Preparation method of 3D printing high-density cell tubular vascular graft

Abstract

The invention relates to a preparation method of a 3D printing high-density cell tubular vascular graft. The preparation method comprises the following steps: sucking a supernatant obtained by centrifuging a cell suspension away and only remaining bottom pure cells; the preparation method comprises the following steps: uniformly stirring and mixing OMA, pig gelatin and an LAP photoinitiator in a Hank's solution to form a hydrogel precursor solution; extruding the pure cells and the hydrogel precursor solution from a three-axis nozzle by using printing equipment, and immersing the pure cells and the hydrogel precursor solution into a calcium chloride solution; lyase is added to degrade the hydrogel precursor solution, the hydrogel precursor solution is completely degraded, and an independent single-layer pipeline structure with only a remaining pure cell layer is obtained. The invention provides a method for manufacturing high-density pure-cell lumen constructs to mimic native blood vessels by using a self-made microfluidic coaxial 3D bioprinting device. And after the support hydrogel on the inner layer and the outer layer is removed, a single-layer tubular structure of high-density MOVAs only limited to pure cells is obtained. The cells in the pure cell vascular constructs exhibit good cell viability and cell function.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic 3D printing, and in particular relates to a method for preparing a 3D printed high-density cell tubular vascular graft. Background Art

[0002] Angioplasty, surgical bypass and stent implantation are currently the commonly used methods for vascular reconstruction. For surgical bypass, the gold standard for treating coronary artery and peripheral vascular disease is still the use of autologous arteries or veins (mainly the internal thoracic artery or saphenous vein). However, the number of healthy autologous blood vessels is limited, surgery is required, and their long-term patency rate is low. Therefore, artificial blood vessels woven from materials such as polyester fibers, polytetrafluoroethylene, and polyurethane are used as substitutes for autologous blood vessels. So far, great progress has been made in the development of various types of artificial blood vessels. In 1986, Weinberg and Bell reported the first artificial blood vessel using a polyester and collagen composite material, marking the first appearance of tissue-engineered blood vessels. In 1990, Parodi et al. successfully repaired abdominal aortic aneurysms using stents made of stainless steel and polyester tube grafts. In 1999, the first artificial artery was implanted in a pig recipient, showing good patency and mechanical durability.

[0003] According to the diameter, artificial blood vessels are divided into large diameter (more than 10 mm), medium diameter (6-10 mm) and small diameter (less than 6 mm). At present, medium and large artificial blood vessels are widely used in clinical practice for aortic replacement or bypass, peripheral vascular bypass, vascular trauma repair, hemodialysis vascular access, coronary artery bypass, etc. Small artificial blood vessels are also widely used in clinical practice, such as peripheral vascular replacement, arteriovenous fistula establishment, heart bypass surgery, etc. However, so far, no small-caliber artificial blood vessels have been launched, and their clinical failure is mainly limited by several factors: the blood compatibility and anticoagulant properties of materials (such as polyurethane materials, etc.) are poor, and fibrin and platelet deposition lead to narrowing of the vascular lumen or even vascular occlusion; the existing materials cannot support the adhesion and growth of vascular endothelial cells well due to the slow blood flow and low blood pressure of small-caliber artificial blood vessels, which easily leads to stenosis of the vascular lumen, and embolism after implantation due to insufficient endothelialization. The long-term patency of small-caliber artificial blood vessels also needs to be solved urgently.

[0004] Three-dimensional (3D) bioprinting technology has become a promising method for the preparation of small blood vessels. By precisely placing biomaterials and living cells (collectively referred to as bioinks) at designated positions, small-sized vascular structures with complex structures and closer to the actual structure can be obtained. Commonly used bioprinting methods include extrusion bioprinting, inkjet bioprinting, laser-assisted bioprinting, and digital light processing bioprinting. Extrusion bioprinting is the most commonly used, low-cost, and easy-to-operate printing method, where bioinks with different viscosities are extruded through a nozzle to form filaments. Currently, the reports mainly focus on the development of different categories of bioinks, such as the biocompatibility, printability, and mechanical properties of the inks. Yu Zhang et al. developed a double-network tough hydrogel bioink composed of energy-consuming ion-crosslinked alginate and elastase-crosslinked gelatin, and prepared single-layer and double-layer hollow conduit structures by microfluidic double-extrusion bioprinting to reconstruct venous and arterial lumen structures. The 3D-printed venous and arterial lumens have key functions such as mechanical properties, permeability, barrier properties, and expression of specific markers. Andrique et al. developed a one-step preparation method for producing functional blood vessels using a microfluidic triple-extrusion device. The obtained lumen has the structure of a blood vessel, with endothelial cells as the inner lining and smooth muscle cells as the outer sheath. This "blood vessel" reaches a steady state within one day and exhibits quiescence, perfusability, and contractility as a functional blood vessel.

