CN111264516B - A kind of blood vessel ice crystal-free cryopreservation solution and blood vessel preservation method - Google Patents

Abstract

The invention discloses a blood vessel ice crystal-free cryopreservation solution and a blood vessel preservation method. The blood vessel ice crystal-free cryopreservation solution comprises 1.0-6.0 mol/L of glycerol, 1.0-7.0 mol/L of formamide, and 0.1-2.5 mol of polyvinylpyrrolidone /L and component X; the component X is one or more of component A, component B or component C; wherein, component A is an NO donor, component B is an endothelin inhibitor or an endothelin antagonist, Component C is an anti-apoptotic agent. The preservation solution of the invention improves the preservation effect of vascular function, especially for the vascular smooth muscle cells and endothelial cells that are most easily damaged by cryopreservation, can effectively protect the vascular smooth muscle cells and endothelial cells, and can effectively protect the vascular smooth muscle cells. The function of cells and endothelial cells, in addition to improving the long-term patency rate after vascular transplantation, greatly guarantees the success rate of vascular clinical transplantation.

Description

A kind of blood vessel ice crystal-free cryopreservation solution and blood vessel preservation method

technical field

The invention belongs to the field of biomedical engineering, and particularly relates to a blood vessel ice crystal-free cryopreservation solution and a blood vessel preservation method.

Background technique

In the past 20 years, the clinical demand for vascular substitutes has increased year by year. The application of allogeneic vascular transplantation can solve various problems such as vascular injury, aneurysm, atherosclerosis, vascular disease complicated by diabetes, various complex heart diseases, and establishment of hemodialysis access. In addition, in the field of organ transplantation, when the vascular anastomosis between the donor organ and the host does not match, additional vascular substitutes need to be transplanted to complete the vascular reconstruction of the transplanted organ. In the past, the invasion of adjacent key blood vessels by malignant tumor tissue was an absolute contraindication to surgery, but with the development of technology, for example, in general surgery, the application of blood vessel transplantation in pancreatic cancer surgery combined with vascular resection and reconstruction can make malignant tumors invading blood vessels get better results. True radical resection. In the Department of Nephrology, the great saphenous vein is transplanted to the forearm and upper arm of uremic patients as a loop transplant to establish permanent vascular access for long-term hemodialysis.

However, according to incomplete statistics, more than 20% of patients who need arterial bypass surgery cannot find suitable blood vessels, and 10% of patients have no blood vessels available. Solving the problem of in vitro long-term preservation of allogeneic vascular tissue has become a key technology to increase the supply of vascular substitutes. The use of traditional cryopreservation methods to preserve vascular tissue can preserve some vascular activity, but due to factors such as cell ice crystal damage during the freezing process, the biological function of blood vessels is reduced - endothelial cell function is damaged, and smooth muscle cells are significantly reduced in contractility. , the long-term patency rate of blood vessels is low, and the results of blood vessel preservation are not ideal. Therefore, it is of great significance to improve the existing vascular preservation technology.

When cells are cryopreserved, the water in the cells will form a large number of ice crystals during the cooling process. The formation of ice crystals will lead to a series of changes in the cells, such as mechanical damage (the cell membrane is punctured by ice crystals), increased electrolyte concentration, osmotic pressure changes, and dehydration. , pH changes, protein denaturation, etc. Adding a cryoprotectant (such as glycerol or dimethyl sulfoxide, etc.) can reduce the freezing point of water in the cytoplasm. During the slow cooling process, the water in the cytoplasm can permeate the cell through the cell membrane and reduce the formation of intracellular ice crystals. , thereby reducing the type and degree of damage that occurs when cells are cryopreserved. By increasing the speed of temperature conduction and the concentration of cryoprotectant, ice crystal-free freezing changes the intracellular and extracellular fluids from liquid to a glass-like amorphous solid state, forming a transparent glassy solid, retaining the normal molecules and ions of the intracellular fluid. distribution, reducing ice crystal formation and improving cell survival.

However, the traditional method of adding cryoprotectant and the method of freezing without ice crystals (cooling, rewarming method) still have shortcomings. High concentrations of cryoprotectants are extremely toxic, causing immeasurable damage to vascular cells. In addition, the vascular structure is composed of endothelial cells, smooth muscle, and adventitia from the inside to the outside. Endothelial cells can prevent the contact of blood components and subendothelial tissue, prevent the activation of intravascular coagulation system and the generation of thrombus, prevent inflammatory cell infiltration, regulate smooth muscle cell migration and proliferation, and the integrity of endothelial cells is important for nitric oxide (NO). Synthesis plays an important role, and NO can effectively promote vasodilation, so the function of endothelial cells is very important. However, during the process of adding and removing cryoprotectants, cryopreservation and rewarming, smooth muscle cells and endothelial cells may also be damaged to varying degrees. Vascular smooth muscle cell damage can result in greatly reduced vasoconstriction and relaxation. After the endothelial cells of the vascular intima are damaged and peeled off, the intima of the blood vessels will be thickened, and the smooth muscle cells will proliferate and migrate under the intima to develop into the main cells, causing the lumen stenosis and affecting the long-term patency rate and other adverse consequences; at the same time, the endothelial cells In addition, the damage of vascular endothelial cells will reduce the secretion of vasodilatory substances such as NO, causing vascular contracture; in addition, the damage of vascular endothelial cells will reduce the secretion of heparin, and the production of less heparin will easily lead to thrombosis, which will affect the long-term patency rate. Therefore, it is also of great significance to reduce the apoptosis of vascular smooth muscle cells and endothelial cells as much as possible during the process of adding, removing cryoprotectants and cryopreservation and resuscitation of blood vessels.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide an ice crystal-free cryopreservation solution for blood vessel preservation.

