Cryopreservation method of biogenic valved blood vessel

Abstract

The invention discloses a cryopreservation method of a biogenic valved blood vessel. The biogenic valved blood vessel is placed in a collagen solution for cryopreservation. Wherein the concentration of the collagen solution is 3 to 5 mg / ml; the ratio of the weight of the biogenic valved blood vessel to the volume of the collagen solution is (1: 2)-(1: 5). The pipeline crack probability of the valved blood vessel obtained by the treatment method provided by the invention is remarkably reduced, the valve leaflet is not torn when a durability test is carried out for 200 million times, and the valve still works normally through a pulsating flow test.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for cryopreservation of biological valved vessels. BACKGROUND

[0002] Biological valved vessels can be divided into xeno-valved vessels and allogeneic valved vessels. Xeno-valved vessels are taken from mammalian tissues such as pigs or cows, and after chemical treatment, they have the advantages of low immunogenicity and abundant raw material supply, can provide a suitable passage for right ventricular outflow tract and valve function, and can effectively improve the hemodynamics of the heart. Compared with xeno-valved vessels, allogeneic valved vessels are taken from human donors, and after sterilization and low-temperature storage, they have the advantages of high biocompatibility and no need for anticoagulation, and can be used to replace diseased, damaged, deformed or failed autologous or prosthetic pulmonary valve. In the ROSS operation, they can also be used to replace autologous pulmonary valve.

[0003] Currently, the processing methods of allogeneic valved vessels mainly include sterilization, decellularization and low-temperature storage.

[0004] Regarding sterilization, Delmo Walter EM team (Delmo Walter EM, et al. HSR Proc Intensive Care Cardiovasc Anesth. (2012) 4: 97-108.) reported that antibiotics were used for sterilization of valved vessels, and the antibiotics included amikacin, flucytosine, vancomycin, ciprofloxacin and metronidazole. M. Esther RV team (M. Esther RV, et al. 2004, 77(1): 0-190.) reported that the antibiotics included cefoxitin, lincomycin, polymyxin B and vancomycin.

[0005] Regarding decellularization, Bader A team (Bader A, et al. Eur J Cardiothorac Surg 1998; 14: 279-284.) reported that when decellularization was performed, the valved vessels were placed in PBS buffer solution, and the PBS buffer solution did not contain Ca 2+ and Mg 2+, 0.02% ethylenediaminetetraacetic acid (EDTA), RNase A (20 mg / ml) and DNase (0.2 mg / ml) at 37 °C in 5% CO2for 24 h. Lichtenberg A team (Lichtenberg A, et al. Biomaterials 2006; 27: 4221-9.) reported that when decellularizing, the valved vessel was placed in a 0.5% sodium deoxycholate (SDC) and 0.5% sodium dodecyl sulfate (SDS) solution for 48 h, followed by 2 cycles of washing in distilled water (12 h / cycle), and then 8 cycles of washing in Ringer lactate solution, with continuous shaking at room temperature. Lichtenberg A team also reported that when decellularizing, the valved vessel was placed in a 0.5% SDC and 0.5% SDS solution for 24 h. The valved vessel was washed 6 times (12 h / cycle) in a PBS buffer solution containing penicillin and streptomycin (100 mg / mL, P / S).

[0006] Regarding cryopreservation, Kirklin JW team (Kirklin JW, et al. Ann Thorac Surg. 1987; 44: 598-606.) reported that the valved vessel was placed in RPMI 1640 complete medium (containing 10% dimethyl sulfoxide and 10% fetal bovine serum) cooled to -80 °C (-1 °C / min), and then stored in gaseous phase liquid nitrogen for a long time.

[0007] The valved vessel obtained by the method disclosed in the above-mentioned existing literature has the following problems: the mechanical properties of the conduit and the leaflet are poor, after resuscitation, the conduit has a high probability of cracking, the leaflet is prone to tearing, and the durability of the valve is poor. SUMMARY

[0008] To solve the above technical problems, the present application provides a cryopreservation method for a biological valved vessel.

[0009] As an aspect of the present application, a cryopreservation method for a biological valved vessel is provided, wherein the biological valved vessel is placed in a collagen solution for cryopreservation; wherein the concentration of the collagen solution is 3-5 mg / ml; and the weight of the biological valved vessel and the volume of the collagen solution are in a ratio of 1:2-1:5.

[0010] In a specific embodiment, the biological valved vessel is subjected to the following treatment before being placed in the collagen solution:

[0011] (1) The biological valved vessel is soaked in an antibiotic-containing medium;

[0012] (2) The biological valved vessel soaked in the antibiotic-containing medium is subjected to decellularization;

[0013] (3) again placed in the antibiotic-added medium for soaking.

[0014] Further, in some embodiments, the biological valved vessel is treated before being placed in the collagen solution as follows:

[0015] (1) the valved vessel is soaked in the antibiotic-added medium at 2-10°C for 18-36h;

[0016] (2) the valved vessel obtained after sterilization in the first step is soaked in the decellularization solution at 4-10°C for 24-48h, then the valved vessel is taken out of the decellularization solution and rinsed with normal saline;

[0017] (3) the valved vessel obtained in the second step is soaked in the antibiotic-added medium at 2-10°C for 24-48h, then the valved vessel is taken out of the antibiotic-added RPMI 1640 complete medium and rinsed with RPMI 1640 complete medium to obtain the valved vessel after secondary sterilization.

[0018] Further, the donor animal of the biological valved vessel is a mammal such as a pig, a sheep, a cow or a human.

[0019] Further, in the steps (1) and (3), the weight of the valved vessel and the volume of the antibiotic-added medium are in the ratio of 1:25-1:40.

[0020] Further, the steps (1) and (3) are performed in a shaking table, and the rotation speed of the shaking table is 100-200rpm.

