Bionic valve type organ obtained through 3D biological printing and corresponding 3D printing ink

Abstract

The invention belongs to the field of biological tissue and organ 3D printing, and discloses a biomimetic valve type organ obtained through 3D biological printing and corresponding 3D printing ink, and the 3D printing ink for forming the biomimetic valve type organ is biological ink obtained by dispersing a photocurable biological material in a phosphate buffer solution, then adding a photoinitiator solution, uniformly mixing, discharging bubbles, and then adding cell spheres to obtain the photoinitiator. The cell ball is a valve interstitial cell ball with a micron-scale diameter, which is obtained by culturing valve interstitial cells by using an agarose mold in the presence of a cell culture medium; and the agarose mold is provided with micron-scale holes. According to the method, the loading mode of the valvular interstitial cells is improved, the cell balls serve as the minimum printing unit, damage to the cells in the 3D printing period is avoided, the interaction between the cells is enhanced, the cells are stimulated to secrete the extracellular matrix, and extracellular matrix remodeling is completed.

Description

Technical Field

[0001] The present invention belongs to the field of 3D printing of biological tissues and organs, and more specifically, relates to a bionic valve organoid obtained by 3D bio-printing and a corresponding 3D printing ink. Background Art

[0002] Heart valve disease, as one of the major diseases threatening human health, has causes including congenital malformations, rheumatic fever, infective endocarditis and valve calcification. Among them, valve stenosis or insufficiency caused by valve calcification is the most common type of lesion. At present, the specific mechanism of valve calcification is not fully understood and further in-depth research is needed. At the same time, there is a lack of effective means to effectively prevent valve calcification. Current clinical treatment mainly relies on valve replacement surgery. Existing replacement valves mainly include mechanical valves and biological valves, but each has its own shortcomings. After mechanical valve replacement surgery, patients are prone to thrombosis, so they need to take anticoagulants for a long time, which brings inconvenience to the patient's quality of life. For some infants with congenital valve defects, mechanical valves are not suitable because they are too large or cannot grow with the body. On the other hand, biological valves will gradually degenerate over time and are prone to calcification. Usually, replacement surgery is required again within 10 to 15 years, which brings more inconvenience to patients. When studying the mechanism of biological valve calcification, the use of living biological valves is not only costly, but also has large individual differences and very complicated experimental operations. Therefore, when screening means or drugs to prevent and treat valve calcification, there is an urgent need for valve organoids that are large in number, have little variability, are low in cost, and are easy to operate. Currently, tissue-engineered valves and valve organoids are widely considered to be one of the most promising valve substitutes.

[0003] Tissue-engineered valves are similar to natural tissues. The construction process is to implant seed cells into bioprinted scaffold materials, and rely on cell proliferation, migration, and degradation and remodeling of the extracellular matrix to gradually form durable, anti-anticoagulation-free, anti-calcification, low immunogenicity, and regenerative valve tissue. Methacryloyl gelatin (GelMA), a hydrogel widely used in tissue engineering, combines the excellent biocompatibility and biodegradability of natural gelatin, and has photocrosslinking properties after methacryloyl modification, making it suitable for 3D bioprinting.

[0004] 3D bioprinting technology can formulate personalized printing plans according to different needs, and control complex tissues in space to reproduce the heterogeneity of tissues. In addition, it can also strictly control the structure to achieve the operability of the mechanical properties and degradation properties of the material. As an important branch of 3D bioprinting, extrusion 3D printing technology has become one of the most commonly used printing technologies in tissue engineering research due to its simple process and high cell loading. Bioprinting ink is a material containing biocompatible components and has good shear thinning properties, which plays an important role in 3D bioprinting. Bioprinting inks with different components are suitable for different types of tissue engineering, and can effectively encapsulate living cells so that they are better and evenly dispersed in the hydrogel formed by the ink.

[0005] The research group of the inventor of the present invention previously reported an electrostatic direct writing degradable polymer fiber composite bioactive hydrogel scaffold for a bionic valve (see Chinese patent application 202310974870.8) and a multi-nozzle 3D bioprinting scaffold for a bionic valve and its application (see Chinese patent application 202311512619.6). Although they can solve some problems in the preparation of bionic valves to a certain extent, considering the printability of printing ink, high-density cell suspensions (such as more than 5×10 6 cells per milliliter of ink) for printing, which leads to insufficient cell-cell interactions and ultimately affects the speed and process of extracellular matrix remodeling.

[0006] At present, the application of 3D bioprinting technology has received increasing attention, but in actual operation, it is often limited by cell density and cell growth environment. Too high cell density will affect the printability of bio-inks such as GelMA, resulting in reduced printing quality. At the same time, a series of cells with high growth environment requirements, including hiPS-VIC, may cause reduced cell viability or even death due to the high shear force at the printing needle during the printing process, which undoubtedly has a significant impact on the construction of high-quality tissue engineering valves. Summary of the invention

[0007] In view of the above defects or improvement needs of the prior art, the object of the present invention is to provide a bionic valve organoid obtained by 3D bioprinting and a corresponding 3D printing ink, wherein the loading method of the valve interstitial cells is improved, and the cell sphere is used as the minimum printing unit to avoid damage to the cells during 3D printing, strengthen the interaction between cells, stimulate the secretion of extracellular matrix, and complete the remodeling of the extracellular matrix.

[0008] To achieve the above object, according to one aspect of the present invention, a 3D printing ink for forming a biomimetic valve organoid is provided, which is obtained by dispersing a photocurable biomaterial in a phosphate buffered saline (PBS) to obtain a bio-ink, then adding a photoinitiator solution, mixing evenly, expelling bubbles, and then adding a cell spheroid to obtain;

[0009] The cell spheres are obtained by culturing valvular interstitial cells (VIC) in an agarose mold in the presence of a cell culture medium, thereby obtaining valvular interstitial cell spheres with a diameter of micrometers, each of which is enriched with valvular interstitial cells; the agarose mold has micrometer-level pores.

[0010] As a further preferred embodiment of the present invention, the diameter of the cell sphere is 130-450 μm; accordingly, each cell sphere contains 500-8000 valve interstitial cells;

[0011] Preferably, the valvular interstitial cells are valvular interstitial cells derived from human induced pluripotent stem cells (hiPS-VIC).

