A method for decellularization of pericardial tissue for bioprosthetic valve production

Abstract

The present application belongs to the technical field of biological valve, and relates to a pericardial tissue decellularization method for biological valve preparation, which comprises the following steps: first, using a phased decompression method to decompress to a target vacuum state; then, performing vacuum-assisted decellularization treatment on the pericardial tissue; then, performing washing treatment on the pericardial tissue after the decellularization treatment; finally, performing pressure treatment on the pericardial tissue after the washing treatment, so as to complete the cell removal of the pericardial tissue; and the washing treatment is performed in a periodic pressure difference auxiliary washing mode. The present application can effectively remove cell components, effectively maintain the integrity of the extracellular matrix, and reduce residues in the decellularization process.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological valve, and relates to a pericardial tissue decellularization method for biological valve preparation. BACKGROUND

[0002] Valvular heart disease is a serious cardiovascular disease worldwide, which is characterized by high morbidity and mortality, and the morbidity is further increasing with the aggravation of population aging. The common clinical treatment method is to perform heart valve replacement surgery. At present, the artificial valve used for clinical replacement mainly includes mechanical valve and biological valve. Although the mechanical valve has a long service life, it is easy to cause thrombosis after implantation, and the patient needs to take anticoagulant drugs for life. Compared with the mechanical valve, the biological valve has more advantages in clinical practice, which simulates the natural heart valve in structure and function, has good hemodynamic performance, and does not need long-term anticoagulant therapy, thereby avoiding many risks and inconveniences.

[0003] The pericardial tissue is a dense connective tissue mainly composed of collagen fibers and elastic fibers, which has excellent mechanical strength and biocompatibility required by the artificial valve. At present, the biological valve prepared from bovine pericardium and porcine pericardium has been widely used in clinical practice, and other sources of pericardial tissue have also been included in the research scope. After implantation, the biological valve needs to withstand hundreds of millions of opening and closing cycles in the blood environment. The blood environment and high frequency movement require the decellularized pericardial tissue to have good biocompatibility and mechanical properties, otherwise it is easy to cause inflammatory reaction, thrombosis, calcification and other problems of the biological valve, which seriously affects the service life.

[0004] In the decellularization method of pericardial tissue, the chemical method still occupies the dominant position in actual use, but the long-term contact of chemical reagents with the tissue will damage the extracellular matrix, and there is also the problem of reagent residue. In addition, the enzyme treatment often used in combination with the chemical method will also damage the extracellular matrix after long-term contact with the tissue. Therefore, at present, the physical methods such as stirring, osmotic pressure, freeze-thaw cycle, high hydrostatic pressure, ultrasound, high pressure transmembrane and vacuum treatment are mainly used to assist the chemical method and the chemical method-enzyme treatment for decellularization, and are usually applied before or during the chemical method or enzyme treatment, so as to improve the material transmission and tissue penetration efficiency, reduce the incubation time of the tissue in the chemical reagent and enzyme solution, and thus better protect the extracellular matrix. In order to remove the residual cell components and chemical reagents after decellularization of the pericardial tissue, the cleaning process mainly uses distilled water or PBS buffer solution for soaking or flushing, and part of the cleaning process uses ultrasonic or oscillation to assist the cleaning.

[0005] However, the current physical methods for decellularizing pericardial tissue still have obvious limitations in improving the efficiency of reagent penetration (shortening the processing period), maintaining the integrity of the three-dimensional structure of the extracellular matrix, and other key indicators, making it difficult to achieve a good balance between removing cellular components and maintaining the integrity of the extracellular matrix. In addition, during the cleaning process after decellularization, the existing methods have problems of insufficient internal cleaning of the tissue and too long cleaning time.

[0006] In document 1 (Enhanced vascular regeneration with chemically / physically treated bovine / human pericardium in rodents [J]. Journal of Surgical Research, 2018, 222: 167-179.), bovine pericardium is continuously stirred in an aqueous solution containing sodium deoxycholate (SDC) and sodium dodecyl sulfate (SDS), using detergents to destroy cell membrane structures and dissolve lipid and protein components to achieve decellularization. Then, it is rinsed in a continuous flow of deionized water to remove lysates and residual reagents. However, stirring can promote the diffusion of chemical reagents, but the diffusion effect is limited, and it is easy to damage the extracellular matrix (ECM).

[0007] In document 2 (Calcification of decellularized and alpha-galactosidase-treated bovine pericardial tissue in an alpha-gal knock-out mouse implantation model: Comparison with primate pericardial tissue [J]. EUROPEAN JOURNAL OF CARDIO-THORACIC SURGERY, 2016, 49(3): 894-900.), bovine pericardial tissue is soaked in a hypotonic solution containing sodium dodecyl sulfate (SDS), washed with distilled water, then soaked in a hypotonic solution of triton X-100, then treated with an isotonic solution, and then washed with distilled water. Finally, the tissue is treated with a hypertonic solution and then washed with PBS buffer. This scheme uses SDS to destroy cell membranes and dissolve cellular components, and Triton X-100 to further remove lipids and membrane proteins; the gradient solution (hypotonic, isotonic, and hypertonic) promotes cell fragment detachment through osmotic pressure differences, and finally removes residual substances through washing.

[0008] In document 3 (A comprehensive comparison of bovine and porcine decellularized pericardia: new insights for surgical applications[J]. BIOMOLECULES, 2020, 10(3): 371.), porcine pericardium and bovine pericardium were decellularized according to the TRICOL protocol, using protease inhibitors, alternating hypotonic / hypertonic solutions and detergents for decellularization, and then digested with endonuclease to obtain decellularized bovine pericardium and porcine pericardium. This scheme prevents autolysis by protease inhibitors, dissolves cell membranes and lipids with detergents, breaks cell structures by osmotic pressure difference with alternating hypotonic / hypertonic solutions, and degrades residual DNA and other genetic materials with endonuclease, finally achieving cell removal.

[0009] However, both document 2 and document 3 use the method of osmotic pressure to cause cell lysis by low and high osmotic solutions, which results in a longer processing time and increases the risk of extracellular matrix damage.

[0010] In document 4 (Efficient decellularization for bovine pericardium with extracellular matrix preservation and good biocomPatibility[J]. Interactive Cardiovascular and Thoracic Surgery, 2018, 26(5): 768-776.), bovine pericardium was subjected to five freeze-thaw cycles and washed with deionized water; then the bovine pericardium was immersed in a buffer containing sodium deoxycholate (SDC) and sodium dodecyl sulfate (SDS) for treatment. This scheme destroys cell structure through freeze-thaw cycles, dissolves cell membranes and lipids with SDC and SDS, and removes cell debris and reagent residues by washing. However, freeze-thaw cycles can also cause ECM damage while destroying cells by generating ice crystals.

[0011] In document 5 (In vitro tissue reconstruction using decellularized pericardium cultured with cells for ligament regeneration[J]. Polymers, 2022, 14(12):2351.), the pericardium was packed into a plastic bag containing physiological saline and sealed, high hydrostatic pressure was applied to destroy the cells using an HHP system, and then the pericardium was soaked in a solution containing DNase for treatment, and finally washed with ethanol and citric acid, respectively. This scheme destroys the cell membrane structure by high hydrostatic pressure, DNase degrades residual DNA, and then ethanol washes to remove proteins and lipids, and citric acid removes residual metal ions to further clean the extracellular matrix. However, high hydrostatic pressure technology can lyse cells, but can also cause protein denaturation and damage to the ECM structure, and the cost is relatively high.