[0021] Further, in the steps (1) and (3), the antibiotic includes any four of amikacin, ceftazidime, linezolid, vancomycin, secnazole, metronidazole and 5-flucytosine.

[0022] Further, in the step (2), the decellularization solution includes two or more reagents of 0.5-1.5% (v / v) triton X-100, 0.5-1.5% (w / v) sodium dodecyl sulfate and 0.5-1.5% (w / v) sodium deoxycholate.

[0023] Further, the medium is RPMI 1640 complete medium.

[0024] Further, the biological valved vessel is an aortic valved vessel or a pulmonary valved vessel.

[0025] The patent provides a processing method of biological valved vessel, the valved vessel donor animal can be a pig, a sheep, a cow, a human or other mammals, after the valved vessel of the donor animal is obtained by dissection, the myocardial length of the valved vessel is limited to at least 0.5 cm away from the annulus, and the conduit length is limited to at least 0.5 cm away from the annulus. The valved vessel can be an aortic valved vessel and / or a pulmonary valved vessel, the aortic valved vessel includes an aortic valve and an aortic conduit, which can be used to repair congenital and acquired valvular lesions, the pulmonary valved vessel includes a pulmonary valve and a pulmonary conduit, which can be used to replace autologous or prosthetic pulmonary valve lesions, injuries, deformities or failures, and can be used to replace autologous pulmonary valve during ROSS surgery.

[0026] According to GB / T 12279.1-2024, for artificial heart valves that have proven failure modes that will cause gradual degradation of valve function, testing should be performed for at least 200 million cycles. The conduit crack probability of the valved vessel obtained by the processing method of the present application is low, and when the durability test is 200 million times, the valve leaflet does not appear to be torn, and the valve is still working normally through the pulsatile flow test. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A schematic diagram of the pig-derived valved vessel after warming resuscitation, wherein, Figure 1 A is the valved vessel obtained in Example 15; Figure 1 B is the valved vessel obtained in Example 16; Figure 1 C is the valved vessel obtained in Example 7; Figure 1 D is the valved vessel obtained in Example 8.

[0028] Figure 2 A schematic diagram of the pig-derived valved vessel after warming resuscitation, wherein, DETAILED DESCRIPTION

[0029] In the process of completing the present application, the inventors first refer to the antibiotics reported by Delmo Walter EM team or M. Esther R V team to sterilize the valved vessels, refer to the method reported by Bader A team or Lichtenberg A team to decellularize the valved vessels, and refer to the method reported by Kirklin JW team to store the valved vessels at low temperature (corresponding to Examples 1-6). After warming and resuscitation, it is found that the valved vessels obtained in Examples 1, 3 and 5 have surface-attached impurities, and the conduits are cracked. Although the valved vessels obtained in Examples 2, 4 and 6 have no surface-attached impurities, there is a problem of high probability of conduit cracking. In the durability test of 200 million times, the leaflets are all torn. In the durability test of 1 billion times, the regurgitation percentage of the valve is close to 50, and the valve fails. According to GB / T 12279.1-2024, for artificial heart valves whose failure mode has been proven to cause gradual degradation of valve function, the test should be carried out for at least 2 billion cycles.

[0030] Subsequently, based on the above experimental results, the inventors improved the experimental steps, the types of antibiotics and other processing conditions (corresponding to Examples 7-14) and found that the problems of high probability of conduit cracking, tearing of the leaflets in the durability test of 200 million times, and regurgitation percentage of the valve close to 50 in the durability test of 1 billion times, and valve failure were not solved.

[0031] In an experimental process (corresponding to Examples 15-16), the inventors soaked the valved vessels obtained after secondary sterilization in the third step in a collagen solution and froze them, and unexpectedly found that the probability of conduit cracking of the valved vessels was significantly reduced. In the durability test of 2 billion times, the leaflets were not torn, and through the pulsatile flow test, it was found that the valve still worked normally. The inventors verified this phenomenon through repeated experiments (Example 16). Subsequently, based on this finding, the inventors optimized the parameters of the whole process (Examples 17-26) and found that when the concentration of the collagen solution was controlled to be 3-5 mg / ml and the weight of the valved vessels and the volume ratio of the collagen solution were 1:2-1:5, the conduits of the valved vessels were not cracked, the leaflets of the valved vessels were not torn in the durability test of 2 billion times, and the valve still worked normally in the durability test of 2 billion times.

[0032] The anatomical method and specifications of the valved vessels obtained in the following examples are as follows: (1) obtaining a donor heart; (2) isolating a pulmonary artery valved vessel. The myocardial length of the valved vessel is limited to at least 0.5 cm away from the annulus, and the conduit length is limited to at least 0.5 cm away from the annulus.

[0033] I. Examples 1-6

[0034] 1. Example 1

[0035] A method for processing biological valved vessels, comprising the following steps:

[0036] S1, according to the report of Delmo Walter EM team, at 4℃, the pig valved vessels (pig age > half a year, weight in 20-30 kg) are soaked in sterile filtered nutrient tissue culture medium containing antibiotics for 24h. The antibiotics include amikacin, flucytosine, vancomycin, ciprofloxacin and metronidazole, and the specific proportion is that 0.6mg of amikacin, 1.5mg of flucytosine, 0.6mg of vancomycin, 150μg of ciprofloxacin and 0.6mg of metronidazole are added in 1 liter of sterile filtered nutrient tissue culture medium (Hank's balanced salt solution, 9.6g / l).