[0012] As a further preferred embodiment of the present invention, the agarose mold has a dot matrix of micropores, any one of which has a circular opening with a diameter of 350 to 1000 μm, and smoothly transitions to the bottommost hemispherical concave surface within 250 to 500 μm, and the diameter of the hemispherical surface is 150 to 500 μm;

[0013] On the plane where the micro-holes of the agarose mold dot matrix are located, the distance between the projections of the centers of two adjacent holes is 500-1250 μm;

[0014] The plane where the micro-holes of the agarose mold dot matrix are located is 5 to 8 mm lower than the surrounding area of ​​the agarose mold;

[0015] When forming cell spheres using an agarose mold, the outside of the agarose mold is placed in a phosphate buffered saline (PBS), and the height difference between the periphery of the agarose mold and the plane where the agarose mold lattice micropores are located can prevent the phosphate buffered saline (PBS) from entering the lattice micropores.

[0016] As a further preferred embodiment of the present invention, the amount of cell spheroids added satisfies that 2000 to 5000 cell spheroids are contained in each milliliter of 3D printing ink.

[0017] As a further preference of the present invention, the photoinitiator in the photoinitiator solution is one or more of α-ketoglutaric acid (α-KGA), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP); preferably phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP).

[0018] As a further preferred embodiment of the present invention, the photocurable biomaterial is methacrylated gelatin (GelMA);

[0019] Preferably, the methacryloyl gelatin (GelMA) is prepared by placing gelatin (Gelatin) in deionized water or PBS, stirring at a temperature of 40°C to 70°C to completely dissolve it, then adding methacrylic anhydride or glycidyl methacrylate, continuing to stir the reaction at a temperature of 40°C to 70°C, and then dialyzing the reaction product with a dialysis bag and freeze-drying it;

[0020] More preferably, the stirring speed is 400-800 rpm; the amount of methacrylic anhydride or glycidyl methacrylate is 0.4-1.2 mL per gram of gelatin; the reaction time after adding methacrylic anhydride or glycidyl methacrylate is 1-24 h; the molecular cutoff of the dialysis bag is 8000-14000 Da, and the dialysis time is 2-14 days.

[0021] According to another aspect of the present invention, the present invention provides a bionic valve organoid obtained by 3D bio-printing, which is obtained by loading the above-mentioned 3D printing ink into the nozzle of the 3D bio-printer and then 3D printing according to a preset shape.

[0022] As a further preferred embodiment of the present invention, the printing parameters used in the 3D printing are as follows:

[0023] The nozzle uses a 22-27G caliber printing needle, the nozzle temperature is 16℃~24℃, the moving speed is 3~7mm / s, and the extrusion speed is 0.25~0.7mm 3 / s, the nozzle's idle retraction volume is 0.2-0.6mm, and the lifting volume is 1-2mm;

[0024] The number of printing layers is 1 to 3;

[0025] UV irradiation is performed once for each printed layer; the UV intensity used is 5-15mW / cm 2 , each irradiation time is 3 to 15 seconds; and, after the last printing layer is pre-crosslinked by ultraviolet light irradiation, ultraviolet light irradiation is performed again to completely crosslink; preferably, the ultraviolet wavelength used for ultraviolet light irradiation is 405nm.

[0026] As a further preferred embodiment of the present invention, the preset shape is a shape corresponding to a cross-net structure.

[0027] According to another aspect of the present invention, the present invention provides the use of the above-mentioned 3D printing ink for forming biomimetic valve organoids in the preparation of valve tissue and / or valve organoids, or the use of the above-mentioned biomimetic valve organoids in the preparation of valve tissue.

[0028] Through the above technical solutions conceived by the present invention, compared with the prior art, the present invention simultaneously utilizes cell spheroid technology and 3D bioprinting technology to construct bionic valve 3D printed organoids, which can effectively solve the problem of slow extracellular matrix reconstruction during the construction of 3D valve printed organoids. The present invention greatly increases the cell density in a small area (inside the cell spheroid) by using cell spheroids enriched with valve interstitial cells, and can provide cells with sufficient cell-to-cell interaction environment without affecting the printability of biological ink (such as GelMA), stimulating them to secrete the required extracellular matrix components, and ultimately improving the speed and process of extracellular matrix reconstruction. At the same time, the use of cell spheroids to construct 3D printing inks in the present invention can effectively solve the problem of low cell activity or survival rate during the construction of 3D valve printed organoids; the outer layer of the cell spheroid can protect the internal cells, thereby greatly reducing the shear force on the internal cells of the cell spheroid during 3D bioprinting, passing through the printing needle, and avoiding the internal cells from being affected and dying rapidly. In addition, for bio-inks that need to be cooled in advance, that is, bio-inks with a phase transition temperature of 37°C or even below room temperature (such as 10% GelMA), the technology proposed in the present invention can also prevent most of the cells inside the cell spheres from being stimulated by rapid cooling, thereby avoiding the problem of reduced vitality.

[0029] The cell sphere in the present invention is preferably prepared by an agarose mold with a specific hole structure, which optimizes the process of cell sphere forming. The cell sphere forming technology reported in the prior art can often only form planar cell aggregates, and cannot form a three-dimensional aggregate structure suitable for printing. The present invention is provided with a hole structure of target size on the agarose mold with a smooth transition structure (that is, with the height change, the cross section of the transition structure smoothly transitions from a hollow circle with a large diameter to a hemispherical concave surface of a target diameter, and the hemispherical concave surface is tangent to the transition structure; the diameter of the hemispherical concave surface is smaller than the diameter of the hollow circle with a large diameter at the starting position, and the cell sphere formed by the agarose mold, the diameter of the cell sphere is often slightly smaller than the diameter of the hemispherical concave surface, taking the hemispherical diameter as 500 μm as an example, the diameter of the formed cell sphere does not exceed 500 μm), and the inclined surface of the transition structure is used to form the holes for cell spheres to collect more cells, avoid too many cells from falling into the holes, and improve the cell utilization rate of the cell sphere. In addition, it is also possible to facilitate the formation of three-dimensional cell spheres with different radii according to different printing needles by means of a special hole bottom spherical structure with a controllable radius. In addition, considering that there will be problems such as easy drying of culture medium and difficulty in collecting cell spheres during cell sphere forming, the present invention preferably deepens the depth of agarose mold (compared to the surroundings, the plane where the lattice micropores are located is concave 5-8mm), which not only slows down the evaporation of culture medium during cultivation, but also facilitates the operator to directly and quickly collect the cell spheres therein by blowing the culture medium in the mold by 1000 μL pipette guns, and when valvular interstitial cells (i.e., forming cell spheres) can be preferably cultivated in agarose molds, PBS buffer is added around the entire mold, PBS buffer is made to surround the entire mold (the height difference between the plane where the agarose mold lattice micropores are located can block PBS from entering the lattice micropores), and the problem of easy evaporation of the culture medium therein is avoided by placing the mold in a 37°C incubator.

