CN111264516A - Cryo-free blood vessel preservation solution and blood vessel preservation method - Google Patents
Cryo-free blood vessel preservation solution and blood vessel preservation method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/069—Vascular Endothelial cells
- C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2523/00—Culture process characterised by temperature
Abstract
The invention discloses a blood vessel ice-free cryopreservation liquid and a blood vessel preservation method, wherein the blood vessel ice-free cryopreservation liquid comprises 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 preservation solution provided by the invention improves the preservation effect on the functions of blood vessels, particularly can effectively protect blood vessel smooth muscle cells and endothelial cells which are most easily damaged by freezing, has the function of effectively protecting the blood vessel smooth muscle cells and the endothelial cells, improves the long-term patency rate after blood vessel transplantation, and greatly ensures the success rate of clinical transplantation of the blood vessels.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and particularly relates to a blood vessel ice-free cryopreservation liquid and a blood vessel preservation method.
Background
Over the last 20 years, the clinical need for vascular substitutes has increased year by year. By applying allogeneic blood vessel transplantation, various problems of blood vessel injury, aneurysm, atherosclerosis, diabetic complicated vasculopathy, various complex heart diseases, establishment of hemodialysis access and the like can be solved. In addition, in the field of organ transplantation, when the vascular anastomosis between the donor organ and the host is not matched, it is also necessary to implant additional vascular substitutes to complete revascularization of the transplanted organ. In the past, the invasion of malignant tumor tissues to adjacent key blood vessels is absolute contraindication of operations, but with the technical development, for example, in general surgery, the blood vessel transplantation is applied to pancreatic cancer operations combined with resection reconstruction of blood vessels, so that the malignant tumors invading the blood vessels can be truly and radically resected. In nephrology, great saphenous vein is transplanted to forearm and upper arm of uremia patient for loop type transplantation to establish permanent blood vessel 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 are even free of blood vessels. Solving the problem of long-term in vitro preservation of allogeneic vascular tissues becomes a key technology for increasing the supply of vascular substitutes at present. The vessel tissue is preserved by using the traditional cryopreservation method, although partial vessel activity can be preserved, the biological function of the vessel is reduced due to factors such as cell ice crystal damage and the like in the freezing process, the endothelial cell function is damaged, the contraction capacity of smooth muscle cells is obviously reduced, the long-term patency rate of the vessel is low, and the vessel preservation result is not ideal. Therefore, the method has great significance for improving the existing blood vessel preservation technology.
When the cells are frozen, a large amount of ice crystals are formed in the cell moisture in the cooling process, and the formation of the ice crystals can cause a series of changes in the cells, such as mechanical damage (cell membranes are punctured by the ice crystals), electrolyte concentration increase, osmotic pressure change, dehydration, pH value change, protein denaturation and the like. The freezing point of water in cytoplasm can be reduced by adding freezing protective agent (such as glycerol or dimethyl sulfoxide), and water in cytoplasm can permeate out of cells through cell membrane in slow cooling process to reduce formation of ice crystal in cells, thereby reducing damage type and degree of cells during freezing. The ice crystal-free freezing process has increased temperature conducting speed and concentration of cryoprotectant to convert the intracellular and extracellular fluids from liquid state to non-crystallized solid state similar to glass state to form transparent glass solid, maintain the normal molecular and ion distribution of intracellular fluid, reduce ice crystal formation and raise cell survival rate.
However, the traditional method of adding cryoprotectants and the ice crystal-free freezing method (temperature reduction and rewarming method) have the defects. High concentrations of cryoprotectants produce extremely high toxicity and cause immeasurable damage to vascular cells. In addition, the vascular structure is, from inside to outside, endothelial cells, smooth muscle, adventitia in order. Endothelial cells can prevent blood components from contacting with subendothelial tissues, prevent the activation of a blood coagulation system and the generation of thrombus in blood vessels, prevent inflammatory cell infiltration, and regulate the migration and proliferation of smooth muscle cells, and the integrity of the endothelial cells plays an important role in the synthesis of Nitric Oxide (NO), and the NO can effectively promote vasodilation, so the function of the endothelial cells is very important. However, smooth muscle cells and endothelial cells may also be damaged to varying degrees during the addition and removal of cryoprotectants and during cryopreservation and rewarming. Vascular smooth muscle cell injury can cause a substantial decrease in vasoconstrictor diastolic function. After endothelial cells of the vascular intima are damaged and peeled off, the vascular intima can be thickened, smooth muscle cells proliferate and migrate to the vascular intima to become main cells, and the adverse effects of the narrow lumen, the influence on the long-term patency rate and the like are caused; meanwhile, the damage of endothelial cells can reduce the secretion of vasodilating substances such as NO and the like, so as to cause vascular contracture; in addition, the vascular endothelial cells are damaged, so that the heparin secretion is reduced, and the generation of heparin is less, so that thrombus is easily formed, and the long-term patency rate is influenced. Therefore, how to reduce the apoptosis of vascular smooth muscle cells and endothelial cells as far as possible in the processes of adding blood vessels, removing cryoprotectants and freezing recovery is also of great significance.