[0037] S2, according to the report of Bader A team (Bader A, et al. Eur J Cardiothorac Surg 1998; 14: 279-284.), at 37℃, the valved vessels obtained in S1 are placed in PBS buffer solution under 5% CO2 condition, and are subjected to decellularization treatment for 24h. The PBS buffer solution is free of Ca 2+ and Mg 2+ , containing 1% Triton X-100, 0.02% EDTA, RNase A (20mg / ml) and DNase (0.2mg / ml). After decellularization, the valved vessels are taken out of the decellularization solution and are rinsed with physiological saline;

[0038] S3, according to the report of Kirklin JW team (Kirklin JW, et al. Ann Thorac Surg. 1987; 44: 598-606.), the valved vessels obtained in S2 are placed in a sterile bag, 10% dimethyl sulfoxide (DMSO) and 10% fetal bovine serum (FBS) are added to the sterile bag, the sterile bag is sealed, and then is placed in a programmed cooling instrument, cooled to-80℃ (cooling rate is-1℃ / min), and then is stored in gaseous liquid nitrogen for a long time.

[0039] Three valved vessels are obtained by the method of Example 1.

[0040] 2, Example 2

[0041] The pig-derived valved conduits (pig age > half a year, weight 20-30 kg) were obtained in Example 2. The main difference between Example 2 and Example 1 is that in S1, Example 2 added cefoxitin 240 mg, lincomycin 120 mg, polymyxin B 100 mg and vancomycin 50 mg per liter of sterile filtered nutrient medium (Hank's balanced salt solution, 9.6 g / l) according to the report of M. Esther R V team. Three valved conduits were obtained by the method of Example 2.

[0042] 3. Example 3

[0043] The main difference between Example 3 and Example 1 is that the goat-derived valved conduits (goat age > 1 year, weight 46-58 kg) were obtained in Example 3. In S2, Example 3 placed the valved conduits in 0.5% SDC and 0.5% SDS solution for 48 h, followed by 2 cycles of distilled water washing (12 h / cycle), and then 8 cycles of washing with Ringer lactate solution, continuously oscillating at room temperature. Three valved conduits were obtained by the method of Example 3.

[0044] 4. Example 4

[0045] The main difference between Example 4 and Example 2 is that the goat-derived valved conduits (goat age > 1 year, weight 46-58 kg) were obtained in Example 4. In S2, Example 4 placed the valved conduits in 1% SDC and 1% SDS solution for 48 h, followed by 2 cycles of distilled water washing (12 h / cycle), and then 8 cycles of washing with Ringer lactate solution, continuously oscillating at room temperature. Three valved conduits were obtained by the method of Example 4.

[0046] 5. Example 5

[0047] The main difference between Example 5 and Example 1 is that the bovine-derived valved conduits (bovine age > 2 years, weight 400-600 kg) were obtained in Example 5. In S2, Example 5 placed the valved conduits in 1% SDC and 1% SDS solution for 24 h. PBS buffer solution containing penicillin and streptomycin (100 mg / mL, P / S) was washed 6 times (12 h / time). Three valved conduits were obtained by the method of Example 5.

[0048] 6. Example 6

[0049] The main difference between Example 6 and Example 2 is that Example 6 obtains bovine source valved conduits (bovine age > 2 years, body weight 400-600 kg). In S2, Example 6 refers to the report of Lichtenberg A team, and the valved conduit is placed in 1% SDC and 1% SDS solution for 24 h. PBS buffer solution containing penicillin and streptomycin (100 mg / mL, P / S) is washed 6 times (12 h / time). Three valved conduits are obtained by the method of Example 6.

[0050] 7. Detection

[0051] The warming recovery method of the valved conduit: the valved conduit stored at low temperature in S3 is soaked in a water bath at 37°C for 20-30 min. After thawing, the valved conduit is taken out of the sterile bag, soaked in RPMI 1640 complete culture medium for 3 min, and then washed twice with RPMI 1640 complete culture medium.

[0052] After the valved conduits obtained in Examples 1-6 are warmed and recovered, visual observation shows that the conduits and leaflets of the valved conduits obtained in Examples 1, 3 and 5 are attached with impurities, and the conduits of the valved conduits are cracked. The conduits of the valved conduits obtained in Examples 2, 4 and 6 are cracked. The cracking conditions of the conduits of the valved conduits obtained in Examples 2, 4 and 6 are recorded, and the results are shown in Table 1 below:

[0053] Table 1: Cracking conditions of the conduits of the valved conduits obtained in Examples 2, 4 and 6

[0054] Group Number of pipes in which cracks appeared Example 2 2 Example 4 2 Example 6 3

[0055] As can be seen from Table 1, although the surfaces of the valved conduits obtained in Examples 2, 4 and 6 are not attached with impurities, at least two conduits are cracked in the three valved conduits obtained in each example, and there is a problem of high probability of conduit cracking.

[0056] The valved conduits obtained in Examples 2 and 4 that do not have conduit cracking are selected, and the durability test of the valved conduits that do not have conduit cracking is carried out according to the provisions of “GB / T 12279.1-2024”, “ISO 5840-1:2021” and “ISO 5840-2:2021”, the tearing of the leaflets is observed, and the number of durability tests when the leaflets are torn is recorded.

[0057] The valved conduit is subjected to pulsatile flow experiment at 0 times of durability test (before durability test), 20 million times (equivalent to 0.5 years of use of the valve), 50 million times (equivalent to 1.25 years of use of the valve) and 100 million times (equivalent to 2.5 years of use of the valve) of durability test, respectively, and the regurgitation percentage of the valve is recorded.

[0058] Table 2: Durability test and pulsatile flow test results of the valved vessels obtained in Example 2, 4

[0059]

[0060]

[0061] As can be seen from Table 2, the leaflets of the valved vessels obtained in Example 2, 4 were torn in the durability test of 20 million times, and the regurgitation percentage of the valves was close to 50 and the valves failed in the pulsatile flow test of 1 billion times.

[0062] In summary, it can be seen that the valved vessels obtained by the methods reported in the prior art have the problems of impurities attached to the valved vessels, high probability of cracks in the pipeline, easy tearing of the leaflets in the durability test, and the regurgitation percentage of the valves close to 50 and the valves failing in the pulsatile flow test of 1 billion times.

