Sterilizing solution, and pretreatment method and preservation method of same-type valved pipeline

Abstract

The invention provides a sterilizing solution, and a pretreatment method and a preservation method of a same valved pipeline, and belongs to the technical field of medical treatment. The invention provides a sterilizing solution which takes TCM199 as a solvent and comprises the following components: third-generation cephalosporin, amphotericin B, polypeptide antibiotics and aminoglycoside antibiotics. The sterilizing solution disclosed by the invention is reasonably compounded, and has the advantages of protecting the activity of a valvular canal and avoiding transplantation-related infectious endocarditis. The invention further provides a pretreatment method of the allogeneic valvular ducts, different treatment methods are adopted for the allogeneic valvular ducts from donors with different death times or heart transplantation times through a classification and time period division method, transplantation related infection is avoided, and meanwhile the treatment efficiency is improved. And the activity of the allogeneic valved pipeline can be protected as much as possible.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on October 24, 2023, with application number 202311384582.3 and invention name “Method for preparing decellularized valved pipes”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of medical technology, and in particular relates to a sterilizing liquid, a pretreatment method of a homologous valved conduit, and a preservation method thereof. Background Art

[0003] A valved conduit is a vascular conduit with a valve (pulmonary valve or aortic valve), which usually ranges from 5mm above the valve to 5mm below the valve. Depending on the source, valved conduits can be divided into homologous (including autologous and allogeneic) and xenogeneic valved conduits. For patients with severe valvular regurgitation or severe stenosis, the current treatment option is valve replacement, and valve replacement can choose mechanical valves, bioprosthetic valves and homologous valves. The homologous valve here refers to the aforementioned homologous valved conduit. The advantages of allogeneic valved conduits are: excellent hemodynamic performance, reduced risk of thromboembolism without the need for anticoagulation, minimal blood cell damage and low incidence of postoperative endocarditis.

[0004] Traditionally, allogeneic valved conduits need to be sterilized before use. The current sterilization method is to soak in antibiotics uniformly. The antibiotic formula is a balanced salt solution of ceftazidime, lincomycin, polymyxin B, and vancomycin for 24 hours. However, the above treatment may cause the activity and durability of allogeneic valved conduits to deteriorate.

[0005] Therefore, the pretreatment of allogeneic valved conduits derived from human cadavers is crucial. How to avoid transplantation-associated infection while protecting the activity of allogeneic valved conduits as much as possible is an urgent problem that needs to be solved. Summary of the invention

[0006] The purpose of the present application is to provide a sterilizing solution, a pretreatment method for homologous valved conduits and a preservation method thereof. The pretreatment method of the present application can protect the activity of the homologous valved conduits as much as possible while avoiding the occurrence of transplant-related infections.

[0007] The present application provides a sterilizing solution, which uses TCM199 as a solvent and includes the following components in concentrations: 1-4 g / L of a third-generation cephalosporin, 5-20 mg / L of amphotericin B, 0.5-2 g / L of a polypeptide antibiotic, and 200-600 mg / L of an aminoglycoside antibiotic; the pH value of the sterilizing solution is 6.6-7.

[0008] Preferably, the polypeptide antibiotic includes vancomycin or teicoplanin; the vancomycin includes vancomycin hydrochloride for injection; the aminoglycoside antibiotic includes amikacin; the amikacin includes amikacin sulfate; the third-generation cephalosporin includes ceftazidime for injection or ceftriaxone for injection; and the amphotericin B includes amphotericin B for injection.

[0009] Preferably, the composition comprises the following concentrations: 1-2 g / L ceftazidime for injection, 5-10 mg / L amphotericin B, 0.5-1.0 g / L vancomycin hydrochloride for injection and 200-300 mg / L amikacin.

[0010] Preferably, the reagent for adjusting the pH value of the sterilization solution includes 5% sodium bicarbonate injection.

[0011] The present application also provides a pretreatment method for an allogeneic valved conduit, wherein the interval time between the death of the donor and the acquisition of the allogeneic valved conduit is ≤24h; the allogeneic valved conduit is pretreated as follows according to the interval time: when the interval time is ≤6h, the allogeneic valved conduit is cleaned; the reagent used for the cleaning includes 0.9% sodium chloride injection; when the interval time is 6h<≤24h, the allogeneic valved conduit is cleaned and then immersed in the sterilizing solution described in the above scheme for sterilization; the sterilization temperature is 0-4°C, and the sterilization time is 20-24h.

[0012] The present application also provides a product for treating tetralogy of Fallot, comprising a homologous valved conduit after prognostic treatment obtained by the pretreatment method described in the above scheme, antibiotics for routine prevention of infection during surgery, antibiotics for prevention of drug-resistant bacterial infection, broad-spectrum antibiotics and antibiotics for heart transplantation.

[0013] Preferably, the antibiotics used for routine prevention of surgical infection include cefuroxime sodium; the antibiotics used to prevent drug-resistant bacterial infection include cefoperazone sulbactam sodium for injection; the broad-spectrum antibiotics include moxifloxacin hydrochloride injection; the antibiotics for heart transplantation include teicoplanin for injection, meropenem for injection and micafungin sodium for injection.

[0014] The present application also provides a method for preserving the homologous valved conduit after pretreatment obtained by the pretreatment method described in the above scheme, comprising the following steps: placing the homologous valved conduit after pretreatment in a cryoprotectant bag, freezing it at -70 to -90°C for 12 to 24 hours, and then transferring it to liquid nitrogen for storage.

[0015] Preferably, the cryopreservation protection solution is based on TCM199 culture medium and also includes dimethyl sulfoxide with a volume concentration of 10% to 15%.

[0016] Preferably, after being transferred into liquid nitrogen for storage, the process further comprises thawing; the thawing temperature is 37° C.; and the thawing time is 10 to 30 minutes.

