Artificial heart valve, artificial heart valve tissue calcification model as well as preparation method and application of artificial heart valve tissue calcification model

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses an artificial valve tissue calcification model and a preparation method and application thereof. The preparation method comprises the following steps: providing human valvular interstitial cells; the method comprises the following steps: providing a hydrogel system, wherein the hydrogel system comprises type I collagen, fibrinogen and Matrigel matrigel; constructing an engineered heart valve tissue strip by taking the hydrogel system as a skeleton structure between valve interstitial cells; and culturing the engineered heart valve tissue strip by using a calcification induction culture solution, so that the valve tissue generates a calcification phenotype. The model provided by the invention can generate a valve calcification phenotype which is similar to that after human heart valve calcification and is represented by valve interstitial cell activation, extracellular matrix remodeling such as collagen mass secretion and calcium deposition region generation. The model provided by the invention is low in cost, can be produced in batches, meets the research on the pathogenesis of extracorporeal valve calcification, and makes up for the dilemma that no artificial valve calcification model is available for drug research and development at present.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an artificial heart valve, an artificial heart valve tissue calcification model, and a preparation method and application thereof. Background Art

[0002] At present, the research on valve calcification mainly relies on cell models and animal models. Source of cell models: mainly rely on primary valvular endothelial cells and valvular interstitial cells. The valvular cells available in clinical practice are generally from patients with valvular stenosis. A small number of normal human valves come from valves removed after heart transplantation. This type of sample is difficult to obtain. Human valvular interstitial cells have low proliferation ability, instability between different sampling batches, and easy activation during cell culture. Planar cultured valvular interstitial cells lack complete valve structure and cell type interaction, resulting in few experimental parameters, few detectable indicators, and lack of tissue function in planar valve cell models, which restricts the research on valve-related diseases. Source of animal models: usually mice, rabbits and pigs. Although animal models have the advantage of studying valvular calcification under natural hemodynamic conditions, conventional model animals such as mice and rats do not have a three-layer valve structure similar to that of the human body, and large animal models generally have disadvantages such as long cycles, high modeling costs, and species differences.

[0003] Therefore, developing a method that can stably and efficiently obtain and reproduce the pathological remodeling process and dysfunctional phenotype after heart valve calcification is the key to breaking through the bottleneck of clinical application of in vitro calcified valve models based on human cells. Summary of the invention

[0004] The purpose of the present invention is to address the above-mentioned deficiencies in the existing art, and to provide an artificial heart valve, an artificial heart valve tissue calcification model, and a preparation method and application thereof. A new artificial valve muscle tissue (Engineering Valve Tissue) calcification model is developed based on two cell sources, human primary valve interstitial cells and human pluripotent stem cell-derived valve interstitial cells, and natural extracellular matrix type I collagen. The purpose is to solve the problem of heart valve calcification drug development caused by the lack of human heart tissue calcification model and to serve as a research model for the mechanism of human heart valve calcification process.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A first aspect of the present invention provides a method for preparing an artificial heart valve, comprising the following steps:

[0007] S1. Provide human valvular interstitial cells;

[0008] S2. Providing a hydrogel system, wherein the hydrogel system comprises valve hydrogel culture solution A and valve hydrogel culture solution B, wherein the valve hydrogel culture solution A is used for cell resuspending, and the valve hydrogel culture solution B comprises type I collagen, fibrinogen and Matrigel matrix glue;

[0009] S3. Resuspend the human valvular interstitial cells with the valve hydrogel culture solution A and add the valve hydrogel culture solution B to form a valve cell hydrogel solution. Transfer the valve cell hydrogel solution to an engineered valve microtissue molding mold, culture in an incubator, and solidify to obtain an engineered heart valve tissue strip, i.e., an artificial heart valve.

[0010] Furthermore, the human valvular interstitial cells are obtained by any one of the methods (a), (b) and (c), specifically:

[0011] (a) digesting human valve tissue overnight with type I collagenase to obtain human primary valve interstitial cells and then amplifying and culturing them in vitro to obtain valve interstitial cells;

[0012] (b) when human induced pluripotent stem cells are differentiated to the cardiac progenitor cell stage, a small molecule compound that can directly or indirectly activate the WNT-BMP signaling pathway is added to the culture medium to direct the differentiation of the stem cells into valvular interstitial cells;

[0013] (c) When human embryonic stem cells are differentiated into cardiac progenitor cells, substances that can directly or indirectly activate the WNT-BMP signaling pathway are added to the culture medium to direct the differentiation of the stem cells into valvular interstitial cells.

