Acquisition and preservation method of allogenic porcine pulmonary artery valved ducts

Abstract

The invention belongs to the technical field of medical tissue acquisition and preservation, and particularly relates to an acquisition and preservation method of an allogenic porcine pulmonary artery valved conduit. According to the acquisition method disclosed by the invention, the valved conduit is acquired from the isolated heart obtained from the Wuzhishan pig, the matching degree is better, the growth potential is better, and the development requirement of a patient is favorably met; particularly, when the valved pipelines are obtained from the isolated heart of the Wuzhishan pig, the number of the valved pipelines is large, the sources are rich, the manufacturing cost of the valved pipelines can be greatly reduced, and the cost is lower. On the basis, the invention also provides a preservation method of the pipeline with the valve, and the preservation method not only reasonably preserves the pipeline with the valve, but also detects virus and bacterial infection conditions of the pipeline with the valve so as to eliminate pollution of pathogenic microorganisms to the pipeline with the valve.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical tissue acquisition and preservation, and particularly relates to a method for acquiring and preserving a xenogeneic porcine pulmonary artery valved conduit. Background Art

[0002] A valved conduit is an important medical device used to repair or replace blood vessels damaged by diseases and the heart valves connected thereto. Such a conduit can not only provide a blood flow passage, but also ensure unidirectional blood flow through the self - contained valve to prevent backflow. Valved conduits are widely used in the treatment of complex cardiovascular diseases, such as repairing right ventricular outflow tract defects (such as tetralogy of Fallot) in congenital heart diseases or replacing damaged aortas.

[0003] Valved conduits can be classified into the following categories according to their sources: ① Allogeneic conduits: derived from human donors and usually used for transplantation after cryopreservation treatment. ② Synthetic conduits: prepared from artificial materials (such as polyester fiber), which are highly durable but lack biological activity and are prone to thrombus formation. ③ Xenogeneic valved conduits: derived from animal (such as pig or cow) tissues and transplanted into the human body after decellularization or anti - calcification treatment.

[0004] In practical applications, xenogeneic valved conduits (i.e., xenotransplantation) have gradually become the core solution to the problem of donor shortage due to their sufficient donor sources and processing potential. However, most of the existing valved conduits still have problems with durability, high cost, and poor matching. The reasons for the durability problem are: ① Immune rejection reaction: Even after decellularization or gene editing, xenogeneic tissues may still cause immune rejection reactions after entering the human body. This reaction can cause inflammation, calcification, or functional degradation of tissues, thereby shortening the service life of valved conduits. ② Biological degradation: Biological tissues will degenerate in the human body due to the influence of the biochemical environment, such as calcification or fibrosis. Especially in a high-pressure cardiovascular environment, this degradation will cause the valve function of the valved conduit to gradually lose, increasing the risk of reoperation. The high cost is due to: ① Complex production process: Taking xenogeneic valved conduits as an example, their production requires multiple high-precision processes, including decellularization, antigen removal, structural repair, and sterilization. In addition, to ensure safety, strict immunogenicity and biomechanical properties testing are also required, which significantly increases production costs. ② Strict donor screening: Whether it is the same donor or a xenogeneic donor, a strict screening procedure is required to ensure that the tissue is free of pathogen contamination or potential immune risks. For example, the breeding environment of xenogeneic donors (such as Wuzhishan pigs) needs to be highly controlled to avoid potential infection risks, which further increases costs. ③ High transportation and storage costs: Valved conduits need to be stored at low temperatures (usually -80°C to -196°C) before transplantation, and the entire transportation process requires a cold chain, which places high demands on the storage equipment and logistics system and increases the economic burden. The most important thing is that most valved conduits currently still have the problem of poor matching, especially for infants and young children after transplantation. As they grow older, the blood vessels upstream and downstream of the valved conduits will grow and the diameter will increase. However, due to the loss of growth of the valved conduits themselves, there will be a problem of matching during transplantation but relatively narrow valved conduits several years after transplantation. For example, the pulmonary valve of a one-year-old baby is about 6mm. Even if a 16mm or 18mm diameter valved conduit is transplanted, a few years later when the baby grows to 12 years old, the normal diameter will be 20-24mm. However, the 16 or 18mm diameter valved conduit is relatively narrow at this time, which may cause a series of clinical complications, such as heart failure.