[0063] The inventors further improved the experimental steps and optimized the treatment conditions such as the type of antibiotics in order to solve the above problems. See Examples 7-14 for specific improvements.

[0064] II. Examples 7-14

[0065] 1. Example 7

[0066] In the first step, the valved vessels obtained after dissection from a pig source (pig age > half a year, body weight 20-30 kg) were soaked in RPMI 1640 complete medium with antibiotics at 4°C with a shaking speed of 200 rpm for 24 h.

[0067] The weight of the valved vessels obtained after dissection was measured, and the ratio of the weight of the valved vessels to the volume of RPMI 1640 complete medium with antibiotics was 1:30. The antibiotics included amikacin 0.5 mg / ml, vancomycin 0.6 mg / ml, secnidazole 0.3 mg / ml, and 5-fluorocytosine 1.0 mg / ml. Amikacin is an antibiotic covering gram-negative bacilli, vancomycin is an antibiotic covering gram-positive bacilli, secnidazole is a nitroimidazole antibiotic, and 5-fluorocytosine is an antifungal antibiotic.

[0068] After sterilization, the valved vessels were taken out of the RPMI 1640 complete medium with antibiotics, and the second step and microbial detection were performed simultaneously.

[0069] Microbial detection step: take the excess fat tissue on the surface of the vascular flap and put it into the microbial culture medium. After 14 days of culture, observe whether bacteria grow. Microbial detection can ensure that the microbial detection is negative, and there is no fungus (aspergillus, candida, penicillium, etc.), bacteria (pseudomonas, escherichia coli, acinetobacter, klebsiella, etc.).

[0070] Second step, at 4°C, the vascular flap obtained after sterilization in the first step is soaked in the decellularization solution for 24h (the solution is changed every 8-12h), then the vascular flap is taken out of the decellularization solution and rinsed with physiological saline.

[0071] The decellularization solution includes 1% (v / v) Triton X-100, 1% (w / v) SDS, and 1% (w / v) SDC. The preparation method of the decellularization solution is as follows: weigh SDS, SDC, and Triton X-100, and dissolve them in physiological saline to make the concentration of Triton X-100, SDS, and SDC in the decellularization solution all 1%.

[0072] Third step, at 4°C in a shaking table, the shaking table rotates at 200rpm, the vascular flap obtained in the second step is soaked in RPMI 1640 complete medium with antibiotics for 24h.

[0073] The vascular flap is taken out of the RPMI 1640 complete medium with antibiotics, and rinsed with RPMI 1640 complete medium (the total rinsing time is not more than 24h, and the rinsing solution is changed every 8-12h), to obtain the vascular flap after secondary sterilization.

[0074] Fourth step, the vascular flap after secondary sterilization obtained in the third step is placed in a sterile bag, and RPMI 1640 complete medium containing 10% DMSO is added to the sterile bag. The weight ratio of the vascular flap to the RPMI 1640 complete medium containing 10% DMSO is 1:2. After the sterile bag is sealed, it is placed in a programmed temperature controller, and the temperature is reduced to -80°C at a speed of -1°C / min, and then the sterile bag is transferred to the gas phase liquid nitrogen for low temperature storage.

[0075] 2. Examples 8-10

[0076] The main difference between examples 8-10 and the method of example 7 is that the antibiotics in the RPMI 1640 complete medium with antibiotics are different, see the following table for details:

[0077] Table 3: Main differences between the methods of examples 7-10

[0078]

[0079]

[0080] Each of the examples obtained 3 valved vessels.

[0081] 3. Detection

[0082] The method for warming up the valved vessels: the valved vessels in the sterile bag were soaked in a 37℃ water bath for 20-30min for thawing. After thawing, the valved vessels were taken out of the sterile bag, soaked in RPMI 1640 complete culture medium for 3min, and then washed twice with RPMI 1640 complete culture medium.

[0083] After warming up the valved vessels obtained in Examples 7-10, the cracks in the conduits of the valved vessels were observed by visual observation. The results are shown in the following table:

[0084] Table 4: The conduit crack situation of the valved vessels obtained in Examples 7-10

[0085] Group Number of pipes in which cracks appeared Example 7 2 Example 8 3 Example 9 2 Example 10 2

[0086] As can be seen from Table 4, the valved vessels obtained in Examples 7-10 still have the problem of high probability of conduit crack.

[0087] The valved vessels obtained in Examples 7, 9 and 10 without conduit cracks were selected, and the durability test was carried out on the valved vessels without conduit cracks according to the provisions of GB / T 12279.1-2024, ISO 5840-1:2021 and ISO 5840-2:2021. The tearing of the leaflets was observed, and the number of durability tests when the leaflets were torn was recorded.

[0088] At 0 times, 20 million times, 50 million times and 100 million times of the durability test, the valved vessels were subjected to pulsatile flow test, the regurgitation was observed, and the regurgitation percentage was recorded.

[0089] Table 5: Durability test and pulsatile flow test results of the valved vessels obtained in Examples 7, 9 and 10

[0090]

[0091]

[0092] As can be seen from Table 5, the valved vessels obtained in Examples 7, 9 and 10 were torn at 20 million times or 50 million times of the durability test. Through the pulsatile flow test, it was found that the regurgitation percentage of the valve exceeded 50 at 100 million times of the durability test, and the valve failed.

[0093] In summary, the experimental steps were improved, the types of antibiotics were optimized, the probability of pipe cracking was high, the leaf was easy to tear, and the valve failure problem was not solved when the durability test was 1 billion times.

[0094] 4. Examples 11-14

[0095] The inventors further optimized the experimental parameters and conditions, and the specific differences of Examples 11-14 are shown in the table below:

[0096] Table 6: Main differences of the methods of Examples 11-14

[0097]

[0098]

[0099] Three valved vessels were obtained for each example.