[0014] Furthermore, the preparation process of the valve hydrogel culture solution A is as follows: Low Glucose DMEM, 10% FBS, 1% PS, final concentration 2μg / mL vitamin B12, and final concentration 1mg / mL aminoacetic acid are mixed to obtain A1 solution, and then 48μL A1 solution is mixed with 2.4μL 50U / mL thrombin to obtain; the preparation process of the valve hydrogel culture solution B is as follows: 20μL 7.5mg / ml Collagen I, 0.42μl 1M NaOH, 20μL 10mg / ml Fibrinogen, and 10μL Matrigel are mixed.

[0015] Furthermore, the tensile modulus of the artificial heart valve is 38.2-78.68 mN / mm, and the elongation is 24.45-52.75%.

[0016] The second aspect of the present invention is to provide an artificial heart valve prepared by the above-mentioned preparation method.

[0017] The third aspect of the present invention is to provide the use of the artificial heart valve prepared as described above in preparing a heart valve disease model.

[0018] The fourth aspect of the present invention is to provide a method for preparing an artificial heart valve tissue calcification model, wherein the artificial heart valve is cultured in a calcification induction culture medium so that the valve tissue produces a calcification phenotype, thereby obtaining an artificial heart valve tissue calcification model.

[0019] Furthermore, the calcification inducing culture medium contains dexamethasone, β-glycerophosphate, ascorbic acid and calcium chloride.

[0020] The fifth aspect of the present invention is to provide an artificial heart valve tissue calcification model prepared by the above-mentioned preparation method.

[0021] The sixth aspect of the present invention is to provide the application of the above-mentioned artificial heart valve tissue calcification model in the screening or detection of heart valve tissue calcification drugs.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention provides a method for preparing an artificial heart valve tissue calcification model, which is a new artificial valve muscle tissue (Engineering Valve Tissue) calcification model developed based on two cell sources, human primary valve interstitial cells and human pluripotent stem cell-derived valve interstitial cells and natural extracellular matrix type I collagen. On the one hand, this in vitro model reproduces the structural and functional characteristics of adult heart valves. On the other hand, after induction of calcification drugs, the engineered valve tissue can show a phenotype of valve interstitial cell damage-calcification similar to that in animals, and has good potential for constructing a new myocardial-valve joint tissue model and drug screening. At the same time, the two stress stimulation methods developed can mechanically stimulate the engineered valve tissue at the in vitro tissue level, and can add mechanical and biomechanical stimulation in addition to biochemical factors to achieve the extended application of the model and bionic functionality.

[0024] (2) The hiPSC-VIC differentiation system developed in the present invention can construct a human heart valve tissue calcification model at low cost and in batches, thereby solving the problem of the lack of suitable valve organoids in the current aortic valve calcification research, and further making up for the dilemma that the existing drug screening system does not have a human heart valve calcification model for related drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a flow cytometric analysis of the differentiation efficiency of induced pluripotent stem cells (iPSCs) into cardiac progenitor cells (CPCs). ISL1 is a marker for CPCs, and the differentiation efficiency is 82.1%;

[0026] Figure 2 This is a flow cytometric test efficiency diagram of cardiac progenitor cells (CPC) differentiation into valvular endothelial cells (VEC), CD144 is a marker of VEC, and the differentiation efficiency is 55.1%;

[0027] Figure 3 This is a flow cytometric detection efficiency diagram of the differentiation of valvular endothelial cells (VEC) into valvular interstitial cells (VIC). VIM is a marker of VEC, and the differentiation efficiency is 70.6%;

[0028] Figure 4 This is a flow cytometric analysis of the differentiation efficiency of induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMs). cTNT is a marker of CMs, and the differentiation efficiency is 68.9%;

[0029] Figure 5a Light microscopy images of long-term culture of hydrogel systems with different concentrations;

[0030] Figure 5b It is a statistical diagram of tissue width size of hydrogel systems with different concentrations;

[0031] Figure 6 Immunofluorescence images of dead and live staining of 0Collagen I, 0.5Collagen I, 1.5Collagen I and 3.0Collagen I;