[0005] It can be seen that although the existing valved conduit technology has made significant progress in functionality and donor sources, it still faces challenges of insufficient durability, high cost and poor matching. In particular, the problem of poor matching limits its widespread promotion in clinical practice, especially in medical environments with limited resources. Summary of the invention

[0006] The object of the present invention is to provide a method for obtaining and preserving a xenogeneic porcine pulmonary artery valved conduit. The valved conduit obtained by using the obtaining and preserving method of the present invention has better matching degree and good growth potential, which helps to meet the development needs of patients.

[0007] The present invention provides a method for obtaining a valved conduit. The valved conduit includes a pulmonary artery valved conduit and / or an aortic valved conduit, and the method comprises the following steps: determining the positions of the pulmonary artery and the aorta of an excised heart, and separating the pulmonary artery and the aorta from the gap between the pulmonary artery and the aorta;

[0008] Cutting open the myocardial tissue at 0.8 - 1.5 cm below the pulmonary valve between the pulmonary artery and the right ventricle to obtain the pulmonary artery valved conduit;

[0009] Cutting open the myocardial tissue at 0.8 - 1.5 cm below the aortic valve between the aorta and the left ventricle to obtain the aortic valved conduit;

[0010] The excised heart is obtained from Wuzhishan pigs.

[0011] As a preferred embodiment, the obtaining method further includes post - processing the valved conduit: removing the outer membrane of the pulmonary artery valved conduit, retaining 5 - 10 mm of myocardial tissue under the valve annulus, and removing the extra - myocardial fat at the same time;

[0012] Removing the outer membrane of the aortic valved conduit, and respectively retaining 5 - 10 mm of the left coronary artery and the right coronary artery.

[0013] As a preferred embodiment, the post - processing is carried out in a physiological saline environment.

[0014] The present invention also provides a method for preserving a valved conduit, which comprises the following steps: taking arterial wall tissue from the valved conduit to obtain the valved conduit after material taking and the arterial wall tissue; the valved conduit is the valved conduit obtained by using the obtaining method described in the above solution;

[0015] Placing the valved conduit after material taking into a first preservation device containing a cryopreservation solution, exhausting air and then sealing it for the first time; the cryopreservation solution includes TCM199 tissue culture medium and dimethyl sulfoxide;

[0016] Placing the first preservation device after the first sealing into a second preservation device and sealing it for the second time;

[0017] Placing the second preservation device after the second sealing into a third preservation device and sealing it for the third time;

[0018] The third preservation device after the third sealing is placed at 4°C for the first preservation. After 0.5 - 1.5 h, the third preservation device is placed at -80°C for the second preservation. After 48 - 72 h, the third preservation device is placed in liquid nitrogen for preservation;

[0019] The arterial wall tissue and the TCM199 tissue culture medium are respectively subjected to virus detection and bacterial culture; if the result of the virus detection and / or bacterial culture is positive, the valved conduit is discarded; if the results of the virus detection and bacterial culture are negative, the valved conduit continues to be preserved in liquid nitrogen.

[0020] As a preferred embodiment, the volume ratio of the TCM199 tissue culture medium to dimethyl sulfoxide is 9:1.

[0021] As a preferred embodiment, the volume of the cryopreservation solution in the first preservation device is 20 - 30 mL.

[0022] As a preferred embodiment, the first preservation device, the second preservation device and the third preservation device respectively include biological storage bags.

[0023] As a preferred embodiment, the first sealing method includes sealing with a sealing strip; the second and third sealing methods respectively include heat sealing; during the second sealing, a heat insulation film is covered at the sealing opening of the second preservation device.

[0024] The present invention also provides a thawing method for the cryopreserved valved conduit, including the following steps: removing the second preservation device and the third preservation device of the valved conduit preserved by the preservation method described in the above solution to obtain the valved conduit wrapped with the first preservation device;

[0025] After soaking the valved conduit wrapped with the first preservation device in iodophor for 2 - 3 min, the valved conduit wrapped with the first preservation device is put into physiological saline at 37°C for thawing until the liquid state in the first preservation device is an ice - water mixture state, and then the valved conduit is taken out;

[0026] After confirming the integrity of the valved conduit, the valved conduit is soaked and washed in a sterile TCM199 solution at 37°C and then stored in a sterile TCM199 solution at 37°C.