[0100] 5. Detection of Examples 11-14

[0101] Method for warming and resuscitating valved vessels: The valved vessels in sterile bags were soaked in a 37℃ water bath for 20-30min. After thawing, the valved vessels were taken out of the sterile bags, soaked in RPMI 1640 complete culture medium for 3min, and then washed twice with RPMI 1640 complete culture medium.

[0102] After warming and resuscitating the valved vessels obtained in Examples 11-14, the occurrence of pipe cracking in the valved vessels was observed by visual observation, and the results are shown in the table below:

[0103] Table 7: Occurrence of pipe cracking in valved vessels obtained in Examples 11-14

[0104] Group Number of pipes in which cracks appeared Example 11 2 Example 12 2 Example 13 2 Example 14 1

[0105] As can be seen from Table 7, compared with Examples 1-10, the probability of pipe cracking in the valved vessels obtained in Examples 11-14 was reduced, but there was still a problem of high probability of pipe cracking.

[0106] The valved vessels obtained in Examples 11-14 without pipe cracking were selected, and the durability test was performed on the valved vessels without pipe cracking according to the provisions of GB / T 12279.1-2024, ISO 5840-1:2021 and ISO 5840-2:2021. The tearing of the leaf was observed, and the number of durability tests when the leaf was torn was recorded. The valved vessels were taken for pulsatile flow experiments at 0 times, 20 million times, 50 million times and 1 billion times of durability test, respectively, and the regurgitation percentage of the valve was recorded. The average regurgitation percentage of two valved vessels of Example 14 was calculated.

[0107] Table 8: Durability test and pulsatile flow test results of the valved vessels obtained in Examples 11-14

[0108]

[0109] As can be seen from the above Table 8, in the durability test of 500 million times, the leaflets of the valved vessels obtained in Examples 11-14 all tore, and in the pulsatile flow experiment of the valved vessels in the durability test of 1 billion times, the regurgitation percentage of the valve was close to 50, and the valve failed.

[0110] In summary, in the valved vessels obtained in Examples 11-14, the mechanical properties of the conduit and the leaflets were poor, the probability of cracks in the conduit was high after resuscitation, the leaflets were prone to tear, and through the durability test and the pulsatile flow test, it was found that in the durability test of 1 billion times, the regurgitation percentage of the valve was close to 50, and the valve failed. The problems were not solved.

[0111] During an experiment, the inventors accidentally found that the mechanical properties of the valved vessels obtained by immersing the second sterilized valved vessels obtained in the third step in a collagen solution were improved, the probability of cracks in the conduit was significantly reduced, and the valve still worked normally in the durability test of 2 billion times. See Examples 15 and 16 for details.

[0112] III. Examples 15-16

[0113] The main difference between Example 15 and Example 7 is that in the fourth step, the second sterilized valved vessels obtained in the third step are immersed in a 4 mg / ml collagen solution (without adding RPMI 1640 complete medium) for freezing, and the weight ratio of the valved vessels to the volume of the collagen solution is 1:3.

[0114] The main difference between Example 16 and Example 8 is that in the fourth step, the second sterilized valved vessels obtained in the third step are immersed in a 4 mg / ml collagen solution (without adding RPMI 1640 complete medium) for freezing, and the weight ratio of the valved vessels to the volume of the collagen solution is 1:4.

[0115] Preparation of the collagen solution: Referring to Example 5 of the Chinese patent application for invention with publication number CN118048704A, collagen fibers are prepared, and the collagen fibers are dissolved in a PBS buffer solution with pH 7.4 to obtain a collagen solution with a specified concentration.

[0116] The valved vessels in the sterile bag are immersed in a 37°C water bath for 20-30 min for thawing. After thawing, the valved vessels are taken out of the sterile bag, immersed in RPMI 1640 complete medium for 3 min, and then washed twice with RPMI 1640 complete medium.

[0117] After the warmed resuscitation of the valved vessels obtained from Example 15 and Example 16, the inventors observed the cracking of the conduits of the valved vessels by visual observation, and unexpectedly found that no cracking occurred in the conduits of the valved vessels. As shown in Figure 1 Figure 1 A is the valved vessel obtained from Example 15, Figure 1 B is the valved vessel obtained from Example 16. Figure 1 C is the valved vessel obtained from Example 7. Figure 1 D is the valved vessel obtained from Example 8.

[0118] According to the provisions of GB / T 12279.1-2024, ISO 5840-1:2021 and ISO 5840-2:2021, the durability test was performed on the valved vessels obtained from Example 15 and Example 16, and the tearing of the leaflets was observed. It was found that the leaflets of the valved vessels obtained from Example 15 and Example 16 were not torn after 200 million times of testing.

[0119] At 0 times, 20 million times, 50 million times, 100 million times and 200 million times of the durability test, the valved vessels were subjected to pulsatile flow experiments, the regurgitation percentage of the valves was recorded, and the average regurgitation percentage of the three valved vessels was calculated.

[0120] Table 9: Durability test and pulsatile flow test results of valved vessels obtained from Example 15-16

[0121]

[0122] In Example 15 and Example 16, the inventors soaked the valved vessels obtained after the second sterilization in the third step in a 4 mg / ml collagen solution, and unexpectedly found that the conduits of the obtained valved vessels had no cracks, the leaflets of the valved vessels were not torn after 200 million times of durability test, and the valves still worked normally after 200 million times of durability test.

[0123] Based on the results of Example 15 and Example 16, the inventors further optimized the experimental steps, the type of antibiotics and other treatment conditions. See Examples 17-26 for details.

[0124] Four, Examples 17-26

[0125] 1. Example 17

[0126] In the first step, the porcine valved vessels obtained after dissection were soaked in RPMI 1640 complete medium with antibiotics at 4°C under the condition of 200 rpm shaking speed for 18 h. The weight ratio of the valved vessels (before treatment) to the volume of RPMI 1640 complete medium with antibiotics was 1:25.