[0012] In document 6 (Pericardial tissue for cardiovascular application: an in-vitro evaluation of established and advanced production processes[J]. JOURNAL OF MATERIALS SCIENCE-MATERIALS IN MEDICINE, 2018, 29(11): 172.), the bovine pericardium was soaked in a buffer containing sodium deoxycholate (SDC) and sodium dodecyl sulfate (SDS) for decellularization, while ultrasonic treatment was applied, and then washed with PBS buffer to remove residual reagents. This scheme dissolves cell membrane lipids and proteins with SDC and SDS to destroy cell structure, and ultrasonic cavitation enhances cell fragmentation efficiency and promotes detergent penetration, and finally PBS washes to remove cell debris and detergent residues. However, when ultrasonic assisted decellularization, if the ultrasonic power is too high or the duration is too long, the strong mechanical shear force and cavitation effect will damage the extracellular matrix. In addition, the controllability of ultrasonic treatment is limited, and its effect is closely related to power density, frequency, probe arrangement and other factors, making it difficult to completely reproduce the results of each treatment, with low repeatability. If the ultrasonic power or duration is insufficient, the ultrasonic distribution is uneven or the cavitation effect is insufficient, which may result in insufficient destruction of cell membrane, nucleus and other structures and insufficient penetration of detergents, resulting in residual cell debris or DNA, ultimately leading to incomplete decellularization.

[0013] In the patent application with the application publication number US20220354991A1, the pericardial tissue is placed in a transmembrane liquid flow device, and the buffer solution and deionized water are made to pass through the tissue by applying high pressure to destroy the cells, and the cells are removed. Subsequently, the tissue is placed between two glass plates and pressure is applied for compression to reduce the tissue pores, and then the tissue is washed. However, the solution is made to penetrate the tissue by high pressure for decellularization, which can make the solution fully penetrate the tissue, but also easily leads to the destruction of the extracellular matrix structure (such as the increase of pore size).

[0014] In document 7 (Resveratrol-loaded decellularized ovine pericardium: ECM introduced for tissue engineering [J]. Biotechnology and Applied Biochemistry, 2024, 71(2): 387-401.), the sheep pericardium is decellularized by soaking in sodium dodecyl sulfate (SDS) at room temperature, and is placed in a vacuum environment for a certain time. After decellularization, distilled water is used for washing. This scheme dissolves the cell membrane and nuclear membrane by SDS to release the cell contents, and at the same time, the negative pressure is used to promote the penetration of the detergent into the deep layer of the tissue to accelerate the discharge of cell fragments and residues, and finally distilled water is used for washing to remove cell fragments and chemical residues. However, vacuum-assisted decellularization can accelerate the entry of decellularization reagents into the interior of the tissue by negative pressure, but will lead to the increase of the pore size and the pore size of the extracellular matrix.

[0015] In the patent with the granted publication number US11185611B2, a physical method of periodic pressure change is disclosed, which is combined with chemical methods and enzyme treatment for decellularization. The pressure difference formed by the pressure change is used to destroy the cell membrane to promote the release of cell contents, and at the same time, the penetration of the decellularization solution is promoted, and then the sample is soaked in PBS buffer for oscillation rinsing. However, the periodic pressure difference is difficult to ensure the uniform distribution of chemical reagents in the tissue in the process of assisting decellularization, and the operation of the equipment is complex.

[0016] Therefore, there is a need for a pericardial tissue decellularization method for the preparation of biological valves, which can efficiently remove cell components while effectively maintaining the integrity of the extracellular matrix and reducing residues in the decellularization process. SUMMARY

[0017] The purpose of the present application is to solve the problems existing in the prior art, and to provide a pericardial tissue decellularization method for the preparation of biological valves, which can efficiently remove cell components while effectively maintaining the integrity of the extracellular matrix and reducing residues in the decellularization process. Figure 1As shown, the decellularization process of pericardial tissue is completed based on three stages of staged decompression vacuum assisted decellularization, periodic pressure difference assisted washing, and post-pressing treatment, aiming to achieve the effects of efficient decellularization, small extracellular matrix damage, and few decellularization residues.

[0018] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0019] A pericardial tissue decellularization method for biological valve preparation, first using a staged decompression method to decompress to a target vacuum state, then vacuum assisted decellularization treatment is performed on the pericardial tissue (including bovine pericardium, porcine pericardium, donkey pericardium, sheep pericardium, etc.), then the pericardial tissue after decellularization treatment is washed, and finally the pericardial tissue after washing is subjected to pressure treatment to complete the removal of cells from the pericardial tissue.

[0020] The washing treatment is performed in a periodic pressure difference assisted washing manner.

[0021] As a preferred technical scheme:

[0022] The pericardial tissue decellularization method as described above, the decellularization process of the pericardial tissue is carried out in a decellularization device; the temperature in the decellularization device during vacuum assisted decellularization treatment, washing treatment and pressure treatment is the same, which is 4-37℃, and is selected according to different species of pericardial tissue and decellularization reagents and other factors.

[0023] The pericardial tissue decellularization method as described above, the staged decompression method refers to decompression from normal pressure (standard atmospheric pressure) to a target vacuum state in stages, and at least two stages of staged decompression are included in the decompression process, and a period of time is maintained at each stage of pressure to ensure stability.

[0024] The pericardial tissue decellularization method as described above, the pressure in the decellularization device at the target vacuum state is 100Pa-0.05MPa, and is further preferably less than 1000Pa.

[0025] The pericardial tissue decellularization method as described above, the decompression range of each adjacent two pressure stages is 1-52KPa, and the stable time of each stage of pressure in the decompression process is 15min; according to the pressure value of the decompression stage, the decompression rate can be divided into three stages, such as Figure 7 As shown, the decompression rate from 101.3kPa to 50kPa is 1000Pa / s, the decompression rate from 50kPa to 10kPa is 500Pa / s, and the decompression rate from 10kPa to 0.1kPa is 100Pa / s.

[0026] The pericardial tissue decellularization method for biological valve preparation as described above, the vacuum-assisted decellularization treatment time is 12-96h (not including the time from normal pressure to target pressure, the vacuum-assisted decellularization time is calculated from reaching the target pressure). The decellularization treatment time under normal pressure is selected according to the decellularization scheme and different species of pericardial tissue, etc. Under the same conditions (except for pressure), the treatment time required for vacuum-assisted decellularization can be reduced to at most 50% of the treatment time under normal pressure under the same conditions.

[0027] The pericardial tissue decellularization method for biological valve preparation as described above, when the periodic pressure difference assisted washing is used, the number of cycles of pressure change is not less than 5, and a cycle of periodic pressure change is: first reduce the pressure in the decellularization device from normal pressure to target pressure, maintain for 15min, and then restore to normal pressure, maintain for 45min; the target pressure is in the range of 0.07-0.09MPa; the time of one cycle is 60min, and the number of cycles can be adjusted according to the decellularization scheme and different species of pericardial tissue, etc.

[0028] The pericardial tissue decellularization method for biological valve preparation as described above, the preliminary washing is performed before the periodic pressure difference assisted washing, and the preliminary washing refers to soaking washing under normal pressure using a washing solution, and the soaking time is 15min.

[0029] The pericardial tissue decellularization method for biological valve preparation as described above, the washing solution used in the preliminary washing and the periodic pressure difference assisted washing is the same, which is distilled water, deionized water, phosphate buffer (PBS buffer), ethanol solution with a volume concentration of 70% or sodium chloride aqueous solution with a concentration of 0.9% (w / v).