The second purpose of the present invention is to provide an ice crystal-free cryopreservation method for blood vessels, which injects an ice crystal-free cryopreservation solution step by step before cryopreservation and programmatically controls the temperature during cryopreservation, so as to preserve fresh blood vessel tissue.

To achieve the above-mentioned purpose of the prior art, the application adopts the following technical solutions:

A blood vessel ice crystal-free cryopreservation solution, comprising glycerol 1.0-6.0mol/L, formamide 1.0-7.0mol/L, polyvinylpyrrolidone 0.1-2.5mol/L and component X; the component X is component A, component B One or more of the components or the C components; wherein, the A component is an NO donor, the B component is an endothelin inhibitor or an endothelin antagonist, and the C component is an anti-apoptotic agent.

Preferably, the blood vessel ice crystal-free cryopreservation solution of the present invention comprises 2.0-5.0 mol/L of glycerol, 2.5-5.5 mol/L of formamide, 0.1-1.0 mol/L of polyvinylpyrrolidone and component X; the component X is one or more of A component, B component or C component; wherein, A component is an NO donor, B component is an endothelin inhibitor or endothelin antagonist, and C component is an anti-apoptotic agent.

In some embodiments, the component X of the present invention is the A component, the B component and the C component.

In some embodiments, component A is preferably 0.05-2 mg/L nitroglycerin, 0.1-5 mg/L isosorbide dinitrate, 0.5-10 mg/L morphoamine or 0.3-7 mg/L nicorandil One or more of ; more preferably 0.05-1 mg/L of nitroglycerin. The inventors found that adding component B can improve the preservation quality of blood vessels.

In some embodiments, component B is preferably 5-50 mg/ml ET-1 converting enzyme inhibitor CGS26303, 10-100 mg/ml ETA receptor antagonist PD155080, 50-200 mg/ml ETA receptor antagonist BQ -123, 6-80mg/ml of one or more of ETA/ETBA receptor antagonist RO46-2005; more preferably 20-40mg/ml of ET-1 convertase inhibitor CGS26303. The inventors found that adding component B can improve the preservation effect of blood vessels.

In some embodiments, the C component is preferably one or more of 5-100 μmol/L Caspase inhibitor Z-VAD, 1-50 μmol/L Z-DEVD; more preferably 30-50 μmol/L Z -DEVD. The inventors found that adding component C can improve the preservation quality of blood vessels.

As a further improvement of the present invention, the glycerol content is 3.10 mol/L.

As a further improvement of the present invention, the content of formamide is 3.70 mol/L.

As a further improvement of the present invention, the content of polyvinylpyrrolidone is 0.5mol/L.

As a further improvement of the present invention, other components of the ice-free cryopreservation solution are solvents, and further preferably, the solvents are Euro-Collin solution.

The present invention also provides a blood vessel preservation method, wherein the ice crystal-free cryopreservation solution of the present invention is introduced into the blood vessel tissue in a step-by-step manner.

In some embodiments, the step-by-step method of introducing the ice-free cryopreservation solution of the present invention is to step by step for 10 to 25 minutes under the condition of -5 to +22° C., and increase the amount into the vascular tissue by 4 to 15 steps. Add the ice-free cryopreservation solution of the present invention.

In some preferred embodiments, the step-by-step method of introducing the ice-free cryopreservation solution in the present invention is to add ice-free cryopreservation to the vascular tissue incrementally in 6 steps at 2-4° C. in 15 min steps. Preservation solution, 15min each time, introduce ice crystal-free cryopreservation solution into vascular tissue.

In some embodiments, in the blood vessel preservation method of the present invention, after the ice crystal-free cryopreservation solution of the present invention is introduced in a step-by-step manner, the temperature is first lowered at a rate of -20°C to -70°C/min for the first time. to -80°C to -120°C, and then lower the temperature for the second time to -120°C to -180°C at a rate of -2°C to -40°C/min, and store at this temperature.

In some preferred embodiments, the first cooling in the present invention is cooling at a rate of -38°C to -50°C/min to -90°C-110°C; further preferably, the temperature is lowered at a rate of -43±2°C/min to -100°C.

In some preferred embodiments, the second cooling rate of the present invention is -2°C～-10°C/min. The second cooling rate is -120°C～-150°C; more preferably, it is -3±0.2°C/ Cool down to -135°C at a rate of minutes.

The present invention also provides a vascular resuscitation method, which utilizes a step-by-step method to elute the ice crystal-free cryopreservation solution of the present invention from vascular tissue.

In some embodiments, the step-by-step method of eluting the ice crystal-free cryopreservation solution of the present invention is to freeze blood vessels without ice crystals in steps of 6 to 25 minutes at -5 to +22° C. Preservation solution replacement and elution.