[0032] Figure 7 The statistical graphs of live and dead staining of 0 Collagen I, 0.5 Collagen I, 1.5 Collagen I and 3.0 Collagen I;

[0033] Figure 8 This is a HE staining image of a human aortic valve section;

[0034] Fig. 9 Schematic diagram of the process of making artificial heart valve tissue: put a paper frame into the customized PDMS mold, add the hydrogel-cell mixture, wait for the hydrogel to form, take out the paper frame and the hydrogel together and put them into the culture medium for cultivation;

[0035] Fig.10 To carry out long-term culture and light microscopy recording of engineered valve tissue (21 days);

[0036] Fig.11 Physical pictures, cytoskeleton staining (F-actin) and HE staining of normal valves, primary source engineered valve tissues and differentiated source engineered valve tissues;

[0037] Fig.12The distribution ratio of the nucleus orientation angle of HE-stained normal valves, primary engineered valve tissues and differentiated engineered valve tissues;

[0038] Fig.13 Schematic diagram of the construction of engineered valve tissue strips, in which a is a physical image of the engineered valve tissue strip after a period of culture, b is an optical microscope image of the engineered valve tissue strip, and c is a full-length image of the engineered valve tissue strip;

[0039] Fig.14 Figure 3: MOVAT and Alizarin red staining of tissue sections of human valve tissue, artificial valve tissue (human primary interstitial cells) and artificial valve tissue (human differentiated interstitial cells) after calcification induction;

[0040] Fig.15 Statistical graph showing the percentage of collagen-producing areas in human valve tissue, artificial valve tissue (human primary interstitial cells) and artificial valve tissue (human differentiated interstitial cells) after calcification induction;

[0041] Fig.16 It is a statistical graph showing the percentage of calcium deposition areas in human valve tissue, artificial valve tissue (human primary interstitial cells) and artificial valve tissue (human differentiated interstitial cells) after calcification induction;

[0042] Fig.17 A physical picture of the in vitro mechanical stretching device and a picture of the culture of artificial valve tissue in the chamber of the mechanical stretching device. DETAILED DESCRIPTION

[0043] In order to explain the technical solution and beneficial effects of the present invention more clearly, the present invention is described in detail below in conjunction with the accompanying drawings and embodiments. It should be clear that the reference drawings described are only some embodiments of the present invention and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0044] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, with reference to "Molecular Cloning Experiment Guide (4th Edition)" (Chinese version), published by Science Press; "Cell Culture (2nd Edition)", Xi'an World Book Publishing Company, and "Stem Cell Technology" (Chinese version), Bruce M. Carlson, USA, published by Science Press. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0045] The primary cell line information used in the present invention is: Human iPSC line WTC (Coriell Institute, GM25256).

[0046] The present invention is a new artificial valve muscle tissue (Engineering Valve Tissue) calcification model developed based on two cell sources, human primary valve interstitial cells and human pluripotent stem cell-derived valve interstitial cells, and natural extracellular matrix type I collagen. On the one hand, this in vitro model reproduces the structural and functional characteristics of adult heart valves. On the other hand, after induction of calcification drugs, the engineered valve tissue can show a phenotype of valve interstitial cell damage-calcification similar to that in animals, and has good potential for constructing a new myocardial-valve joint tissue model and drug screening. At the same time, it is also proposed to promote calcification of engineered heart valve tissue stress stimulation at the in vitro tissue level, providing a research model that can serve as a mechanism for the calcification process of human heart valves.

[0047] The information of the reagents used in the specific embodiment of the present invention is shown in Table 1.

[0048] Table 1. Reagent information

[0049]

[0050] Example 1

[0051] This embodiment provides a method for preparing an artificial valve. The specific steps are as follows:

[0052] (1) Providing human valvular interstitial cells,

[0053] Method 1: Human valvular interstitial cells digested from human aortic valves were cultured and expanded (provided by Wuhan Union Hospital). The specific process was to wash the non-calcified aortic valve leaflets with PBS and cut them into 1 mm 3 The small pieces were digested with 1mg / ml type I collagenase at 37°C for 20min. The valve endothelial cells (VEC) on the valve surface were gently scraped with a throat swab, and the remaining valves were digested for 16 hours (1 / 3 high glucose and 1% PS were added to the collagenase). The isolated hVICs were resuspended and cultured in high glucose DMEM supplemented with 10% FBS, 200mg / mL AC-mg and 1% PS. The hVICs used in the in vitro experiments were all 2-6 generations.

