Composite ice inhibitor, vitrification protective agent and blood vessel cryopreservation method

Abstract

The invention discloses a composite ice inhibitor, a vitrification protective agent and a blood vessel cryopreservation method, and belongs to the field of cryopreservation or vitrification preservation of vascularized tissues and organs. According to the volume of 100 mL of the composite ice inhibiting agent, the composite ice inhibiting agent is prepared from 10 to 500 g of polyvinyl alcohol, 10 to 500 g of polyglycerol, 10 to 500 g of trehalose, 10 to 500 g of L-proline, 10 to 500 g of polyvinylpyrrolidone, 10 to 500 g of polyethylene glycol 6000 and the balance of ultrapure water, and the total volume of the composite ice inhibiting agent is 100 mL. According to the volume of 1L of the vitrification protective agent, the vitrification protective agent contains 1.65 to 2.2 mol of dihydric alcohol, 2.3 to 3.1 mol of dimethyl sulfoxide, 2.3 to 3.1 mol of formamide, 12 to 57 mmol of a pH buffer solution, 0.39 to 1.93 mmol of a composite ice inhibitor and the balance of ultrapure water, wherein the total volume is 1L; the compound ice inhibiting agent has the advantages that the solution damage is reduced, the ice inhibiting performance is better, and the damage to cells caused by extra osmotic pressure is reduced under the minimum use metering. The composite ice inhibitor is especially suitable for cryopreservation of endothelial cells, smooth muscle cells and blood vessels.

Description

Technical Field

[0001] The invention relates to the field of freezing or vitrification preservation of vascularized tissues and organs, and in particular to a composite ice inhibitor, a vitrification protective agent and a blood vessel freezing preservation method. Background Art

[0002] Cardiovascular disease is by far the leading cause of disability and death worldwide. Patients with severe cardiovascular disease often require bypass surgery to avoid tissue damage. To date, the most successful vascular conduits for coronary or peripheral vascular surgery are the patient's own blood vessels, usually the great saphenous vein in the leg. Engineered blood vessels or decellularized blood vessels can also be used. Although engineered blood vessels and decellularized blood vessels are easier to obtain and have the characteristics of immediacy than autologous / allogeneic blood vessels, they encounter more serious problems such as acute thrombosis and graft stenosis in actual applications. Even if it can be alleviated by inoculating appropriate cells, small-caliber blood vessels (<6mm) are still prone to intimal hyperplasia.

[0003] Among autologous blood vessels, arteries have a better long-term patency rate than veins and are the first choice for arterial transplantation. However, due to various factors, such as the internal mammary artery can only be transplanted to one diseased coronary artery, the radial artery can be transplanted to multiple diseased coronary arteries in sequence, but the length is limited and it is prone to spasm, the use of arterial transplantation is limited. For this reason, we believe that suitable cryopreserved blood vessels, if the storage volume is sufficient, will have immediacy and will facilitate clinical treatment. Therefore, cryopreserving blood vessels and maintaining good cell activity is the current research difficulty in the field of cryobiology, and it is also an important way for clinical vascular transplantation treatment.

[0004] With the deepening of clinical research, vascular cryopreservation should not be limited to preserving the integrity of vascular structure, but also need to consider the impact of cell survival status on postoperative expectations. Slow freezing method is widely used in cell cryopreservation, but this method cannot avoid ice crystal formation, and it cannot stably guarantee the integrity of tissue / organ structure. In order to avoid ice crystal formation and damage to cells and structures inside tissues and organs, high concentrations of vitrified protective agents (6-9M) are usually used to load protective agents through a process of increasing concentration gradient. However, during the process of loading protective agents, cells of tissues and organs are in contact with protective agents for a long time (about 1-2h), which may cause cell death in severe cases, which will limit the time for tissues / organs to recover function in a short period of time after transplantation. Summary of the invention

[0005] In view of this, the present invention provides a composite ice inhibitor, a vitrification protective agent and a blood vessel cryopreservation method to solve the problems raised in the above-mentioned background technology. By using a new vitrification protective agent and an unloading liquid to cryopreserve the blood vessels, biologically active blood vessels can be obtained, and the blood vessels have potential for clinical application.