In some preferred embodiments, the step-by-step method for eluting the ice crystal-free cryopreservation solution of the present invention is to reduce the amount of ice crystals in vascular tissue in 6 steps at 2-4° C. in 5 min steps. Cryopreservation solution, 5min each time.

In some embodiments, in the vascular resuscitation method of the present invention, during resuscitation, after the frozen sample is taken out of the storage environment, the temperature is first heated to -70°C to -120°C at a rate of 10°C to 50°C/min. °C, and then the temperature is raised to 1-10 °C for the second time at a speed of 140-350 °C/min, and then the ice crystal-free cryopreservation solution of the present invention is eluted from the vascular tissue in a stepwise manner.

In some embodiments of the present invention, the first temperature increase of the present invention is to increase the temperature to -100°C at a rate of 30±0.2°C/min.

In some embodiments of the present invention, the second temperature increase of the present invention is a rapid rewarming to 4°C at a rate of 225±15°C/min.

The present invention also provides a blood vessel preservation and resuscitation method, which is a combination of the above blood vessel preservation method and resuscitation method.

The present invention also provides a method for using the ice crystal-free cryopreservation solution of the present invention: when vascular tissue is preserved, the ice crystal-free cryopreservation solution of the present invention is introduced into the blood vessel tissue in a step-by-step manner; Specifically, the ice crystal-free cryopreservation solution of the present invention is incrementally added to the blood vessel tissue in steps of 10 to 25 minutes under the condition of -5 to +22° C. in steps of 4 to 15 minutes.

In some preferred embodiments, the step-by-step method is to add ice crystal-free cryopreservation solution to the vascular tissue incrementally in 6 steps at 2-4°C in 15min steps, 15min each time , and introduce ice crystal-free cryopreservation solution into vascular tissue.

In some embodiments, in the method of use of the present invention, after the ice-free cryopreservation solution of the present invention is introduced in a step-by-step manner, the temperature is lowered to a temperature of -20°C to -70°C/min for the first time. -80°C～-120°C, and then lower the temperature to -120°C～-180°C for the second time at the rate of -2°C～-40°C/min, and store at this temperature.

In some preferred embodiments, the first cooling in the present invention is cooling at a rate of -38°C to -50°C/min to -90°C-110°C; further preferably, the temperature is lowered at a rate of -43±2°C/min to -100°C.

In some preferred embodiments, the second cooling rate of the present invention is -2°C～-10°C/min. The second cooling rate is -120°C～-150°C; more preferably, it is -3±0.2°C/ Cool down to -135°C at a rate of minutes.

Advantages of the present invention relative to the prior art:

(1) the preservation solution of the present invention improves the preservation effect of vascular function, especially for the vascular smooth muscle cells and endothelial cells most easily damaged by cryopreservation, the existing vascular cryopreservation technology lacks its protection, the present invention The preservation solution can effectively protect vascular smooth muscle cells and endothelial cells, and has the function of effectively protecting vascular smooth muscle cells and endothelial cells, which is beneficial to improve the long-term patency rate after vascular transplantation. The ultimate test of the efficacy of vascular preservation is the long-term patency rate after transplantation. Regardless of the various indicators in the laboratory examination, the true function of the blood vessels is whether they can be unobstructed and how long they can remain unobstructed after transplantation. The preservation solution of the invention improves the long-term patency rate after vascular transplantation, is the highest index unmatched by other experimental effects, and greatly ensures the success rate of clinical vascular transplantation.

(2) Step-by-step introduction of ice crystal-free cryopreservation solution before freezing, so that the preservation solution can be evenly distributed, fully infiltrate the cells and structures inside and outside the vascular tissue, and reduce the damage of ice crystals to the tissue cell structure; the cooling rate and recovery heating rate are optimized during the freezing process. It can greatly reduce the formation of ice crystals in vascular tissue during cryopreservation and resuscitation. Compared with the existing conventional cryopreservation vascular technology, it can more effectively protect the spatial structure of vascular tissue, improve the success rate of transplantation and maintain function after transplantation, and further improve vascular transplantation. The post-long-term patency rate further improves the vascular preservation effect.

(3) Gradually reduce the content of ice crystal-free cryopreservation fluid in blood vessels during the resuscitation process, and fully reduce the toxic effect on blood vessels.

Description of drawings

Figure 1 Microscopic observation of the size and location of ice crystals in the carotid artery segment of rabbits that were routinely frozen and frozen without ice crystals;

Fig. 2 Comparison of contraction responses of fresh blood vessels, conventional cryo-preserved blood vessels, and ice-free cryo-preserved blood vessels to a group of stimulant drugs (norepinephrine, phenylephrine);

Figure 3 Dose/contraction response curves of fresh vessels, cryopreserved vessels and cryopreserved vessels to a panel of stimulant drugs (norepinephrine, phenylephrine).

Detailed ways

The present invention will be further described below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not intended to limit the present invention. All simple improvements to the preparation method of the present invention under the concept of the present invention It belongs to the protection scope of the present invention. The following examples do not specify the experimental method of specific conditions, usually according to the well-known means in the art. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The specific experimental method is described in the effect experiment.

Example 1:

1. Take 3.10mol glycerol, 3.70mol formamide, 0.5mol polyvinylpyrrolidone, 0.5mg/l nitroglycerin, 30mg/l CGS26303, 40μmol/l Z-DEVD, mix well and add Euro-Collin solution to make up to 1L.