[0030] Taking the ratio of adding 2000 to 5000 cell spheres per milliliter of printing ink as an example (the diameter of each cell sphere is in the micrometer range, and each cell sphere contains 500 to 8000 valve interstitial cells), the present invention can also increase the equivalent loading capacity of cells in 3D bioprinting (compared with 1×10 6 cells / mL density).

[0031] Valvular interstitial cells (hiPS-VIC) derived from human induced pluripotent stem cells currently show many potentials in valve tissue engineering applications. Compared with the common porcine aortic valvular interstitial cells (PAVIC) and allogeneic human aortic valvular interstitial cells (HAVIC) in current valve tissue engineering and organoids, hiPS-VIC can be derived from autologous cells and can be free from the influence caused by rejection, but hiPS-VIC is easy to die in conventional cell printing (i.e., single cell printing), which limits its application. The present invention is particularly suitable for hiPS-VIC, which can overcome the drawbacks of hiPS-VIC being easy to die in conventional cell printing processes, so that the advantages of hiPS-VIC can be effectively exerted, such as the risk of reducing immune rejection of xenogeneic or allogeneic cells. At the same time, considering that hiPS-VIC is induced by induced pluripotent stem cells (iPSC), it can be amplified on a large scale at the stem cell stage and induced into VIC to meet the large-scale demand for cells in tissue engineering and organoid structures. Based on the 3D printing ink formed by the present invention, using hiPS-VIC can further increase the cell density in 3D bioprinting, strengthen the connection between cells, and promote the process of extracellular matrix reconstruction. Taking the inventor’s previous research results, Chinese patent applications 202310974870.8 and 202311512619.6 as examples, they cannot use high-density cell suspensions (more than 5×10 6 In the present invention, cell spheres are used as the cell existence form in 3D bioprinting, which effectively improves the local cell density, promotes cell-to-cell interaction, and ultimately accelerates the speed and process of extracellular matrix remodeling without affecting printability. In addition, in the above-mentioned preliminary studies, when cells with high growth environment requirements such as hiPS-VIC are selected for printing, the viability of some cells is often reduced or even killed due to excessive shear stress at the printing needle, which also reduces the equivalent cell density to a certain extent, and then has a certain impact on the process of extracellular matrix remodeling. The present invention uses the form of cell spheres to keep the vast majority of cells alive and healthy, ensuring high cell density and viability in the region, and promoting the process of extracellular matrix remodeling.

[0032] Specifically, compared with the prior art, the present invention can achieve the following beneficial effects:

[0033] (1) The present invention utilizes cell spheroids and 3D bioprinting technology, and utilizes the three-dimensional structure of cell spheroids to strengthen the complex cell-cell and cell-extracellular matrix interactions after printing, so that the printed cell components can form a complex valve tissue structure in a shorter time, thereby accelerating the subsequent matrix reconstruction process, and finally completing the construction of a valve bionic structure in a histological sense. In comparison, single-cell printing requires a longer time to form a valve bionic structure.

[0034] (2) The present invention uses cell spheroid technology to solve the problem of low cell viability or low survival rate in existing cell-carrying 3D bioprinting technology, where single cells are subjected to excessive shear force when passing through the printing needle. The present invention uses 3D bioprinting based on cell spheroids, which can reduce or even prevent the internal cells from being subjected to excessive shear force by protecting the inner cells with the outer cells of the cell spheroid, thereby enhancing the survival rate of cells after printing and accelerating the process of extracellular matrix reconstruction.

[0035] (3) The cell sphere preparation method and cell sphere preparation mold of the present invention optimize the process of cell sphere forming, solve the problem that in the existing process of preparing cell spheres, the aggregates are limited to two-dimensional structures, the cell spheres are difficult to collect, and it is difficult to accurately control the diameter of the cell spheres. This special structure allows the diameter of the formed cell spheres to be pre-controlled according to the inner diameter of the printing needle, and promotes its formation of a three-dimensional structure, which is more in line with the needs of 3D bioprinting valve tissue engineering. In addition, it is preferred to set a deeper groove (that is, the plane where the micropores of the agarose mold dot matrix are located is sunken compared to the surroundings of the agarose mold, and the sinking depth can be preferably 5 to 8 mm), which can prevent the rapid evaporation of the cell culture medium during the culture of the cell spheres; this special circular groove array with a narrow bottom and a wide top facilitates the 1000 μL pipette gun tip to directly absorb the prepared cell spheres, which is convenient for their rapid collection.

[0036] (4) The present invention can especially use hiPS-VIC, which can overcome the disadvantage that hiPS-VIC is easy to die in the conventional cell printing process (as illustrated in Comparative Example 1 below, the conventional single-cell printing process is easy to damage cells and reduce cell activity). Based on the present invention, hiPS-VIC is applied to bionic valve 3D bioprinted organoids, which effectively solves the rejection problem caused by PAVIC or HAVIC due to xenogeneic or allogeneic in tissue engineering bionic valves in the past. The present invention uses hiPS-VIC and finds that it can secrete extracellular matrix more quickly and in larger quantities in the form of cell spheres than PAVIC cell spheres, and perform effective extracellular matrix remodeling, which is also not reported in the prior art; therefore, the present invention further solves the above two problems of small cell sources and slow amplification, so that in valve tissue engineering or organoid 3D printing that requires a large number of cells, it will not be difficult to quickly and effectively reconstruct the extracellular matrix due to insufficient cells, affecting the final effect.