[0054] Method 2: Human pluripotent stem cells or human embryonic stem cells are induced to differentiate into valvular interstitial cells by regulating the WNT-BMP signaling pathway in stages through small molecules. The specific process is to inoculate human pluripotent stem cells into a 6-well plate coated with Matrigel (Corning) at a density of 3.0×10 6 When the iPSC confluence reached 80%-90%, versene was used for passage at 2.5×10 4 / cm 2The cells were seeded at a density of 1.50 μg / ml in a culture dish coated with matrigel. The cells were cultured in mTeSR for 2-3 days. When the confluence reached 60%-70%, differentiation was initiated. The RPMI1640 medium supplemented with 2% B27(-INS) and 200μg / ml AC-mg was replaced with 10μM CHIR and 10ng / ml BMP4. After 24h, the medium was replaced with RPMI1640 medium supplemented with 2% B27(-INS), 200μg / ml AC-mg, 20ng / ml bFGF and 50ng / ml BMP4. The medium was cultured for 48h, and the medium was changed every 24h. At this time, the cells were differentiated to the cardiac progenitor cell stage. The cells were digested with TrypLE and cultured at 3×10 4 / cm 2 The cells were seeded at a density of 100 μg / ml in a matrigel-coated culture dish. The cells were cultured in RPMI1640 medium supplemented with 2% B27(-INS), 200 μg / ml AC-mg, 100 ng / ml VEGF, 10 ng / ml BMP4, and 10 ng / ml TGFβ1 for 6 days, with the medium changed every 48 hours. At this time, the cells differentiated into the valvular endothelial cell stage. The cells were digested with TrypLE and cultured at 3×10 4 / cm 2 The cells were seeded at a density of 100 μg / ml in a culture dish coated with matrigel, cultured in a high-glucose DMEM medium supplemented with 5% fetal bovine serum and 200 μg / ml AC-mg for 5 days, and the medium was replaced every two days to finally obtain valvular interstitial cells.

[0055] like Figure 1-Figure 3 As shown, the flow cytometric efficiency diagram of induced pluripotent stem cells (iPSCs) differentiating into cardiac progenitor cells (CPC), ISL1 is a marker of CPC, and the differentiation efficiency is 82.1%; the flow cytometric efficiency diagram of cardiac progenitor cells (CPC) differentiating into valvular endothelial cells (VEC), CD144 is a marker of VEC, and the differentiation efficiency is 55.1%; the flow cytometric efficiency diagram of valvular endothelial cells (VEC) differentiating into valvular interstitial cells (VIC), VIM is a marker of VEC, and the differentiation efficiency is 70.6%.

[0056] (2) Constructing a hydrogel system,

[0057] The hydrogel system provided by the present invention comprises valve hydrogel culture A solution and valve hydrogel culture B solution.

[0058] The preparation process of valve hydrogel culture solution A is as follows: Low Glucose DMEM, 10% FBS, 1% PS, final concentration 2μg / mL vitamin B12, and final concentration 1mg / mL aminoacetic acid are mixed to obtain solution A1, and then 48μL of solution A1 is mixed with 2.4μL thrombin (50U / mL) to obtain hydrogel culture solution A.

[0059] The preparation process of valve hydrogel culture solution B is as follows: 20 μL Collagen I (7.5 mg / ml), 0.42 ul NaOH (1 M), 20 μL Fibrinogen, and 10 μL Matrigel are mixed to form hydrogel culture solution B.

[0060] In the development process of the hydrogel system, pure gel group, pure type I collagen (0.5mg / ml, 1.5mg / ml and 3.0mg / ml), fibrinogen hydrogel (Fibrinogen, Matrigel and Thrombin) and type I collagen (1.5mg / ml and 3mg / ml) mixed with fibrinogen hydrogel were set up. The specific operation is as follows: First, 6.66μl, 20μl and 40μl of high concentration Collagen I (7.5mg / ml) were mixed with 0.14μl, 0.42μl and 0.84μl of 1M NaOH respectively, and the pH of the Collagen I solution was adjusted to neutral, about 7.4. At the same time, the entire process of taking and mixing was operated on ice to ensure that the Collagen I solution would not solidify. Then, the evenly mixed neutral collagen I solution was mixed with 93.34 μl of the valve interstitial cell suspension, and the mixture was repeatedly blown to mix well, and then added to the strip valve microtissue mold.