[0006] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention discloses a composite ice depressant, which contains 10-500g of polyvinyl alcohol, 10-500g of polyglycerol, 10-500g of trehalose, 10-500g of L-proline, 10-500g of polyvinyl pyrrolidone, and 10-500g of polyethylene glycol 6000, based on 100mL of the composite ice depressant, and is supplemented to 100ml with ultrapure water.

[0007] In a second aspect, the present invention discloses a vitrification protective agent, which contains 1.65-2.2 mol of glycol, 2.3-3.1 mol of dimethyl sulfoxide, 2.3-3.1 mol of formamide, 12-57 mmol of pH buffer, 0.39-1.93 mmol of composite ice inhibitor, based on the volume of 1L of vitrification protective agent, and ultrapure water is added to make up to 1L; the composite ice inhibitor is the composite ice inhibitor described in the first aspect of the present invention.

[0008] As a further embodiment of the present invention: the diol is ethylene glycol or propylene glycol.

[0009] As a further solution of the present invention: based on the volume of 1 L of the vitrification protective agent, 0-200 mmol of glucose is also included.

[0010] As a further embodiment of the present invention: the pH buffer is at least one of K2HPO4, KH2PO4, K2HPO4, NaHCO3, KH2PO4 and Na2HPO4.

[0011] In a third aspect, the present invention discloses a method for vitrification preservation of blood vessels, comprising the following steps: S1. Isolate fresh blood vessels, wash them, place them in a vitrification protective medium at 4-6°C, and perform gradient loading at concentrations of 12.5%, 25%, 50%, 75%, and 100%, each time for 12-18 minutes; S2. Take the blood vessel out of the vitrification protective agent, wipe the surface dry, put it into liquid nitrogen vapor to cool down for 10-20 minutes, then put the blood vessel into a cryopreservation tube and transfer it to liquid nitrogen for storage; The vitrification protective agent used in step S1 is the vitrification protective agent described in the second aspect of the present invention.

[0012] As a further solution of the present invention: the method further comprises step S3: rewarming and unloading the protective agent.

[0013] As a further solution of the present invention: the rewarming and unloading protective agent specifically comprises: taking out the blood vessel, transferring it into an unloading solution at 20-30°C for 4-5 seconds, waiting for the blood vessel to be completely thawed, and then transferring it into a PBS solution at 4-6°C for immersion for use.

[0014] As a further solution of the present invention: based on the volume of 1L of unloading liquid, the unloading liquid includes 12-57mmol of the pH buffer, 0-200mmol of glucose and 0.39-1.93mmol of the composite ice inhibitor; the composite ice inhibitor adopts the composite ice inhibitor according to claim 1.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The composite ice inhibitor (CII) of the present invention is composed of six components: polyvinyl alcohol (PVA), polyglycerol (PGL), trehalose (Trehalose), L-proline (L-Pro), polyvinyl pyrrolidone (PVP-K12), and polyethylene glycol-6000 (PEG6000). While reducing solution damage (including protective agent toxicity damage and osmotic damage), it also has good ice inhibition performance. Under the minimum usage dosage (each component does not exceed 5%, w / v), it reduces the damage to cells caused by additional osmotic pressure. The composite ice inhibitor is particularly suitable for the cryopreservation of endothelial cells, smooth muscle cells, and blood vessels.