[0037] In summary, the present invention utilizes cell spheres, with cell spheres as the minimum printing unit, to increase local cell density, promote the process of cell-cell interaction and extracellular matrix reconstruction, and avoid the problem of difficulty in extracellular matrix reconstruction due to low overall cell density; the activity and survival rate of cells are improved by protecting the inner cells from excessive shear force through the outer layer of cell spheres. The 3D bioprinted organoids of the present invention include 3D bioprinted scaffolds with photocurable hydrogel materials as carriers, and cell components with cell spheres as the existing form (that is, the cell components therein are in the form of cell spheres), with good structural stability and cell adaptability, good biological activity, can effectively promote extracellular matrix reconstruction and cell proliferation, and have great application value in valve repair, valve calcification pathogenesis research, and valve calcification treatment drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the result of H NMR analysis of GelMA used in Examples 5 and 6.

[0039] Figure 2 This is a design drawing for preparing a light-curable resin mold in Example 1, wherein: Figure 2 (a) is a three-dimensional schematic diagram of the mold. Figure 2 (b) is the three-view drawing of the mold. Figure 2 (c) is an enlarged schematic diagram of the hole structure.

[0040] Figure 3 This is a physical picture of a cell ball mold of a 3D bioprinted organoid of a biomimetic valve obtained in Example 1, wherein: Figure 3 (a) is a light-curing resin mold. Figure 3 (b) is a PDMS mold. Figure 3 (c) in the figure is an agarose mold.

[0041] Figure 4 This is a real picture of a 3D bioprinted organoid of a bionic valve obtained in Example 7. The scale in the figure represents 2 mm.

[0042] Figure 5 The live / dead staining image and matrix secretion staining image of the bionic valve 3D bioprinted organoid obtained in Example 7, wherein: Figure 5 (a) in the figure is the corresponding live / dead staining image. Figure 5 (b) and (c) are the corresponding extracellular matrix secretion staining images.

[0043] Figure 6 This is a staining image of extracellular matrix secretion obtained by using PAVIC cell spheres to perform bionic valve 3D bioprinting on organoids in Example 9.

[0044] Figure 7The live / dead staining image and extracellular matrix secretion staining image obtained by using hiPS-VIC single cells to perform biomimetic valve 3D bioprinting on organoids in Comparative Example 1, wherein: Figure 7 (a) in the figure is the corresponding live / dead staining image. Figure 7 (b) is the corresponding extracellular matrix secretion staining image. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0046] The printing ink matrix involved in the present invention can be prepared with reference to the prior art (such as Chinese patent application CN202311512619.6), for example, it can be synthesized according to the following steps:

[0047] Place gelatin in deionized water and stir at a constant temperature until it is completely dissolved. Then slowly add methacrylic anhydride or glycidyl methacrylate and continue the reaction for a certain period of time. After the reaction is completed, the product is placed in a dialysis bag, dialyzed for several days, and then freeze-dried to obtain GelMA; GelMA is mixed in PBS at a certain concentration, a photoinitiator solution is added, mixed thoroughly and bubbles are removed (bubbles can be discharged by centrifugation), and finally the ink for 3D printing is obtained.

[0048] Specifically, for printing ink, gelatin can be type A pig skin gelatin (Gelatin from porcineskin, Type A), the temperature of the sol gelatin can be set at 40-70°C, the stirring speed can be set at 400-800rpm, the amount of methacrylic anhydride or glycidyl methacrylate added can be set at 0.4-1.2mL per gram of gelatin, the reaction time can be set at 1-24h, the molecular cutoff of the dialysis bag can be set at 8000-14000Da, and the dialysis time can be set at 2-14 days; the dissolved concentration can be set at 0.05-0.15g / mL, and the photoinitiator can be one of α-ketoglutaric acid (α-KGA), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), and its final concentration can be set at 300-500μg / mL.

[0049] The preparation method of GelMA used in the following examples includes the following steps:

[0050] (1) Add 10 g of gelatin into 100 ml of deionized water and stir at 450 rpm at 60°C until it is completely dissolved to form a uniform solution.

[0051] (2) While stirring, slowly add 4 ml of methacrylic anhydride.

[0052] (3) After reacting for 10 h, add deionized water for dilution.

[0053] (4) The product was placed in a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed for 7 days.

[0054] (5) The dialyzed solution is freeze-dried to obtain GelMA.

[0055] Figure 1 The H NMR spectrum of the prepared product GelMA is shown. 1 H NMR analysis results. It can be seen that at about 5.5ppm, GelMA has two more peaks than gelatin, indicating that these are different types of hydrogen atom signals on both sides of the double bond in methacrylic anhydride. This phenomenon proves that the olefinic carbonyl group has been successfully modified on gelatin.

[0056] In addition to the above preparation method, GelMA can also be prepared by other methods known in the prior art. Of course, commercially available products can also be directly used.

[0057] The cell spheroid mold used in the present invention can be prepared, for example, according to the following steps:

[0058] Using the software SolidWorks, a mold designed according to the requirements was drawn, and then printed with a photocurable resin. Then polydimethylsiloxane (PDMS) was used for casting, and after multiple vacuum negative pressures in a vacuum drying oven, the bubbles in the PDMS were exhausted, and then placed in a 4°C refrigerator for a period of time to further exhaust the bubbles, and then the photocurable resin containing PDMS was placed in an oven until the inside was completely solidified; after complete solidification, the PDMS was removed to obtain a new mold, which was sterilized, and agarose solution was poured into the mold. After it was cooled and solidified, it was taken out to obtain the designed agarose cell sphere mold.

[0059] Specifically, the printing parameters of the photocurable resin can be: layer thickness 0.03-0.05 mm, printing rise height 7-10 mm, motor speed 4.5-5.5 mm / s, light off delay 5-8 s, initial layer exposure time 25-30 s, initial layer exposure number 1-5 layers, regular layer printing exposure time 0.5-1.5 s, ultraviolet light wavelength 405 nm, standard light intensity 8000 μW / cm 2. PDMS can use a PDMS to curing agent mass ratio of 10:1 to 15:1. In the curing process, the parameters can be placed at 4°C for 4 to 48 hours, the oven temperature can be 35 to 40°C, and the curing time can be 12 to 120 hours. In the curing process of agarose, the following parameters can be used: agarose liquid temperature can be 70 to 90°C, agarose concentration can be 1 to 5 w / v%, cooling time can be 5 to 20 minutes, and the temperature after cooling can be 25 to 40°C.

[0060] Using the prepared agarose cell sphere mold, the preparation of cell spheres can be carried out according to the following steps:

[0061] Add VIC cell suspension to the agarose mold, culture in a 37°C, 5% CO2 cell culture incubator until the cell spheres are tightly aggregated, and then use a pipette to blow in the agarose mold several times to suck out and collect.