2. Take an appropriate amount of the solution in step 1 and cool it to 2-4°C. Add the cooled solution into the human vascular tissue to be frozen in 6 steps at 2-4°C, and complete the introduction within 90 minutes (step by step at 2-4°C for 15 minutes, follow 6 steps to Add ice crystal-free cryopreservation solution incrementally to vascular tissue, 15 min each time).

3. Cool the human vascular tissue introduced into the cryopreservation solution in the previous step to -100°C at a rate of -43°C/min, and then cool the sample to -135°C at a rate of -3±0.2°C/min. Store at -135°C for at least 24h; after that, samples can be stored at -135°C for a long time.

4. Take out the blood vessel sample to be resuscitated, recover to -70°C at a rate of 30±0.2°C/min, and then return to 4°C at a rate of 225±15°C/min;

5. Under the condition of 2-4°C, step by step for 5 minutes, reduce the amount of ice-free cryopreservation solution to the vascular tissue in 6 steps, replace and elute the ice-free cryopreservation solution within 30 minutes each time for 5 minutes.

Example 2:

1. Take 3.10mol glycerol, 3.70mol formamide, 0.5mol polyvinylpyrrolidone, 0.5mg/l nitroglycerin, mix well, add Euro-Collin solution and make up to 1L.

2. Take an appropriate amount of the solution in step 1 and cool it to 2-4°C. Add the cooled solution into the human vascular tissue to be frozen in 6 steps at 2-4°C, and complete the introduction within 90 minutes (step by step at 2-4°C for 15 minutes, follow 6 steps to Add ice crystal-free cryopreservation solution incrementally to vascular tissue, 15 min each time).

3. Cool the human vascular tissue in the previous step to -100°C at a rate of -43°C/min, then cool the sample to -135°C at a rate of -3±0.2°C/min, and store it at this temperature for at least 24h ; Samples can be stored at -135°C for a long time thereafter.

4. Take out the blood vessel sample to be resuscitated, return to -70°C at a rate of 30±0.2°C/min, and then return to 4°C at a rate of 225±15°C/min.

5. Under the condition of 2-4°C, step by step for 5 minutes, reduce the amount of ice-free cryopreservation solution to the vascular tissue in 6 steps, replace and elute the ice-free cryopreservation solution within 30 minutes each time for 5 minutes.

Example 3:

1. Mix 3.10 mol of glycerol, 3.70 mol of formamide, 0.5 mol of polyvinylpyrrolidone, and 30 mg/l of CGS26303, and add Euro-Collin solution to make up to 1 L.

2. Take an appropriate amount of the solution in step 1 and cool it to 2-4°C. Add the cooled solution into the human vascular tissue to be frozen in 6 steps at 2-4°C, and complete the introduction within 90 minutes (step by step at 2-4°C for 15 minutes, follow 6 steps to Add ice crystal-free cryopreservation solution incrementally to vascular tissue, 15 min each time).

3. Cool the human vascular tissue introduced into the cryopreservation solution in the previous step to -100°C at a rate of -43°C/min, and then cool the sample to -135°C at a rate of -3±0.2°C/min. Store at -135°C for at least 24h; after that, samples can be stored at -135°C for a long time.

4. Take out the blood vessel sample to be resuscitated, return to -70°C at a rate of 30±0.2°C/min, and then return to 4°C at a rate of 225±15°C/min.

5. Under the condition of 2-4°C, step by step for 5 minutes, reduce the amount of ice-free cryopreservation solution to the vascular tissue in 6 steps, replace and elute the ice-free cryopreservation solution within 30 minutes each time for 5 minutes.

Example 4:

1. Take 3.10 mol of glycerol, 3.70 mol of formamide, 0.5 mol of polyvinylpyrrolidone, and 40 μmol/l Z-DEVD. After mixing, add Euro-Collin solution to dilute to 1 L.

2. Take an appropriate amount of the solution in step 1 and cool it to 2-4°C. Add the cooled solution into the human vascular tissue to be frozen in 6 steps at 2-4°C, and complete the introduction within 90 minutes (step by step at 2-4°C for 15 minutes, follow 6 steps to Add ice crystal-free cryopreservation solution incrementally to vascular tissue, 15 min each time).

3. Cool the human vascular tissue introduced into the cryopreservation solution in the previous step to -100°C at a rate of -43°C/min, and then cool the sample to -135°C at a rate of -3±0.2°C/min. Store at -135°C for at least 24h; after that, samples can be stored at -135°C for a long time.

4. Take out the blood vessel sample to be resuscitated, recover to -70°C at a rate of 30±0.2°C/min, and then return to 4°C at a rate of 225±15°C/min; at 2-4°C, within 90min Replace and elute ice-free cryopreservation solution.

5. Under the condition of 2-4°C, step by step for 5 minutes, reduce the amount of ice-free cryopreservation solution to the vascular tissue in 6 steps, replace and elute the ice-free cryopreservation solution within 30 minutes each time for 5 minutes.

Control group 1: A conventional cryoprotectant solution was provided, in volume percent, containing 1.0 MDMSO.