[0005] The development of 3D-printed small-diameter artificial blood vessels mainly focuses on the research of the composition design of biomaterials, the scaffold structure of biomaterials, the cell suspension embedded in biomaterials, etc. Using biomaterial-based scaffolds faces several challenges, such as biomaterials interfering with cell-cell interactions, the potential immunogenicity of biomaterials and their degradation products, the mismatch between the rate of scaffold degradation and new tissue formation, and the non-uniformity and low density of embedded cells, which affect the biological functions of the printed artificial blood vessels. Normal animal tissues are composed of closely interconnected high-density cells to achieve the required physiological functions. The use of biomaterial scaffolds weakens the connections between cells, affecting the formation of the extracellular matrix network and cell functions. Summary of the Invention

[0006] The present invention provides a method for manufacturing a lumen construct of high-density pure cells to mimic natural blood vessels using a self-made microfluidic coaxial 3D bioprinting device. After removing the support hydrogels of the inner and outer layers, a single-layer tubular structure of high-density MOVAs composed only of pure cells is obtained. The cells in the pure cell vascular construct exhibit good cell viability and cell functions.

[0007] A method for preparing a 3D-printed high-density cell tubular vascular graft, comprising the following steps:

[0008] Step S1: Collecting pure cells used: Digest the cells from the culture flask to make a cell suspension, place it in a centrifuge for centrifugation. The cells settle at the bottom of the centrifuge tube. Aspirate the supernatant, leaving only the pure cells at the bottom. Aspirate the pure cells into syringe A with a 1 - 2 ml syringe for standby;

[0009] Step S2: Preparation of hydrogel precursor solution: Stir and mix 1.2 - 4.8 wt% oxidized alginate methacrylate composite material (OMA), 0.1 - 0.8 wt% porcine gelatin, and 0.1 - 0.5 wt% LAP photoinitiator in Hank's solution to form a hydrogel precursor solution. Transfer the hydrogel precursor solution to syringe B and syringe C;

[0010] Step S3: Printing: Use a printing device to extrude pure cells and the hydrogel precursor from a triaxial nozzle to obtain fibers with a three - layer coaxial structure. Subsequently, immerse the fibers in an open container filled with 3% calcium chloride solution below; the hydrogel precursor solution will rapidly gelify upon contact with the 3% calcium chloride solution to support the pure cell layer;

[0011] Step S4: Post - printing treatment: Place the printed fibers under UV ultraviolet irradiation for 15 - 60 s to cross - link the OMA hydrogel; put the cross - linked fibers into cell culture medium and place them in a cell culture incubator for long - term culture;

[0012] Step S5: Generation of pure cell lumen: After culturing for 1 - 4 weeks, add 0.2 - 1% lytic enzyme to degrade the hydrogel precursor solution, and place it in a cell culture incubator for 1 - 5 hours. The hydrogel precursor solution is completely degraded, obtaining an independent single - layer pipe structure with only the pure cell layer remaining.

[0013] The 3D - printed small - diameter artificial blood vessel is mainly based on the extrusion - type 3D - printed bioink (a mixture of cells and biomaterials) to form a lumen. To solve problems such as the interference of biomaterials in the bioink of the cell - and - biomaterial mixture with the interaction between cells, the potential immunogenicity of biomaterials and their degradation products, the mismatch between the degradation rate of biomaterials and the formation rate of new tissues, and the non - uniformity and low density of cells embedded in biomaterials, which affect the biological functions of the printed artificial blood vessels. The present invention demonstrates the biofabrication of pure cells and high - cell - density independent vascular tissues for functional artificial blood vessels, and provides a strategy for the preparation of artificial blood vessels and the development of vascular models for cardiovascular and atherosclerosis disease research.