[0062] Specifically, the parameters for preparing cell spheres can be: each cell sphere contains 500 to 8000 cells.

[0063] The agarose cell sphere mold can be prepared by printing the initial mold with a photocurable resin, and then casting and demolding PDMS and agarose in sequence (i.e., the target agarose mold is obtained by printing with a photocurable resin and casting and demolding twice; since PDMS is a relatively soft material, and the agarose mold has a relatively low strength and is relatively brittle, the two demoldings can easily separate the agarose mold from the PDMS). The preparation of the agarose cell sphere mold and the cell sphere may include the following steps:

[0064] a1: Use photocurable resin to print a mold with special dot matrix micro holes;

[0065] a2: Use polydimethylsiloxane (PDMS) to cast and demould in a light-curing resin mold. During this process, the bubbles in the PDMS need to be emptied by vacuum negative pressure to obtain a PDMS mold printed with a special dot pattern;

[0066] a3: Heat the sterilized 1-5wt% (e.g., 3wt%) agarose to above 70°C, pour it into the sterilized PDMS mold, wait for it to cool to room temperature and solidify, then demold it to obtain an agarose mold with special dot matrix micropores;

[0067] a4: Disperse 500 to 8000 hiPS-VIC cells per well in a suitable amount of complete cell culture medium (e.g., DMEM high glucose medium + 5% FBS + 1% P / S) into the agarose mold and incubate at 37°C, 5% CO 2 After culturing for 24 hours under the same conditions, hiPS-VIC cell spheres with a diameter of 130 to 450 μm were obtained.

[0068] Subsequently, the prepared cell spheres can be added to the printing ink at a ratio of 2000 to 5000 cell spheres per milliliter, the printing pattern and printing parameters can be set, printing can be performed, and ultraviolet light can be used for photocuring to obtain a 3D bio-printed organoid of a bionic valve.

[0069] That is, the printing ink is loaded into the nozzle of the 3D bioprinter, and printing is performed according to a preset model file; for example, a cross-net structure can be obtained by printing a light-cured hydrogel material on a plane; for another example, the printing pattern can be a rectangular or triangular prism-shaped layer-by-layer accumulation. Taking the rectangular shape as an example, the pattern design and printing parameters can be specifically as follows:

[0070] On the one hand, the pattern design can be generated by a biological 3D printer. For example, the length of the cuboid can be 10 to 30 mm and the width can be 10 to 30 mm. The nozzle prints 1 to 6 layers to obtain the target scaffold.

[0071] On the other hand, the nozzle uses a printing needle with a caliber of 22 to 27G, the nozzle temperature is 16°C to 24°C, the moving speed is 3 to 7mm / s, and the extrusion speed is 0.25 to 0.7mm 3 / s, the retraction amount of the nozzle is 0.2-0.6mm, and the lifting amount is 1-2mm. The UV lamp used for UV treatment in the following embodiments is the UV lamp built into the print head, with a wavelength of 405nm and a light intensity of 5-15mW / cm 2 (e.g. 5mW / cm 2 、10mW / cm 2 、15mW / cm 2 After each layer is printed, it is pre-crosslinked by ultraviolet light exposure for 3 to 15 seconds. After the pre-crosslinking of the last printed layer is completed, it will be irradiated with ultraviolet light again to achieve complete crosslinking.

[0072] The following are specific embodiments:

[0073] Example 1 A method for preparing a cell spheroid mold for 3D bioprinting bionic valve organoids, comprising the following steps:

[0074] (1) Preparation of light-curing resin mold: Using SolidWorks software, first draw a Figure 2 The mold shown has a unique hole design, such as Figure 2As shown in (c). The overall macrostructure of the mold is in the shape of a "hui" character. The inner circle is square with a side length of 16 mm, the hollow depth of the inner circle part is 6 mm, and the bottom thickness is 2.5 mm. The outer circle is square with a side length of 32 mm, the hollow depth of the outer circle part is 9.5 mm, and the bottom thickness is 1 mm. And, a hole structure is designed on the bottom surface of the inner circle part. The top of each hole is a circular opening, which is connected to the hole by a conical structure downward (of course, the conical structure can also adopt other structural designs that smoothly transition from a large-diameter circular opening to a small-diameter hemispherical shape; for example, the cross-section of the transition structure can be other shapes such as curves in addition to straight lines). The diameter of the circular opening is 500 μm, and the distance between the centers of any two circular openings is 750 μm. The conical structure starts from the circular hole above, and gradually shrinks to a circular shape with a diameter of about 180 μm within about 320 μm downward, and is tangent to the hemispherical groove below. The tip of the cone is a hemispherical hole with a diameter of 200 μm. Using the Creality HALOT-MAGE PRO resin 3D printer, print according to the following printing parameters: printing layer thickness 0.03 mm, printing lifting height 8 mm, motor speed 5 mm / s, lamp-off delay 6 s, initial layer exposure time 29 s, initial layer exposure number of layers 4 layers, conventional layer printing exposure time 1.1 s, using ultraviolet light wavelength 405 nm, standard light intensity 8000 μW / cm 2 . After printing, clean it thoroughly with absolute ethanol to obtain a photocured resin mold (as shown in (a) of Figure 3 ).

[0075] (2) Preparation of PDMS mold: Prepare PDMS with a mass ratio of PDMS to curing agent of 10:1. After mixing evenly, first perform centrifugation at 5000 rpm for 5 min to remove the air bubbles therein, and then add it to the above-mentioned photocured resin mold. After multiple vacuum negative pressures in a vacuum drying oven, remove the air bubbles in the PDMS, and then place it in a 4°C refrigerator for 36 h to further exhaust the air bubbles therein. Subsequently, place the photocured resin containing PDMS in an oven at 40°C and keep it at a constant temperature for 120 h until it is completely cured inside. After it is completely cured, remove it to obtain a PDMS mold (as shown in (b) of Figure 3 ).

[0076] (3) Preparation of agarose mold: After sterilizing the PDMS mold, pour a 3 wt% agarose solution (prepared with UP water, 90°C) into the mold. After it cools to room temperature and cures, take it out to obtain the designed agarose cell sphere mold (as shown in (c) of Figure 3 ).