[0016] The present invention uses a novel vitrification protective agent and an unloading liquid to cryopreserve blood vessels, thereby obtaining biologically active blood vessels with potential for clinical application. The novel protective agent in the preservation method of the present invention has a synergistic effect with the one-step unloading method, and is more helpful in maintaining cell viability than the traditional gradient unloading protective agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a table showing the corresponding relationship between different protective agent concentrations and cell viability when 7 different formulations of vitrification protective agents are used in the preservation method in the embodiment; Figure 2 When the protective agents of Formula 6 and Formula 7 are used for preservation, after staining with crystal violet, the cell adhesion state maintenance state diagram at different protective agent concentrations, and the relationship diagram between the cell adhesion rate and the protective agent concentration; Figure 3 The photographs are of ice crystals and blood vessels of blood vessel slices loaded with traditional protective agents and the protective agent of the present invention under liquid nitrogen vapor cooling conditions; Figure 4 The photographs are of ice crystals observed by cryomicroscope on blood vessel slices loaded with traditional protective agents and the protective agent of the present invention; Figure 5The figure is a graph showing the change in the survival ability of vascular cells over the days of in vitro culture of fresh vascular rings, vascular rings treated with a traditional protective agent (VS55) and vascular rings treated with the protective agent of the present invention; Figure 6 Pathological sections of fresh blood vessels, blood vessels preserved with traditional protective agents, and blood vessels preserved with the protective agent of the present invention before and after blood vessel transplantation; Figure 7 The statistical table is a table of pulsatility index (PI), end-diastolic velocity / peak systolic velocity (ED / PS), and stenosis index (RI) of fresh blood vessels, blood vessels preserved by traditional protective agents, and blood vessels preserved by the protective agent of the present invention after transplantation surgery; Figure 8 It is a statistical table of stenosis diagrams of fresh blood vessels, blood vessels preserved by traditional protective agents, and blood vessels preserved by the protective agent of the present invention after transplantation surgery; Fig. 9 The comparison of cell migration, cell viability and cell adhesion rate after conventional gradient unloading and one-step unloading of the present invention is shown; Fig.10 Comparison of cell viability, survival, and adhesion rate test results after one-step unloading using unloading solutions with different formulations. DETAILED DESCRIPTION

[0018] For ease of understanding of the present invention, the present invention will be described more fully below in conjunction with specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0020] Any range described in the present invention includes the end value and any numerical value between the end values ​​and any sub-range formed by the end value or any numerical value between the end values.

[0021] Example 1 Screening of vitrification protective agent The present invention is configured with 7 kinds of vitrification protective agents, the formulas are as follows: Protective agent 1: propylene glycol (3.1 mol / L), dimethyl sulfoxide (3.1 mol / L), formamide (3.1 mol / L), KCl (15 mmol / L), glucose (195 mmol / L), pH buffer pair (42 mmol / L K2HPO4 and 15 mmol / L KH2PO4); Protective agent 2: propylene glycol (3.1 mol / L), dimethyl sulfoxide (3.1 mol / L), formamide (3.1 mol / L), KCl (15 mmol / L), glucose (195 mmol / L), pH buffer pair (42 mmol / L K2HPO4 and 15 mmol / L KH2PO4), CII (the dosage range of PVA, PGL, Trehalose, L-Pro, PVP-K12, and PEG6000 was 1-50 g / L); Protective agent 3: ethylene glycol (3.1 mol / L), dimethyl sulfoxide (3.1 mol / L), formamide (3.1 mol / L), KCl (15 mmol / L), glucose (195 mmol / L), pH buffer pair (42 mmol / L K2HPO4 and 15 mmol / L KH2PO4); Protective agent 4: propylene glycol (3.1 mol / L), dimethyl sulfoxide (3.1 mol / L), formamide (3.1 mol / L), KCl (2.7 mmol / L), NaCl (102 mmol / L), pH buffer pair (2 mmol / L KH2PO4; 10 mmol / L Na2HPO4), CII (the dosage range of PVA, PGL, Trehalose, L-Pro, PVP-K12, and PEG6000 is 1-50 g / L); Protective agent 5: propylene glycol (3.1 mol / L), dimethyl sulfoxide (3.1 mol / L), formamide (3.1 mol / L), glucose (195 mmol / L), KCl (2.7 mmol / L), NaCl (102 mmol / L), pH buffer pair (2 mmol / L KH2PO4; 10 mmol / L Na2HPO4), CII (the dosage range of PVA, PGL, Trehalose, L-Pro, PVP-K12, and PEG6000 is 1-50 g / L); Protective agent 6: propylene glycol (3.1 mol / L), dimethyl sulfoxide (3.1 mol / L), formamide (3.1 mol / L), glucose (195 mmol / L), KCl (2.7 mmol / L), pH buffer pair (2 mmol / L KH2PO4; 10 mmol / L Na2HPO4), CII (PVA, PGL, Trehalose, L-Pro, PVP-K12, PEG6000, the dosage of all ingredients is in the range of 1-50 g / L); Protective agent 7: propylene glycol (3.1 mol / L), dimethyl sulfoxide (3.1 mol / L), formamide (3.1 mol / L), glucose (195 mmol / L), pH buffer pair (2 mmol / L KH2PO4; 10 mmol / L Na2HPO4), CII (the dosage range of PVA, PGL, Trehalose, L-Pro, PVP-K12, and PEG6000 is all 1-50 g / L).