[0030] The pericardial tissue decellularization method for biological valve preparation as described above, the pressure of the pressurization treatment is 0.2-0.8MPa, preferably 0.2-0.5MPa, and the pressurization holding time is 1-6h, preferably 1-4h.

[0031] Invention principle:

[0032] The present application is based on three stages of decellularization process of pericardial tissue, including vacuum-assisted decellularization, periodic pressure difference assisted washing and pressurization treatment, aiming to achieve the effects of efficient decellularization, small extracellular matrix damage and less decellularization residues. First, vacuum-assisted decellularization improves the decellularization efficiency and makes the removal of cell components more complete. Compared with non-vacuum-assisted decellularization, the vacuum-assisted decellularization effectively reduces the time required for decellularization, and the DNA content in the decellularized tissue is less than 50 ng dsDNA / mg ECM dry weight, which meets the standard requirement for complete decellularization. The stage-by-stage decompression method makes the vacuum application process more gentle, reduces the arrangement disorder and fracture of fibers in the ECM caused by severe decompression, and the stage-by-stage decompression combined with subsequent pressurization treatment can make the structural integrity of the ECM better and the damage smaller, which helps to alleviate the problems of damage to the structural integrity of the pericardial tissue and the decline of mechanical properties. Then, in the post-decellularization treatment, the implementation of effective washing program is crucial to remove the residual cell fragments and chemical reagents in the tissue, which can effectively avoid the formation of cytotoxic microenvironment. When the tissue is exposed to a negative pressure environment for a long time, the pore size and porosity of the decellularized tissue increase, and washing at this time is more helpful to the efficient removal of residual substances. The periodic pressure difference assisted washing adopted in the present application promotes the penetration and discharge of the washing solution in the tissue through the dynamic change of the pressure field, thereby improving the efficiency and effect of washing. Compared with conventional soaking or flushing, the periodic pressure difference washing can effectively reduce the residual substances in the tissue within the same washing time. Finally, pressurization treatment can effectively alleviate the problem of tissue swelling caused by long-term negative pressure treatment, promote the decrease of pore size and porosity, and make the extracellular matrix structure closer to the natural state.

[0033] The present application effectively maintains the integrity of the extracellular matrix while efficiently removing cellular components by the synergistic cooperation of three stages: vacuum-assisted decellularization, periodic pressure difference-assisted washing, and pressurization treatment. Specifically, on the one hand, vacuum-assisted decellularization promotes the penetration of decellularization reagents through negative pressure, shortening the decellularization time and making the cell removal more complete. However, this process leads to tissue swelling and increased porosity. This tissue swelling creates favorable conditions for subsequent washing, and periodic pressure changes further expand the washing advantages brought by the increased tissue porosity. The present application significantly improves the removal efficiency and effectiveness of residual decellularization reagents and residual cellular components by using the method of periodic pressure reduction, which promotes the dynamic penetration and discharge of liquid in the expanded pores. On the other hand, the periodic pressure reduction in vacuum-assisted decellularization helps to reduce the damage to the extracellular matrix. The efficiency of vacuum decellularization and periodic pressure difference washing comes at the cost of tissue swelling, and pressurization treatment is used to specifically repair this side effect. After washing, pressure is applied to further compact the ECM fiber network, restoring the density of the ECM and simultaneously restoring the mechanical stability.

[0034] In addition, the tissue swelling problem caused by negative pressure treatment is an advantage in the decellularization process, making it easier for the decellularization solution to penetrate the tissue and for the exchange of substances such as cell fragments within the tissue with the outside world. In the washing process, it also shows advantages, which is beneficial for the cleaning of residual decellularization solution and cell fragments. However, in subsequent applications, the tissue swelling and loose fiber structure caused by long-term negative pressure treatment may lead to the following problems after the decellularized pericardium is made into a biological valve:

[0035] 1. Structural integrity is damaged and mechanical properties are reduced;

[0036]

[0036] First, the pericardial tissue fiber gap increases and the arrangement becomes loose, which weakens the mechanical strength of the material. The mechanical stretching data in the paper show that the ultimate tensile strength and modulus are lower in the vacuum-assisted decellularization group compared to the group without vacuum. In subsequent use, the biological valve is prone to fatigue damage or tearing when subjected to long-term blood flow impact;

[0037] Second, the porosity of the swollen tissue increases, but the uneven porosity may affect the uniform penetration of cross-linking agents such as glutaraldehyde, leading to uneven cross-linking density, which in turn causes the non-cross-linked areas of the valve leaflets to be more prone to degradation and mechanical weakness;

[0038] Third, the swelling of the decellularized pericardial tissue leads to a disorderly arrangement of fibers and uneven thickness, as well as mechanical performance differences, which may affect the dimensional stability of the biological valve and the coordination of the valve leaflet opening and closing.

[0039] 2. Reduced resistance to calcification;

[0040] Due to the increase of fiber gap, loose arrangement, more easily lead to calcification site exposure, aggravate the valve calcification.

[0041] 3. Long-term durability challenges;

[0042] The superimposed effects of mechanical performance degradation, blood flow impact injury and calcification tendency can significantly shorten the service life of the biological valve.

[0043] And the pressurization treatment after washing of the application can effectively alleviate the tissue swelling problem caused by long-term negative pressure treatment, promote the decrease of tissue pore size, and ensure the excellent performance of the biological valve made of decellularized pericardium.

[0044] Advantages:

[0045] (1) The application cooperates the three stages of phase reduction vacuum assisted decellularization, periodic pressure difference assisted washing and pressurization treatment, effectively maintains the integrity of the extracellular matrix while efficiently removing cell components, and reduces residues during decellularization.

[0046] (2) The application is decellularized by vacuum assistance combined with chemical methods and enzyme treatment, which can more completely remove cells and other immune substances, requires less time, and reduces extracellular matrix damage.

[0047] (3) The application makes the vacuum application process more moderate by the phase reduction method, reduces the damage of rapid pressure reduction to the fibers in the extracellular matrix, and at the same time, the periodic pressure difference assisted washing helps the penetration and discharge of the washing solution in the tissue, improves the washing efficiency and effect.

[0048] (4) The application can effectively reduce the tissue swelling phenomenon caused by long-term negative pressure exposure through pressurization treatment, so that the structure and morphology of the extracellular matrix are closer to the natural state. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 The figure is a schematic diagram of the pressure-time relationship in the process of the pericardial tissue decellularization method for biological valve preparation of the application; in the figure, a is a schematic diagram of the pressure-time relationship in the process of vacuum assisted decellularization of pericardial tissue, b is a schematic diagram of the pressure-time relationship in the process of washing treatment of the decellularized pericardial tissue, and c is a schematic diagram of the pressure-time relationship in the process of pressurization treatment of the washed pericardial tissue;

[0050] Figure 2 The figure is a schematic diagram of the device for the pericardial tissue decellularization method for biological valve preparation of the application;

[0051] Figure 3Figure 1 is a schematic diagram of the cross-section of the H&E staining of the pericardial tissue after cell removal in Example 1, the pericardial tissue after cell removal in Comparative Example 1, and the natural pericardium; in the figure, a is a schematic diagram of the cross-section of the H&E staining of the natural pericardial tissue, b is a schematic diagram of the cross-section of the H&E staining of the pericardial tissue after cell removal in Comparative Example 1, and c is a schematic diagram of the cross-section of the H&E staining of the pericardial tissue after cell removal in Example 1;