[0061] like Figure 5a and 5b Long-term culture light microscopy images of hydrogel systems with different concentrations are shown, and Figure 6 and Figure 7The tissue width statistics of the hydrogel systems with different concentrations shown in the figure show that, due to the relatively high gel strength of pure type I collagen, it began to break and shrink on the second day of culture, among which the pure type I collagen group (0.5mg / ml, 1.5mg / ml and 3.0mg / ml) broke completely on the tenth day, and only the microtissues cultured in the type I collagen (1.5mg / ml and 3mg / ml) mixed fibrinogen hydrogel had normal morphology. The cell survival and cell elongation of the gel system were subsequently detected. Interstitial cells were able to grow normally in these four gels with different concentrations, with a tissue width of about 300μm and a cell survival rate of about 99%. Compared with the high concentration type I collagen (3mg / ml) mixed fibrinogen hydrogel, the low concentration type I collagen (1.5mg / ml) mixed fibrinogen hydrogel had a low shrinkage degree. Among them, the microtissue cell survival ratio constructed by the Fibrinogen, Matrigel and 1.5mg / ml Collagen I hydrogel systems was the highest, and the subsequent microtissues constructed all adopted this gel system.

[0062] (3) Preparation of engineered heart valve tissue strips

[0063] like Figure 8 The figure shows HE staining of a human aortic valve section. HE staining shows that the nuclei of the valve interstitial cells in the fibrous layer present a uniform orientation (nuclear deflection <15°), indicating that the interstitial cells in the valve have the characteristics of oriented arrangement. Therefore, we constructed a strip-shaped engineered valve tissue, and the tensile modulus of the artificial heart valve was 38.2-78.68mN / mm, and the elongation was 24.45-52.75%.

[0064] like Fig. 9 The figure shows a schematic diagram of the process of making artificial heart valve tissue. A paper frame is placed in a customized PDMS mold, and a hydrogel cell mixture is added. After the hydrogel is formed, the paper frame and the hydrogel are taken out and placed in a culture medium for culture. Specifically, the human valve interstitial cells are resuspended in valve hydrogel culture solution A and valve hydrogel culture solution B containing CollagenI, Fibrinogen and Matrigel is added to form a valve cell hydrogel solution. The valve cell hydrogel solution is transferred to the engineered valve microtissue molding mold, and the engineered valve tissue strips are obtained after culture and solidification in an incubator. Fig. 9 As shown, the figure is a flow chart for constructing engineered valve tissue strips in a PDMS mold.

[0065] like Fig.10As shown in the figure, the engineered valve tissue was cultured for a long time and recorded under light microscopy (21 days). The engineered valve tissue can still maintain complete tissue morphology. It was found that the constructed strip-shaped valve organoids also had obvious valve interstitial cell orientation characteristics after 3 weeks of culture. Next, we sectioned and stained the constructed valve microtissues from the two cell sources, as shown in the figure. Fig.11 and Fig.12 As shown in the figure, the valve organoids can be stably maintained and the arrangement and orientation of the cells are basically consistent, which is consistent with the cell orientation characteristics in the fibrous layer of the valve tissue. At the same time, the cytoskeleton (F-actin) of the valve microtissue was stained and the nuclear orientation was counted. The cells were arranged tightly, the cytoskeleton was arranged neatly, and the arrangement direction was consistent with the direction of the tissue, indicating that the valve microtissue we constructed has similar structural and cell types to the valves in the human body in terms of organizational structure.