[0077] The corresponding agarose mold has a dot matrix of micro-holes. The top of any hole is a circular opening with a diameter of 500 μm, and gradually narrows to a circle with a diameter of 180 μm within 320 μm, tangent to the hemispherical groove with a diameter of 200 μm at the bottom. The holes are repeatedly arranged in a square array with a spacing of 750 μm between the centers of adjacent holes (a total of 441 holes).

[0078] Example 2 A method for preparing a cell spheroid mold for 3D bioprinting biomimetic valve organoids, comprising the following steps:

[0079] (1) Preparation of light-curing resin mold: Using SolidWorks software, a “U”-shaped mold was first drawn. The inner circle was a square with a side length of 16 mm, a hollow depth of 6 mm, and a bottom thickness of 2.5 mm; the outer circle was a square with a side length of 32 mm, a hollow depth of 9.5 mm, and a bottom thickness of 1 mm; and a hole structure was designed on the bottom surface of the inner circle. The top of each hole was a circular opening, and the bottom was a conical structure with a hole. The holes are connected (of course, the conical structure can also be designed with other structures that smoothly transition from a large-diameter circular opening to a hemispherical small diameter), the diameter of the circular opening is 750μm, and the distance between the centers of any two circular openings is 1000μm; the conical structure starts with the circular hole above, and gradually shrinks to a circle with a diameter of about 270μm within about 560μm downward (that is, tangent to the hemispherical groove below); the tip of the cone is a hemispherical hole with a diameter of 300μm. Use the Chuangxiang 3D HALOT-MAGE PRO light-curing printer and print according to the following printing parameters: printing layer thickness 0.05mm, printing rise height 7mm, motor speed 5.5mm / s, light off delay 7s, initial layer exposure time 25s, initial layer exposure number 3 layers, regular layer printing exposure time 1.5s, ultraviolet light wavelength 405nm, standard light intensity 8000μW / cm 2 .

[0080] (2) Preparation of PDMS mold: PDMS and curing agent are prepared in a mass ratio of 15:1. After mixing evenly, centrifuge at 5000 rpm for 5 minutes to remove bubbles. Then add it to the above-mentioned photocurable resin mold, and perform vacuum negative pressure in a vacuum drying oven for multiple times to remove bubbles in the PDMS. Then place it in a 4°C refrigerator for 36 hours to further remove bubbles. Then, place the photocurable resin containing PDMS in a 40°C oven at a constant temperature for 120 hours until the inside is completely cured. After it is completely cured, remove it and obtain the PDMS mold.

[0081] (3) Preparation of agarose mold: After the PDMS mold is sterilized, 5 wt % agarose solution (prepared with UP water, 90° C.) is poured into the mold. After it is cooled to room temperature and solidified, it is taken out to obtain the designed agarose cell sphere mold.

[0082] The corresponding agarose mold has dot matrix micro-holes, with the top of any hole opening in a circle with a diameter of 750 μm, and gradually narrowing to a circle with a diameter of 270 μm within 450 μm, tangent to the hemispherical groove with a diameter of 300 μm at the bottom. The holes are repeatedly arranged in a square array (a total of 225 holes) with a spacing of 1000 μm between the centers of adjacent holes.

[0083] Example 3 A method for preparing cell spheres for 3D bioprinted biomimetic valve organoids, comprising the following steps:

[0084] (1) After trypsin digestion and counting of the third-generation hiPS-VIC cells (provided by Hubei University) with a growth confluence of 80%, the total number of cells required was calculated at a cell density of 1000 hiPS-VIC cells per well, which in this case was 4.41×10 5 The total amount of cells was dissolved in an appropriate amount of complete culture medium (ie, DMEM high glucose + 5% FBS + 1% P / S; the same below), pipetted evenly and then planted in the agarose mold prepared in Example 1.

[0085] (2) Place the mold in a round culture dish, add an appropriate amount of PBS around the agarose mold, and cover the culture dish to reduce evaporation of the culture medium. Place the culture dish at 37°C and 5% CO 2 Culture in a cell culture incubator for 24 h.

[0086] (3) After the cell spheres are spontaneously formed and compacted, remove the mold, blow it slightly with a 1000 μL pipette, and then transfer it to a centrifuge tube for later use. The agarose mold can be blown with culture medium 2 to 3 times, and the culture medium after blowing is collected. Collect as many cell spheres as possible. The resulting cell spheres have a diameter of 150 μm.

[0087] Example 4 A method for preparing cell spheres for 3D bioprinted biomimetic valve organoids, comprising the following steps:

[0088] (1) After trypsin digestion and counting of third-generation human induced pluripotent stem cell-derived valvular interstitial cells (hiPS-VIC) with a confluence of 80%, the total number of cells required was calculated at a cell density of 2000 hiPS-VIC per well, which in this case was 4.5×10 5 The total amount of cells was dissolved in an appropriate amount of complete culture medium, pipetted evenly, and then planted into the agarose mold prepared in Example 2.

[0089] (2) Place the mold in a round culture dish, add an appropriate amount of PBS around the agarose mold, and cover the culture dish to reduce evaporation of the culture medium. Place the culture dish at 37°C and 5% CO 2 Culture in a cell culture incubator for 24 h.

[0090] (3) After the cell spheres are spontaneously formed and compacted, remove the mold, blow it slightly with a 1000 μL pipette, and then transfer it to a centrifuge tube for later use. The agarose mold can be blown with culture medium 2 to 3 times, and the culture medium after blowing is collected. Collect as many cell spheres as possible. The diameter of the obtained cell spheres is 280 μm.

[0091] Example 5 A method for preparing a printing ink for 3D bioprinting bionic valve organoids, comprising the following steps:

[0092] GelMA was dissolved in PBS at a concentration of 0.1 g / mL. After pasteurization, the sterilized photoinitiator LAP was added to a final concentration of 300 μg / mL. After the solution was mixed evenly, the bubbles were removed by vacuum and the cell spheroids prepared in Example 3 were added at a ratio of 5000 cell spheroids per milliliter.

[0093] Example 6 A method for preparing a printing ink for 3D bioprinting bionic valve organoids, comprising the following steps:

[0094] GelMA was dissolved in PBS at a concentration of 0.05 g / mL. After pasteurization, the sterilized photoinitiator LAP was added to a final concentration of 200 μg / mL. After the solution was mixed evenly, the bubbles were removed by vacuum, and the cell spheroids prepared in Example 4 were added thereto at a ratio of 2000 cell spheroids per milliliter.