[0027] As a preferred embodiment, the number of times of the soaking and washing is 3 times, 5 min each time.

[0028] Beneficial effects:

[0029] The present invention provides a method for obtaining a valved conduit. The valved conduit obtained by the method is taken from an excised heart of a Wuzhishan pig, has better matching degree and good growth potential, and is helpful to meet the development needs of patients. Especially when obtaining the valved conduit from the excised heart of a Wuzhishan pig, the quantity is large and the source is abundant, which can greatly reduce the cost of the valved conduit and lower the cost.

[0030] On this basis, the present invention also provides a method for preserving a valved conduit. In addition to reasonably preserving the valved conduit, the preservation method also detects the virus and bacteria infection conditions of the valved conduit to exclude the contamination of the valved conduit by pathogenic microorganisms. Brief Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0032] Figure 1 It is a schematic diagram of the positions of the pulmonary artery and the aorta in Example 1; wherein PA represents the pulmonary artery and Ao represents the aorta;

[0033] Figure 2 It is a schematic diagram of the gap between the pulmonary artery and the aorta and the separation of the pulmonary artery and the aorta in Example 1; wherein PA represents the pulmonary artery and Ao represents the aorta;

[0034] Figure 3 It is a schematic diagram of obtaining the pulmonary artery valved conduit in Example 1; wherein PA represents the pulmonary artery and RVOT represents the right ventricular outflow tract;

[0035] Figure 4 It is a schematic diagram of obtaining the aortic valved conduit in Example 1; wherein PA represents the pulmonary artery and Ao represents the aorta;

[0036] Figure 5 It is a schematic diagram of measuring the valved conduit in Example 1;

[0037] Figure 6 It is a diagram of the valve diameter after the valved conduit transplantation;

[0038] Figure 7 It is the survival rate of children within 2 years after the valved conduit transplantation. Detailed Embodiments

[0039] The present invention provides a method for obtaining a valved conduit. The valved conduit includes a pulmonary artery valved conduit and / or an aortic valved conduit, and the method comprises the following steps: determining the positions of the pulmonary artery and the aorta of the excised heart, and separating the pulmonary artery and the aorta from the gap between the pulmonary artery and the aorta;

[0040] At a position 0.8 - 1.5 cm below the pulmonary valve between the pulmonary artery and the right ventricle, the myocardial tissue is incised to obtain the pulmonary valved conduit;

[0041] At a position 0.8 - 1.5 cm below the aortic valve between the aorta and the left ventricle, the myocardial tissue is incised to obtain the aortic valved conduit;

[0042] The source of the isolated heart is the Wuzhishan pig.

[0043] The present invention determines the positions of the pulmonary artery and the aorta of the isolated heart, and separates the pulmonary artery and the aorta from the gap between the pulmonary artery and the aorta. As an implementation manner, the source of the isolated heart in the present invention is the Wuzhishan pig. The Wuzhishan pig is mainly produced in the Wuzhishan area of Hainan Province, China. At present, artificial cultivation has been realized, and it has the characteristics of wide source and large quantity, which can further reduce the cost of the valved conduit. Moreover, the Wuzhishan pig has a relatively small body size, and the size of its valved conduit is similar to that of an infant valve, and has good compatibility. After transplantation, it can grow with the growth and development of the infant, avoiding secondary surgery caused by valve mismatch due to growth and development.

[0044] After separating the pulmonary artery and the aorta, in the present invention, at a position 0.8 - 1.5 cm below the pulmonary valve between the pulmonary artery and the right ventricle, the myocardial tissue is incised to obtain the pulmonary valved conduit; at a position 0.8 - 1.5 cm below the aortic valve between the aorta and the left ventricle, the myocardial tissue is incised to obtain the aortic valved conduit. As an implementation manner, the present invention dissects from the front to incise the myocardial tissue. In the present invention, the front refers to the front in the anatomical sense, that is, the side of the heart close to the abdomen. As an implementation manner, when obtaining the pulmonary valved conduit in the present invention, the pulmonary valved conduit is obtained by dissecting from the back, and the periphery of the right ventricular outflow tract incision is enlarged, keeping more than 0.5 cm away from the pulmonary valve. In the present invention, the back refers to the back in the anatomical sense, that is, the side of the heart close to the back.