[0127] ​The antibiotics include amikacin 0.5 mg / ml, linezolid 0.6 mg / ml, secnidazole 0.5 mg / ml and 5-flucytosine 2 mg / ml.

[0128] After sterilization, the valved vessel is taken out of the antibiotic-added RPMI 1640 complete medium, and the second step and microbial detection are performed.

[0129] Microbial detection step: the excess fat tissue on the surface of the valved vessel is placed in a microbial culture medium, and whether bacteria grow is observed after 14 days of culture. Microbial detection can ensure that the microbial detection is negative, and there is no fungus (aspergillus, candida, penicillium, etc.), bacteria (pseudomonas, escherichia coli, acinetobacter, klebsiella, etc.).

[0130] Second step, the valved vessel obtained after sterilization in the first step is soaked in the decellularization solution at 4°C for 24 h under the condition that the shaking speed of the shaker is 200 rpm, and then the valved vessel is taken out of the decellularization solution and rinsed with physiological saline. The decellularization solution is replaced every 8-12 h. The decellularization solution includes 1% (v / v) Triton X-100 and 1% (w / v) SDS.

[0131] Third step, the valved vessel obtained in the second step is soaked in the antibiotic-added RPMI 1640 complete medium at 4°C for 24 h under the condition that the shaking speed of the shaker is 200 rpm, and the antibiotics are the same as in the first step.

[0132] Then, the valved vessel is taken out of the antibiotic-added RPMI 1640 complete medium, and the valved vessel is rinsed with RPMI 1640 complete medium (the total rinsing time is not more than 24 h, and the rinsing solution is replaced every 8-12 h) to obtain the valved vessel after secondary sterilization.

[0133] Fourth step, the valved vessel after secondary sterilization obtained in the third step is placed in a sterile bag, and 3 mg / ml of collagen solution is added to the sterile bag, and the weight ratio of the valved vessel to the volume of the collagen solution is 1:5. After the sterile bag is sealed, it is placed in a programmed temperature controller, and the temperature is reduced to -80°C at a speed of -1°C / min, and then the sterile bag is transferred to the gas phase liquid nitrogen for low-temperature storage.

[0134] Three valved vessels are obtained by the method of Example 17.

[0135] 2, Example 18

[0136] First step, the dissected sheep-derived valved vessels were soaked in the antibiotic- added RPMI 1640 complete medium at 10°C with a shaking speed of 100 rpm for 18 h. The weight of the valved vessels (before treatment) and the volume of the antibiotic- added RPMI 1640 complete medium were in a ratio of 1:30.

[0137] The antibiotics included amikacin 0.5 mg / ml, linezolid 0.6 mg / ml, metronidazole 1.0 mg / ml, and 5-flucytosine 2.0 mg / ml.

[0138] After sterilization, the valved vessels were taken out of the antibiotic- added RPMI 1640 complete medium, and the second step and microbial detection were performed.

[0139] Microbial detection step: the excess fat tissue on the surface of the valved vessels was placed in a microbial culture medium, and whether bacteria grew was observed after 14 days of culture. The microbial detection ensured that the microbial detection was negative, and there was no fungus (aspergillus, candida, penicillium, etc.), bacteria (pseudomonas, escherichia coli, acinetobacter, klebsiella, etc.).

[0140] Second step, the valved vessels obtained after sterilization in the first step were soaked in the decellularization solution at 10°C with a shaking speed of 100 rpm for 24 h, and then the valved vessels were taken out of the decellularization solution and rinsed with physiological saline. The decellularization solution was replaced every 8-12 h. The decellularization solution included 0.5% (w / v) SDS and 1% (w / v) SDC.

[0141] Third step, the valved vessels obtained in the second step were soaked in the antibiotic- added RPMI 1640 complete medium at 10°C with a shaking speed of 100 rpm for 48 h. Then the valved vessels were taken out of the antibiotic- added RPMI 1640 complete medium and rinsed with RPMI 1640 complete medium (the total rinsing time was not more than 24 h, and the rinsing solution was replaced every 8-12 h) to obtain the valved vessels after secondary sterilization.

[0142] Fourth step, the valved vessels after secondary sterilization obtained in the third step were placed in a sterile bag, 3 mg / ml of collagen solution was added to the sterile bag, and the weight of the valved vessels and the volume of the collagen solution were in a ratio of 1:3. After the sterile bag was sealed, it was placed in a programmed temperature controller, and the temperature was reduced to -80°C at a speed of -1°C / min. Then the sterile bag was transferred to the gas phase liquid nitrogen for low-temperature storage.

[0143] Three valved vessels were obtained by the method of Example 18.

[0144] 3, Example 19

[0145] First step, at 10℃, the bovine source of valve vessels obtained by dissection were soaked in RPMI 1640 complete culture medium with antibiotics for 24h. The weight of valve vessels (before treatment) and the volume ratio of RPMI 1640 complete culture medium with antibiotics = 1:40.

[0146] The antibiotics include ceftazidime 1.0mg / ml, vancomycin 0.8mg / ml, metronidazole 1.0mg / ml and 5-fluorocytosine 1.0mg / ml. The sterilization was carried out in a shaker with a rotation speed of 100-200rpm. After sterilization, microbial detection was carried out, and the microbial detection was negative, ensuring no fungi and bacteria. The fungi include aspergillus, candida, penicillium, etc., and the bacteria include pseudomonas, escherichia coli, acinetobacter, klebsiella, etc.

[0147] Second step, at 4℃, the valve vessels obtained after sterilization in the first step were soaked in the decellularization solution for 24h, and the solution was changed every 8-12h, then the valve vessels were taken out from the decellularization solution and rinsed with physiological saline. The decellularization solution includes 0.5% (v / v) Triton X-100 and 1.5% SDS.

[0148] Third step, at 4℃, the valve vessels obtained in the second step were soaked in RPMI 1640 complete culture medium with antibiotics in a shaker with a rotation speed of 100-200rpm for 36h, and the antibiotics were the same as the first step.