[0022] After gradient loading and unloading of the protective agent, the cell viability of the adherent cells was detected. The test results are shown in the attached figure. Figure 1 As shown, the detection process is as follows: Gradient loading of protective agent: The common carotid artery (hereinafter referred to as the blood vessel) was surgically removed from the rat's neck and placed in Hank's solution (containing sodium heparin) at 4°C to clean the adherent mucosa and blood. Then, the blood vessel was placed in a vitrified protective agent at 4°C for gradient loading (the gradient was 12.5%, 25%, 50%, 75%, and 100%), and each step was left to stand for 15 minutes.

[0023] Rewarming and removal of the protective agent were performed using traditional gradient unloading: 50% protective agent → 25% protective agent → 12.5% ​​protective agent → PBS, with each step standing for 15 minutes.

[0024] Combination Figure 1 Explanation and analysis: Protectant 1 is the traditional protectant VS55; Protectant 2 is based on the formula of Protectant 1 and CII is added. By comparing the two, it can be seen that CII has the effect of reducing the damage of the protectant. The difference between Protectant 3 and Protectant 1 is that Protectant 3 replaces the propylene glycol in Protectant 1 with ethylene glycol in equal amounts. By comparing the two, it can be seen that the toxicity of ethylene glycol is lower than that of propylene glycol. The difference between Protectant 4 and Protectant 1 is that the EC buffer in Protectant 1 is replaced with PBS as the buffer. The difference between Protectant 5 and Protectant 4 is that Protectant 5 adds more glucose, and the difference between Protectant 6 and Protectant 5 is that Protectant 6 removes the sodium chloride component in the buffer in PBS.

[0025] Based on the above results, a new buffer (Z-Buffer, ZB) adapted to the use conditions of CII was optimized, namely the pH buffer pair in formula 7 (2 mmol / L KH2PO4; 10 mmol / L Na2HPO4). This buffer had little difference in cell viability compared with the NaCl-removed group, that is, there was little difference between protective agents 6 and 7.

[0026] The vascular adherent cells treated with protectant 6 and protectant 7 were then stained with crystal violet and observed, and the cell adhesion rates were counted. The results are as follows: Figure 2As shown, in terms of cell adhesion rate, the cell adhesion rate retained by the Z-Buffer group is better, that is, the formula of protective agent 7 is better than the formula of protective agent 6.

[0027] Since the toxicity of ethylene glycol is lower than that of propylene glycol, the protective agent 7 formula was further optimized by replacing the propylene glycol in the protective agent 7 formula with ethylene glycol to obtain the final protective agent formula of the present invention, and the protective agent was named LDSCII.

[0028] Evaluation of ice crystal inhibition in solutions and tissues: 1. Comparison of ice crystal content by naked eye observation Three groups of comparative experiments were set up: the standard traditional vitrification protective agent VS55 (8.4M), 75% VS55 with the same molar concentration as LDSCII (6.3M), and LDSCII (6.3M). Among them, VS55 (8.4M) contains 3.1M dimethyl sulfoxide, 3.1M formamide and 2.2M propylene glycol, and the molar concentration of dimethyl sulfoxide, formamide and ethylene glycol in LDSCII (6.3M) is 75% of that in VS55 (8.4M).