[0045] After obtaining the pulmonary valved conduit and the aortic valved conduit, as an implementation manner, the present invention further includes post-processing the valved conduit: removing the adventitia of the pulmonary valved conduit, retaining 5-10 mm of myocardial tissue under the valve annulus, and at the same time removing the fat outside the myocardium; removing the adventitia of the aortic valved conduit, and retaining the left coronary artery and the right coronary artery of 5-10 mm respectively. As an implementation manner, the present invention retains the left coronary artery and the right coronary artery of 5 mm respectively. As an implementation manner, the post-processing is carried out in a physiological saline environment. As an implementation manner, the post-processed pulmonary valved conduit and aortic valved conduit can be soaked and temporarily stored in physiological saline. As an implementation manner, after obtaining the valved conduit, the present invention further includes measuring the diameter of the valved conduit, the length of the main pulmonary artery, the upper half circumference of the valve annulus, the anterior-posterior and left-right diameters of the mitral valve, the diameter of the anterior leaflet, the length of the papillary muscle chordae tendineae, the diameter of the tricuspid valve leaflet, and the length of the papillary muscle chordae tendineae.

[0046] The present invention also provides a method for storing a valved conduit, including the following steps: taking arterial wall tissue from the valved conduit to obtain the valved conduit after sampling and the arterial wall tissue; the valved conduit is the valved conduit obtained by using the acquisition method described in the above-mentioned solution;

[0047] Placing the valved conduit after sampling in a first storage device containing a cryopreservation solution, exhausting the air, and then sealing it for the first time; the cryopreservation solution includes TCM199 tissue culture medium and dimethyl sulfoxide;

[0048] Placing the first storage device after the first sealing in a second storage device and sealing it for the second time;

[0049] Placing the second storage device after the second sealing in a third storage device and sealing it for the third time;

[0050] Placing the third storage device after the third sealing at 4°C for the first storage, after 0.5-1.5 h, placing the third storage device at -80°C for the second storage, and after 48-72 h, placing the third storage device in liquid nitrogen for storage;

[0051] Performing virus detection and bacterial culture on the arterial wall tissue and the TCM199 tissue culture medium respectively; if the results of the virus detection and / or bacterial culture are positive, discarding the valved conduit, and if the results of the virus detection and bacterial culture are negative, continuing to store the valved conduit in liquid nitrogen.

[0052] The present invention places the valved conduit after material collection into a first storage device containing cryopreservation solution. After exhausting air, it is sealed for the first time. As an implementation manner, the cryopreservation solution of the present invention can be composed of TCM199 tissue culture medium and dimethyl sulfoxide, and the volume ratio of the TCM199 tissue culture medium to dimethyl sulfoxide is 9:1. As an implementation manner, the first storage device can be a biological storage bag. As an implementation manner, the volume of the cryopreservation solution in the first storage device is 20 - 30 mL. In a specific embodiment of the present invention, 20 - 30 mL of the preservation solution is required to preserve one valved conduit. As an implementation manner, the first sealing method can be sealing with a sealing strip.

[0053] After the first sealing, the present invention places the first storage device after the first sealing into a second storage device and seals it for the second time. As an implementation manner, the second storage device can be a biological storage bag. As an implementation manner, the second sealing method can be heat-sealing, such as using a heat-sealing machine for sealing. As an implementation manner, when performing the second sealing, a heat-insulating film is covered at the sealing part of the second storage device.

[0054] As an implementation manner, after the second sealing, the present invention places the second storage device after the second sealing into a third storage device and seals it for the third time. As an implementation manner, the third storage device can be a biological storage bag. As an implementation manner, the third sealing method can be heat-sealing, such as using a heat-sealing machine for sealing.

[0055] After the third sealing, the present invention places the third storage device after the third sealing under the condition of 4°C for the first storage. After 0.5 - 1.5 h, the third storage device is placed under the condition of -80°C for the second storage. After 48 - 72 h, the third storage device is placed in liquid nitrogen for storage. As an implementation manner, the time for the first storage can be 1 h.