[0149] Then, the valve vessels were taken out from the RPMI 1640 complete culture medium with antibiotics, rinsed with RPMI 1640 complete culture medium (the total rinsing time was not more than 24h, and the rinsing solution was changed every 8-12h), and the valve vessels were obtained after secondary sterilization.

[0150] Fourth step, the valve vessels obtained after secondary sterilization in the third step were placed in a sterile bag, 5mg / ml collagen solution was added to the sterile bag, and the weight of valve vessels and the volume of collagen solution = 1:4. After the sterile bag was sealed, it was placed in a programmed temperature controller, and the temperature was reduced to-80℃ at a speed of-2℃ / min, and then the sterile bag was transferred to gas phase liquid nitrogen for low temperature storage.

[0151] Three valve vessels were obtained by the method of Example 19.

[0152] 4, Example 20

[0153] First step, the human source of valve vessel obtained by dissection was soaked in the RPMI 1640 complete culture medium with antibiotics at 8℃ and the speed of 200 rpm for 24 h. The antibiotics were the RPMI 1640 complete culture medium with antibiotics, and the weight of the valve vessel (before treatment) and the volume of the antibiotics were in the ratio of 1:35.

[0154] The antibiotics included 1.0 mg / ml of ceftazidime, 0.5 mg / ml of vancomycin, 0.8 mg / ml of secnazole and 3.0 mg / ml of 5-fluorocytosine.

[0155] After sterilization, the valve vessel was taken out of the RPMI 1640 complete culture medium with antibiotics, and the second step and the microbial detection were performed.

[0156] Microbial detection step: the excess fat tissue on the surface of the valve vessel was put into a microbial culture medium, and whether bacteria grew was observed after 14 days of culture. The microbial detection ensured that the microbial detection was negative, and there was no fungus (aspergillus, candida, penicillium, etc.), bacteria (pseudomonas, escherichia coli, acinetobacter, klebsiella, etc.).

[0157] Second step, the valve vessel obtained after sterilization in the first step was soaked in the decellularization solution at 8℃ for 24 h, and then the valve vessel was taken out of the decellularization solution and rinsed with normal saline. The decellularization solution was replaced every 8-12 h, and the decellularization solution included 1.5% (v / v) Triton X-100 and 1% (w / v) SDC.

[0158] Third step, the valve vessel obtained in the second step was soaked in the RPMI 1640 complete culture medium with antibiotics at 8℃ in a shaking table at the speed of 200 rpm for 24 h, and the antibiotics were the same as those in the first step.

[0159] Then, the valve vessel was taken out of the RPMI 1640 complete culture medium with antibiotics, rinsed with the RPMI 1640 complete culture medium (the total rinsing time was not more than 24 h, and the rinsing solution was replaced every 8-12 h), and a twice-sterilized valve vessel was obtained.

[0160] Fourth step, the twice-sterilized valve vessel obtained in the third step was put into a sterile bag, 5 mg / ml of collagen solution was added to the sterile bag, and the weight of the valve vessel and the volume of the collagen solution were in the ratio of 1:2. After the sterile bag was sealed, it was placed in a programmed temperature controller, and the temperature was reduced to -80℃ at the speed of -3℃ / min, and then the sterile bag was transferred to the gas phase liquid nitrogen for low-temperature storage.

[0161] Three valve vessels were obtained by the method of Example 20.

[0162] 5, Example 21

[0163] The main difference between the method of Example 21 and the method of Example 17 is that in the fourth step, the collagen solution concentration is 4 mg / ml, and the ratio of the weight of the valved vessel to the volume of the collagen solution is 1 :6. Three valved vessels were obtained using the method of Example 21.

[0164] 6. Example 22

[0165] The main difference between the method of Example 22 and the method of Example 17 is that in the fourth step, the collagen solution concentration is 4 mg / ml, and the ratio of the weight of the valved vessel to the volume of the collagen solution is 1 : 1. Three valved vessels were obtained using the method of Example 22.

[0166] 7. Example 23

[0167] The main difference between the method of Example 23 and the method of Example 18 is that in the fourth step, the collagen solution concentration is 2 mg / ml, and the ratio of the weight of the valved vessel to the volume of the collagen solution is 1 :3. Three valved vessels were obtained using the method of Example 23.

[0168] 8. Example 24

[0169] The main difference between the method of Example 24 and the method of Example 18 is that in the fourth step, the collagen solution concentration is 3 mg / ml, and the ratio of the weight of the valved vessel to the volume of the collagen solution is 1 :7. Three valved vessels were obtained using the method of Example 24.

[0170] 9. Example 25

[0171] The main difference between the method of Example 25 and the method of Example 19 is that in the fourth step, the collagen solution concentration is 5 mg / ml, and the ratio of the weight of the valved vessel to the volume of the collagen solution is 1 : 1. Three valved vessels were obtained using the method of Example 25.

[0172] 10. Example 26

[0173] The main difference between the method of Example 26 and the method of Example 20 is that in the fourth step, the collagen solution concentration is 7 mg / ml, and the ratio of the weight of the valved vessel to the volume of the collagen solution is 1 :3. Three valved vessels were obtained using the method of Example 26.

[0174] 11. Inspection

[0175] The valved vessels in the sterile bag were thawed by immersion in a water bath at 37°C for 20-30 min. After thawing, the valved vessels were removed from the sterile bag, immersed in RPMI 1640 complete medium for 3 min, and washed twice with RPMI 1640 complete medium.

[0176] As Figure 2As shown, after HE staining of the valved vessels obtained in Examples 17, it was found that the pulmonary artery conduit and valve cells were completely removed, the inner layer cells of the conduit were not lysed, and solid cell nuclei were present; the valve nuclei were completely washed clean.