[0017] The present application provides a sterilizing solution, which uses TCM199 as a solvent and includes the following components: a third-generation cephalosporin, amphotericin B, polypeptide antibiotics, and aminoglycoside antibiotics. After a person dies, as the time of death increases, the bacteria colonized in the body, such as Escherichia coli, Salmonella typhi, and other Gram-negative bacilli, will multiply in large numbers, and the risk of valve infection will be greatly increased. The sterilizing solution of the present application can not only effectively resist drug-resistant Gram-positive cocci, but also effectively resist common Gram-negative bacilli such as Escherichia coli and Salmonella typhi, and has a broad-spectrum antibacterial property. In addition, the sterilizing solution of the present application can not only effectively sterilize, but also reduce damage to the same type of valved conduit, protect the activity of the same type of valved conduit as much as possible, and thus ensure the clinical effect after valved conduit transplantation. From the comparison results of clinical outcomes after homologous valved conduit transplantation treated with the sterilization solution formula of this application and the traditional sterilization solution formula, it can be seen that the probability of arrhythmia, heart failure, infective endocarditis and valvular failure (stenosis or moderate or above regurgitation) after homologous valved conduit transplantation treated with the sterilization solution formula of this application is significantly reduced compared with the traditional formula. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0019] Figure 1 This is a flow chart of Example 1. DETAILED DESCRIPTION

[0020] The present application provides a sterilizing solution, which uses TCM199 as a solvent and includes the following components in concentrations: 1-4 g / L of a third-generation cephalosporin, 5-20 mg / L of amphotericin B, 0.5-2 g / L of a polypeptide antibiotic, and 200-600 mg / L of an aminoglycoside antibiotic; the pH value of the sterilizing solution is 6.6-7.

[0021] In the present application, the sterilizing solution uses TCM199 as solvent and is preferably composed of the following components in the following concentrations: third-generation cephalosporin 1-4 g / L, amphotericin B 5-20 mg / L, polypeptide antibiotics 0.5-2 g / L and aminoglycoside antibiotics 200-600 mg / L.

[0022] In the specific implementation of the present application, the polypeptide antibiotic includes vancomycin or teicoplanin; the vancomycin includes vancomycin hydrochloride for injection; the aminoglycoside antibiotic includes amikacin; the amikacin includes amikacin sulfate; the third-generation cephalosporin includes ceftazidime for injection or ceftriaxone for injection; and the amphotericin B includes amphotericin B for injection.

[0023] In the specific implementation of the present application, the sterilizing solution includes the following components in concentrations: 1-2 g / L of ceftazidime for injection, 5-10 mg / L of amphotericin B, 0.5-1 g / L of vancomycin hydrochloride for injection, and 200-300 mg / L of amikacin.

[0024] In one embodiment of the present application, the sterilization solution is composed of the following components in concentrations: 1 g / L ceftazidime for injection, 5 mg / L amphotericin B for injection, 0.5 g / L vancomycin hydrochloride for injection, and 200 mg / L amikacin sulfate.

[0025] In the specific implementation process of the present application, the reagent for adjusting the pH value of the sterilization solution includes 5% sodium bicarbonate injection.

[0026] The present application also provides a pretreatment method for an allogeneic valved conduit, wherein the interval time between the death of the donor and the acquisition of the allogeneic valved conduit is ≤24h; the allogeneic valved conduit is pretreated as follows according to the interval time: when the interval time is ≤6h, the allogeneic valved conduit is cleaned; the reagent used for the cleaning includes 0.9% sodium chloride injection; when the interval time is 6h<≤24h, the allogeneic valved conduit is cleaned and then immersed in the sterilizing solution described in the above scheme for sterilization; the sterilization temperature is 0-4°C, and the sterilization time is 20-24h.

[0027] The interval between the death of the donor and the acquisition of the allogeneic valved conduit is related to the death time of the donor. The present application adopts different treatment methods for allogeneic valved conduits from cadavers at different death times, which can protect the activity of the allogeneic valved conduits as much as possible while avoiding transplant-related infection.

[0028] In the specific implementation of the present application, the homograft valved conduit is an allogeneic valved conduit.

[0029] During the specific implementation of this application, the donor has no infectious diseases such as tuberculosis, hepatitis, syphilis, AIDS, malaria, leprosy, history of sepsis and tumor; big triple positive, small triple positive, and single HBsAg positivity are excluded; single HBsAb positivity is not excluded, single HBcAb positivity or HBcAb+HBsAb positivity is not excluded, but requires a note; no large-area burns, autoimmune disease, history of cardiac surgery, valvular disease, cardiomyopathy, untreated active lung disease, rheumatic fever, bacterial endocarditis, prolonged closed CPR, and serious diseases of unknown cause; age is under 60 years old, and age over 50 years old requires a note.

[0030] In the specific implementation of the present application, the homologous valved conduit is taken from a fresh heart or a cadaveric heart replaced by a heart transplant recipient. In the present application, the fresh heart or a cadaveric heart replaced by a heart transplant recipient has no arterial wall damage, coronary artery calcification, valve closure stenosis or regurgitation.

[0031] The homologous valved conduit of the present application can be obtained from a fresh heart replaced by a heart transplant recipient, or from a cadaveric heart, thereby expanding the range of valved conduit material selection and solving the problem of donor shortage of human homologous pulmonary artery valved conduits.

[0032] In the specific implementation process of the present application, the cold ischemia time of the fresh heart is ≤24h.

[0033] In the present application, when the interval between the death of the donor and the acquisition of the allogeneic valved conduit is ≤6h, the allogeneic valved conduit is only cleaned without sterilization, which can improve the vascular compliance of the valved conduit and its similarity to the natural valved conduit, and reduce the damage to the valved conduit caused by antibiotic disinfection during the treatment process. In addition, the allogeneic valved conduit of the present application can be customized according to the recipient's heart, and the valved conduit can be trimmed to a suitable length, avoiding direct use of antibiotics to damage the valve during the cleaning process, and has the advantage of extremely short cold ischemia time.

[0034] The present application has no particular restrictions on the method of obtaining a fresh heart or a cadaveric heart replaced by a heart transplant recipient, and the method of obtaining a cadaveric valved conduit known in the art can be used.

[0035] Prior to the cleaning, the present application also includes sampling the same type of valved conduit to detect whether there is bacterial contamination.

[0036] After the cleaning, the present application also includes removing the outer membrane of the cleaned homogeneous valved conduit, removing the extramyocardial fat, and soaking it in a container; the container includes a stainless steel barrel; the solution used for soaking includes 0.9% sodium chloride solution (normal saline); the soaking time is 1 to 2 minutes; the soaking temperature is 20 to 30°C, preferably 20 to 25°C.