[0056] The arterial wall tissue and the TCM199 tissue culture medium are respectively subjected to virus detection and bacterial culture. As an implementation manner, the size of the arterial wall tissue can be 0.2 - 1 g. As an implementation manner, the amount of the TCM199 tissue culture medium can be 5 mL. As an implementation manner, the virus detection and bacterial culture are carried out with reference to the standards in

HealthCareEDftQoM.Guide to the quality and safety of tissues and cells for human application. 5th Edition ed. 2022.

[0057] The present invention also provides a thawing method for a cryopreserved valved conduit, comprising the following steps: removing the second storage device and the third storage device of the valved conduit stored by the storage method according to the above technical solution to obtain a valved conduit wrapped with the first storage device;

[0058] After soaking the valved conduit wrapped with the first storage device in iodophor for 2 - 3 min, the valved conduit wrapped with the first storage device is put into physiological saline at 37°C for thawing until the liquid state in the first storage device is an ice - water mixture state, and then the valved conduit is taken out;

[0059] After confirming the integrity of the valved conduit, the valved conduit is soaked and washed in sterile TCM199 tissue culture medium at 37°C and then stored in sterile TCM199 tissue culture medium at 37°C. The TCM199 tissue culture medium of the present invention can protect the cells of the valved conduit and prevent them from dying.

[0060] As an implementation manner, the number of times of soaking and washing can be 3 times, 5 min each time. Soaking the valved conduit wrapped with the first storage device in iodophor in the present invention can achieve disinfection and prevent the transmission of pathogenic microorganisms. The advantage of thawing the valved conduit wrapped with the first storage device in physiological saline at 37°C in the present invention is that 37°C is consistent with the human physiological environment. Thawing with 37°C physiological saline can reduce the damage to the valved conduit caused by temperature changes; and physiological saline is an isotonic solution, which can maintain the cell osmotic pressure and keep the cell integrity.

[0061] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0062] Example 1

[0063] A method for obtaining a valved conduit, comprising the following steps:

[0064] 1. Preparation before obtaining valved conduit

[0065] 1. Prepare the pathological specimen bags in advance and send them to the ethylene oxide room for disinfection;

[0066] 2. Prepare the equipment package and packaging package in advance and sterilize them under high temperature and high pressure in the supply room;

[0067] 3. 50 minutes before operation, turn on the ultraviolet irradiation on the clean bench and the operating room for 20 minutes, turn off the ultraviolet irradiation and turn on the fan for 10 minutes;

[0068] 4. Acquisition of the heart:

[0069] (1) Open the chest in the middle, open the skin and subcutaneous tissue layer by layer, split the sternum, and use a spreader to spread the sternum;

[0070] (2) Remove the thymus, cut the pericardium, and expose the pulmonary artery trunk and ascending aorta;

[0071] (3) Separate the tissues around the heart, retain the main pulmonary artery to the pulmonary artery bifurcation, and retain at least 2 cm of the ascending aorta;

[0072] 5. Remove the heart completely, taking care not to damage the supravalvular and subvalvular tissues of the pulmonary artery and aortic valve;

[0073] 6. Place the heart in a sterile plastic bag (single layer) and have an assistant move it from the operating room to the cell room;

[0074] 7. The trimmer brushes his hands and enters the cell room, puts on surgical gown and sterile gloves with the help of an assistant;

[0075] 8. Open the dressing package, take the middle sheet and treatment towel and lay them in 4 to 6 layers from outside to inside and from left to right;

[0076] 9. The assistant opens the outer packaging of the instrument package, and the trimmer opens the inner packaging, takes out the stainless steel tube and instruments, and places them neatly on the sterile table. The assistant fills 2 bags of 0.9% saline solution (500 mL) for the trimmer, and pours them into a stainless steel bucket for later use;

[0077] 10. The assistant pulls down the clean bench operation window, leaving it 30cm high;

[0078] 11. The assistant holds the edges of the packaging bag containing the donor heart with both hands and turns the opening of the packaging bag outward (note that the inner side of the packaging bag must not be touched); the trimmer takes out the donor heart and places it in a pre-prepared kidney dish.

[0079] 12. Use a 2 mL syringe to draw out the remaining liquid in the inner plastic bag, cover the needle and the protective cap, and place it aside.

[0080] 13. Rinse the heart, gently squeeze to drain the blood and normal saline in the heart cavity, hold the heart with one hand and lift the rinsing basin with the other hand to pour the rinsing liquid into the medical waste bag.