[0052] Figure 4 Figure 2 is a schematic diagram of the mechanical properties of the pericardial tissue after cell removal in Example 1, Comparative Example 1, and Comparative Example 3, and the natural pericardium; in the figure, a is a schematic diagram of the ultimate tensile strength of the natural pericardium, the pericardial tissue after cell removal in Example 1, Comparative Example 1, and Comparative Example 3, and b is a schematic diagram of the elastic modulus of the natural pericardium, the pericardial tissue after cell removal in Example 1, Comparative Example 1, and Comparative Example 3;

[0053] Figure 5 Figure 3 is a SEM diagram of the surface micro-morphology of the pericardial tissue after cell removal in Example 1, Comparative Example 1, and Comparative Example 3, and the natural pericardium; in the figure, a is a SEM diagram of the surface micro-morphology of the natural pericardium, b is a SEM diagram of the surface micro-morphology of the pericardial tissue after cell removal in Comparative Example 1, c is a SEM diagram of the surface micro-morphology of the pericardial tissue after cell removal in Comparative Example 3, and d is a SEM diagram of the surface micro-morphology of the pericardial tissue after cell removal in Example 1;

[0054] Figure 6 Figure 4 is a schematic diagram of the cell survival rate in the cytotoxicity test of the pericardial tissue in Example 1, Comparative Example 2, the control group, and the natural pericardial tissue;

[0055] Figure 7 Figure 5 is a schematic diagram of the decompression rate curve of the decompression device in the application;

[0056] Figure 2 In the figure, 1 is a high-pressure gas source, 2 is a constant-temperature water source, 3 is a pressure container, 4 is a sandwich layer, 5 is an inner container, 6 is a metal mesh, 7 is a pressure gauge, 8 is a pressure valve, and 9 is a vacuum pump. DETAILED DESCRIPTION

[0057] The application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content of the application, and these equivalent forms also fall within the scope of the appended claims of the application.

[0058] To ensure that the properties of the substances used in the embodiments are fully disclosed, the manufacturers and brands of the substances are written down, and the products of other manufacturers and brands that meet the definition of the application are also feasible.

[0059] The sources of the substances in each example are as follows:

[0060] Sodium dodecyl sulfate (SDS): the manufacturer is Shanghai Maikelin Biochemical Technology Co., Ltd., and the purity is analytical pure;

[0061] Sodium deoxycholate (SDC): the manufacturer is Shanghai Maikelin Biochemical Technology Co., Ltd., and the purity is 98%;

[0062] Deoxyribonuclease I (bovine pancreas): the manufacturer is Shanghai Yuan Ye Biological Technology Co., Ltd., and the product number is S10073;

[0063] Ribonuclease RNase A: the manufacturer is Shanghai Yuan Ye Biological Technology Co., Ltd., and the product number is R21094;

[0064] Tris-HCl buffer: the manufacturer is Wuhan Saivier Biological Technology Co., Ltd., and the product number is G2153-1L.

[0065] The test methods of the relevant performance indicators in each example are as follows:

[0066] Residual DNA content: the pericardial tissue after cell removal in each example and each comparative example was used as a sample, and then DNeasy Blood & Tissue Kit (Qiagen) was used for determination, and the specific process was as follows (the ATL buffer, AL buffer, Buffer AW1, Buffer AW2, and Buffer AE used in the following test process were all from the DNeasy Blood & Tissue Kit kit of QIAGEN Company in Germany):

[0067] (1) The sample was cut into small pieces (<25 mg) and loaded into a 2 ml collection tube, 180 μL of ATL buffer (Buffer ATL) was added, 20 μL of proteinase K was added, and after oscillation and mixing, it was incubated at 56°C until the tissue was completely lysed, and the oscillation was interrupted during the incubation, each time for 15 seconds, 200 μL of AL buffer (Buffer AL) was continuously added, and then incubated at 56°C for 10 min, and then 200 μL of anhydrous ethanol was added and oscillated and mixed to obtain a mixture;

[0068] (2) The mixture was transferred to a DNeasy Mini column in a 2 ml collection tube using a pipette, centrifuged at 8000 rpm for 1 min, and the filtrate and collection tube were discarded. The column was then placed in a new 2 ml collection tube, and 500 μL of wash solution 1 (Buffer AW1) was added. The mixture was centrifuged at 8000 rpm for 1 min, and the filtrate and collection tube were discarded. The column was then placed in a new 2 ml collection tube, and 500 μL of wash solution 2 (Buffer AW2) was added. The mixture was centrifuged at 14000 rpm for 3 min, and the filtrate and collection tube were discarded. The column was then transferred to a new 2 ml collection tube, and 200 μL of elution buffer (Buffer AE) was added to the center of the column membrane to elute the DNA. The mixture was incubated at 25°C for 1 min, and then centrifuged at 8000 rpm for 1 min to collect the DNA solution. Finally, the DNA concentration at 260 nm was measured using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific), and the DNA amount was calculated based on the dry weight of the sample, expressed in ng / mg.

[0069] Porosity: The pericardial tissue after cell removal in each example and each comparative example was used as a sample, and then SEM images were taken. The porosity was calculated using image analysis software ImageJ.

[0070] Elastic modulus and ultimate tensile strength: The pericardial tissue after cell removal in each example and each comparative example, as well as the natural pericardium, were used as samples. Then, the uniform thickness part of the sample was cut into a 20 mm x 5 mm rectangular sample along the direction parallel to the orientation of most of the fiber bundles. The uniaxial tensile mechanical properties of the sample were tested using a universal material testing machine (microcomputer-controlled electronic universal material testing machine CTM2050, Shanghai Xieqiang Instrument Manufacturing Co., Ltd.). The sample was stretched until it broke during the stretching process. The elastic modulus (EM) and ultimate tensile strength (UTS) were calculated based on the test data, and the results were averaged. The stretching speed was set to 12.5 mm / min, and the gauge length was set to 10 mm.

[0071] Cell survival rate: The pericardial tissue after cell removal in each example and each comparative example and the natural pericardium were taken as samples, and then the samples were cut to 0.5 cm x 0.5 cm in size after thorough washing and sterilization, and three parallel samples were set for each group. After ultraviolet sterilization was completed, the samples were placed in a 24-well plate, 0.5 mL of complete medium was added to each well, and soaked for three days; after soaking was completed, the extract was filtered using a 0.22 μm filter, and 100 μL of the extract was taken from each well and transferred to a 96-well plate, and mixed with 100 μL of complete medium to make a total volume of 200 μL, a concentration of 50% (v / v) of the extract, and then fibroblasts (HFFs) were inoculated in the 96-well plate at a density of 1 w / mL as a treatment group; the control group contained 200 μL of complete medium, and fibroblasts (HFFs) were inoculated in the 96-well plate at a density of 1 w / mL; the blank group only contained 200 μL of complete medium without inoculating cells; after 48 h and 72 h of culture, the absorbance (i.e. OD value) at 450 nm wavelength of the treatment group, the control group and the blank group was measured by CCK-8 method under an enzyme marker, and then the cell survival rate was calculated according to the measured results; wherein the calculation formula of the cell survival rate is as follows:

[0072] Cell survival rate (%) = [(OD 处理组 - OD 空白组 ) / (OD 对照组 - OD 空白组 ) ] x 100%).