[0177] After warming resuscitation of the valved vessels obtained in Examples 17-26, visual observation found that the conduits of the valved vessels obtained in Examples 17-20 had no cracks, and the conduits of the valved vessels obtained in Examples 21-26 had cracks as follows:

[0178] Table 10: Conduit crack situation of valved vessels obtained in Examples 21-26

[0179] Group Number of pipes in which cracks appeared Example 21 1 Example 22 1 Example 23 1 Example 24 1 Example 25 1 Example 26 1

[0180] As can be seen from the above table, the probability of conduit cracking of the valved vessels obtained in Examples 21-26 is significantly reduced, and the conduits of the valved vessels obtained in Examples 17-20 have no cracks compared with Examples 21-26.

[0181] According to the provisions of “GB / T 12279.1-2024”, “ISO 5840-1:2021” and “ISO 5840-2:2021”, the durability test was performed on the valved vessels obtained in Examples 17-20 and the valved vessels obtained in Examples 21-26 without conduit cracking, the tearing of the leaflets was observed, and the number of durability tests when the leaflets were torn was recorded. Pulsatile flow experiments were performed on the valved vessels at 0 times, 20 million times, 50 million times, 100 million times and 200 million times of durability test, and the average regurgitation percentage of the valve was recorded.

[0182] Table 11: Durability test and pulsatile flow test results of valved vessels obtained in Examples 17-26

[0183]

[0184] As can be seen from Table 11, the leaflets of the valved vessels obtained in Examples 17-20 were not torn after 200 million times of durability test.

[0185] Compared with Examples 17-20, the valved vessels obtained in Examples 21-26 had torn leaflets at 100 million times of durability test, the regurgitation percentage of the valve was significantly increased, and the regurgitation percentage of the valve was close to 50 at 200 million times of durability test, and the valve failed.

[0186] The valved vessels obtained by the method of Examples 15-20, the valved vessels were not torn at 200 million times of durability test, and it was found that the valve could still work normally at 200 million times of durability test.

[0187] Compared with Examples 21-26, the concentration of the collagen solution of Examples 15-20 is 3-5 mg / ml, the weight of the valved vessel and the volume ratio of the collagen solution = 1:2-1:5.

[0188] In summary, the inventors surprisingly found that when the concentration of the collagen solution is controlled to be 3-5 mg / ml, the weight of the valved vessel and the volume ratio of the collagen solution = 1:2-1:5, the cryopreservation and recovery of the valved vessel is good, the conduit does not crack, the leaflet of the valved vessel is not torn in the durability test of 200 million times, and the valve is still working normally in the durability test of 200 million times.

[0189] The above describes preferred embodiments of the present application in detail. It should be understood that those skilled in the art can make many modifications and changes without creative labor based on the concept of the present application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiment based on the prior art according to the concept of the present application shall be within the protection scope defined by the claims.

Claims

1. A method for cryopreservation of biogenic valved blood vessels, characterized in that, The biological valvular blood vessel is placed in a collagen solution for freezing; wherein the concentration of the collagen solution is 3-5 mg / ml; and the weight of the biological valvular blood vessel and the volume of the collagen solution are in a ratio of 1:2-1:

5.

2. The method for cryopreservation of biological valved vessels according to claim 1, characterized in that, Before being placed in the collagen solution, the biological valvular blood vessel is treated as follows: (1) the biological valvular blood vessel is soaked in an antibiotic-containing culture medium; (2) the biological valvular blood vessel soaked in the antibiotic-containing culture medium is decellularized; (3) the biological valvular blood vessel is soaked in the antibiotic-containing culture medium again.

3. The method for cryopreservation of biogenic valved vascular grafts according to claim 2, characterized in that, Before being placed in the collagen solution, the biological valvular blood vessel is treated as follows: (1) the valvular blood vessel is soaked in an antibiotic-containing culture medium at 2-10℃ for 18-36h; (2) the valvular blood vessel obtained after sterilization in the first step is soaked in a decellularization solution at 4-10℃ for 24-48h, then the valvular blood vessel is taken out of the decellularization solution and rinsed with normal saline; (3) the valvular blood vessel obtained in the second step is soaked in an antibiotic-containing culture medium at 2-10℃ for 24-48h, then the valvular blood vessel is taken out of the antibiotic-containing RPMI 1640 complete culture medium and rinsed with RPMI 1640 complete culture medium to obtain the valvular blood vessel after secondary sterilization.

4. The method of cryopreservation of biologic valved conduit according to claim 3, wherein, The donor animal of the biological valvular blood vessel is a mammal such as a pig, a sheep, a cow or a human.

5. The method of claim 3, wherein the biological valved conduit is a biological pulmonary valved conduit. In steps (1) and (3), the weight of the valvular blood vessel and the volume of the antibiotic-containing culture medium are in a ratio of 1:25-1:

40.

6. The method of cryopreservation of biologic valved conduit according to claim 3, wherein Steps (1) and (3) are carried out in a shaking bed, and the rotation speed of the shaking bed is 100-200 rpm.

7. The method of claim 3, wherein the biological valved conduit is a biological pulmonary valve. In steps (1) and (3), the antibiotic includes any four of amikacin, ceftazidime, linezolid, vancomycin, secnazole, metronidazole and 5-flucytosine.

8. The method of claim 3, wherein the biological valved conduit is a biological pulmonary valve. In step (2), the decellularization solution includes two or more reagents of 0.5-1.5% (v / v) triton X-100, 0.5-1.5% (w / v) sodium dodecyl sulfate and 0.5-1.5% (w / v) sodium deoxycholate.

9. The method of claim 3, wherein the biological valved conduit is a biological pulmonary valve. The culture medium is RPMI 1640 complete culture medium.

10. The method of cryopreservation of biologic valved conduit according to claim 1, wherein, The biological valvular blood vessel is an aortic valvular blood vessel or a pulmonary valvular blood vessel.

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