At room temperature, this vascular cryoprotectant was added to the blood vessels to be cryopreserved, and then the blood vessels were transferred to the blood vessels containing 1.0 MDMSO to be frozen for 1 minute, and then placed in a programmed cooling device, and lowered to -90 °C at a speed of -1 °C/min. Then transfer to -196°C liquid nitrogen for long-term storage.

Control group 2: fresh blood vessels without freezing treatment. After blood vessels are obtained, they are placed in Euro-Collin solution and stored at 2-10°C for no more than 12 hours.

Effect experiment:

Effect Experiment 1 Vascular Function and Effect Experiment of Vascular Transplantation

After the blood vessels were treated with the example and the control group, experiments on the effect of blood vessel function and blood vessel transplantation were carried out. Vasoconstrictor and diastolic function examines the response of blood vessels to different doses of vasoconstrictive and diastolic drugs, as well as the susceptibility of blood vessels to these drugs. The recovery of vascular function takes the recovery of vasoconstriction as the main index, and the vasodilation and the sensitivity to drugs as reference. Vascular transplantation was autologous transplantation of carotid arteries from New Zealand rabbits, and one carotid artery was transplanted to the same site on the other side after the treatment of the example and the control group. The survival rate after vascular transplantation refers to the vascular patency after 3 months of vascular transplantation as an index. The recovery of vascular function and the survival rate after vascular transplantation are shown in Table 1.

Table 1 Recovery of vascular function and survival rate after vascular transplantation

Effect experiment 2 Detection of ice crystal formation in conventional freezing and ice-free freezing during the freezing process

Take two blood vessel groups as examples to illustrate: these two groups are control group 1 (traditional cell cryopreservation agent (1.0 mol DMSO) cryopreservation) and example 1 without ice crystal cryopreservation solution group.

Healthy New Zealand rabbits weighing 2 kg were selected, and the allogeneic blood vessels to be transplanted were taken from the carotid arteries of healthy New Zealand rabbits. Use ice-free cryopreservation solution and traditional cell cryopreservation agent (1.0mol DMSO) respectively to complete the step-by-step cryogen introduction according to the method described in Example 1, and then perform programmed cooling, and select ice-free and conventional freezing at -90°C. The rabbit arterial segment samples were sectioned for microscopic observation, as shown in Figure 1, in the control group 1, ice crystal regions of different sizes distributed on the extracellular matrix of the blood vessel wall (B), and white irregular-sized voids in the figure. (thin arrow) is left when ice crystals occupy, from the innermost endothelial layer to the middle smooth muscle layer, so that the outermost blood vessel outer layer is covered with ice crystals (thin arrow), making the structure of cryovascular obvious Changes occurred, as the outer layer was looser with larger ice crystals and the vascular endothelium and muscle layers were tighter with smaller ice crystals. However, the formation of ice crystals in the gaps between the layers (thick arrows) destroys the overall function of blood vessels, especially the formation under the endothelium (thick arrows) is of great significance to whether the integrity of the endothelial layer can be maintained after vascular transplantation. Integrity will cause red blood cells, platelets to accumulate, and blood vessels to block. However, the blood vessels frozen without ice crystals in Example 1 had no ice crystals (A). The integrity of vascular tissue preserved with ice crystal-free cryopreservation solution is higher than that of vascular tissue preserved with traditional cell cryopreservation agent. The cells and tissues in blood vessels preserved with ice crystal-free cryopreservation solution are in good shape and low in damage. It can be seen that in the blood vessels of control group 1, ice crystal regions of different sizes are clearly distributed on the extracellular matrix of the blood vessel wall, which makes the structure of the frozen blood vessels obviously distorted, while the frozen blood vessels without ice crystals in Example 1 have no ice crystals. significantly improved.

Effect Experiment 3 Detection of Rabbit Carotid Artery Function

Take three blood vessel groups as an example to illustrate: these three groups are the blood vessels cryopreserved by the traditional cell cryopreservation agent after resuscitation (control group 1), and the blood vessels cryopreserved without ice crystals in Example 1 (hereinafter referred to as Example 1). Mechanical tests were performed together with fresh blood vessels (control group 2).

1000ml of Krebs Henseleit Buffer (KHB) solution was injected into the isolated vascular tissue perfusion system for experiment. The constant temperature perfusion was adjusted to 37°C, and the tension sensor was calibrated and zeroed. 10ml of KHB solution was injected into the perfusion tank, and 5% O ₂ and CO ₂ were continuously filled. Gently insert the vascular ring between the two probes connected to the sensor, adjust the distance so that the initial tension is 4g, balance for 1 hour, and continuously adjust the distance between the probes to keep the initial tension at 4g, and replace the tank every 15 minutes. KHB solution once. 75ul of 10% KCL solution was added successively to a final concentration of 10-90 mmol/L, and the maximum contractile force after each addition was recorded respectively. Change the KHB solution twice, add 10ul of norepinephrine or phenylephrine (concentrations are ^10-10 , ^10-9 , ^10-8 , ^10-7 , ^10-6 , ^{10-5 ,} 10- ⁴ mol/L), and recorded the maximum contraction force of each time. Finally, after the maximum contractile force remained stable, 10ul of 10 ^-4 mol/L calcium ionophore A23187 or 10 ^-4 mol/L sodium nitroprusside was added to record the maximum post-diastolic vascular tension.