[0014] 1. Adjustable structure: According to requirements, the number of layers of the coaxial nozzle, the size of different layers, the injection rate of the injection pump, and different biomaterial inks can be adjusted to obtain coaxial fibers with different structural sizes and types.

[0015] 2. Low cost: The nozzle used in this invention is self-made, and other printing parts are easily available. The entire printing device can be assembled by oneself.

[0016] 3. High efficiency: The entire printing process takes no more than 5 minutes and will not affect the cell viability in the cell layer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic structural diagram of the microfluidic coaxial 3D printing device of the present invention.

[0018] Figure 2 It is a schematic diagram of coaxial 3D printing nozzles of different sizes and the printed tubular structure model.

[0019] Figure 3 It is an optical microscope picture of a pure NIH3T3 cell lumen structure printed by the microfluidic coaxial 3D printing device.

[0020] Figure 4 It is a physical picture of a pure NIH3T3 cell lumen structure printed by the microfluidic coaxial 3D printing device.

[0021] Figure 5 It is a cell viability test picture of a pure MOVAs cell lumen structure printed by the microfluidic coaxial 3D printing device. DETAILED DESCRIPTION OF THE INVENTION

[0022] The existing extrusion 3D printing lumen tissue has the following technical problems:

[0023] 1. Commonly used extrusion 3D printing lumen tissues all use a mixed bioink of biomaterials and cells. This ink introduces biomaterials, which will interfere with the interconnection and communication between cells.

[0024] 2. During the printing process, the printing rates of the composite hydrogel materials used in the outer layer and the inner layer should match that of the pure cells in the middle layer. The pure cell layer in the middle should not be affected by the flow rates of the outer layer and the inner layer, resulting in a thinner cell layer density.

[0025] In order to overcome the defects existing in the existing extrusion method, the bioprinting of the present invention uses a middle layer of high-density pure cells, and the outer layer and the inner layer are biomaterials as support layers (which can be degraded and removed). The middle high-density pure cell layer simulates the in-vivo environment of cells, increasing the contact and communication between cells. The composite hydrogel materials used in the outer layer and the inner layer are degradable materials or biomaterials that will degrade in response to lytic enzymes, and can maintain the structure in the early cell culture medium. When the required time comes, lytic enzymes are used for degradation.

[0026] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings:

[0027] 1. Self-made microfluidic coaxial 3D printing device:

[0028] Preparation of multi-axis nozzle ( Figure 2 ): Use a blunt needle. The model numbers are: the inner layer is 16 - 27G, the middle layer is 12 - 20G, and the outer layer is 8 - 16G. Figure 2 Shows coaxial 3D printing nozzles of different sizes and the printed tubular structure models: where a is inner layer 16G / middle layer 12G / outer layer 8G, b is inner layer 23G / middle layer 18G / outer layer 14G; c is inner layer 27G / middle layer 20G / outer layer 16G.

[0029] Three-axis nozzle: Insert the small-sized needle into the medium-sized needle, and then insert this set of small / medium-sized needles into the large-sized needle. Cut off the excess length of the stainless steel tube parts of the large / medium / small needles as needed, and then polish the cutting edges of these needles with coarse sandpaper and fine sandpaper to ensure that when the large / medium / small needles are stacked together, their cutting edges are on the same plane, that is, two or three coaxial concentric circles are formed. Then, for the three-axis nozzle, drill a small through-hole in the tail pipe wall of the large-sized needle and the medium-sized needle (for the coaxial nozzle, only drill a small through-hole in the tail pipe wall of the large needle), then insert a blunt needle as the feeding channel, let the printing ink flow into the large / medium-sized needle through the small hole, and fix them together with 5-minute quick-drying epoxy resin. Concentrically fix the large / medium-sized needle with the feeding channel and the small needle with 5-minute quick-drying epoxy resin, and at the same time ensure that the tips of these three needles are coaxial, and there is no gap at the contact positions of the tails of the large needle and the medium-sized needle and the contact positions of the tails of the medium-sized needle and the small needle. Check the center alignment of the fabricated multi-axis nozzle and whether there are potential leakage defects in the feeding channel position. The number of layers can be increased as needed.