Disclosure of Invention
The invention aims to provide an ice-free crystal cryopreservation liquid for vascular preservation.
The invention also aims to provide a vascular ice-free cryopreservation method for injecting the ice-free cryopreservation liquid step by step before cryopreservation and controlling the temperature in a programmed manner in the cryopreservation process, which is used for preserving fresh vascular tissues.
In order to achieve the above purpose of the prior art, the following technical solutions are adopted in the present application:
a blood vessel non-crystal frozen preservation solution comprises 1.0-6.0mol/L of glycerin, 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.
Preferably, the blood vessel ice-free cryopreservation liquid comprises 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; wherein component A is NO donor, component B is endothelin inhibitor or endothelin antagonist, and component C is anti-apoptosis agent.
In some embodiments, ingredient X of the present invention is ingredient a, ingredient B, and ingredient C.
In some embodiments, the component A is preferably one or more of 0.05-2mg/L nitroglycerin, 0.1-5mg/L isosorbide dinitrate, 0.5-10mg/L molsidomine or 0.3-7mg/L nicorandil; further preferably 0.05 to 1mg/L of nitroglycerin. The inventors found that the addition of the component B improves the preservation quality of blood vessels.
In some embodiments, component B is preferably one or more of 5-50mg/ml of ET-1 invertase inhibitor CGS26303, 10-100mg/ml of ETA receptor antagonist PD155080, 50-200mg/ml of ETA receptor antagonist BQ-123, 6-80mg/ml of ETA/ETBA receptor antagonist RO 46-2005; more preferably 20-40mg/ml of the ET-1 convertase inhibitor CGS 26303. The inventors found that the preservation effect of blood vessels can be improved by adding the component B.
In some embodiments, component C is preferably one or more of 5-100. mu. mol/L Caspase inhibitor Z-VAD, 1-50. mu. mol/L Z-DEVD; further preferably 30 to 50. mu. mol/L of Z-DEVD. The inventors found that the addition of the component C improves the preservation quality of blood vessels.
As a further improvement of the invention, the glycerol content is 3.10 mol/L.
As a further development of the invention, the formamide content is 3.70 mol/L.
As a further improvement of the invention, the content of polyvinylpyrrolidone is 0.5 mol/L.
In a further improvement of the present invention, the other component of the non-ice crystal cryopreservation liquid is a solvent, and more preferably, the solvent is a Euro-Collin solution.
The invention also provides a blood vessel preservation method, which is used for introducing the ice-free crystal cryopreservation liquid into a blood vessel tissue in a stepping mode.
In some embodiments, the step-by-step method for introducing the non-ice crystal cryopreservation liquid is to add the non-ice crystal cryopreservation liquid into the vascular tissue in 4-15 steps in a step-by-step manner of 10-25 min at-5 to +22 ℃.
In some preferred embodiments, the step-by-step mode for introducing the ice-free crystal cryopreservation liquid is to add the ice-free crystal cryopreservation liquid to the vascular tissue in 6 steps in 15min step-by-step manner at 2-4 ℃, and introduce the ice-free crystal cryopreservation liquid to the vascular tissue 15min at a time.
In some embodiments, after introducing the cryo-preservation solution without ice crystal in a stepwise manner, the method for preserving blood vessels of the present invention first reduces the temperature to-80 ℃ to-120 ℃ at a rate of-20 ℃ to-70 ℃/min, then reduces the temperature to-120 ℃ to-180 ℃ at a rate of-2 ℃ to-40 ℃/min, and preserves the blood vessels at the temperature.