[0081] 14. Repeat the rinsing once to obtain the heart.

[0082] II. Obtaining the valved conduit, the steps are as follows:

[0083] 1. Place the heart obtained in Step 1, distinguish the structures, and determine the positions of the pulmonary artery (PA) and the aorta (Ao), as Figure 1 shown.

[0084] 2. Carefully cut open the gap between the pulmonary artery and the aorta, and separate the pulmonary artery and the aorta, as Figure 2 shown.

[0085] 3. Dissect from the front (the side of the heart close to the abdomen), and cut open the myocardial tissue 1 cm below the pulmonary valve, as Figure 3 shown.

[0086] 4. Dissect the valved pulmonary artery conduit from the back (the side of the heart close to the back), expand the periphery of the right ventricular outflow tract incision, and keep a safe distance from the pulmonary valve (PV) (separation can be carried out from the inside to the outside or from the outside to the inside), as Figure 4 shown, where the left figure is the schematic diagram of separation from the outer layer and the right figure is the schematic diagram of separation from the inner layer;

[0087] ① The separation method of the aortic valved conduit is the same as that of the pulmonary artery (repeat Steps 3 and 4), and keep the left and right coronary arteries 5 - 10 mm.

[0088] ② It is not recommended to directly extend the incision in Step 4 to the back. It should be cut open both in the front and the back and then gradually separated.

[0089] 5. Pour 50 mL of normal saline from the stainless steel cylinder into the kidney dish, place the removed valved conduit into it, remove the adventitia of the aorta, carefully separate the left and right coronary artery branches, cut off the left and right coronary arteries while keeping 5 mm; take out the valved pulmonary artery conduit, carefully remove the adventitia, trim the myocardium under the valve ring, keep 5 mm, remove the fat outside the myocardium, and soak it in the stainless steel cylinder filled with normal saline.

[0090] 6. Measure the diameter of the valved conduit with a probe (or stainless steel ruler), and measure the length of the main pulmonary artery, the upper half circumference of the valve annulus, the anterior-posterior and left-right diameters of the mitral valve, the diameter of the anterior leaflet, the length of the papillary muscle chordae tendineae, the diameter of the tricuspid valve leaflets, and the length of the papillary muscle chordae tendineae with a stainless steel ruler. Have the assistant record them one by one, as Figure 5 shown.

[0091] Example 2

[0092] A method for processing and preserving a valved conduit is as follows:

[0093] 1. The trimmer changes into sterile gloves and lays 2 layers of dressings on the sterile laminar flow bench;

[0094] 2. The assistant opens the outer package of the packaging bag, and the trimmer takes out the packaging bag and places it on the laminar flow bench for standby;

[0095] 3. The assistant prepares 2 50 mL syringes for the trimmer;

[0096] 4. The assistant lights the alcohol lamp, removes the packaging at the mouth of the TCM199 tissue culture medium bottle, picks up 1 piece of alcohol gauze with forceps, wipes the mouth of the bottle, tilts it at 30 degrees, rotates the bottle body, and passes it over the outer flame of the alcohol lamp for 2 - 3 circles;

[0097] 5. The assistant takes the TCM199 tissue culture medium to the edge of the laminar flow bench, opens the bottle mouth facing the laminar flow bench, keeps the bottle cap in the same direction as the bottle mouth and close to the bottle mouth, paying attention to aseptic operation;

[0098] 6. The trimmer withdraws 45 mL of TCM199 tissue culture medium with a 50 mL syringe;

[0099] 7. The assistant repeats steps 5 - 6 to open DMSO, and the trimmer withdraws 5 mL and mixes it with 45 mL of TCM199 tissue culture medium to prepare a 10% DMSO cryopreservation solution;

[0100] 8. The assistant places the heat sealer next to the laminar flow bench and turns on the power supply;

[0101] 9. Use non - traumatic forceps to take out the valved conduit, cut small pieces of arterial wall and put them into a stainless steel bucket (for virus detection and bacterial culture).