[0073] Example 1

[0074] A pericardial tissue decellularization method for biological valve preparation, the steps are as follows:

[0075] (1) Preparation of raw materials;

[0076] Pericardial tissue: fresh bovine pericardium;

[0077] PBS buffer: phosphate buffer, composed of 137 mM NaCl (sodium chloride), 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and water, pH 7.4;

[0078] Solution of chemical reagent: 1% (w / v) concentration, solvent is ultrapure water, and chemical reagent is sodium dodecyl sulfate (SDS);

[0079] Washing solution: PBS buffer;

[0080] Preservation solution: PBS buffer;

[0081] Metal mesh: 10cm x 10cm, 3mm aperture;

[0082] Decellularization devices: such as Figure 2 As shown, the decellularization device includes a high-pressure gas source 1, a constant-temperature water source 2, a pressure vessel 3, a metal mesh 6, a pressure gauge 7, a pressure valve 8, and a vacuum pump 9. The pressure vessel 3 is internally divided into two parts: a jacket 4 and an inner liner 5, with the metal mesh 6 placed inside the inner liner. The high-pressure gas source 1 and the vacuum pump 9 are each connected to the inner liner 5 through a pressure valve 8, which can adjust the pressure inside the inner liner. The pressure valve 8 is used to adjust and control the pressure inside the inner liner. The constant-temperature water source 2 is connected to the jacket 4 of the pressure vessel 3.

[0083] (2) Pretreatment of pericardial tissue;

[0084] Pericardial tissue was rinsed with PBS buffer to remove blood and external fat. The rinsed pericardial tissue was then stored in PBS buffer at 4°C for later use. Meanwhile, the tissue was examined, and areas with pores or uneven thickness were discarded. Regular tissue was selected and cut to obtain pre-treated pericardial tissue.

[0085] (3) The pretreated pericardial tissue was placed in the inner liner of a 5L decellularization device, and 3.5L of chemical reagent solution was added. After placing the metal mesh and sealing the decellularization device, the constant temperature water source was adjusted so that the temperature in the inner liner reached 25℃. The pressure inside the decellularization device was reduced from normal pressure to a vacuum state of 1000Pa in two stages. Then, vacuum-assisted decellularization was carried out for 30 hours (excluding the time of the decompression process). The decompression was operated by controlling the vacuum pump. The stage decompression value of the first stage was 51.3KPa, and the stage decompression value of the second stage was 49KPa. The duration of each stage was 15min.

[0086] (4) Washing treatment;

[0087] (4.1) After vacuum-assisted decellularization is completed, the pressure inside the device is restored to normal, the decellularization solution is removed, and then 3.5L of washing solution is added for soaking and washing under normal pressure and temperature of 25℃; the soaking and washing is performed 3 times, and the soaking and washing time is 15min each time.

[0088] (4.2) After soaking and washing, add 3.5L of washing solution and seal the decellularization device. Then, perform periodic pressure difference assisted washing under the conditions of atmospheric pressure and 25℃. During periodic pressure difference assisted washing, the pressure change cycle is 10 times. The washing solution is replaced every 5 cycles. One periodic pressure change is as follows: first, reduce the pressure in the decellularization device from atmospheric pressure to 0.07MPa and maintain it for 15min, then restore it to atmospheric pressure and maintain it for 45min.

[0089] (5) After the washing is completed, the pressure is first restored to normal pressure, then PBS buffer is added to the device, and the pressure vessel is sealed, then the pericardial tissue after washing treatment is pressurized at a pressure of 0.5 MPa and a temperature of 25 DEG C for 3 h, and then restored to normal pressure, finally the pericardial tissue after pressurization is washed with PBS buffer, and placed in a storage solution at a temperature of 4 DEG C for wet storage, that is, the cell removal of the pericardial tissue is completed.

[0090] The pericardial tissue after cell removal (the mechanical properties thereof are shown in Table 1, and the surface micro-morphology SEM image is shown in Figure 1d) has a residual DNA content of 35.1 ng / mg, a porosity of 6.46%, an elastic modulus of 49.19 MPa, a ultimate tensile strength of 18.52 MPa, a cell survival rate of 72% after 48 h, and a cell survival rate of 85% after 72 h. Figure 4 Figure 5 The pericardial tissue after cell removal obtained by the above method has a residual DNA content of less than 50 ng dsDNA / mg ECM dry weight, which meets the standard requirement of complete decellularization.

[0091] In terms of small extracellular matrix damage, H&E staining and SEM images show a dense extracellular matrix structure and regular fiber arrangement and less porosity; the elastic modulus and ultimate tensile strength are close to those of natural pericardial tissue, indicating that the matrix damage is small and the mechanical properties are basically retained.

[0092] In terms of less residual components after decellularization, the residual decellularization reagent is less, and the cell survival rate in the cytotoxicity test is significantly higher than that in the soaking and washing condition, and the cytotoxicity test shows good cell compatibility.

[0093] In terms of less residual components after decellularization, the residual decellularization reagent is less, and the cell survival rate in the cytotoxicity test is significantly higher than that in the soaking and washing condition, and the cytotoxicity test shows good cell compatibility.

[0094] Comparative Example 1

[0095] A pericardial tissue decellularization method for biological valve preparation, which is basically the same as Example 1, except that in step (3), the vacuum-assisted decellularization treatment is not performed by using a staged decompression method, but directly decompressed from normal pressure to a vacuum state of 1000 Pa at the same decompression rate as Example 1, and then vacuum-assisted decellularization treatment is performed.

[0096] The pericardial tissue after cell removal (the mechanical properties thereof are shown in Table 1, and the surface micro-morphology SEM image is shown in Figure 1d) has a residual DNA content of 35.1 ng / mg, a porosity of 6.46%, an elastic modulus of 49.19 MPa, a ultimate tensile strength of 18.52 MPa, a cell survival rate of 72% after 48 h, and a cell survival rate of 85% after 72 h. Figure 4 Figure 5 ​​The porosity of the pericardial tissue after cell removal was 10.63%, the elastic modulus was 35.26 MPa, and the ultimate tensile strength was 12.61 MPa.

[0097] Comparing Example 1 with Comparative Example 1, the porosity of the pericardial tissue after cell removal in Comparative Example 1 increased, and the elastic modulus and the ultimate tensile strength decreased. This is because the staged decompression method used in Example 1 makes the vacuum application process more gentle, reduces the arrangement disorder and fracture of fibers in ECM caused by severe decompression, and the staged decompression combined with subsequent pressure treatment can make the structural integrity of ECM better and the damage smaller, which helps to alleviate the problems of damage to the structural integrity of pericardial tissue and the decrease of mechanical properties. In Comparative Example 1, the staged decompression method was not used during the vacuum-assisted decellularization, which ultimately led to the decrease of the elastic modulus and the ultimate tensile strength of the pericardial tissue after cell removal and the increase of the porosity.

[0098] The pericardial tissue after cell removal in Example 1 and Comparative Example 1 and the natural pericardium were subjected to H&E staining as samples. The specific process was as follows: the samples were first dehydrated, completely dried, and then embedded in paraffin, and tissue sections were prepared using a microtome. Then, the tissue sections were stained with hematoxylin (manufacturer: Shanghai Maikelin Biochemical Technology Co., Ltd., model: H810910) and eosin staining agent (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd., model: R30115). The changes in the internal structure and composition of the tissue were observed under a microscope, and the results are shown in FIG. 2. Figure 3 As can be seen from the figure, the natural bovine pericardium has a dense tissue structure composed of ECM (purple) and cell nuclei (blue). No obvious residual cell nuclei were observed in the pericardial tissue after cell removal in Example 1 and Comparative Example 1. Compared with the pericardial tissue after cell removal in Comparative Example 1, the pericardial tissue after cell removal in Example 1 had a more dense extracellular matrix structure and a more regular fiber arrangement, which was close to that of the natural pericardial tissue.