[0037] The present application also provides a product for treating tetralogy of Fallot, comprising a homologous valved conduit after prognostic treatment obtained by the pretreatment method described in the above scheme, antibiotics for routine prevention of infection during surgery, antibiotics for prevention of drug-resistant bacterial infection, broad-spectrum antibiotics and antibiotics for heart transplantation.

[0038] During the specific implementation of this application, when the interval time is ≤2h, the product also includes a first antibiotic for preventing infection; when 2h<interval time ≤4h, the product also includes a second antibiotic for preventing infection; when 4h<interval time ≤6h, the product also includes a third antibiotic for preventing Gram-positive coccal infection; when 6h<interval time ≤8h, the product also includes antibiotics for routine surgical infection prevention; when 8h<interval time ≤12h, the product also includes broad-spectrum antibiotics; when 12h<interval time ≤24h, the product also includes antibiotics for heart transplantation.

[0039] During the specific implementation of this application, the antibiotics used for routine prevention of surgical infection include cefuroxime sodium; the antibiotics used to prevent drug-resistant bacterial infection include cefoperazone sulbactam sodium for injection; the broad-spectrum antibiotics include moxifloxacin hydrochloride injection; the antibiotics for heart transplantation include teicoplanin for injection, meropenem for injection and micafungin sodium for injection.

[0040] In the specific implementation of this application, the product also includes 0.9% sodium chloride injection, which is used to dissolve the antibiotics described in the above scheme.

[0041] In one embodiment of the present application, the interval between the death of the donor and the acquisition of the allogeneic valved conduit is ≤2h. After the allogeneic valved conduit is thoroughly cleaned and the bacterial culture is negative after the valved conduit is acquired, the possibility of infection is very low. The patient (recipient) is given antibiotics prophylactically for a Class I incision for more than 4 hours after surgery: 1.5g of Cefuroxime Sodium for Injection (Ancoxin) + 100ml of 0.9% Sodium Chloride Injection intravenously twice a day. The use of antibiotics after surgery does not exceed 48h.

[0042] In another embodiment of the present application, if the interval between the death of the donor and the acquisition of the allogeneic valved conduit is greater than 2 hours and ≤ 4 hours, the probability of infection increases, and the patient (recipient) is given antibiotics prophylactically for a Class I incision of more than 4 hours after surgery: 3g of cefoperazone sulbactam sodium (Supsen) for injection + 100ml of 0.9% sodium chloride injection intravenously, once every 12 hours. The postoperative antibiotic use time does not exceed 48 hours.

[0043] In another embodiment of the present application, the interval between the death of the donor and the acquisition of the allogeneic valved conduit is greater than 4h and ≤6h, the probability of infection is further increased, and the possibility of infection with Gram-positive cocci such as Staphylococcus epidermidis is increased. The patient (recipient) is given antibiotics according to the infected incision after surgery: 0.4g of moxifloxacin hydrochloride injection (Baifule) is intravenously injected once a day. Antibiotics are used for 5 to 7 days after surgery, and the patient's body temperature and other vital signs are dynamically monitored. On the 1st, 3rd, and 5th days after surgery, venous blood is collected for routine blood tests, blood biochemistry, procalcitonin, erythrocyte sedimentation rate and other infection indicators. If signs of infection occur, blood culture is sent in time, and the use of antibiotics is adjusted according to the drug sensitivity results.

[0044] In another embodiment of the present application, the interval between the death of the donor and the acquisition of the allogeneic valved conduit is greater than 6 hours and less than or equal to 8 hours, and the probability of infection is high. The patient (recipient) is given antibiotics prophylactically for more than 4 hours of Class I incision after surgery: 3g of cefoperazone sulbactam sodium (Supsen) for injection + 100ml of 0.9% sodium chloride injection intravenously once every 12 hours. The postoperative antibiotic use time does not exceed 48 hours.

[0045] In another embodiment of the present application, the interval between the death of the donor and the acquisition of the allogeneic valved conduit is greater than 8 hours and less than or equal to 12 hours, and the probability of infection is very high. The patient (recipient) is given antibiotics according to the infected incision after surgery: 0.4g of moxifloxacin hydrochloride injection (Baifule) is intravenously injected once a day. Antibiotics are used for 5 to 7 days after surgery, and the patient's body temperature and other vital signs are dynamically monitored. On the 1st, 3rd, and 5th days after surgery, venous blood is collected for routine blood tests, blood biochemistry, procalcitonin, erythrocyte sedimentation rate and other infection indicators. If signs of infection occur, blood is sent for culture in time, and the use of antibiotics is adjusted according to the drug sensitivity results.

[0046] In another embodiment of the present application, the interval between the death of the donor and the acquisition of the allogeneic valved conduit is greater than 12h and less than or equal to 24h, and the probability of infection is very high. The patient (recipient) is given antibiotics according to the standard of heart transplantation after surgery: 0.4g of teicoplanin (Galixin) for injection + 100ml of 0.9% sodium chloride injection intravenously once a day; 1g of meropenem (Meping / Beineng) for injection + 100ml of 0.9% sodium chloride injection intravenously once every 8h; 50mg of micafungin sodium for injection + 100ml of 0.9% sodium chloride injection intravenously once a day. Antibiotics are used for 7 to 10 days after surgery, and the patient's body temperature and other vital signs are dynamically monitored. On the 1st, 3rd, and 5th days after surgery, venous blood is collected for blood routine, blood biochemistry, procalcitonin, erythrocyte sedimentation rate and other infection indicators. If signs of infection occur, blood culture is sent in time, and the use of antibiotics is adjusted according to the drug sensitivity results.

[0047] This application adopts different treatment methods for allogeneic valved conduits at different times according to the time from death to transplantation. Through the method of classification and time division, different treatment methods can be fully achieved for allogeneic valved conduits from corpses at different times of death, while avoiding transplant-related infection and protecting the activity of allogeneic valved conduits as much as possible.

[0048] The present application also provides a method for preserving the homologous valved conduit after pretreatment obtained by the pretreatment method described in the above scheme, comprising the following steps:

[0049] The homologous valved tube after the pre-treatment is placed in a cryoprotectant bag, frozen at -70 to -90°C for 12 to 24 hours, and then transferred to liquid nitrogen for storage.