[0029] Observations were conducted at the droplet and vascular tissue levels. Observation methods: 1) Droplet: Take 2 μL of protective agent solution and drop it on a glass slide, then place the glass slide on a gauze, and finally place the gauze in liquid nitrogen vapor above the liquid nitrogen surface. 2) Vascular tissue: Take a 0.5 cm to 1 cm blood vessel, incubate it with a vitrified protective agent, wipe off the surface solution with filter paper, then place the blood vessel on a gauze, and finally place the gauze in liquid nitrogen vapor above the liquid nitrogen surface.

[0030] By the attached Figure 3 It can be seen that the LDSCII (6.3M) protective agent of the present invention has no ice crystals visible to the naked eye under liquid nitrogen vapor cooling conditions. In vascular tissue, by comparison, it can be clearly observed that the traditional group without adding CII (composite ice inhibitor) produces obvious white ice crystals.

[0031] 2. Cryomicroscopy observation To further confirm the effect of ice crystal inhibition, use cryo-microscopy to observe the effect on the protective agent solution and ice crystal formation within the blood vessels, and follow the following steps: 1) Liquid samples Mix 75% VS55 (6.3M) or VSCII (6.3M) with 10μm spherical silica, take 3μL and drop it on a coverslip, and cover the drop with another coverslip to stabilize the liquid surface thickness at 10μm for easy observation of ice crystals. Wash the coverslips and wipe them dry with lens paper. In order to be as close to the actual operation process as possible, use the fastest cooling and heating rates of the cryo-microscope to process the samples. Take the first 3-4 ice crystals observed to calculate the radius change curve with temperature. Record the actual temperature change over time during the test.

[0032] 2) Blood vessel slice samples After the blood vessels were sliced ​​into 20 μm slices on a cryostat, the slices were loaded with a gradient of protective agent (the process refers to the adherent cell viability test). After loading, the blood vessel slices were sucked out with a plastic dropper and placed on a cover glass. After absorbing the excess protective agent, another cover glass was used to cover it. In order to restore the actual thermal history of the cryopreservation operation, the glass slide with the blood vessel slices was cooled in liquid nitrogen vapor, quickly placed on the sample stage of a pre-cooled (-150°C) cryo-microscope, and after standing for about 5 minutes, the temperature was raised at the fastest heating rate of the instrument. The actual temperature change over time was recorded during the test.

[0033] As attached Figure 4 As shown in the figure, the protective agent solution (75% VS55) without the composite ice inhibitor showed obvious ice crystals during the cooling process. During the rewarming process, there were many obvious ice crystal shadows in the blood vessel slices of the protective agent solution. In the test results of the present invention, no ice crystals were found to be formed inside the blood vessels, and ice crystals mainly occurred in the solution outside the blood vessel slices.

[0034] Example 2 Determination of one-step unloading method Fresh blood vessels were placed in Hank's solution (containing sodium heparin) at 4°C to clean the adherent mucosa and blood, and then placed in the vitrified protective agent LDSCII (6.3M) at 4°C for gradient loading (the gradient was 12.5%, 25%, 50%, 75%, and 100%), and each step was left to stand for 15 minutes. The blood vessels were taken out of the LDSCII (6.3M) protective agent and directly placed in a 4-6°C PBS solution to remove the protective agent.

[0035] To perform a multi-step uninstall as a control, follow these steps: LDSCII (6.3M) protective agent gradient loading: fresh blood vessels were placed in Hank's solution (containing sodium heparin) at 4°C to clean the adherent mucosa and blood, and then the blood vessels were placed in the vitrified protective agent LDSCII (6.3M) at 4°C for gradient loading (the gradient was 12.5%, 25%, 50%, 75%, 100%), and each step was left to stand for 15 minutes. Multi-step unloading (i.e. gradient unloading) was carried out according to 50% LDSCII protective agent → 25% LDSCII protective agent → 12.5% ​​LDSCII protective agent → PBS, and each step was left to stand for 15 minutes. By setting different concentration gradients for the first step of unloading, the effects of different unloading concentrations on cell viability and adhesion rate were compared.

