal) on the MIMETAS plate holder in the incubator, allowing the cells in the upper perfusion channel to settle and adhere to the ECM gel. The cell attachment time is 25 minutes.

[0041] 6) After the cells have attached, add 50 µL of complete endothelial cell culture medium to the outlet (well 2) of the upper perfusion channel. Observe under a microscope to ensure that the culture medium completely fills the channel and that there are no air bubbles at the inlet (well 1) and outlet (well 2). If there are air bubbles, gently remove them with the tip of a pipette or the needle of a syringe.

[0042] 7) Place the dish containing the chip on the OrganoFlow shaker in the incubator. After leveling the shaker, set the shaker program to a tilt angle of 7° and an angle switching time interval of 8 minutes.

[0043] 8) During the culture process, every 1-2 days, discard the culture medium from the inlet (well 1) and outlet (well 2) of the upper perfusion channel and replace it with fresh endothelial cell complete culture medium. At the same time, pay close attention to the water level in the incubator tray and keep the incubator humid.

[0044] 9) Starting from the second day after cell inoculation, use a live cell imaging system to take bright field images every 24 hours to measure the degree of endothelial cell fusion on the upper and lower surfaces of the lumen. Figure 6 , 7 The image shows a blood vessel that was successfully inoculated.

[0045] Example 3 In this embodiment, the organoid chip prepared in Example 2 is applied, and the application process includes: 1. Visualization of the luminal structure of three-dimensional tubular blood vessels using fluorescent dyes The entire staining process was carried out on a shaker at room temperature. The shaker parameters were set as follows: tilt angle of 5° and angle switching time interval of 5 minutes.

[0046] 1) Before fixation begins, the Lionheart intelligent live-cell imaging system records the bright-field state of the cells.

[0047] 2) Aspirate the culture medium from all wells in the chip. The 1-2 μL of culture medium remaining in the microfluidic channels will not affect fixation.

[0048] 3) Add 50 µL of 4% paraformaldehyde to the inlet (well 1) and outlet (well 2) of the blood vessel. Fix for 20 min. Examine under a microscope to check for cell detachment.

[0049] 4) Discard the 4% paraformaldehyde, then add 50 µL PBS and wash 3 times, 5 min each time.

[0050] 5) Add 50 µL of immunostaining permeabilization solution to the inlet (hole 1) and outlet (hole 2) of the vascular access, and place on a shaker for 10-20 min at room temperature.

[0051] 6) Discard the immunostaining permeabilization solution, and add 50 µL of PBS solution containing Hoechst 33342 (1:2000) and F-actin (1:100) probes to the inlet (well 1) and outlet (well 2) of the vascular channel, respectively. Incubate at room temperature for 1 h.

[0052] 7) Take 2D images of blood vessels. The excitation wavelength of Hoechst 33342 is 346 nm and the emission wavelength is 460 nm; the excitation wavelength of F-actin is 540 nm and the emission wavelength is 565 nm.

[0053] On day 8 of endothelial cell growth, blood vessels were fixed with 4% paraformaldehyde, cell nuclei were labeled with HOECHST, and the cytoskeleton was labeled with F-actin. Fluorescence imaging of the blood vessels was then performed using live-cell imaging. Results are as follows: Figure 8 As shown, the curved side of the blood vessel adheres to the Collagen I gel, and the endothelial cells on the upper, lower, and lateral sides of the vessel are tightly connected, without invading into the ECM. This indicates that HUVECs have formed a dense 3D blood vessel within the channel.

[0054] 2. Lower channel cell seeding Wells 5 and 6 can be left empty without adding cells; alternatively, they can be modified and then inoculated with vascular endothelial cells or barrier cells such as Caco-2 or Bewo for scientific research.

[0055] Example 4 In this embodiment, the organoid chip of the present invention and the commercially available chip (MIMETAS, OrganoPlate) are compared. @The cost of 3-lane is calculated as shown in Table 1 below: Table 1 Comparison of Cost Accounting As can be seen from Table 1, the chip cost of the present invention is significantly lower than the commercial price.