[0050] Compared with the conventional freezing conditions of cooling at a rate limit of 1°C per minute, the present application directly freezes the same type of valved tube at -80°C for 12 to 24 hours. During the freezing process, the temperature drops by an average of 4 to 5°C per minute, shortening the freezing time, which is not conducive to ice crystal formation and minimizing cell damage during the storage process. In addition, the storage method of the present application does not require pre-cooling and can also reduce cell damage.

[0051] Before freezing, the present application preferably also includes sampling the same type of valved tube to detect whether there is bacterial contamination. If bacterial contamination is found, the valved tube cannot be used; during the detection process, the freezing bag containing the same type of valved tube is preferably stored at 4°C; the storage time at 4°C is preferably ≤60min, more preferably 30-60min.

[0052] In the specific implementation process of the present application, the cryopreservation protection solution is based on TCM199 culture medium and also includes dimethyl sulfoxide (DMSO) with a volume concentration of 10% to 15%; more preferably, the cryopreservation protection solution is composed of TCM199 culture medium and DMSO, and the volume concentration of DMSO in the cryopreservation protection solution is 10%. The present application uses a cryopreservation protection solution of TCM199 culture medium + DMSO dimethyl sulfoxide to replace the traditional cryopreservation protection solution containing propylene glycol, thereby reducing biological toxicity.

[0053] In the specific implementation of the present application, the cryopreservation bag has multiple layers, preferably 3 layers; the cryopreservation bag includes a sterile plastic bag. In the specific implementation of the present application, the homologous valved pipe is encapsulated in the innermost layer of the cryopreservation bag containing cryopreservation protection solution.

[0054] After placing the homologous valved tube in a cryoprotectant bag, the present application preferably uses an ice box to transport the homologous valved tube to a freezer at -90 to -70°C, more preferably at -80°C.

[0055] The present application performs rapid freezing at -90 to -70°C, so that the temperature quickly passes through the phase transition point, and the liquid components in the cells are quickly transformed into a glassy state, thereby reducing the formation of ice crystals.

[0056] After being transferred into liquid nitrogen for storage, the present application preferably further comprises thawing; the thawing temperature is 37° C.; the thawing time is 10 to 30 minutes, preferably 15 to 20 minutes.

[0057] Before thawing, when the freezing bag described in the above scheme is three-layer, the present application preferably further comprises opening the outer two layers of packaging bags and soaking them in iodine before thawing.

[0058] Compared with conventional thawing at room temperature (20-30°C), the thawing temperature in this application is set at 37°C, which can shorten the thawing time, achieve the effect of rapid rewarming, reduce ice crystal formation, and thus reduce cell damage.

[0059] In the present application, the thawing preferably includes thawing in a water bath in physiological saline.

[0060] After the thawing, the present application preferably further comprises immersing the thawed homologous valved conduit in TCM199 culture medium.

[0061] In the present application, the soaking times are preferably 3 times, and the soaking time for each time is preferably 5 minutes.

[0062] After the soaking, the present application preferably also includes placing the soaked homologous valved conduit into a new TCM199 culture medium and handing it over to the instrument nurse to wait for transplantation.

[0063] In order to further illustrate the present application, a sterilization solution, a pretreatment method for the same type of valved tube and a preservation method thereof provided by the present application are described in detail below in combination with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present application.

[0064] Example 1

[0065] See the flowchart Figure 1 .

[0066] 1. Equipment and consumables:

[0067] 1.1. Equipment: laminar flow biosafety cabinet, liquid nitrogen storage tank, 4℃ refrigerator, -80℃ low-temperature refrigerator, heat sealing machine.

[0068] 1.2. Consumables and reagents: bacterial culture bottles, low-temperature resistant polyethylene freezing bags, 0.9% saline 500ml / bag, 50mL syringe, TCM199 culture medium, DMSO.

[0069] 2. Preparation:

[0070] 2.1. Donor selection: The donor comes from a fresh heart of a recipient who has participated in a heart transplant operation. The patient criteria follow the following requirements:

[0071] (1) No history of infectious diseases such as tuberculosis, hepatitis, syphilis, AIDS, malaria, leprosy, sepsis, and tumors (positive HBsAg, positive HBsAb, positive HBcAb, or HBcAb+HBsAb are not excluded, but require remarks);

[0072] (2) No extensive burns, autoimmune diseases, history of cardiac surgery, valvular disease, cardiomyopathy, untreated active lung disease, rheumatic fever, bacterial endocarditis, prolonged closed CPR, or serious illness of unknown etiology;

[0073] (3) Excluding nucleic acid positive;

[0074] (4) Those under 60 years old, but those over 50 years old need to make a note;

[0075] (5) Cold ischemia time ≤ 24 h.

[0076] 2.2. Disinfection:

[0077] (1) Prepare all consumables in advance and send them to the ethylene oxide room for disinfection;

[0078] (2) Prepare the sampling instruments and corresponding packaging packages in advance and sterilize them under high temperature and high pressure in the supply room;

[0079] (3) 50 minutes before the operation, turn on the ultraviolet light for 20 minutes in the laminar flow biosafety cabinet and the operating room, then turn off the ultraviolet light and turn on the fan for 10 minutes.

[0080] 2.3. Preparation before taking the valved tube:

[0081] (1) The surgeon removes the heart and places it in a sterile plastic bag (single layer), which is then moved from the operating room to the cell room by an assistant;

[0082] (2) The trimmer brushes his hands and enters the cell room, puts on surgical gown and sterile gloves with the help of an assistant;

[0083] (3) Open the pavement bag, take the middle sheet and treatment towel and lay them out in 4 to 6 layers from outside to inside and from left to right;

[0084] (4) The assistant opens the outer packaging of the instrument package, and the trimmer opens the inner packaging, takes out the stainless steel barrel and the instruments, and places them neatly on the sterile table of the laminar flow biosafety cabinet. Open 2 bags of 500 mL 0.9% saline and pour them into the stainless steel barrel for later use;

[0085] (5) The assistant pulls down the operating window of the laminar flow biosafety cabinet to leave it 30 cm high;

[0086] (6) The assistant holds the edge of the packaging bag containing the donor heart with both hands and turns the bag mouth outward (be careful not to touch the inside of the packaging bag); the trimmer takes out the donor heart and puts it into the sterilized curved tray;

[0087] (7) Use a 2 ml syringe to extract the remaining liquid in the inner plastic bag, cover the needle and protective cap and set aside;