[0036] See also Fig. 9 ,from Fig. 9 As shown in A, one-step unloading can lead to the appearance of migrating cells on the third day, while multi-step unloading cannot observe this phenomenon on the third day, which proves that the one-step unloading method is helpful to improve the cell migration ability; Fig. 9 In B, the horizontal axis is the first step unloading concentration, 0M corresponds to PBS solution, 1.05M, 2.1M, 3.15M, and 4.2M are the molar concentrations of the protective agent; the normal group is endothelial cells without any treatment. Fig. 9 B shows that, through the verification of the adhesion rate, a high-span unloading gradient is beneficial to retaining cell viability. So far, the one-step unloading method has been clarified, and the unloading solution formula will be optimized.

[0037] Example 3 Screening of unloading liquid formula In order to give full play to the advantages of one-step unloading, the effects of three different unloading solutions were compared from three perspectives: cell viability, cell survival (AO / EB staining detection), and cell adhesion rate, and three unloading solution formulas were set: Unloading solution 1: 2 mM KH2PO4; 10 mM Na2HPO4; 195 mM glucose; 102mmol / L sodium chloride.

[0038] Unloading solution 2: PVP k12 (0.1%, w / v), PEG 6000 (0.1%, w / v), Trehalose (1%, w / v), PGL (0.1%, w / v), PVA (1%, w / v), L-Pro (1%, w / v); 2 mM KH2PO4; 10 mM Na2HPO4; 195 mM glucose; 102mmol / L sodium chloride.

[0039] Unloading solution 3: PVP k12 (0.1%, w / v), PEG 6000 (0.1%, w / v), Trehalose (1%, w / v), PGL (0.1%, w / v), PVA (1%, w / v), L-Pro (1%, w / v); 2 mM KH2PO4; 10 mM Na2HPO4; 195 mM glucose.

[0040] Place fresh blood vessel tissue in Hank's solution (containing sodium heparin) at 4°C to clean the adherent mucosa and blood. Then place the blood vessel in LDSCII vitrification protective agent at 4°C for gradient loading (gradient is 12.5%, 25%, 50%, 75%, 100%), and let it stand for 15 minutes at each step. Take the blood vessel out of the LDSCII protective agent, transfer it to the unloading solution at 25°C for 4 seconds, wait until the blood vessel is completely thawed, and then transfer it to the PBS solution at 4°C.

[0041] Normal group: endothelial cells without any treatment.

[0042] The cell viability was detected using the CCK-8 cell viability detection kit, and the cell survival status after treatment was observed using the dead / live cell staining kit. The cell adhesion rate under bright field was counted. The results are as follows: Fig.10 As shown, Fig.10 In (II), red fluorescence indicates dead cells, and green fluorescence indicates living cells. Fig.10 It can be seen that the vascular cells have better cell viability after one-step unloading with unloading solution 3. It should be pointed out that the formula of unloading solution 1 is based on PBS solution with glucose added. From the perspective of cell viability, the unloading effect of unloading solution 1 is better than that of PBS solution.

[0043] Therefore, it is determined that the unloading solution 3 is the unloading solution formula of the present invention. The following Example 4 is a process of treating blood vessels using the protective agent, unloading solution formula and one-step unloading method determined by the present invention, as well as the detection of the survival of vascular cells.

[0044] Example 4 The present invention uses the following method to treat blood vessels: Step 1: Protective agent loading: The common carotid artery (hereinafter referred to as the blood vessel) was surgically removed from the rat's neck and placed in Hank's solution (containing sodium heparin) at 4°C to clean the adherent mucosa and blood. Then, the blood vessel was placed in LDSCII vitrified protective agent at 4°C for gradient loading (the gradient was 12.5%, 25%, 50%, 75%, and 100%), and each step was left to stand for 15 minutes.

[0045] Step 2, cooling and storage: Take out the blood vessel from the vitrification protective agent, wipe the surface of the vitrification protective agent with filter paper, and place it on the gauze. Place the gauze on the surface of liquid nitrogen, use liquid nitrogen vapor to cool the blood vessel, and after 15 minutes of equilibrium, use liquid nitrogen pre-cooled tweezers to put the blood vessel and the gauze under the blood vessel into a liquid nitrogen pre-cooled cryopreservation tube, and transfer it to a liquid nitrogen tank for storage.