[0030] Assembly of the microfluidic coaxial 3D printing device ( Figure 1 ): This self-made microfluidic coaxial 3D printing device includes a nozzle (multi-axis nozzle), a catheter (connecting the nozzle and the syringe), an injection pump (controlling the printing speed of the material), a syringe (containing the printing material), an adjustable bracket (adjusting the height of the nozzle), and an open container (containing the solution for crosslinking).

[0031] A preparation method for 3D printing a high-density cell tubular vascular graft, comprising the following steps:

[0032] Step S1: Preparation of printing ink: Collect the pure cells used: Digest the cells from the culture flask to make a cell suspension, put it into a centrifuge for centrifugation, the cells deposit at the bottom of the centrifuge tube, suck away the supernatant and only leave the pure cells at the bottom, and suck the pure cells into a syringe with a 1 - 2 ml syringe for standby (try to avoid introducing air bubbles during this process).

[0033] Step S2: Preparation of outer and inner hydrogel precursor solutions: A hydrogel precursor solution was formed by stirring and mixing 1.2 - 4.8 wt% oxidized alginate methacrylate composite materials (2% or 4% oxidized and 40% alginate methacrylate), 0.1 - 0.8 wt% porcine gelatin (as a rheology modifier and cell adhesion component), and 0.1 - 0.5 wt% LAP photoinitiator in Hank's solution (the order of reagent addition was: gelatin was first completely dissolved in Hank's solution at 50 °C, the LAP photoinitiator was added, and then OMA was added with strong stirring at 37 °C). The hydrogel precursor solution was transferred to a 10 ml syringe.

[0034] Step S3: Printing process: Combining Figure 1 with the microfluidic coaxial 3D printing device shown, first fix the nozzle on the bracket. Syringe A filled with pure cells was installed on injection pump A. One end of a silicone hose with an inner diameter of 0.3 mm - 0.5 mm was connected to the syringe connector of the syringe filled with pure cells, and the other end was connected to the middle layer inlet of the nozzle. Two syringes B and C filled with the hydrogel precursor solution were respectively installed on injection pumps B and C. One end of a silicone hose with an inner diameter of 0.3 mm - 0.5 mm was connected to the syringe connector of the syringe filled with the hydrogel precursor, and the other end was connected to the outer layer inlet B and the inner layer inlet C of the nozzle. A 3% calcium chloride (CaCl2) solution (triggering the rapid gelation of alginate gel) was placed in the container directly below the nozzle. Adjust the injection flow rates of each injection pump: 20 - 170 μl / min (outer layer), 30 - 200 μl / min (middle cell layer), and 10 - 90 μl / min (inner layer). At the same time, turn on injection pumps A, B, and C to start printing. Three - layer coaxial - structured fibers will be extruded from the nozzle, and then the fibers will be immersed in the 3% CaCl2 solution below. The outer and inner hydrogel precursor solutions will rapidly gelate upon contact with the 3% CaCl2 solution to support the middle pure cell layer. Figure 3 Figure 7 shows an optical microscope image of a pure NIH3T3 cell lumen structure printed using a microfluidic coaxial 3D printing device, where a is a side view (the outermost transparent part is the outer layer, and the black part is the cell layer); b is a cross - sectional view (the outermost transparent part is the outer layer, the middle black part is the cell layer, and the innermost transparent part is the inner layer). Figure 4 Figure 8 shows a physical picture of a pure NIH3T3 cell lumen structure printed using a microfluidic coaxial 3D printing device.

[0035] Step S4: Post - printing treatment: Place the printed fibers under UV ultraviolet irradiation for 15 - 60 s to cross - link the OMA hydrogel. Put the cross - linked fibers into cell culture medium and place them in a cell culture incubator for long - term culture (change the culture medium every 2 days: 30% fetal bovine serum, 70% cell culture medium, 0.5% double antibody).