[0102] 10. Put the valved conduit into the innermost bag, add 20 - 30 mL of cryopreservation solution, expel the air in the bag, fold the sealing strip at the bag mouth inwards for 2 - 3 circles, and seal the packaging bag;

[0103] 11. Put it into the second - layer packaging bag, cover a heat - insulating film at the bag mouth, place it at the heat sealer, and have the assistant press the heat sealer button 3 times continuously;

[0104] 12. The trimmer takes out the package with the heat-insulating film. After 5 s, pinch the side facing the plastic bag and tear off the heat-insulating film. Take it back to the laminar flow hood and check for any liquid leakage. Note that when sealing, do not touch the heat sealer.

[0105] 13. Cut off the excess plastic bag at the sealed part, put it into the third-layer plastic bag, and directly place it at the heat sealer. Ask the assistant to press the heat sealer button, and after cooling, ask the assistant to take it off.

[0106] 14. The assistant uses a low-temperature marker pen to mark the valve number and date on the outermost packaging bag. At the same time, stick an adhesive plaster for marking, put it into the 4 °C refrigerator, and record the time.

[0107] 15. Inject the initially drawn TCM199 tissue culture medium and the small piece of arterial wall taken respectively into the bacterial culture bottle, tighten the bottle cap, hand the 2 culture bottles to the assistant, send them to the laboratory department for virus detection and bacterial culture. The virus detection and bacterial culture are carried out according to the standards in

HealthCare EDftQoM.Guide to the quality and safety of tissues and cells for human application. 5th Edition ed. 2022.

[0108] 16. Check the items, classify and process all medical wastes as required, put them into medical garbage bags, domestic garbage bags and sharp containers respectively. Send the samples to be tested to the laboratory department, and send the used dressings and instruments to the supply room for disinfection.

[0109] 17. (After about 1 h) Take out the valved conduit in the 4 °C refrigerator and transfer it to the -80 °C refrigerator.

[0110] 18. After 12 h, transfer the valved conduit to liquid nitrogen for storage.

[0111] 19. Pay attention to the results from the laboratory department. If the laboratory reports that the sample culture result is positive, immediately discard the valved conduit, and all data should be entered into the computer for storage.

[0112] Example 3

[0113] Thawing and use of the valved conduit are as follows:

[0114] 1. Carefully take out the cryopreserved valved conduit from the liquid nitrogen storage tank, and immediately transfer it to the operating room with a portable liquid nitrogen tank.

[0115] 2. Carefully open the outer packaging of the allogeneic valved conduit and place it on the operating table ("put it on the sterile table").

[0116] 3. Immerse the inner packaging completely in iodophor for 2 - 3 min to ensure that the liquid can cover the entire packaging.

[0117] 4. Remove the inner package from the povidone-iodine solution and immediately place it in physiological saline at 37°C for thawing;

[0118] 5. Observe the liquid state in the inner package. When the liquid reaches the ice-water mixture state, take out the valved conduit and carefully check its integrity, including whether there are cracks, damages or other abnormalities in the valve leaflets and the pipe wall;

[0119] 6. Prepare sterile TCM199 solution at 37°C and soak the valved conduit 3 times, 5 minutes each time. After each soaking, a new nutrient solution should be replaced to ensure thorough cleaning.

[0120] 7. After soaking, store the AVC in TCM199 solution at 37°C for further processing and implant preparation before surgery.

[0121] Example 4

[0122] Transplantation of the valved conduit

[0123] Transplant the valved conduit of Example 3 into the child. Perform right ventricular outflow tract reconstruction surgery on the child about 180 - 200 days after birth. Observe the valve diameter of the child after transplantation and the valve diameter of a healthy infant. The results of the valve diameter of one child and a healthy infant are shown in Figure 6 In Table 1, the actual valve diameter in the figure represents the valve diameter of the transplanted patient, and the standard diameter of the table membrane represents the valve diameter of a healthy infant.

[0124] Table 1 Statistics of the diameter after implantation of the xenogeneic valved conduit

[0125]

[0126]

[0127] According to Table 1 and Figure 6 It can be seen that after transplantation of the valved conduit prepared by the method of the present invention, it can grow normally and has a small gap compared with healthy infants.

[0128] In subsequent follow-up experiments, it was found that for xenotransplantation using the valved conduit obtained by the method of the present invention, the survival rate of children within 2 years is about 99% (see Figure 7, the sample size was 70), which was higher than 95% of the records in the prior art

Romeo JLR et al. Long-term clinical outcome and echocardiographic function of homografts in the right ventricular outflow tract. Eur J Cardiothorac Surg 2019; 55: 518-26.