[0046] Step 3: Rewarming and unloading the protective agent: Use tweezers pre-cooled with liquid nitrogen to clamp the gauze and place it in the 25°C unloading solution. After about 4-5 seconds, when the vascular tissue is completely thawed, transfer it to a 4°C PBS solution to remove the protective agent.

[0047] The traditional method group served as a control, and the blood vessels were treated with the following methods: Protective agent loading: The common carotid artery (hereinafter referred to as the blood vessel) was surgically removed from the rat's neck and placed in Hank's solution (containing sodium heparin) at 4°C to clean the adherent mucosa and blood. Then, the blood vessel was placed in VS55 vitrified protective agent at 4°C for gradient loading (the gradient was 12.5%, 25%, 50%, 75%, and 100%), and each step was left to stand for 15 minutes.

[0048] Cooling and storage: Take out the blood vessel from the vitrification protective agent, wipe the surface of the vitrification protective agent with filter paper, and place it on the gauze. Place the gauze on the surface of liquid nitrogen, use liquid nitrogen vapor to cool the blood vessel, and after 15 minutes of equilibrium, use liquid nitrogen pre-cooled tweezers to put the blood vessel and the gauze under the blood vessel into a liquid nitrogen pre-cooled cryopreservation tube, and transfer it to a liquid nitrogen tank for storage.

[0049] Rewarming and unloading protective agent: Use tweezers pre-cooled with liquid nitrogen to clamp the gauze, and place it in 50% VS55 protective agent → 25% VS55 protective agent → 12.5% ​​VS55 protective agent → PBS for gradient unloading, and let it stand for 15 minutes at each step.

[0050] The fresh blood vessel group consisted of common carotid arteries removed from rat necks by surgery without any treatment.

[0051] The vascular cell viability test is divided into the following three parts: 1. In vitro culture of vascular rings The vascular rings were embedded with rat tail type I collagen, and then immersed in DMEM containing 2.5% rat serum and placed in an incubator for culture. Follow-up observation was conducted for about 14 days, and photos were taken for record. Figure 5 As shown, the vascular cells detected by in vitro culture of the vascular ring treated by the preservation method of the present invention have long-term survival ability.

[0052] 2. Vascular pathology analysis: Use 4% paraformaldehyde to fix the vascular tissue at 4°C overnight. Then dehydrate the fixed blood vessels with gradient ethanol, make them transparent with xylene, and embed them in paraffin at 52-54°C. Cut the paraffin blocks into 3μm thickness and stick them on anti-slip slides. Figure 6 As shown in the figure, by observing the vascular pathological sections before and after transplantation, it can be clearly found that the smooth muscle cells in the traditional method group have died and more severe stenosis has occurred, which is similar to the problems reported in the decellularized vascular tissue. The statistics of the degree of stenosis are compared in detail in the transplantation surgery section.

[0053] Depend on Figure 5 It can be seen that by long-term culture of frozen blood vessels, by observing the time when intravascular cell migration occurs, it can be determined whether the intravascular cells have long-term survival ability. The present invention can start the migration of vascular cells on the third day, just like normal blood vessels.

[0054] 3. Vascular transplantation effect: 1) Transplantation surgery The rat carotid artery was transplanted by single knot anastomosis. Amoxicillin and warfarin were added to the drinking water of the rats, with the final concentrations of 4 mg / L and 400 mg / L, respectively, and warfarin was no longer added after two weeks.

[0055] 2) Doppler ultrasound After the transplantation, a portable digital color ultrasound diagnostic instrument was used to detect and record the pulsatility index (PI); stenosis index (RI); end-diastolic velocity / peak systolic velocity (ED / PS). The test was performed every 7 days and observed for 42 days. After each test, the changes in vascular stenosis indicators were counted and the proportion of normal rats was recorded.