[0036] Step S5: Generation of pure cell lumen: After culturing for the required time (1 - 4 weeks), 0.2 - 1% lyase is added to degrade the inner and outer OMA hydrogels. Then it is placed in a cell culture incubator for 1 - 5 hours. The inner and outer OMA hydrogels are completely degraded, and an independent monolayer tubular structure with only a pure cell layer remaining is obtained. Figure 5 Cell viability test diagrams of pure MOVAs cell lumen structures printed by a microfluidic coaxial 3D printing device at different time periods; among them, a is the side view of the live (green) and dead (red) cell staining of the printed pure MOVAs lumen on the 2nd day of culture (blue is the cell nucleus); b is the sectional view of the live (green) and dead (red) cell staining of the printed pure MOVAs lumen on the 4th day of culture (blue is the cell nucleus); c is the immunofluorescence staining image of albumin (green) and cell nucleus (blue) of the printed pure MOVAs lumen on the 14th day of culture.

[0037] Example 1:

[0038] 1. Preparation of hydrogel precursor solution: The inner and outer hydrogels are composed of a 2.4% OMA complex (2% oxidized alginate and 40% methacrylated alginate) by mass fraction, a 0.4% porcine-derived gelatin (as a rheology modifier and cell adhesion component) by mass fraction, and a 0.5% photoinitiator LAP. The solvent is Hank’s solution. First, gelatin is completely dissolved in Hank’s solution at 50 °C, then the photoinitiator LAP is added, and subsequently the OMA complex is added and vigorously stirred at 37 °C to form a slightly viscous bioink. MOVAs cells cultured in a culture flask are collected as the bioink for the middle layer, and then the cell suspension is centrifuged to remove the cell culture medium and microbubbles to obtain approximately 1 ml of pure MOVAs cells for one-time bioprinting. A 3% calcium chloride solution is used as a crosslinking agent to crosslink the gelatin in the hydrogel precursor solution.

[0039] 2. 3D printing steps: Use a self-made triple coaxial microfluidic coextrusion 3D printing device for printing. It consists of three layers: the inner layer, the middle layer, and the outer layer. The diameter of the blunt needle for each layer can be adjusted according to actual needs. The models of the blunt needles used for the nozzle are: 25G for the inner layer, 18G for the middle layer, and 14G for the outer layer. After assembling these three needles coaxially, connect the inlet of each layer to a hose, and then connect the hose to a syringe, which is fixed on an injection pump. Use a 3% calcium chloride solution to crosslink the printed tubular structure. Injection flow rates: 120 μl / min (outer layer), 140 μl / min (middle cell layer), and 60 μl / min (inner layer). Use a 3% calcium chloride bath at 37 °C to trigger the rapid gelation of the alginate gel. Immediately after bioprinting (less than 5 minutes), irradiate the printed sample with ultraviolet light for 30 seconds to crosslink the OMA, thereby forming the OMA hydrogel for the inner and outer layers to support the cell-only middle layer. Transfer it to MOVAs cell growth medium and culture it long-term at 37 °C, changing the culture solution every 2 days (30% fetal bovine serum, cell culture medium, 0.5% double antibody).

[0040] 3. Preparation of pure cell lumen: After culturing the three-layer fiber structure obtained above for 1 week, add 0.5% lyase to degrade the OMA hydrogels of the inner and outer layers. Place it in a cell culture incubator for 2 hours, and the OMA hydrogels of the inner and outer layers are completely degraded, obtaining an independent single-layer pipe structure with only OVAs remaining.

[0041] Example 2

[0042] 1. Preparation of hydrogel precursor solution: The hydrogels for the inner and outer layers are composed of a 2.4% OMA complex (2% oxidized alginate and 40% methacrylated alginate) by mass fraction, a 0.4% porcine-derived gelatin (as a rheology modifier and cell adhesion component) by mass fraction, and a 0.5% photoinitiator LAP by mass fraction. The solvent is Hank’s solution. First, completely dissolve the gelatin in Hank’s solution at 50 °C, then add the photoinitiator LAP, and then add the OMA complex and stir vigorously at 37 °C to form a slightly viscous bioink. Collect NIH3T3 cells cultured in a culture flask as the bioink for the middle layer, and then centrifuge the cell suspension to remove the cell culture medium and tiny bubbles to obtain about 1 ml of pure NIH3T3 cells for one-time bioprinting. Use a 3% calcium chloride solution as a crosslinking agent to crosslink the gelatin in the hydrogel precursor solution.