[0095] Example 7 A printing scheme for a 3D bioprinted stent of a biomimetic valve, using a SUNP BIOMAKER 4 3D bioprinter, and the printing ink is the printing ink prepared in Example 5. The scheme specifically includes the following contents:

[0096] (1) Printing pattern: The bottom of the cuboid is 20 mm long and 20 mm wide, the printing layer height is 0.21 mm, the printing needle diameter is 25G, and the number of printing layers is 2 (each layer represents one layer printed along the x-axis and y-axis directions on the plane).

[0097] (2) Printing parameters: The nozzle temperature of the print head is set to 20°C, the moving speed is 3 mm / s, and the extrusion speed is 0.25 mm 3 / s, the nozzle retraction amount is 0.2mm, the nozzle lifting amount is 2mm, the wavelength of the UV lamp attached to the nozzle is 405nm, and the light intensity is 5mW / cm 2, the illumination time is 12s.

[0098] (3) Printing structures and UV cross-linking on cell culture well plates.

[0099] Figure 4 This is a real picture of a multi-nozzle 3D printed bio-printed stent for a bionic valve. It can be seen from the picture that the stent has a clear morphology and a complete structure.

[0100] Figure 5 (a) shows the biomimetic valve 3D bioprinted organoids in complete culture medium (DMEM high glucose + 5% FBS + 1% P / S), 37°C, 5% CO 2 After 5 days of culture, the live / dead staining of valve cells, green (Calcein-AM staining) is live cells, and red (PI staining) is dead cells. It can be seen from the figure that most cells survive, and the present invention effectively improves the survival rate of valve cells in the 3D bioprinting process.

[0101] Figure 5 (b) Figure 5 (c) shows the biomimetic valve 3D bioprinted organoids in complete culture medium (DMEM high glucose + 5% FBS + 1% P / S), 37°C, 5% CO 2 After 5 days of culture, the valvular cells secreted type 1 collagen (Col-Ⅰ) and type 3 collagen (Col-Ⅲ) respectively. Figure 5 In (b), green (FITC staining) represents Col-Ⅰ, and blue (DAPI staining) represents cell nuclei; Figure 5 In (c), green (Cy3 staining) is Col-Ⅲ, and blue (DAPI staining) is the cell nucleus. It can be seen from the figure that the extracellular matrix secretion of valve cells is vigorous. The present invention effectively enhances the extracellular matrix remodeling ability of cells by increasing the local cell density.

[0102] Example 8 A printing scheme for a 3D bioprinted stent of a biomimetic valve, using a SUNP BIOMAKER 4 3D bioprinter, the printing ink is the printing ink prepared in Example 6, and the scheme specifically includes the following contents:

[0103] (1) Printing pattern: The bottom of the cuboid is 14 mm long and 14 mm wide, the printing layer height is 0.34 mm, the printing needle diameter is 22G, and the number of printing layers is 4 (each layer represents one layer printed along the x-axis and y-axis directions on the plane).

[0104] (2) Printing parameters: The nozzle temperature of the print head is set to 24°C, the moving speed is 6 mm / s, and the extrusion speed is 0.55 mm 3 / s, the nozzle retraction amount is 0.35mm, the nozzle lifting amount is 1mm, the nozzle is equipped with a UV lamp with a wavelength of 405nm and a light intensity of 15mW / cm 2 , the illumination time is 3s.

[0105] (3) Printing structures and UV cross-linking on cell culture well plates.

[0106] Example 9

[0107] This example uses the same cell sphere preparation process as Example 3, except that the cells used are third-generation porcine aortic valve cells (PAVIC). The process for obtaining the cells is as follows:

[0108] (1) Immediately after killing the pigs at the slaughterhouse, the aortic valves were taken and rinsed three times in 10 μL / mL penicillin / streptomycin (PBS solution). Afterwards, the valves were stored in antibiotic-free, serum-free DMEM high-glucose medium, ensuring that the entire process did not exceed 20 minutes.

[0109] (2) After arriving at the laboratory, the valve was immediately washed again three times with PBS containing 10 μL / mL penicillin / streptomycin in a clean bench, and the valve surface was gently wiped with a sterile cotton swab to remove the endothelial cells on the surface. The valve was cut into 1×1 mm 2 small pieces of tissue.

[0110] (3) Place the small piece of valve tissue obtained in step (2) into type I collagenase (2 mg / mL) that has been incubated at 37°C for 30 min, add 50% volume of DMEM high-glucose culture medium, and digest for 6 to 8 hours. When 60% of the small piece of tissue is digested into flocs and the collagenase solution becomes turbid, terminate the digestion immediately.

[0111] (4) The digested suspension was centrifuged at 1000 rpm for 3 min, and then the cells were resuspended in DMEM high-glucose medium containing 5% FBS and 10 uL / ml penicillin / streptomycin, and continued to be cultured in a culture dish.

[0112] (5) Cell culture conditions were 37°C and 5% CO 2 The medium was changed every 2 days and the cells were passaged when the cell density reached 80%.

[0113] (6) The PAVIC at the third generation was digested with trypsin and counted for later use.

[0114] PAVIC was used to prepare cell spheres according to the procedures of Examples 3, 5, and 7, and finally 3D bioprinting was completed. In complete medium (DMEM high glucose + 5% FBS + 1% P / S), 37°C, 5% CO 2 After 5 days of culture, Col-Ⅰ staining was performed to characterize extracellular matrix remodeling.

[0115] The results are as follows Figure 6 As shown, green (FITC staining) is Col-Ⅰ, and blue (DAPI staining) is the cell nucleus. It is not difficult to see that the printed PAVIC cell sphere has a certain amount of extracellular matrix secretion, showing a relatively good matrix remodeling process. Of course, the remodeling effect is not as good as that of Example 7 ( Figure 5 In addition, during the preparation of cell spheres, it was found that the total amount of PAVIC cells was less than that of hiPS-VIC cells in the same size culture dish after the same culture time, and its cell proliferation and expansion rate was lower than that of PAVIC.