[0088] 3. Preparation of valved conduit:

[0089] 3.1. Heart cleaning and trimming of valved tubes:

[0090] (1) Flush the heart with 0.9% saline solution, gently squeeze to drain the blood and saline from the heart cavity, gently support the heart with one hand, hold the flushing basin with the other hand and pour the flushing solution into the medical waste bag; repeat twice;

[0091] (2) Separate the papillary muscles of the mitral and tricuspid valves, cut the ventricular muscle 5 to 10 mm below the valve ring, cut the atrium 5 to 10 mm above the valve ring, and cut the ventricular muscle 5 to 10 mm below the main pulmonary valve ring. Give the cut ventricular muscle and atrium to the assistant;

[0092] (3) Pour 50 ml of normal saline from the stainless steel cylinder into the curved plate, remove the aortic adventitia, carefully separate the left and right coronary artery branches, and cut off the left and right coronary arteries with 5 mm left;

[0093] (4) Free the main and pulmonary artery junction, separate the main and pulmonary artery valved conduit, and soak the valved conduit in a stainless steel tube, which should be covered at any time.

[0094] (5) Remove the pulmonary artery valved conduit, carefully remove the outer membrane, trim the myocardium under the valve ring, leaving 5 mm, remove the fat outside the myocardium, and soak it in a stainless steel cylinder;

[0095] (6) Use a stainless steel ruler to measure the length of the main pulmonary artery, the upper half circumference of the valve ring, the anterior-posterior and lateral 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. An assistant should record each measurement, especially the presence of arterial wall damage and coronary artery calcification.

[0096] 3.2. Cryopreservation of valved tubes:

[0097] (1) The trimmer changes into sterile gloves, adds two layers of bedding material to the laminar flow biosafety cabinet, and takes out the cryopreservation bag and two 50 mL syringes for use;

[0098] (2) Take TCM199 culture medium, disinfect the bottle mouth with alcohol, and burn it with an alcohol lamp for 2 to 3 circles;

[0099] (3) Take DMSO, disinfect the bottle mouth with alcohol, and burn it with the outer flame of an alcohol lamp for 2 to 3 circles;

[0100] (4) Open the TCM199 medium inside the biosafety cabinet and draw 45 mL with a syringe; open the DMSO inside the biosafety cabinet and draw 5 mL with a syringe, add it to the beaker, and prepare a DMSO cryopreservation protection solution with a volume concentration of 10%;

[0101] (5) Use non-destructive forceps to remove the valves one by one, cut small pieces of the arterial wall and place them in a stainless steel bucket;

[0102] (6) Place the valves in the innermost sterile plastic bag, add 20-30 ml of cryopreservation protection solution, expel the air in the bag, fold the plastic seal strip at the bag opening inward 2-3 times, seal the sterile plastic bag, place it in the second sterile plastic bag, cover the bag opening with a heat-insulating film, place it in the heat sealer, and start it three times in a row;

[0103] (7) The trimmer takes out the package with the insulation film, and after 5 seconds, pinches the side facing the plastic bag to tear off the insulation film, and takes it back to the biosafety cabinet to observe whether there is any leakage;

[0104] (8) Cut off the excess plastic bag at the seal, put it into a third layer of sterile plastic bag, seal it with a heat sealer, and take it out after cooling;

[0105] (9) Use a low-temperature marker to mark the valve number and date on the outermost packaging bag, apply a rubber band to mark it, and place it in a 4°C refrigerator for 30 to 60 minutes;

[0106] (10) The initially extracted sample solution and the small piece of arterial wall are injected into the bacterial culture bottle, which is tightly closed and sent to the culture room. If bacterial contamination occurs, the valved tube cannot be used.

[0107] (11) The valved tube is transported in an ice box and stored in a -80°C refrigerator;

[0108] (12) After 12 hours, the valve is transferred from the deep-freezer to liquid nitrogen for storage, and the valve number, date, recording time, and liquid nitrogen tank location are marked on the liquid nitrogen tank.

[0109] 3.3. Use valved tube:

[0110] (1) Take out the cryopreservation bag on the sterile table in the operating room, open the two outer packaging bags, soak it in iodine, and then thaw it in 37°C saline;

[0111] (2) After initial thawing, soak in TCM199 medium for 5 min, repeated three times;

[0112] (3) Check the valve for any problems, place it in new TCM199 culture medium and hand it over to the instrument nurse to wait for transplantation.

[0113] Comparative Example 1

[0114] A xenogeneic bioprosthesis, the Medtronic Freestyle stentless bioprosthesis (Medtronic Inc., Minneapolis, MN, USA), was used for transplantation.

[0115] The comparative data of the valved conduit transplantation effects of Example 1 and Comparative Example 1 are shown in Table 1. As can be seen from Table 1, compared with the fresh homologous valved conduit of Example 1, the xenogeneic bioprosthetic valve of Comparative Example 1 is more likely to suffer from valve failure and death during follow-up after surgery.

[0116] Table 1 Clinical outcomes after surgery with fresh homograft valved conduits and xenograft bioprostheses

[0117]

[0118] Comparative Example 2

[0119] Autopsy-derived homograft valved conduits were used for transplantation.

[0120] The comparative data of the valved conduit transplantation effects of Example 1 and Comparative Example 2 are shown in Table 2. As can be seen from Table 2, the transplantation source homologous valved conduit of Example 1 and the traditional source homologous valved conduit of Comparative Example 2 both have an improvement effect on right ventricular function, and the effect of the transplantation source homologous valved conduit is not inferior to that of the traditional source homologous valved conduit, which expands the source of organ transplant donors, and is superior to the traditional source in the degree of improvement of RVEDD.

[0121] Table 2 Comparison of the effects of fresh transplanted homologous valved conduits and traditional valved conduits in Example 1

[0122]

[0123] Comparative Example 3: Traditional temperature-controlled freezing of the same valved pipe

[0124] The conventional temperature-controlled freezing procedure is: -135°C methylbutane bath for 15 min, then cooling to -80°C at -1°C / min.

[0125] The comparative data of the valved conduit transplantation effects of Example 1 and Comparative Example 3 are shown in Table 3. As can be seen from Table 3, rapid freezing is less likely to cause adverse cardiovascular events, valvular stenosis or regurgitation than the temperature-controlled freezing method.