[0099] Comparative Example 2

[0100] A method for preparing pericardial tissue for biological valve preparation, which is basically the same as Example 1, except that step (4.2) does not perform periodic pressure difference assisted washing, and the pericardial tissue after step (4.1) is directly immersed in the washing solution. The immersion time is the same as that in Example 1.

[0101] The residual DNA content in the pericardial tissue after cell removal was 49.5 ng / mg, the cell survival rate after 48 h was 60%, and the cell survival rate after 72 h was 71%.

[0102] The DNA content and cytotoxicity of the pericardial tissue after cell removal in Comparative Example 2 increased, because the periodic pressure difference assisted washing in Example 1 can promote the penetration and discharge of the washing solution in the tissue through the dynamic change of the pressure field, thereby improving the efficiency and effect of washing, and effectively reducing the residual reagents in the tissue. In Comparative Example 2, the residual DNA content and chemical reagent content in the pericardial tissue increased in the same washing time, thereby causing the cytotoxicity to increase and the cell survival rate to decrease.

[0103] The pericardial tissue after cell removal in Example 1 and Comparative Example 2 and the natural pericardium were respectively used as samples for cell survival rate test in cytotoxicity test, and the results are shown in Figure 6 As can be seen from the figure, according to the key quantitative index of determining no potential cytotoxicity of materials in GB / T16886.5-2017, the cell survival rate is ≥70%, but the cell survival rate of the pericardial tissue after cell removal in Comparative Example 2 is less than 70% after 48h of culture, which does not meet the requirement of no potential cytotoxicity, because the chemical reagents are not completely removed, and the toxicity of the residual reagents causes it; the cell survival rate in Example 1 is all greater than 70%, and the cell survival rate after 72h of culture is closer to that of the natural pericardial tissue.

[0104] Comparative Example 3

[0105] A pericardial tissue decellularization method for biological valve preparation, which is basically the same as Example 1, except that in step (5), the pericardial tissue after washing in step (4) is directly flushed with PBS buffer and placed in a preservation solution at a temperature of 4°C for wet preservation, that is, the cell removal of the pericardial tissue is completed.

[0106] The porosity of the pericardial tissue after the above cell removal (the mechanical properties thereof are shown in Figure 4 , and the surface micro-morphology SEM image is shown in Figure 5 ) is 25.51%, the elastic modulus is 34.15MPa, and the ultimate tensile strength is 11.41MPa.

[0107] Comparing Comparative Example 3 and Example 1, the porosity of the pericardial tissue after cell removal in this comparative example is higher, and the elastic modulus and ultimate tensile strength decrease, because the pressure treatment can effectively alleviate the tissue swelling problem caused by long-term negative pressure treatment, and promote the tissue pore size to become smaller and the porosity to decrease, so that the extracellular matrix structure is closer to the natural state. The scaffold in this comparative example lacks pressure treatment, and is more prone to deformation under low stress, which is manifested as a significant decrease in elastic modulus and a decrease in ultimate tensile strength.

[0108] Example 2

[0109] A pericardial tissue decellularization method for biological valve preparation, the steps are as follows:

[0110] (1) Preparation of raw materials;

[0111] Pericardial tissue: fresh bovine pericardium;

[0112] PBS buffer: phosphate buffer, consisting of 137 mM NaCl (sodium chloride), 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and water, pH 7.4;

[0113] Solution of chemical reagents: 1% (w / v) concentration, solvent is ultrapure water, chemical reagent is sodium dodecyl sulfate (SDS);

[0114] Washing solution: 70% volume concentration of ethanol solution;

[0115] Preservation solution: PBS buffer;

[0116] Metal mesh: size of 10 cm x 10 cm, pore size of 3 mm;

[0117] Decellularization device: the decellularization device includes a high-pressure gas source, a constant-temperature water source, a pressure container, a metal mesh, a pressure gauge, a pressure valve and a vacuum pump; the inner part of the pressure container is divided into two parts, a sandwich and an inner container, the metal mesh is placed in the inner container; the high-pressure gas source and the vacuum pump are respectively connected to the inner container through a pressure valve, and the pressure in the inner container can be adjusted; the pressure valve is used to adjust and control the pressure of the inner container; the constant-temperature water source is connected to the sandwich of the pressure container;

[0118] (2) Pretreatment of pericardial tissue;

[0119] The pericardial tissue is washed with PBS buffer to remove blood and external fat, and then the washed pericardial tissue is stored in PBS buffer at 4℃ for standby, while the tissue is inspected, the areas with holes and uneven thickness are discarded, and the regular tissue is selected and cut to obtain the pretreated pericardial tissue;

[0120] (3) the pretreated pericardial tissue is placed in the inner container of the decellularization device with a capacity of 5 L, 3.5 L of a solution of chemical reagents is added, a metal mesh is placed and the decellularization device is sealed, then the temperature of the inner container is adjusted to 25℃, and then the pressure in the decellularization device is reduced from normal pressure to 0.03 MPa in three stages, and vacuum assisted decellularization is performed for 12 h; wherein the pressure reduction is performed by controlling the vacuum pump, the first stage of the three-stage pressure reduction is 25.3 KPa, the second stage of the three-stage pressure reduction is 26 KPa, and the third stage of the three-stage pressure reduction is 20 KPa, and each stage lasts for 15 min;

[0121] (4) washing treatment;

[0122] (4.1) after the vacuum assisted decellularization is completed, the pressure in the device is restored to normal pressure, the decellularization solution is removed, and 3.5 L of a washing solution is added for soaking and washing under the conditions of normal pressure and 25℃; wherein the soaking and washing is performed for 3 times, and each time lasts for 15 min;

[0123] (4.2) after the soaking and washing is completed, 3.5 L of a washing solution is added and the decellularization device is sealed, and then periodic pressure difference assisted washing is performed under the conditions of normal pressure and 25℃; wherein during the periodic pressure difference assisted washing, the number of pressure change cycles is 7, the washing solution is replaced every 5 cycles, and one cycle of pressure change is as follows: the pressure in the decellularization device is reduced from normal pressure to 0.09 MPa, maintained for 15 min, and then restored to normal pressure, and maintained for 45 min;

[0124] (5) after the washing is completed, the pressure is restored to normal pressure, PBS buffer is added to the device, the pressure container is sealed, and then the pericardial tissue after washing is pressurized at 0.2 MPa and 25℃ for 6 h, and then the pressure is restored to normal pressure, finally the pressurized pericardial tissue is washed with PBS buffer and stored in a storage solution at 4℃ for wet storage, thereby completing the cell removal of the pericardial tissue.

[0125] The DNA content in the pericardial tissue after cell removal is 47.5 ng / mg, the porosity is 8.65%, the elastic modulus is 47.50 MPa, the ultimate tensile strength is 17.80 MPa, the cell survival rate after 48 h is 68%, and the cell survival rate after 72 h is 82%.