The experimental results showed that after the blood vessels of control group 1 (conventional freezing) were frozen, the contractile force was significantly decreased, and decreased to 23-26%. The blood vessels of the Example 1 (no ice crystals) group retained the contractile force response of the control group 2 (fresh). 23-26%, while the blood vessels of Example 1 (no ice crystals) retained the contractile response of the blood vessels of the control group 2; the ratio of the blood vessels of the control group 1 (conventional frozen) to the blood vessels of the control group 2 (fresh), p<0.0001;# : compared with the blood vessels of the control group 1 (conventional freezing) group and the blood vessels of the example 1 (no ice crystals) group, p<0.0001. Norepinephrine group: control group 2 vessels n=16; Example 1 (no ice crystals) vessels n=43; control group 1 (conventional frozen) vessels n=20; phenylephrine group: control group 2 (fresh ) vessels n=12; Example 1 (no ice crystals) vessels n=31; Control 1 (conventional freezing) vessels n=20.

In addition, control group 1 vasodilation decreased significantly; Example 1 group relaxation reaction was weaker than control group 2, but better than control group 1, specifically as shown in Table 2, norepinephrine was used as precontraction, sodium nitroprusside ( Non-vascular endothelial cell relaxation factor, acting directly on vascular smooth muscle cells) and calcium ionophore A23187 (vascular endothelial cell relaxation factor, acting through vascular endothelial cells) relaxation. Each drug is divided into three groups, control group 2 vascular pre-constriction and relaxation (fresh pre-constriction/fresh relaxation), Example 1 vascular pre-constriction and relaxation (no-ice pre-constriction/no-ice-dilation), control group 1 vascular pre-constriction and relaxation (frozen precontraction/frozen diastole). In the relaxation caused by the action of sodium nitroprusside, the vasodilation tone of control group 1 was significantly decreased (9%), and the relaxation response of example 1 group (35%) was much better than that of control group 1, which was the same as that of control group 2 (37%). The relaxation induced by calcium ionophore A23187 stimulation was lower in both Example 1 (37%) and Control 1 (28%) than Control 2 (68%). It shows that deep hypothermia has an effect on the passage of vascular endothelial cells. Calcium ionophore A23187 group: Control group 2 vessels n=8; Example 1 vessels n=33; Control group 1 vessel n=11; Sodium nitroprusside group: Control group 2 vessels n=16; Example 1 vessels n=33 ; control group 1 vessel n=11.

Table 2 Control group 2, the pre-contraction and relaxation force of the three groups of blood vessels in Example 1 and control group 1

Generally, from the principle of pharmacology, we use pD ₂ to represent the drug concentration that reaches half of the maximum contractile force, that is, the negative logarithm (-log ₎ of the molar concentration that produces 50% of the maximum effect. more sensitive. Control group 2, Example 1 and Control group 1 are the three groups of blood vessels sensitive to norepinephrine and phenylephrine to more accurately detect the degree of damage to blood vessels after treatment in different ways.

As shown in Table 3, the pD2 values of the three groups of blood vessels in control group 2, example 1 and control group 1 indicate the sensitivity of each of the three groups of blood vessels to drugs. The sensitivity of the blood vessels in the control group 1 decreased significantly after being frozen, while the blood vessels in the freeze-free group retained the same sensitivity as the blood vessels in the control group 2. @: The ratio of blood vessels in control group 1 to those without ice crystals, p<0.01; #: Compared with blood vessels in control group 1 and blood vessels in Example 1, p<0.05. Norepinephrine group: control group 2 vessels n=16; Example 1 vessel n=39; control group 1 vessel n=20. Phenylephrine group: Control group 2 vessels n=12; Example 1 vessel n= 27; control group 1 vessel n=20.

Table 3 Control group 2, the pD ₂ values of the blood vessels of the three groups of example 1 and control group 1

drug control group 2 Example 1 control group 1 Norepinephrine 6.207±0.104 6.082±0.115 5.317±0.312@ Phenylephrine 5.720±0.131 5.617±0.130 5.079±0.257^#

The maximum contraction and relaxation of blood vessels only represent the maximum limit that blood vessels can reach after different treatments and preservation, but they do not respond to drug sensitivity. For example, to a drug, what is the concentration at which the blood vessels are maximally responsive. In other words, if the blood vessels are damaged, a higher drug concentration may be required to achieve the concentration can be achieved. Response sensitivity to drugs can test cellular receptor function, smooth muscle contraction and relaxation sensitivity, and the integrity or impairment of overall vascular function. We divided the contractile force corresponding to each dose of each drug by the maximum contractile force to obtain a percentage, and formed a drug dose/contraction response curve. The results are shown in Figure 3: control group 2 (fresh) blood vessels, control Dose-contraction curves of Group 1 vessels and Example 1 (no ice crystals) vessels to a group of stimulant drugs (norepinephrine, phenylephrine). Drug doses ranged from a minimum dose of ^10-10 mol to a maximum dose of ^10-4 mol, and the corresponding contractile force was obtained as a percentage of the maximum contractile force. From the dose-contraction response curve, it can be clearly seen that after the blood vessels of control group 1 (conventional freezing) are treated, the curve obviously shifts to the right, and the sensitivity of blood vessels to the drug decreases, and a larger dose is required to reach control group 2 (fresh ) of the blood vessels and the contractile force of the blood vessels of Example 1 (no ice crystals). However, the contraction force response of blood vessels in Example 1 (no ice crystals) group was similar to that of control group 2 (fresh) blood vessels, indicating that the sensitivity to the drug was not affected. Norepinephrine group: control group 2 (fresh) blood vessels n=16; Example 1 (no ice crystals) blood vessels n=43; control group 1 (conventional frozen) blood vessels n=20. Phenylephrine group: control group 2 (fresh) vessels n=12; Example 1 (no ice crystals) vessels n=27-39; Control 1 (conventional frozen) vessels n=20.