[0043] 2. 3D printing steps: Use a self-made triple coaxial microfluidic co-extrusion 3D printing device for printing. It consists of three layers: the inner layer, the middle layer, and the outer layer. The diameter of the blunt needle for each layer can be adjusted according to actual needs. The models of the blunt needles used for the nozzles are: 23G for the inner layer, 18G for the middle layer, and 14G for the outer layer. After assembling these three needles coaxially, connect the inlet of each layer to a hose, and then connect the hose to a syringe. The syringe is fixed on an injection pump. Use a 3% calcium chloride solution to crosslink the printed tubular structure. Injection flow rates: 120 μl / min (outer layer), 140 μl / min (middle cell layer), and 80 μl / min (inner layer). Use a 3% calcium chloride bath to trigger the rapid gelation of the alginate gel at 37 °C. Immediately after bioprinting (less than 5 minutes), irradiate the printed sample with ultraviolet light for 30 seconds to crosslink OMA, thereby forming the OMA hydrogel for the inner and outer layers to support the cell-only middle layer. Transfer it to MOVAs cell growth medium and culture it long-term at 37 °C, changing the culture medium every 2 days (30% fetal bovine serum, cell culture medium, 0.5% double antibody).

[0044] 3. Preparation of the pure cell lumen: After culturing the three-layer fiber structure obtained above for 4 weeks, add 0.5% lyase to degrade the OMA hydrogels of the inner and outer layers. Place it in an incubator for 2 hours, and the OMA hydrogels of the inner and outer layers are completely degraded, obtaining an independent single-layer tubular structure with only OVAs remaining.

[0045] The present invention proposes a new strategy for 3D bioprinting pure cell bioinks to prepare small-caliber vascular tissue lumens and mimic the structure of native blood vessels. Specifically, a 3-layer lumen structure is 3D printed using microfluidic coaxial extrusion, with the middle layer being a high-density pure cell bioink of mouse aortic vascular smooth muscle cell line (MOVAs), and the oxidized methacrylated alginates (OMA) composite hydrogel as the core layer and the outer layer to support the high-density pure cell middle layer and maintain the lumen structure of the pure cell middle layer. After 1 week - 1 month of in vitro culture, after degrading the OMA composite hydrogels of the core layer and the outer layer with lyase, a single-layer lumen composed only of MOVAs cells is obtained. The single-layer lumen of MOVAs exhibits high cell viability, obvious F-actin, and abundant albumin, indicating that the extrusion-based bioprinting process and the designed inner and outer supporting OMA composite hydrogel layers have no adverse effects on the survival of high-density cells. This method overcomes the limitation of introducing exogenous biomaterial scaffolds in current 3D printing of small-caliber blood vessels, develops 3D printing of high-density pure cells without any biomaterial scaffolds, and cultivates them into a pure cell lumen in vitro. This method has a wide range of biomedical applications, such as developing precise models for simulating tissues and diseases, drug screening, etc.

Claims

1. A method for preparing a 3D printed high-density cell tubular vascular graft, characterized in that It includes the following steps: Step S1: Collect the pure cells used: Digest the cells from the culture flask to make a cell suspension, put it into a centrifuge for centrifugation. The cells sediment at the bottom of the centrifuge tube. Aspirate the supernatant, leaving only the pure cells at the bottom. Aspirate the pure cells into syringe A with a 1-2 ml syringe for standby; Step S2: Preparation of the hydrogel precursor solution: Stir and mix 1.2-4.8 wt% of oxidized alginate methacrylate composite material, 0.1-0.8 wt% of porcine gelatin, and 0.1-0.5 wt% of LAP photoinitiator in Hank's solution to form a hydrogel precursor solution. Transfer the hydrogel precursor solution to syringe B and syringe C; Step S3: Printing: Extrude the pure cells and the hydrogel precursor from the three-axis nozzle using a printing device to obtain fibers with a three-layer coaxial structure. Subsequently, the fibers are immersed in an open container filled with 3% calcium chloride solution below; The hydrogel precursor solution will rapidly gelate upon contact with the 3% calcium chloride solution to support the pure cell layer; Step S4: Post-printing treatment: Place the printed fibers under UV ultraviolet irradiation for 15-60 s to crosslink the OMA hydrogel; Put the crosslinked fibers into cell culture medium and place them in a cell culture incubator for long-term culture; Step S5: Generation of pure cell lumen: After culturing for 1-4 weeks, add 0.2-1% of lytic enzyme to degrade the hydrogel precursor solution, and place it in a cell culture incubator for 1-5 hours. The hydrogel precursor solution is completely degraded, and an independent single-layer pipeline structure with only the pure cell layer remaining is obtained.