[0126] Example 3

[0127] A pericardial tissue decellularization method for biological valve preparation, the steps are as follows:

[0128] (1) preparation of raw materials;

[0129] Pericardial tissue: fresh bovine pericardium;

[0130] PBS buffer: phosphate buffer solution, consisting of 137 mM NaCl (sodium chloride), 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and water, pH 7.4;

[0131] Solution of chemical reagent: 1% (w / v) of sodium deoxycholate (SDC) in ultrapure water;

[0132] Washing solution: 0.9% (w / v) sodium chloride aqueous solution;

[0133] Preservation solution: PBS buffer;

[0134] Metal mesh: 10 cm x 10 cm in size, 3 mm in aperture;

[0135] Decellularization device: the decellularization device comprises a high-pressure gas source, a constant-temperature water source, a pressure container, a metal mesh, a pressure gauge, a pressure valve and a vacuum pump; the pressure container has two parts, i.e. a sandwich layer and an inner container, and the metal mesh is placed in the inner container; the high-pressure gas source and the vacuum pump are respectively connected to the inner container through a pressure valve, so as to adjust the pressure in the inner container; the pressure valve is used to adjust and control the pressure in the inner container; the constant-temperature water source is connected to the sandwich layer of the pressure container;

[0136] (2) Pretreatment of pericardial tissue;

[0137] The pericardial tissue is washed with PBS buffer solution to remove blood and external fat, and then the washed pericardial tissue is stored in PBS buffer solution at 4℃ for standby, while the tissue is inspected, and the regions with holes and uneven thickness are discarded, and the regular tissue is selected for cutting to obtain the pretreated pericardial tissue;

[0138] (3) The pretreated pericardial tissue is placed in the inner container of the decellularization device with a capacity of 5 L, 3.5 L of the solution of chemical reagent is added, the metal mesh is placed and the decellularization device is sealed, then the constant-temperature water source is adjusted to make the temperature in the inner container reach 4℃, and then the pressure in the decellularization device is reduced from normal pressure to 100 Pa vacuum state in four stages, and then vacuum-assisted decellularization treatment is performed for 30 h; wherein the pressure reduction is operated by controlling the vacuum pump, the stage pressure reduction value of the first stage is 26.2 KPa, the stage pressure reduction value of the second stage is 25 KPa, the stage pressure reduction value of the third stage is 25 KPa, and the stage pressure reduction value of the fourth stage is 25 KPa, and the duration of each stage is 15 min;

[0139] (4) Washing treatment;

[0140] (4.1) After the vacuum-assisted decellularization is completed, the pressure in the device is returned to normal, the decellularization solution is removed, and 3.5 L of a washing solution is added for soaking and washing under the condition of a normal pressure and a temperature of 4°C; wherein the soaking and washing is performed 3 times, and each time the soaking and washing lasts for 15 min;

[0141] (4.2) After the soaking and washing is completed, 3.5 L of a washing solution is added to the decellularization device, which is then sealed, and periodic pressure difference-assisted washing is performed under the condition of a normal pressure and a temperature of 4°C; wherein, during the periodic pressure difference-assisted washing, the pressure changes 5 cycles, and the washing solution is replaced every 5 cycles; one cycle of pressure change is as follows: the pressure in the decellularization device is first reduced from normal pressure to 0.07 MPa, maintained for 15 min, and then returned to normal pressure, and maintained for 45 min;

[0142] (5) After the washing is completed, the pressure is first returned to normal, then PBS buffer is added to the device, which is then sealed in a pressure container, and then the pericardial tissue after the washing treatment is pressurized at a pressure of 0.8 MPa and a temperature of 4°C for 1 h, and then returned to normal pressure; finally, the pressurized pericardial tissue is rinsed with PBS buffer and placed in a storage solution at a temperature of 4°C for wet storage, thereby completing the cell removal of the pericardial tissue.

[0143] The DNA content in the pericardial tissue after the cell removal is 42.3 ng / mg, the porosity is 7.25%, the elastic modulus is 53.50 MPa, the ultimate tensile strength is 19.72 MPa, the cell survival rate after 48 h is 76%, and the cell survival rate after 72 h is 89%.

[0144] Example 4

[0145] A pericardial tissue decellularization method for biological valve preparation, the steps are as follows:

[0146] (1) Preparation of raw materials;

[0147] Pericardial tissue: fresh bovine pericardium;

[0148] PBS buffer: phosphate buffer, composed of 137 mM NaCl (sodium chloride), 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and water, with a pH value of 7.4;

[0149] Solution of chemical reagent: 1% (w / v) concentration, solvent: ultrapure water, chemical reagent: sodium deoxycholate (SDC);

[0150] Washing solution: deionized water;

[0151] Preservation solution: PBS buffer solution;

[0152] Metal mesh: size of 10 cm x 10 cm, aperture of 3 mm;

[0153] Decellularization device: the decellularization device comprises a high-pressure gas source, a constant-temperature water source, a pressure container, a metal mesh, a pressure gauge, a pressure valve and a vacuum pump; the pressure container is internally divided into two parts, i.e. a sandwich layer and an inner container, and the metal mesh is placed in the inner container; the high-pressure gas source and the vacuum pump are respectively connected to the inner container through a pressure valve, so as to adjust the pressure in the inner container; the pressure valve is used for adjusting and controlling the pressure in the inner container; the constant-temperature water source is connected to the sandwich layer of the pressure container;

[0154] (2) Preprocessing of pericardial tissue;

[0155] The pericardial tissue is washed with a PBS buffer solution to remove blood and external fat, and then the washed pericardial tissue is stored in a PBS buffer solution at 4°C for standby, while the tissue is inspected, and the regions with holes and uneven thickness are discarded, and the regular tissue is selected for cutting to obtain the pretreated pericardial tissue;

[0156] (3) The pretreated pericardial tissue is placed in the inner container of the decellularization device with a capacity of 5L, 3.5L of a solution of chemical reagents is added, the metal mesh is placed and the decellularization device is sealed, then the constant-temperature water source is adjusted to make the temperature in the inner container reach 25°C, and then the pressure in the decellularization device is reduced from normal pressure to a vacuum state of 0.05MPa in two stages, and then vacuum-assisted decellularization is performed for 96h; wherein the pressure reduction is operated by controlling the vacuum pump, the first-stage pressure reduction value in the two-stage pressure reduction is 26.3KPa, and the second-stage pressure reduction value is 25KPa, and the duration of each stage is 15min;

[0157] (4) Washing treatment;

[0158] (4.1) After the vacuum-assisted decellularization is completed, the pressure in the device is restored to normal pressure, the decellularization solution is removed, and then 3.5L of a washing solution is added for soaking and washing under the conditions of normal pressure and 25°C; wherein the soaking and washing is performed for 3 times, and the soaking and washing time of each time is 15min;

[0159] (4.2) After the soaking and washing is completed, 3.5L of a washing solution is added to the decellularization device, which is then sealed, and then periodic pressure difference assisted washing is performed under the conditions of normal pressure and 25°C; wherein, during the periodic pressure difference assisted washing, the number of pressure change cycles is 10, the washing solution is replaced every 5 cycles, and one cycle of pressure change is: the pressure in the decellularization device is reduced from normal pressure to 0.08MPa, maintained for 15min, and then restored to normal pressure, and maintained for 45min.

[0160] (5) After the washing is completed, the pressure is first restored to normal pressure, then PBS buffer solution is added to the device, and the pressure vessel is sealed, then the pericardial tissue after the washing treatment is pressurized at a pressure of 0.5 MPa and a temperature of 25°C for 3 h, and then restored to normal pressure, finally the pericardial tissue after the pressurization is washed with PBS buffer solution, and placed in a storage solution at a temperature of 4°C for wet storage, that is, the cell removal of the pericardial tissue is completed.

[0161] The DNA content remaining in the pericardial tissue after the above cell removal is 40.5 ng / mg, the porosity is 7.8%, the elastic modulus is 36.2 MPa, the ultimate tensile strength is 13.17 MPa, the cell survival rate after 48 h is 70%, and the cell survival rate after 72 h is 81%.