The blood vessels of the three groups were fixed with 10% formalin, made into paraffin sections, and stained with hematoxylin and eosin (HE) to observe the pathological changes of blood vessels. The results show:

(1) Control group 2: Endothelial cells have good continuity and orderly arrangement, very few endothelial cells can be found shedding, the nuclei of the middle smooth muscle cells are clear, and the structures of collagen fibers and elastic fibers are normal.

(2) Control group 1: The endothelial cells shed more seriously, the endothelial cells were arranged in irregular order, and the middle smooth muscle, collagen fibers and elastic fibers were partially broken.

(3) Example 1 group: endothelial cells were slightly exfoliated, a few gaps were seen between the endothelial layer and the subendothelial layer, and the structures of the subendothelial layer, the inner elastic membrane and the smooth muscle cells were still good.

Effect Experiment 4 Autologous vascular transplantation rabbit model to detect vascular patency after vascular transplantation

Take the example of 3 groups of blood vessel transplantation to illustrate: these three groups are control group 2, control group 1 (traditional cell cryopreservation agent (1.0mol DMSO) cryopreservation), the ice crystal-free cryopreservation solution group of Example 1 , 9-12 operations were completed in each group using the middle of the rabbit common carotid artery as the anastomosis site of the autologous artery. Vascular patency rate (Table 3) was detected by ultrasound Doppler probe at 2 and 4 weeks after the operation. At 12 weeks after surgery, the specimens were fixed in 10% formalin for grafted vessels. The fixed blood vessel specimens were made into paraffin sections and stained with hematoxylin and eosin (HE) to observe the pathological changes of the transplanted blood vessels. The changes of the intima, media and adventitia of the grafted arteries and the infiltration of monocytes/lymphocytes in the vessel wall were observed after 3 months.

Table 4. Comparison table of vascular patency after transplantation of control group 2 blood vessels, control group 1 blood vessels and Example 1 blood vessels after transplantation for 2 weeks to 3 months

The results are shown in Table 4. The vascular transplantation model shows that the patency of the vascular tissue cryopreserved using the ice-free cryopreservation solution is close to that of fresh vascular tissue after transplantation, indicating that the present invention can increase the patency of the ice-free cryopreserved blood vessel by adding components A, B and C. sex.

Three months after the operation, the grafted blood vessels in each group were observed, and each treatment group had different degrees of lumen hyperplasia and inflammatory reaction. The main tissue components of intimal hyperplasia are proliferating vascular smooth muscle cells and stroma.

Control group 2: The arterial wall was slightly thicker than before transplantation, the lumen was flat and the geometry was basically regular, the lumen stenosis and the degree of wall proliferation were the least, and the vascular smooth muscle proliferation and matrix deposition were less. The patency after vascular tissue transplantation is 90%.

Control group 1: The vessel wall was significantly thickened, the intimal hyperplasia was obvious, the smooth muscle cells proliferated in large quantities, the matrix was deposited, the lumen was irregularly narrowed, and endothelial cell necrosis was seen in different degrees. The patency after vascular tissue transplantation was 33%.

Example 1: The arterial wall was slightly thicker than before transplantation, the lumen was flat and the geometric shape was regular, the degree of lumen stenosis and wall proliferation were lighter than those of the previous two groups, and the proliferation of vascular smooth muscle and matrix deposition were less. The subendothelial layer, inner elastic membrane and smooth muscle cells were structurally intact. The patency of the vascular tissue after transplantation reached 84%, which was close to 90% of the vascular patency of the control group 2.

Claims (17)

1. The blood vessel ice crystal-free cryopreservation liquid is characterized by comprising 1.0-6.0mol/L of glycerol, 1.0-7.0mol/L of formamide, 0.1-2.5mol/L of polyvinylpyrrolidone and a component X; the component X is one or more of a component A, a component B or a component C; wherein component A is NO donor, component B is endothelin inhibitor or endothelin antagonist, and component C is anti-apoptosis agent; the component A is one or more of nitroglycerin of 0.05-2mg/L, isosorbide dinitrate of 0.1-5mg/L, molsidomine of 0.5-10mg/L or nicorandil of 0.3-7 mg/L; the component B is one or more of 5-50mg/ml ET-1 invertase inhibitor CGS26303, 10-100mg/ml ETA receptor antagonist PD155080, 50-200mg/ml ETA receptor antagonist BQ-123 and 6-80mg/ml ETA/ETBA receptor antagonist RO 46-2005; the component C is one or more of Caspase inhibitor Z-VAD of 5-100 μmol/L and Z-DEVD of 1-50 μmol/L.