[0056] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0057] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for constructing a three-dimensional tubular vascular organoid model chip, characterized in that, Includes the following steps: S1, a negative photoresist is coated on a silicon wafer, and a positive mold with a microfluidic network pattern is formed after exposure and development using a mask pattern; the microfluidic network pattern includes: protrusions corresponding to three parallel channels, with the middle protrusion corresponding to the matrix channel and the two sides corresponding to the infusion channels, and grooves for forming phase guide structures are provided between adjacent protrusions. S2, the polydimethylsiloxane PDMS prepolymer is cast and cured in the positive mold and the flat-bottomed container respectively; A lower PDMS chip with matrix channels, infusion channels and phase guide structures on its surface is obtained by peeling it off from the positive mold; a PDMS blank without microchannels is obtained by peeling it off from the flat-bottomed container, and after drilling, an upper PDMS chip and a middle PDMS chip are formed respectively; after air plasma treatment of the bonding surfaces of the upper PDMS chip, the middle PDMS chip and the lower PDMS chip, they are aligned, bonded and cured in sequence; S3, inoculate extracellular matrix (ECM) gel into the matrix channel in the middle of the lower PDMS chip, and let it stand until the ECM gel polymerizes; S4, Inoculate the bottom of the inlet of the perfusion channel with an endothelial cell suspension, and tilt the culture dish containing the chip to allow the endothelial cells in the perfusion channel to settle and adhere to the side wall of the ECM gel. S5. After adding culture medium to the perfusion channel, the chip is placed horizontally on a shaker, and reciprocating fluid perfusion culture is performed by periodically switching the tilt angle of the shaker.

2. The method for constructing a three-dimensional tubular vascular organoid model chip according to claim 1, characterized in that, The process of obtaining the mask pattern includes: A white light interferometer was used to perform a three-dimensional scan on a chip prototype containing the target microfluidic structure to obtain three-dimensional point cloud data including channel width, depth, and the height and width of the phase guide structure. The obtained 3D point cloud data is imported into 3D modeling software for data repair and generates two 2D mask images for photolithography. The first mask image contains three channels and two phase guides, and its height is the phase guide height when used. The second mask contains only three channels, and its height is the chip channel height when used.

3. The method for constructing a three-dimensional tubular vascular organoid model chip according to claim 1, characterized in that, In S2: A 4mm diameter punch is used to punch holes in a 5mm thick PDMS blank after peeling to form the upper PDMS chip; a 1mm diameter punch is used to punch holes in a 2mm thick PDMS blank after peeling to form the middle PDMS chip.

4. The method for constructing a three-dimensional tubular vascular organoid model chip according to claim 1, characterized in that, The ECM gel in S3 was prepared by mixing 1M HEPES, 37g / L NaHCO3 solution and rat tail collagen in a volume ratio of 1:1:

8.

5. The method for constructing a three-dimensional tubular vascular organoid model chip according to claim 1, characterized in that, Between S3 and S4, there is also a channel modification step: the perfusion channel is coated with human fibronectin at a concentration of 0.1 mg / ml, placed at 37°C for 2 hours, and then removed and washed.

6. The method for constructing a three-dimensional tubular vascular organoid model chip according to claim 1, characterized in that, The tilt angle of the shaker is 7°, and the angle switching time interval is set to 8 minutes.

7. A three-dimensional tubular vascular organoid model chip, characterized in that, It is prepared by the construction method according to any one of claims 1-6.

8. A three-dimensional tubular vascular organoid model chip according to claim 7, characterized in that, include: Upper-layer chip, middle-layer chip, and lower-layer chip, among which: The upper chip is 5mm thick and has 3 flow channel inlets and 3 flow channel outlets, each with a diameter of 4mm. The thickness of the middle layer chip is 2mm, and it is provided with 3 flow channel inlets and 3 flow channel outlets that are coaxially connected to the upper layer chip, and the aperture of each channel is 1mm. The lower chip is 2 mm thick and has a microfluidic unit on it. The microfluidic unit includes three parallel channels, with a matrix channel in the middle and two infusion channels on either side of the matrix channel. The matrix channel and each infusion channel are physically separated by a phase guide structure.

9. A three-dimensional tubular vascular organoid model chip according to claim 8, characterized in that, The matrix channel in the lower chip has a width of 350µm, and the infusion channels on both sides have a width of 300µm and a depth of 200µm.

10. A three-dimensional tubular vascular organoid model chip according to claim 8, characterized in that, The matrix channel is filled with cross-linked polymerized extracellular matrix (ECM) gel, and the physical boundary of the ECM gel is confined at the phase guide structure. The inner wall of the perfusion channel is coated with a layer of human fibronectin, and an endothelial cell layer formed by tilting sedimentation is attached at the physical interface adjacent to the ECM gel.

Images