[0162] Example 5

[0163] A pericardial tissue decellularization method for biological valve preparation, the steps are as follows:

[0164] (1) Preparation of raw materials;

[0165] Pericardial tissue: fresh bovine pericardium;

[0166] PBS buffer solution: phosphate buffer solution, composed of 137 mM NaCl (sodium chloride), 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and water, pH 7.4;

[0167] Solution of chemical reagent: 1% (w / v) of sodium dodecyl sulfate (SDS) in ultrapure water;

[0168] Enzyme treatment solution: Tris-HCl buffer solution, composed of 100 mg / L deoxyribonuclease I, 20 mg / L ribonuclease Rnase A and 10 mM Tris-HCl buffer solution;

[0169] Washing solution: PBS buffer solution;

[0170] Storage solution: PBS buffer solution;

[0171] Metal mesh: size of 10 cm x 10 cm, pore size of 3 mm;

[0172] Decellularization device: the decellularization device comprises a high-pressure gas source, a constant-temperature water source, a pressure container, a metal mesh, a pressure gauge, a pressure valve and a vacuum pump; the pressure container is internally divided into two parts, a sandwich and an inner container, and the metal mesh is placed in the inner container; the high-pressure gas source and the vacuum pump are respectively connected to the inner container through a pressure valve, and the pressure in the inner container can be adjusted; the pressure valve is used for adjusting and controlling the pressure in the inner container; the constant-temperature water source is connected to the sandwich of the pressure container;

[0173] (2) Preprocessing of pericardial tissue;

[0174] The pericardial tissue is washed with PBS buffer to remove blood and external fat, and then the washed pericardial tissue is stored in PBS buffer at 4°C for standby, while the tissue is checked, and the regions with holes and uneven thickness are discarded, and the regular tissue is selected for cutting to obtain the pretreated pericardial tissue;

[0175] (3) The pretreated pericardial tissue is placed in the inner container of the decellularization device with a capacity of 5L, 3.5L of a chemical reagent solution is added, a metal mesh is placed and the decellularization device is sealed, then the constant-temperature water source is adjusted to make the temperature in the inner container reach 37°C, and then the pressure in the decellularization device is reduced from normal pressure to 1000Pa vacuum state in two stages, and vacuum-assisted decellularization treatment is performed for 24h; wherein the pressure reduction is operated by controlling the vacuum pump, the first-stage stage pressure reduction value is 50.3KPa, and the second-stage stage pressure reduction value is 50KPa, and the duration of each stage is 15min;

[0176] (4) Enzyme treatment;

[0177] After the vacuum-assisted decellularization is completed, the device returns to normal pressure, and then the pericardial tissue treated by vacuum-assisted decellularization is washed for 15min, and then it is placed in the inner container of the decellularization device, 3.5L of enzyme treatment solution is added, and then it is placed in the metal mesh and sealed, and the constant-temperature water source is adjusted to make the temperature in the inner container reach 37°C, and then vacuum-assisted decellularization treatment is performed for 6h; wherein the pressure in the decellularization device is reduced from normal pressure to 1000Pa vacuum state in two stages during the vacuum-assisted decellularization treatment, and the pressure reduction is operated by controlling the vacuum pump, the first-stage stage pressure reduction value is 50.3KPa, and the second-stage stage pressure reduction value is 50KPa, and the duration of each stage is 15min.

[0178] (5) Washing treatment;

[0179] (5.1) After the enzymatic treatment is completed, the device is returned to normal pressure, the decellularization solution is removed, and 3.5 L of washing solution is added for immersion washing under the conditions of a pressure of normal pressure and a temperature of 37°C; wherein the immersion washing is performed 3 times, and each immersion washing is performed for 15 min;

[0180] (5.2) After the immersion washing is completed, 3.5 L of washing solution is added to the device, which is then sealed, and periodic pressure difference assisted washing is performed under the conditions of a pressure of normal pressure and a temperature of 37°C; wherein, during the periodic pressure difference assisted washing, the number of pressure change cycles is 10, the washing solution is replaced every 5 cycles, and one cycle of pressure change is: the pressure in the decellularization device is first reduced from normal pressure to 0.07 MPa, maintained for 15 min, then returned to normal pressure, and maintained for 45 min;

[0181] (6) After the washing is completed, the pressure is first returned to normal pressure, then PBS buffer is added to the device, the pressure vessel is sealed, and the washed pericardial tissue is pressurized for 3 h at a pressure of 0.5 MPa and a temperature of 37°C, then returned to normal pressure, finally, the pressurized pericardial tissue is rinsed with PBS buffer and placed in a storage solution at a temperature of 4°C for wet storage, thereby completing the cell removal of the pericardial tissue.

[0182] The DNA content in the above-mentioned pericardial tissue after cell removal is 30.3 ng / mg, the porosity is 8.02%, the elastic modulus is 43.3 MPa, the ultimate tensile strength is 16.85 MPa, the cell survival rate after 48 h is 78%, and the cell survival rate after 72 h is 90%.

Claims

1. A method for decellularization of pericardial tissue for bioprosthetic valve production, the method comprising: Firstly, the pericardial tissue is subjected to vacuum assisted decellularization by using a stepwise decompression method to reduce the pressure to a target vacuum state, then subjected to washing treatment, and finally subjected to pressurization treatment to complete the cell removal of the pericardial tissue; ​ The stepwise decompression method refers to decompression from normal pressure to a target vacuum state in stages; the decompression process includes at least two stages of stepwise decompression, and after each stage of stepwise decompression is completed, the pressure is maintained for a period of time; The washing treatment is performed by using a periodic pressure difference assisted washing method; when the periodic pressure difference assisted washing is performed, the number of cycles of pressure change is not less than 5, and one cycle of pressure change is: first, the pressure in the decellularization device is reduced from normal pressure to a target pressure, maintained for 15 min, and then restored to normal pressure, maintained for 45 min; the target pressure is in the range of 0.07-0.09 MPa.

2. The method for decellularizing pericardial tissue for preparing biological valves according to claim 1, characterized in that, The decellularization process of the pericardial tissue is carried out in a decellularization device; the temperature in the decellularization device during vacuum assisted decellularization, washing treatment and pressurization treatment is the same, which is 4-37℃.

3. The method for decellularizing pericardial tissue for preparing biological valves according to claim 2, characterized in that, The pressure in the decellularization device at the target vacuum state is 100 Pa-0.05 MPa.

4. The method for decellularizing pericardial tissue for preparing biological valves according to claim 3, characterized in that, The decompression range of each adjacent two pressure stages is 1-52 KPa, and the stable time of each stage of pressure during the decompression process is 15 min.

5. A method for decellularizing pericardial tissue for preparing biological valves according to claim 4, characterized in that, The vacuum assisted decellularization time is 12-96 h.

6. The method of claim 5, wherein the pericardial tissue is decellularized for the preparation of a bioprosthetic valve. The periodic pressure difference assisted washing is preceded by preliminary washing, which refers to soaking washing using a washing solution under normal pressure, and the soaking time is 15 min.

7. The method of claim 6, wherein the pericardial tissue is decellularized for the preparation of a bioprosthetic valve. The washing solution used in the preliminary washing and the periodic pressure difference assisted washing is the same, which is distilled water, deionized water, phosphate buffer, ethanol solution or sodium chloride aqueous solution.

8. The method of claim 7, wherein the pericardial tissue is decellularized for the preparation of a biological valve. The pressure of the pressurization treatment is 0.2-0.8 MPa, and the pressurization holding time is 1-6 h.

Images