2. The preparation method of a 3D printed high-density cell tubular vascular graft according to claim 1, characterized in that The composite material of oxidized alginate methacrylate in Step S1 above is a mixed solution of 2% or 4% oxidation and 40% alginate methacrylate; 3. The preparation method of a 3D printed high-density cell tubular vascular graft according to claim 1, characterized in that The order of adding reagents in Step S1 above is: Gelatin is first completely dissolved in Hank's solution at 50 °C, add the LAP photoinitiator, and then add the oxidized alginate methacrylate composite material and stir vigorously at 37 °C.

4. The preparation method of a 3D printed high-density cell tubular vascular graft according to claim 1, characterized in that During printing in Step S3 above, first fix the three-axis nozzle on the bracket; Install syringe A filled with pure cells on injection pump A. Connect one end of a silicone hose with an inner diameter of 0.3 mm - 0.5 mm to the syringe joint filled with pure cells, and the other end to the middle layer feed port of the nozzle; Install the 2 syringes B and C filled with the hydrogel precursor solution on injection pump B and injection pump C respectively. Connect one end of a silicone hose with an inner diameter of 0.3 mm - 0.5 mm to the syringe joint filled with the hydrogel precursor, and the other end to the outer layer feed port B and the inner layer feed port C of the nozzle; Adjust the injection flow rates of each injection pump, and simultaneously turn on injection pump A, injection pump B, and injection pump C to start printing.

5. The preparation method of a 3D printed high-density cellular tubular vascular graft according to claim 4, characterized in that The injection flow rates of each injection pump above are: The injection flow rate of injection pump B located on the outer layer is 20-170 μl / min, the injection flow rate of injection pump A located in the middle cell layer is 30-200 μl / min, and the injection flow rate of injection pump C located on the inner layer is 10-90 μl / min.

6. A method for 3D printing a high-density cell tubular vascular graft according to claim 1, characterized in that During the long-term culture process in step S4 above, the culture medium is changed every two days; the culture medium includes: 30% fetal bovine serum, 70% cell culture medium, and 0.5% double antibody.

7. The preparation method of a 3D printed high-density cell tubular vascular graft according to claim 1, characterized in that The printing device used in step S3 above is: The three-axis nozzle is fixed on a bracket for adjusting the nozzle height. The inlet section of the three-axis nozzle is connected to syringes through conduits respectively. Among them, the medium-sized needle is connected to syringe A, the small-sized needle is connected to syringe B, and the large-sized needle is connected to syringe C; each syringe corresponds to an injection pump for controlling the printing speed of the material; the outlet section of the three-axis nozzle is arranged with an open container for containing the solution used for crosslinking.

8. The preparation method of a 3D printed high-density cell tubular vascular graft according to claim 1, characterized in that A blunt needle is used: the inner layer is a small-sized needle of 16-27G, the middle layer is a medium-sized needle of 12-20G, and the outer layer is a large-sized needle of 8-16G; Three-axis nozzle: Insert the small-sized needle into the medium-sized needle, and then insert this set of small / medium-sized needles into the large-sized needle; Cut off the excess length of the stainless steel tube part of the large / medium / small needles, and then polish the cutting edges of these needles with coarse sandpaper and fine sandpaper to ensure that when the large / medium / small needles are stacked together, their cutting edges are on the same plane, forming two or three coaxial concentric circles; For the three-axis nozzle, drill a small through hole in the tail pipe wall of the large-sized needle and the medium-sized needle, then insert a blunt needle as the feeding channel, let the printing ink flow into the large / medium-sized needle through the small hole, and fix them together with fast-drying epoxy resin; Concentrically fix the large / medium-sized needle with the feeding channel and the small needle with fast-drying epoxy resin, and at the same time ensure that the tips of these three needles are coaxial, and there is no gap at the contact position of the tails of the large needle and the medium-sized needle and the contact position of the tails of the medium-sized needle and the small needle.

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