[0066] (4) Cultivation of engineered valves

[0067] The constructed engineered valve tissue strips are placed in a 37°C incubator for incubation and then taken out and placed in valve tissue culture medium for culture. The specific components of the valve tissue culture medium are: High Glucose DMEM, 10% FBS, 1% PS, final concentration of 50μg / mL ascorbic acid, final concentration of 2mg / mL aminoacetic acid (sigma), final concentration of 0.45μM 1-thioglycerol (Gibco), 100X Non-Essential Amino Acid (Gibco), 100X Sodium Pyruvate (Gibco). On the first day, a final concentration of 10μM ROCK inhibitor Y27632 needs to be added, and after 24 hours, the normal tissue culture medium is replaced to remove Y27632. The subsequent culture uses normal myocardial-valve tissue culture medium, and the culture medium is changed every three days. Fig.13 As shown in , b is a light microscopic image of the culture of the engineered valve tissue strip, in which the tissue undergoes remodeling and becomes compact, and c is a microscopic fluorescence image of the engineered valve tissue strip, in which the cells are closely arranged and grow along the direction of the tissue strip.

[0068] Example 2

[0069] This embodiment provides a preparation of an artificial valve tissue calcification model. The specific steps are:

[0070] The artificial valve prepared in Example 1 was subjected to calcification treatment.

[0071] After the engineered valve tissue was built, it was placed in a shaker for dynamic culture and cultured in a calcification induction medium. The calcification induction medium was composed of: High Glucose DMEM, 5% FBS, 1% PS, 50 μg / mL ascorbic acid at a final concentration, 2 mg / mL aminoacetic acid (Sigma), 0.45 μM 1-thioglycerol (Gibco), 100X Non-Essential Amino Acid (Gibco), 100X Sodium Pyruvate (Gibco), 100nM Dexamethasone, 10mM β-Glycerophosphate disodium salt hydrate, and 1.0mM CaCl 2 ; The calcification induction program is to change the calcification induction medium every three days for 14 days; the engineered valve tissue shows the phenotype of valve calcification, which is manifested as disordered cell orientation, increased secretion of matrix components such as type I collagen in the tissue, tissue remodeling, and the formation of calcified nodules, which can be identified by Alizarin red staining. Fig.14 As shown in FIG, MOVAT and Alizarin red staining images of tissue sections of human valve tissue, artificial valve tissue (human primary interstitial cells) and artificial valve tissue (human differentiated interstitial cells) after calcification induction; after statistics, as shown in FIG. Fig.15 and Fig.16 As shown, Fig.15 Statistical graph showing the percentage of collagen-producing areas in human valve tissue, artificial valve tissue (human primary interstitial cells) and artificial valve tissue (human differentiated interstitial cells) after calcification induction; Fig.16 Statistical graph showing the percentage of calcium deposition areas in human valve tissue, artificial valve tissue (human primary interstitial cells) and artificial valve tissue (human differentiated interstitial cells) after calcification induction; it is obvious that the artificial valve tissue (human primary interstitial cells) and artificial valve tissue (human differentiated interstitial cells) after calcification induction produce pathological characteristics similar to those of human calcified valve tissue, specifically matrix remodeling and increased collagen secretion in the calcification group; the calcification group produced obvious calcium deposition compared with the normal group, and the Alizarin Red attachment area was much larger than that of the normal group.

[0072] Example 3

[0073] This embodiment provides a method for simulating the mechanical stimulation of blood flow on the valve by in vitro mechanical stress.

[0074] After the engineered valve tissue is built, stress stimulation is used to promote calcification of the engineered heart valve tissue. The stress stimulation can be mechanical stretching stress stimulation and biomechanical stimulation. In this embodiment, mechanical stimulation is applied to the engineered valve tissue by an in vitro mechanical stretching device. Fig.17As shown, the design and assembly of the in vitro mechanical stretching device, the design and assembly of the stretching culture chamber, and the setting of the stretching parameters are as follows:

[0075] First, a silicone chamber was made by casting for subsequent stretching culture. The silicone chamber is made by pouring silicone (A and B components in a ratio of 1:1) into a mold (3D printing, material is PLA) and curing it in an oven at 65°C for 2 hours. The resulting silicone chamber has six chambers (size 17mm×21mm×15mm) and a thickness of 2.5mm. The chambers are used to culture and stretch tissues. The control cyclic motion system consists of a 42-through stepper motor (42HA48-T8, HANPOSE), a visual controller (DC5-24V-0.6W, Mingtai Intelligent), a stepper motor driver (MT542-AL, Mingtai Intelligent), a guide rail (BSGI-20N guide rail L800MM) and a guide rail adapter (CNC processing). By fixing the stepper motor lead screw on the guide rail adapter, the slide rail is pushed to complete the cyclic motion. The visual controller can adjust the operating frequency, speed, stroke and other parameters of the stepper motor, making the stretching operation easier. The silicone chamber fixings are all CNC machined and made of metal aluminum. Their function is to fix one end of the silicone chamber and the other end on the guide rail, so that the silicone chamber can be stretched synchronously when the stepper motor pushes the guide rail.