[0126] Table 3 Comparison of clinical outcomes after rapid freezing (Example 1) and traditional temperature-controlled freezing of valved conduit transplantation

[0127]

[0128]

[0129] Comparative Example 4: Traditional rewarming method with valve pipe

[0130] The valved pipe is thawed at room temperature (20-30° C.) in the conventional rewarming method, and the rest is the same as in Example 1.

[0131] The comparative data of the valved conduit transplantation effects of Example 1 and Comparative Example 4 are shown in Table 4. As can be seen from Table 4, rapid thawing at 37°C is less likely to cause stenosis and regurgitation than thawing at room temperature.

[0132] Table 4 Comparison of clinical outcomes after rapid rewarming (Example 1) and traditional rewarming methods for valved conduit transplantation

[0133]

[0134] Example 2

[0135] Source of remains: Voluntarily donated remains with no previous heart disease and no death caused by heart or major blood vessel trauma.

[0136] After obtaining the informed consent of the family members or the person has signed the informed consent for body donation:

[0137] (I) Preparation before acquisition

[0138] 1. Prepare the packaging bags of allogeneic valved tubes in advance and send them to the ethylene oxide room for disinfection, and prepare the equipment packages and packaging bags for sampling in advance and sterilize them under high temperature and high pressure in the supply room;

[0139] 2. 50 minutes before operation, turn on the ultraviolet irradiation for 20 minutes in the clean bench and the dedicated operation room, turn off the ultraviolet irradiation and turn on the fan for 10 minutes;

[0140] 3. Before the operation, the echocardiogram results of the deceased donor should be reviewed, and the aortic valve and / or pulmonary valve regurgitation should be no regurgitation or trace regurgitation; the body should be placed in a freezer within 1 hour after death; the interval between the donor's death and the acquisition of the allogeneic valved conduit should be ≤2 hours;

[0141] (II) Acquisition of allogeneic valved conduits

[0142] 4. Push the body to the sterile operating room and disinfect and cover the body according to the requirements of the cardiac surgery midline thoracotomy;

[0143] 5. Perform a midline thoracotomy to expose the heart surface corresponding to the pulmonary artery trunk, conus arteriosus, ascending aorta and left ventricular outflow tract;

[0144] 6. After separating the pulmonary trunk and ascending aorta and determining the position of the aortic valve and pulmonary valve, retain 10 mm of the ascending aorta and pulmonary trunk above the valve (5 mm of the origin of the left and right coronary arteries at the root of the ascending aorta should be retained), cut the heart 5 mm below the valve, and completely remove the aortic valved conduit and pulmonary valved conduit (if the antemortem echocardiogram results show that the valve has no regurgitation or a small amount of regurgitation);

[0145] 7. Remove the allogeneic valved conduit, place it in a sterile plastic bag (single layer), and have the assistant move it from the operating room to the dedicated operating room;

[0146] 3. Treatment and preservation of allogeneic valved conduits

[0147] 8. The operator cleans his hands according to standard aseptic technique before entering the cell room, and puts on surgical gown and sterile gloves with the help of an assistant;

[0148] 9. Open the sterile dressing package, take the middle treatment sheet and treatment towel, and spread 4 to 6 layers of treatment towel from outside to inside and from left to right;

[0149] 10. The assistant opens the outer packaging of the instrument package, and the operator opens the inner packaging, takes out the stainless steel tube and the instrument, and places them neatly on the sterile table. The assistant hands 2 bags of 0.9% sodium chloride injection (500ml) to the operator, and pours them into a stainless steel bucket for later use;

[0150] 11. The assistant pulls down the clean bench operation window, leaving it 30cm high;

[0151] 12. The operator takes out the allogeneic valved conduit from the original packaging bag and puts it into the prepared curved tray;

[0152] Use a 13.2ml syringe to extract the remaining liquid in the inner plastic bag, cover the needle and protective cap and send it for bacterial culture;

[0153] 14. Flush the allogeneic valved conduit twice with 0.9% sodium chloride injection;

[0154] 15. Remove the outer membrane of the valved tube, remove the fat outside the myocardium, and place it in a stainless steel tube filled with 0.9% sodium chloride injection and soak it at room temperature for 1 to 2 minutes;

[0155] 16. Take a stainless steel ruler and measure the length of the valved tube and the upper circumference of the valve ring. The assistant will record them one by one. If there is arterial wall damage or coronary artery calcification, it should be recorded truthfully. Also check the valve closure. If there is stenosis or regurgitation, it should be recorded truthfully and discarded.

[0156] 17. The operator changes into sterile gloves and lays two more layers of treatment towels on the sterile clean bench;

[0157] 18. The assistant opens the outer packaging of the bag, and the trimmer takes out the new packaging bag wrapped inside and puts it on the clean bench for use;

[0158] 19. The assistant hands two new 50ml syringes to the trimmer for use;

[0159] 20. The assistant lights the alcohol lamp and removes TCM199 (Gibco TM , Waltham, MA, US) bottle mouth packaging, use tweezers to take a piece of alcohol gauze, wipe the bottle mouth, tilt 30 degrees, rotate the bottle body, and burn it 2 to 3 times under the outer flame of the alcohol lamp;

[0160] 21. The assistant takes TCM199 to the edge of the clean bench, opens the bottle mouth toward the clean bench, and keeps the bottle cap in the same direction as the bottle mouth and close to the bottle mouth. Pay attention to aseptic operation.

[0161] 22. The operator draws out 45 ml of TCM199 using a 50 ml syringe;

[0162] 23. The assistant repeats steps 21 to 22 to open the DMSO, and the trimmer extracts 5 ml to prepare a 10% DMSO freezing solution;

[0163] 24. Cut a small amount of arterial wall from one end of the valved conduit and place it in a sterile box for bacterial culture;

[0164] 25. Place the valved tubes into the innermost bag, add 20-30 ml of freezing solution, expel the air from the bag, fold the plastic seal strip at the bag mouth inward 2-3 times, and seal the bag;

[0165] 26. Put the second and third layers of packaging bags on the outside, mark the number and date on the outermost packaging bag, put it in a 4℃ refrigerator, and record the time;

[0166] 27. After 60 minutes, transfer the samples from the 4°C refrigerator to a -80°C refrigerator. After all bacterial cultures are negative (about 72 hours), transfer the samples to a liquid nitrogen tank for storage.