2. The vascular ice-crystal-free cryopreservation liquid according to claim 1, comprising 2.0-5.0mol/L of glycerol, 2.5-5.5mol/L of formamide, 0.1-1.0mol/L of polyvinylpyrrolidone and a component X; the component X is one or more of a component A, a component B or a component C.

3. The cryo-preservation solution for blood vessels according to claim 1, wherein the component X comprises the components A, B and C.

4. The cryo-preservation solution for blood vessels according to claim 1, wherein the component A is nitroglycerin at 0.05-1 mg/L; component B is ET-1 invertase inhibitor CGS26303 with concentration of 20-40 mg/ml; the component C is Z-DEVD of 30-50 mu mol/L.

5. The vascular ice-crystal-free cryopreservation liquid as claimed in claim 1, wherein the glycerol content is 3.10 mol/L; the content of formamide is 3.70 mol/L; the content of polyvinylpyrrolidone is 0.5 mol/L.

6. The cryo-preservation solution for blood vessels according to claim 5, wherein the other component of the cryo-preservation solution is a solvent.

7. The vascular ice-crystal-free cryopreservation liquid according to claim 6, wherein the solvent is a Euro-Collin solution.

8. A method for preserving blood vessels, which comprises introducing the non-crystal cryopreservation liquid according to claim 1 into a blood vessel tissue in a stepwise manner; the step-by-step mode is specifically that the non-ice crystal frozen preservation solution of claim 1 is added into the vascular tissue in 4-15 steps in a step-by-step manner for 10-25 min at the temperature of-5 to +22 ℃; after introducing the non-ice crystal cryopreservation liquid of claim 1 in a stepwise manner, the temperature is first reduced to-80 ℃ to-120 ℃ at a rate of-20 ℃ to-70 ℃/min, then is reduced to-120 ℃ to-180 ℃ at a rate of-2 ℃ to-40 ℃/min, and is preserved at the temperature.

9. The method for preserving blood vessels according to claim 8, wherein the stepwise manner is to add the ice-free cryo-preservation solution to the blood vessel tissue in 6 steps in 15min stepwise increments at 2-4 ℃, and the ice-free cryo-preservation solution is introduced into the blood vessel tissue 15min at a time.

10. The method for preserving blood vessels according to claim 8, wherein the first temperature reduction is carried out at a rate of-38 ℃ to-50 ℃/min to-90 ℃ -110 ℃; the second temperature reduction rate is-2 ℃ to-10 ℃/min, and the temperature is reduced to-120 ℃ to-150 ℃.

11. The method for preserving blood vessels according to claim 10, wherein the first cooling is carried out at a rate of-43 ± 2 ℃/min to-100 ℃; the temperature is reduced to-135 ℃ at a rate of-3 +/-0.2 ℃/min for the second time.

12. A method for recovering blood vessels, which comprises eluting the non-crystal cryopreservation solution of claim 1 from blood vessel tissues in a stepwise manner; the step-by-step mode is specifically that the blood vessel ice-free frozen preservation solution is replaced and eluted by 6-25 steps in a step-by-step manner of 2-25 min at the temperature of-5 to +22 ℃; when in recovery, the frozen sample is taken out from the storage environment, the temperature is firstly raised to-70 ℃ to-120 ℃ at the speed of 10-50 ℃/min, then the temperature is raised to 1-10 ℃ at the speed of 140-350 ℃/min, and then the non-crystal frozen preservation solution of claim 1 is eluted from the vascular tissue in a step-by-step mode.

13. The method for recovering blood vessels according to claim 12, wherein the step-by-step mode is specifically that the ice-free crystal cryopreservation solution is reduced and removed from the blood vessel tissue in 6 steps at 2-4 ℃ for 5 min.

14. The method for vascular resuscitation according to claim 12, wherein the first warming is at a rate of 30 ± 0.2 ℃/minute to-100 ℃; the second temperature rise is to rapidly rewarm to 4 ℃ at the speed of 225 +/-15 ℃/min.

15. A method of vascular preservation and resuscitation that is a combination of the method of vascular preservation according to claim 8 and the method of resuscitation according to claim 12.

16. The method of using the non-ice crystal cryopreservation liquid according to claim 1, wherein the non-ice crystal cryopreservation liquid according to claim 1 is introduced into a vascular tissue by a stepwise method when the vascular tissue is preserved; the step-by-step mode is specifically that the ice-free crystal cryopreservation solution of claim 1 is added into the vascular tissue in 4-15 steps in a step-by-step manner for 10-25 min at the temperature of-5 to +22 ℃; after introducing the non-ice crystal cryopreservation liquid of claim 1 in a stepwise manner, the temperature is first reduced to-80 ℃ to-120 ℃ at a rate of-20 ℃ to-70 ℃/min, then is reduced to-120 ℃ to-180 ℃ at a rate of-2 ℃ to-40 ℃/min, and is preserved at the temperature.

17. The method for using an ice-free cryopreservation liquid according to claim 16, wherein the step-by-step manner is to add the ice-free cryopreservation liquid to the vascular tissue in 6 steps in 15min step by step at 2-4 ℃, and introduce the ice-free cryopreservation liquid to the vascular tissue 15min at a time.

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