[0076] Place the human engineered valve prepared in Example 1 into the silicone chamber of an in vitro mechanical stretching device and fix it with a specimen needle, add 2 ml of valve tissue culture medium so that the liquid covers the fixed engineered valve microtissue, turn on the control panel of the mechanical stretching device, set the set parameters, and the length of the engineered valve microtissue is stretched with the movement of the motor, which can simulate the mechanical stimulation of blood flow on the valve.

[0077] In summary, the artificial valve tissue calcification model prepared by the preparation method provided by the present invention can simulate the process of fibrosis and calcification of heart valve tissue. Furthermore, the application method includes culturing the obtained artificial valve tissue model in a culture medium containing a drug to be tested, and judging the drug effect by observing the degree of calcification, which can be used as an in vitro model of valve tissue for drug screening of heart valves.

[0078] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing an artificial heart valve, characterized in that: The following steps are involved: S1. Provide human valvular interstitial cells; S2. Providing a hydrogel system, wherein the hydrogel system comprises valve hydrogel culture solution A and valve hydrogel culture solution B, wherein the valve hydrogel culture solution A is used for cell resuspending, and the valve hydrogel culture solution B comprises type I collagen, fibrinogen and Matrigel matrix glue; S3. Resuspend the human valvular interstitial cells with the valve hydrogel culture solution A and add the valve hydrogel culture solution B to form a valve cell hydrogel solution. Transfer the valve cell hydrogel solution to an engineered valve microtissue molding mold, culture in an incubator, and solidify to obtain an engineered heart valve tissue strip, i.e., an artificial heart valve.

2. The preparation method according to claim 1, characterized in that The human valvular interstitial cells are obtained by any one of the methods (a), (b) and (c), specifically: (a) digesting human valve tissue overnight with type I collagenase to obtain human primary valve interstitial cells and then amplifying and culturing them in vitro to obtain valve interstitial cells; (b) when human induced pluripotent stem cells are differentiated to the cardiac progenitor cell stage, a small molecule compound that can directly or indirectly activate the WNT-BMP signaling pathway is added to the culture medium to direct the differentiation of the stem cells into valvular interstitial cells; (c) When human embryonic stem cells are differentiated into cardiac progenitor cells, substances that can directly or indirectly activate the WNT-BMP signaling pathway are added to the culture medium to direct the differentiation of the stem cells into valvular interstitial cells.

3. The preparation method according to claim 1, characterized in that: The preparation process of the valve hydrogel culture solution A is as follows: Low Glucose DMEM, 10% FBS, 1% PS, final concentration 2μg / mL vitamin B12, and final concentration 1mg / mL aminoacetic acid are mixed to obtain A1 solution, and then 48μL A1 solution is mixed with 2.4μL 50U / mL thrombin to obtain; the preparation process of the valve hydrogel culture solution B is as follows: 20μL 7.5mg / ml Collagen I, 0.42μl 1M NaOH, 20μL 10mg / ml Fibrinogen, and 10μL Matrigel are mixed.

4. The preparation method according to claim 1, characterized in that: The tensile modulus is 38.2-78.68 mN / mm, and the elongation is 24.45-52.75%.

5. An artificial heart valve prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the artificial heart valve as claimed in claim 5 in preparing a heart valve disease model.

7. A method for preparing an artificial heart valve tissue calcification model, characterized in that: The artificial heart valve of claim 5 is cultured in a calcification inducing culture medium so that the valve tissue produces a calcified phenotype, thereby obtaining an artificial heart valve tissue calcification model.

8. The preparation method according to claim 7, characterized in that: The calcification inducing culture medium contains dexamethasone, β-glycerophosphate, ascorbic acid and calcium chloride.

9. An artificial heart valve tissue calcification model prepared by the preparation method according to any one of claims 7 to 8.

10. Use of the artificial heart valve tissue calcification model according to claim 9 in heart valve tissue calcification drug screening or drug testing.

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