[0167] 4. Rewarming and transplantation of allogeneic valved conduits

[0168] 28. When needed, take it out from the liquid nitrogen tank and bring it to the operating room for standby use;

[0169] 29. Discard the outermost two layers of packaging and keep the innermost layer of packaging, and place it on the sterile table;

[0170] 30. Immerse the valved pipe together with the innermost packaging in iodine disinfectant, and replace the iodine disinfectant every 5 minutes for a total of 3 times;

[0171] 31. Immerse the valved tube together with the innermost packaging in 37°C sterile saline until the innermost valved tube and TCM199 soaking solution are converted into an ice-water mixture;

[0172] 32. After changing to ice water mixture, open the innermost packaging, pour the valved tube and TCM199 soaking solution into the stainless steel disinfection bowl, and check the integrity of the valved tube (if damaged, discard immediately);

[0173] 33. Take out the valved tube and place it in 37℃ TCM199 solution to continue rewarming. Change the TCM199 solution every 5 minutes for a total of 3 changes.

[0174] 34. The valved conduit is rinsed in 37°C saline before transplantation;

[0175] 35. Postoperative patients (recipients) are given antibiotics for prevention according to the type I incisions that lasted for more than 4 hours: 1.5g of Cefuroxime Sodium for Injection (Ancoxin) + 100ml of 0.9% Sodium Chloride Injection, intravenously twice a day. The duration of postoperative antibiotic use should not exceed 48 hours.

[0176] Example 3

[0177] If the interval between the death of the donor and the acquisition of the allogeneic valved conduit is greater than 2 hours and less than or equal to 4 hours, step 35 is changed to: After surgery, the patient (recipient) is given antibiotics prophylactically according to a Class I incision of more than 4 hours: 3g of cefoperazone sulbactam sodium for injection (Supsen) + 100ml of 0.9% sodium chloride injection intravenously, once every 12 hours. The duration of antibiotic use after surgery shall not exceed 48 hours.

[0178] Example 4

[0179] If the interval between the death of the donor and the acquisition of the allogeneic valved conduit is greater than 4 hours and less than or equal to 6 hours, step 35 is changed to: antibiotics are given to the patient (recipient) according to the infected incision after surgery: 0.4g of moxifloxacin hydrochloride injection (Baifule) intravenously once a day. Antibiotics are used for 5 to 7 days after surgery, and the patient's body temperature and other vital signs are dynamically monitored. On the 1st, 3rd, and 5th days after surgery, venous blood is collected for routine blood tests, blood biochemistry, procalcitonin, erythrocyte sedimentation rate and other infection indicators. If signs of infection occur, blood is sent for culture in time, and the use of antibiotics is adjusted according to the drug sensitivity results.

[0180] Clinical verification of Examples 2 to 4 found that even without antibiotic treatment within 6 hours, according to clinical data statistics, the probability of postoperative transplant-related infective endocarditis is 0 (n = 60). The data comes from the Pediatric Surgery Center and Animal Center of Fuwai Hospital, Chinese Academy of Medical Sciences. After 60 patients with tetralogy of Fallot received allogeneic valved conduits for right ventricular outflow tract reconstruction, the valved conduits of the transplanted heart and the heart of the deceased within 6 hours after death were not soaked in antibiotics, and then the transplantation was completed. None of the 60 patients developed transplant-related infective endocarditis after surgery. The results are shown in Table 5.

[0181] Table 5 Follow-up results of 60 patients with allogeneic valved conduit transplantation in adult and pediatric surgical centers from 2009 to 2022

[0182] Project Name quantity(%) Follow-up time, median time (interquartile range), years 2.6(1.0～6.9) Adverse clinical events 24(40.0) All-cause death 4(6.7) Death during hospitalization 2(50) Death during follow-up 2(50) Infective endocarditis 0(0) Valved tube failure 20(33.3) Valvular regurgitation 15(75.0) Valvular stenosis 4(20.0) Valvular stenosis with regurgitation 1(5.0)

[0183] Example 5

[0184] If the interval between the death of the donor and the acquisition of the allogeneic valved conduit is greater than 6 hours and less than or equal to 8 hours, add a step between step 15 and step 16, named: step 15A. Step 15A is: add 1g of ceftazidime for injection, 5mg of amphotericin B for injection, 0.5g of vancomycin hydrochloride for injection and 0.2g of amikacin sulfate to 1000ml of TCM199, and add 5% sodium bicarbonate injection to maintain the pH value of the mixed solution between 6.6 and 7.0, completely immerse the valved conduit in the mixed solution, and soak it in a 4°C refrigerator for 24 hours. At the same time, put the "retaining bacterial culture" in step 13 after step 16, named step 16A;

[0185] Step 35 is changed to: Postoperative patients (recipients) are given prophylactic antibiotics for Class I incisions that last more than 4 hours: 3g of cefoperazone sulbactam sodium for injection (Supsen) + 100ml of 0.9% sodium chloride injection intravenously every 12 hours. The duration of postoperative antibiotic use does not exceed 48 hours.

[0186] Example 6

[0187] If the interval between the death of the donor and the acquisition of the allogeneic valved conduit is greater than 8 hours and less than or equal to 12 hours, a step is added between step 15 and step 16, named: step 15A. Step 15A is: 1g of ceftazidime for injection, 5mg of amphotericin B for injection, 0.5g of vancomycin hydrochloride for injection and 0.2g of amikacin sulfate are added to 1000ml of TCM199, and 5% sodium bicarbonate injection is added to maintain the pH value of the mixed solution between 6.6 and 7.0, the valved conduit is completely immersed in the mixed solution, and is placed in a 4°C refrigerator for 24 hours. At the same time, the "retaining bacterial culture" in step 13 is placed after step 16, named step 16A.

[0188] On the premise of "thoroughly cleaning the allogeneic valved conduit and the bacterial culture after the valved conduit is negative", step 35 is changed to: antibiotics are given to the patient (recipient) according to the infected incision after surgery: 0.4g of moxifloxacin hydrochloride injection (Baifule) intravenously once a day. Antibiotics are used for 5 to 7 days after surgery, and the patient's vital signs such as body temperature are dynamically monitored. On the 1st, 3rd and 5th days after surgery, venous blood is collected for routine blood tests, blood biochemistry, procalcitonin, erythrocyte sedimentation rate and other infection indicators. If signs of infection occur, blood culture is sent in time, and the use of antibiotics is adjusted according to the drug sensitivity results.