[0129] In summary, the valved conduit obtained by using the method of the present invention has a better matching degree and good growth potential, which helps to meet the development needs of patients; and when obtaining the valved conduit from the in vitro heart of Wuzhishan pigs, the quantity is large and the source is rich, which can greatly reduce the cost of the valved conduit and the cost is lower.

[0130] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A method for obtaining a valved conduit, wherein the valved conduit includes a pulmonary artery valved conduit and / or an aortic valved conduit, characterized in that: The method comprises the following steps: determining the positions of the pulmonary artery and the aorta of the isolated heart, and separating the pulmonary artery and the aorta from the gap between the pulmonary artery and the aorta; Cut the myocardial tissue 0.8 to 1.5 cm below the pulmonary valve between the pulmonary artery and the right ventricle to obtain the pulmonary artery valved conduit; Cut the myocardial tissue 0.8 to 1.5 cm below the aortic valve between the aorta and the left ventricle to obtain the aortic valve conduit; The isolated heart is obtained from Wuzhishan pig.

2. The acquisition method according to claim 1, characterized in that: The acquisition method further includes post-processing the valved conduit: removing the outer membrane of the pulmonary artery valved conduit, retaining 5 to 10 mm of myocardial tissue under the valve ring, and removing extramyocardial fat; The adventitia of the aortic valved conduit is removed, and 5 to 10 mm of the left coronary artery and the right coronary artery are retained respectively.

3. The acquisition method according to claim 2, characterized in that: The post-treatment was performed in a physiological saline environment.

4. A method for preserving a valved conduit, characterized in that: The method comprises the following steps: taking arterial wall tissue from a valved conduit to obtain a valved conduit and arterial wall tissue after sampling; the valved conduit is a valved conduit obtained by the acquisition method according to any one of claims 1 to 3; Placing the sampled valved tube in a first storage device containing a cryopreservation solution, removing air, and then first sealing the device; the cryopreservation solution includes TCM199 tissue culture solution and dimethyl sulfoxide; placing the first storage device after the first sealing in the second storage device, and sealing it a second time; placing the second storage device after the second sealing in a third storage device, and sealing it a third time; The third sealed storage device is placed at 4° C. for first storage, 0.5 to 1.5 hours later, the third storage device is placed at -80° C. for second storage, and 48 to 72 hours later, the third storage device is placed in liquid nitrogen for storage; The arterial wall tissue and the TCM199 tissue culture fluid are subjected to virus detection and bacterial culture, respectively; if the results of the virus detection and / or bacterial culture are positive, the valved conduit is discarded; if the results of the virus detection and bacterial culture are negative, the valved conduit continues to be stored in liquid nitrogen.

5. The storage method according to claim 4, characterized in that: The volume ratio of the TCM199 tissue culture medium to dimethyl sulfoxide is 9:

1.

6. The storage method according to claim 4 or 5, characterized in that: The volume of the freezing solution in the first storage device is 20 to 30 mL.

7. The storage method according to claim 4, characterized in that: The first storage device, the second storage device and the third storage device each include a biological storage bag.

8. The storage method according to claim 7, characterized in that: The first sealing method includes sealing with a sealing strip; the second sealing and third sealing methods include heat sealing respectively; during the second sealing, a heat insulating film is covered on the sealing portion of the second storage device.

9. A method for thawing a frozen valved pipe, characterized in that: The method comprises the following steps: removing the second preservation device and the third preservation device of the valved conduit preserved by the preservation method according to any one of claims 4 to 8 to obtain the valved conduit containing the first preservation device; After soaking the valved tube wrapped with the first preservation device in iodine for 2 to 3 minutes, the valved tube wrapped with the first preservation device is placed in 37° C. physiological saline for thawing until the liquid in the first preservation device is in a state of ice-water mixture, and then the valved tube is taken out; After confirming the integrity of the valved conduit, the valved conduit was immersed and cleaned in a sterile TCM199 solution at 37°C and then stored in a sterile TCM199 solution at 37°C.

10. The thawing method according to claim 9, characterized in that: The soaking and cleaning was performed 3 times, each time for 5 minutes.

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