[0056] 3) Area of ​​vascular stenosis Pathological sections were obtained by H&E staining. The degree of stenosis was calculated using the following formula: Stenosis degree (%) = (smooth muscle layer inner area - lumen area) / smooth muscle layer inner area × 100% Since it is impossible to obtain a standard circle in vascular sections, the above areas were calculated by measuring the circumference using Image J.

[0057] The changes of the normal rate of rat carotid artery over time were recorded, as shown in Table 1.

[0058] Table 1

[0059] After the transplantation operation, the carotid arteries of the transplanted rats were followed up for about 42 days by Doppler ultrasound detection. It was found that the physiological performance indicators of the blood vessels in the traditional method group began to become abnormal from the 14th day (Table 1) and were significantly worse than those of the present invention from the 28th day (Appendix Figure 7 ), and after 42 days of observation, the stenosis of the traditional method group was higher (Appendix Figure 8 ).

[0060] In summary, it is shown that the use of new protective agents and unloading fluids to cryopreserve blood vessels can produce biologically active blood vessels that have potential for clinical application.

[0061] Although this specification is described according to implementation modes, not every implementation mode includes only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0062] Therefore, the above description is only a preferred embodiment of the present application and is not intended to limit the scope of implementation of the present application; that is, all equivalent changes made according to the scope of the claims of the present application are within the protection scope of the claims of the present application.

Claims

1. A composite ice inhibitor, characterized in that: Based on the volume of 100 mL of the composite ice inhibitor, it contains 10-500 g of polyvinyl alcohol, 10-500 g of polyglycerol, 10-500 g of trehalose, 10-500 g of L-proline, 10-500 g of polyvinyl pyrrolidone, and 10-500 g of polyethylene glycol 6000, and ultrapure water is added to make up to 100 ml.

2. A vitrification protective agent, characterized in that: Based on the volume of 1L of vitrification protective agent, it contains 1.65-2.2 mol of diol, 2.3-3.1 mol of dimethyl sulfoxide, 2.3-3.1 mol of formamide, 12-57 mmol of pH buffer, 0.39-1.93 mmol of composite ice inhibitor, and ultrapure water is added to 1L; Wherein, the composite ice inhibitor is the composite ice inhibitor according to claim 1.

3. The vitrification protective agent according to claim 2, characterized in that: The diol is ethylene glycol or propylene glycol.

4. The vitrification protective agent according to claim 2, characterized in that: Based on 1 L of vitrification protective medium volume, 0-200 mmol of glucose is also included.

5. The vitrification protective agent according to claim 2, characterized in that: The pH buffer is at least one of K2HPO4, KH2PO4, K2HPO4, NaHCO3, KH2PO4 and Na2HPO4.

6. A method for cryopreserving a blood vessel using the vitrification protective agent according to any one of claims 2 to 5, characterized in that: The following steps are involved: S1. Isolate fresh blood vessels, wash them, place them in a vitrification protective medium at 4-6°C, and perform gradient loading at concentrations of 12.5%, 25%, 50%, 75%, and 100%, each time for 12-18 minutes; S2. Take the blood vessel out of the vitrification protective agent, wipe the surface dry, place it in liquid nitrogen vapor to cool down for 10-20 minutes, then place the blood vessel in a cryopreservation tube and transfer it to liquid nitrogen for storage.

7. The method for vitrification preservation of blood vessels according to claim 6, characterized in that: The step S3 is also included: rewarming and unloading the protective agent.

8. The method for vitrification preservation of blood vessels according to claim 7, characterized in that: The rewarming and unloading protective agent specifically comprises: taking out the blood vessel, transferring it into an unloading solution at 20-30° C. for 4-5 seconds, waiting for the blood vessel to be completely thawed, and then transferring it into a PBS solution at 4-6° C. for immersion and standby use.

9. The method for vitrification preservation of blood vessels according to claim 7, characterized in that: Based on the volume of 1L of unloading liquid, the unloading liquid includes 12-57 mmol of the pH buffer, 0-200 mmol of glucose and 0.39-1.93 mmol of the composite ice inhibitor; the composite ice inhibitor is the composite ice inhibitor as claimed in claim 1.

Images