[0189] Example 7

[0190] If the interval between the death of the donor and the acquisition of the allogeneic valved conduit is greater than 12 hours and less than or equal to 24 hours, a step is added between step 15 and step 16, named: step 15A. Step 15A is: 1g of ceftazidime for injection, 5mg of amphotericin B for injection, 0.5g of vancomycin hydrochloride for injection and 0.2g of amikacin sulfate are added to 1000ml of TCM199, and 5% sodium bicarbonate injection is added to maintain the pH value of the mixed solution between 6.6 and 7.0, the valved conduit is completely immersed in the mixed solution, and is placed in a 4°C refrigerator for 24 hours. At the same time, the "retaining bacterial culture" in step 13 is placed after step 16, named step 16A.

[0191] Step 35 is changed to: Postoperative patients (recipients) are given antibiotics according to the standard of heart transplantation: 0.4g of teicoplanin for injection (Galixin) + 100ml of 0.9% sodium chloride injection, intravenous injection, once a day; 1g of meropenem for injection (Meping / Beineng) + 100ml of 0.9% sodium chloride injection, intravenous injection, once every 8 hours; 50mg of micafungin sodium for injection + 100ml of 0.9% sodium chloride injection, intravenous injection, once a day. Antibiotics are used for 7 to 10 days after surgery, and the patient's body temperature and other vital signs are dynamically monitored. On the 1st, 3rd, and 5th days after surgery, venous blood is collected for routine blood tests, blood biochemistry, procalcitonin, erythrocyte sedimentation rate and other infection indicators. If signs of infection occur, blood culture is sent in time, and the use of antibiotics is adjusted according to the drug sensitivity results.

[0192] It has been verified that in Examples 5 to 7, after the valved conduits obtained from the cadaveric hearts 6 to 24 hours after death were treated with antibiotics, the probability of the patient developing transplant-related infective endocarditis after surgery was reduced from the original 15% to 1% to 2%.

[0193] Comparative Example 5

[0194] The formula of the sterilizing solution used in step 15A is a traditional formula (a balanced salt solution containing 2 g / L of ceftazidime, 1.2 g / L of lincomycin, 1 million U / L of polymyxin B and 1 g / L of vancomycin), and the rest is the same as in Example 5, Example 6 or Example 7.

[0195] The comparative results of clinical outcomes after valved conduit transplantation treated with the sterilizing solution formula of the present application and the traditional sterilizing solution formula are shown in Table 6. It can be seen from Table 6 that compared with the traditional formula, the probability of arrhythmia, heart failure, infective endocarditis and valvular failure (stenosis or moderate or above reflux) after the same valved conduit transplantation treated with the sterilizing solution formula of the present application is significantly reduced.

[0196] Table 6 Comparison of clinical outcomes after valved conduit transplantation using the sterilization solution formula of this application and the traditional sterilization solution formula

[0197]

[0198] Although the above-mentioned embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. A sterilizing solution, characterized in that: Using TCM199 as solvent, the following components are included: third generation cephalosporin 1-4 g / L, amphotericin B 5-20 mg / L, peptide antibiotics 0.5-2 g / L and aminoglycoside antibiotics 200-600 mg / L; The pH value of the sterilization solution is 6.6-7.

2. The sterilizing solution according to claim 1, characterized in that: The polypeptide antibiotics include vancomycin or teicoplanin; the vancomycin includes vancomycin hydrochloride for injection; the aminoglycoside antibiotics include amikacin; the amikacin includes amikacin sulfate; the third-generation cephalosporin includes ceftazidime for injection or ceftriaxone for injection; and the amphotericin B includes amphotericin B for injection.

3. The sterilizing liquid according to claim 3, characterized in that: Includes components in the following concentrations: Ceftazidime for injection 1-2g / L, amphotericin B 5-10mg / L, vancomycin hydrochloride for injection 0.5-1g / L and amikacin 200-300mg / L.

4. The sterilizing solution according to claim 1, characterized in that: The reagent for adjusting the pH value of the sterilization solution includes 5% sodium bicarbonate injection.

5. A method for pretreatment of a homologous valved conduit, characterized in that: The interval between the death of the donor and the acquisition of the allogeneic valved conduit is ≤ 24 hours; the allogeneic valved conduit is pretreated as follows according to the interval: When the interval time is ≤6h, the allogeneic valved conduit is cleaned; the cleaning reagent includes 0.9% sodium chloride injection; When the interval time of 6h<≤24h, the allogeneic valved conduit is cleaned and then immersed in the sterilization solution of any one of claims 1 to 4 for sterilization; the sterilization temperature is 0 to 4°C and the sterilization time is 20 to 24h.

6. A product for treating tetralogy of Fallot, characterized in that: The invention comprises a homologous valved conduit obtained by the pretreatment method of claim 5, antibiotics for routine infection prevention during surgery, antibiotics for preventing infection by drug-resistant bacteria, broad-spectrum antibiotics and antibiotics for heart transplantation.

7. The product according to claim 6, characterized in that The antibiotics used for routine infection prevention during surgery include cefuroxime sodium; the antibiotics used to prevent drug-resistant bacterial infections include cefoperazone sulbactam sodium for injection; the broad-spectrum antibiotics include moxifloxacin hydrochloride injection; and the antibiotics for heart transplantation include teicoplanin for injection, meropenem for injection, and micafungin sodium for injection.

8. The method for preserving the homologous valved conduit obtained by the pretreatment method of claim 5, characterized in that: The following steps are involved: The homologous valved tube after the pre-treatment is placed in a cryoprotectant bag, frozen at -70 to -90°C for 12 to 24 hours, and then transferred to liquid nitrogen for storage.

9. The storage method according to claim 8, characterized in that: The cryopreservation protection solution is based on TCM199 culture medium and also includes dimethyl sulfoxide with a volume concentration of 10% to 15%.

10. The storage method according to claim 8, characterized in that: After being transferred into liquid nitrogen for storage, the process further includes thawing; the thawing temperature is 37° C.; and the thawing time is 10 to 30 minutes.

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