[0116] Comparative Example 1

[0117] The printing parameters of this comparative example are consistent with those of Example 7. The difference is that the cell components during printing exist in the form of single cells. hiPS-VIC cells are used. Under the premise of ensuring printability, the cell density is increased to 1.5×10 6 Each milliliter of printing ink. Also in complete medium (DMEM high glucose + 5% FBS + 1% P / S), 37°C, 5% CO 2 After 5 days of culture, live / dead staining was performed to characterize cell survival, and Col-Ⅰ staining was performed to characterize extracellular matrix remodeling.

[0118] Figure 7 (a) is the comparative example after printing in complete medium (DMEM high glucose + 5% FBS + 1% P / S), 37°C, 5% CO 2 After 5 days of culture, the live / dead staining of valve cells. In the figure, green (Calcein-AM staining) represents live cells, and red (PI staining) represents dead cells. It is not difficult to see from the figure that although the cell survival rate is not low, most of the cells are oval in shape and the spreading condition is poor, which indicates that the cell vitality is poor and has been damaged to a certain extent during the printing process.

[0119] Figure 7 (b) is the comparative example after printing in complete medium (DMEM high glucose + 5% FBS + 1% P / S), 37°C, 5% CO 2 After 5 days of culture, the valvular cells secreted type I collagen (Col-Ⅰ) fluorescent staining, where green (FITC staining) is Col-Ⅰ, and blue (DAPI staining) is the cell nucleus. It is not difficult to see that the printed hiPS-VIC cannot quickly secrete extracellular matrix components. Only near the edge of the printed hydrogel (i.e., the part closer to the culture medium) will there be a more obvious matrix remodeling, and the remodeling effect is significantly inferior to that of Example 7 ( Figure 5 ) in the matrix remodeling.

[0120] The above embodiments are only examples. For example, the culture medium used for culturing cells is not limited to the ratio of DMEM high glucose + 5% FBS + 1% P / S. M199, F-12, DMEM / F-12 and other commonly used culture media can also be used. The proportion of FBS can also be changed as needed, and specific growth factors can also be added thereto. For another example, in addition to GelMA, the photocurable biomaterial can also be other photocurable biomaterials.

[0121] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A 3D printing ink for forming biomimetic valve organoids, characterized in that: The bio-ink is obtained by dispersing a photocurable biomaterial in phosphate buffered saline (PBS), then adding a photoinitiator solution, mixing well, removing bubbles, and then adding cell spheroids; The cell spheres are obtained by culturing valvular interstitial cells (VIC) in an agarose mold in the presence of a cell culture medium, thereby obtaining valvular interstitial cell spheres with a diameter of micrometers, each of which is enriched with valvular interstitial cells; the agarose mold has micrometer-level pores.

2. The 3D printing ink according to claim 1, characterized in that: The diameter of the cell sphere is 130-450 μm; accordingly, each cell sphere contains 500-8000 valve interstitial cells; Preferably, the valvular interstitial cells are valvular interstitial cells derived from human induced pluripotent stem cells (hiPS-VIC).

3. The 3D printing ink according to claim 1, characterized in that: The agarose mold has a dot matrix of micro holes, any one of which has a circular opening with a diameter of 350 to 1000 μm and smoothly transitions to a hemispherical concave surface at the bottom within 250 to 500 μm, and the diameter of the hemispherical surface is 150 to 500 μm; On the plane where the micro-holes of the agarose mold dot matrix are located, the distance between the projections of the centers of two adjacent holes is 500-1250 μm; The plane where the micro-holes of the agarose mold dot matrix are located is 5 to 8 mm concave compared to the surrounding area of ​​the agarose mold; When forming cell spheres using an agarose mold, the outside of the agarose mold is placed in a phosphate buffered saline (PBS), and the height difference between the periphery of the agarose mold and the plane where the agarose mold lattice micropores are located can prevent the phosphate buffered saline (PBS) from entering the lattice micropores.

4. The 3D printing ink according to claim 1, characterized in that: The amount of cell spheres added is sufficient to ensure that 2000 to 5000 cell spheres are contained in each milliliter of 3D printing ink.

5. The 3D printing ink according to claim 1, characterized in that: The photoinitiator in the photoinitiator solution is one or more of α-ketoglutaric acid (α-KGA), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP); preferably phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP).

6. The 3D printing ink according to claim 1, characterized in that: The photocurable biomaterial is methacrylated gelatin (GelMA); Preferably, the methacryloyl gelatin (GelMA) is prepared by placing gelatin (Gelatin) in deionized water or PBS, stirring at a temperature of 40°C to 70°C to completely dissolve it, then adding methacrylic anhydride or glycidyl methacrylate, continuing to stir the reaction at a temperature of 40°C to 70°C, and then dialyzing the reaction product with a dialysis bag and freeze-drying it; More preferably, the stirring speed is 400-800 rpm; the amount of methacrylic anhydride or glycidyl methacrylate is 0.4-1.2 mL per gram of gelatin; the reaction time after adding methacrylic anhydride or glycidyl methacrylate is 1-24 h; the molecular cutoff of the dialysis bag is 8000-14000 Da, and the dialysis time is 2-14 days.

7. A bionic valve organoid obtained by 3D bioprinting, characterized in that: The method is to load the 3D printing ink as described in any one of claims 1 to 6 into the nozzle of a 3D bioprinter, and then 3D print the bioprinted product according to a preset shape.

8. The bionic valve organoid according to claim 7, characterized in that: The printing parameters used in the 3D printing are as follows: The nozzle uses a 22-27G caliber printing needle, the nozzle temperature is 16℃~24℃, the moving speed is 3~7mm / s, and the extrusion speed is 0.25~0.7mm 3 / s, the nozzle's idle retraction volume is 0.2-0.6mm, and the lifting volume is 1-2mm; The number of printing layers is 1 to 3; UV irradiation is performed once for each printed layer; the UV intensity used is 5-15mW / cm 2 , each irradiation time is 3 to 15 seconds; and, after the last printing layer is pre-crosslinked by ultraviolet light irradiation, ultraviolet light irradiation is performed again to completely crosslink; preferably, the ultraviolet wavelength used for ultraviolet light irradiation is 405nm.

9. The bionic valve organoid according to claim 7, characterized in that: The preset shape is a shape corresponding to the cross-net structure.

10. Use of the 3D printing ink for forming biomimetic valve organoids as claimed in any one of claims 1 to 6 in the preparation of valve tissue and / or valve organoids, or use of the biomimetic valve organoids as claimed in any one of claims 7 to 9 in the preparation of valve tissue.

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