In some preferred embodiments, the first temperature reduction of the invention is carried out at a rate of-38 ℃ to-50 ℃/minute to-90 ℃ to 110 ℃; further preferably, the temperature is reduced to-100 ℃ at a rate of-43 +/-2 ℃/min.
In some preferred embodiments, the second temperature reduction rate of the present invention is a second temperature reduction rate of-2 ℃ to-10 ℃/minute to-120 ℃ to-150 ℃; further preferably, the temperature is reduced to-135 ℃ at a rate of-3 +/-0.2 ℃/min.
The invention also provides a vascular resuscitation method, which utilizes a step-by-step mode to elute the ice crystal-free cryopreservation liquid from vascular tissues.
In some embodiments, the step-by-step mode of eluting the non-ice crystal frozen preservation solution is to replace and elute the blood vessel non-ice crystal frozen preservation solution by 6 to 25 steps in 2 to 25min step by step under the condition of-5 to +22 ℃.
In some preferred embodiments, the step-by-step mode for eluting the non-ice crystal cryopreservation solution is to gradually reduce and remove the non-ice crystal cryopreservation solution into the vascular tissue in 6 steps at 2-4 ℃ for 5 min.
In some embodiments, in the method for recovering blood vessels, after a cryopreserved sample is taken out of a storage environment during recovery, the temperature is first raised to-70 ℃ to-120 ℃ at the speed of 10-50 ℃/min, then raised to 1-10 ℃ at the speed of 140-350 ℃/min, and then the non-crystal cryopreservation liquid is eluted from the blood vessel tissue in a step-by-step manner.
In some embodiments of the invention, the first temperature ramp of the present invention is a ramp to-100 ℃ at a rate of 30 ± 0.2 ℃/minute.
In some embodiments of the invention, the second temperature ramp of the present invention is a rapid ramp back to 4 ℃ at a rate of 225 ± 15 ℃/minute.
The invention also provides a blood vessel preservation method and a recovery method, which are the combination of the blood vessel preservation method and the recovery method.
The invention also provides a using method of the ice-free crystal cryopreservation liquid, which comprises the following steps: when the vascular tissue is preserved, introducing the ice-free crystal cryopreservation liquid into the vascular tissue in a stepping mode; the step-by-step mode is specifically that the ice-free crystal cryopreservation liquid 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 ℃.
In some preferred embodiments, the step-by-step mode is specifically to add the ice-free cryo-preservation solution to the vascular tissue in 6 steps in 15min step-by-step manner under the condition of 2-4 ℃, and the ice-free cryo-preservation solution is introduced into the vascular tissue 15min each time.
In some embodiments, after introducing the non-crystal cryopreservation liquid of the invention in a stepwise manner, the non-crystal cryopreservation liquid is first cooled to-80 ℃ to-120 ℃ at a rate of-20 ℃ to-70 ℃/min, then cooled to-120 ℃ to-180 ℃ at a rate of-2 ℃ to-40 ℃/min, and then stored at the temperature.
In some preferred embodiments, the first temperature reduction of the invention is carried out at a rate of-38 ℃ to-50 ℃/minute to-90 ℃ to 110 ℃; further preferably, the temperature is reduced to-100 ℃ at a rate of-43 +/-2 ℃/min.
In some preferred embodiments, the second temperature reduction rate of the present invention is a second temperature reduction rate of-2 ℃ to-10 ℃/minute to-120 ℃ to-150 ℃; further preferably, the temperature is reduced to-135 ℃ at a rate of-3 +/-0.2 ℃/min.
The advantages of the invention over the prior art are:
(1) the preservation solution provided by the invention improves the preservation effect of the vascular function, and particularly for vascular smooth muscle cells and endothelial cells which are most easily damaged by freezing, the existing vascular freezing preservation technology lacks protection of the vascular smooth muscle cells and the endothelial cells. The efficacy of the final test after vessel preservation is the long-term patency rate after transplantation. Regardless of various indexes checked in a laboratory, the blood vessel really shows the self-function of not being unobstructed after transplantation and how long the blood vessel can be unobstructed. The preserving fluid improves the long-term patency rate after the blood vessel transplantation, is the highest index which cannot be compared with other experimental effects, and greatly ensures the success rate of the blood vessel clinical transplantation.
(2) Leading in ice crystal-free frozen preservation solution step by step before freezing, so that the preservation solution is uniformly distributed and fully infiltrates cells and structures inside and outside the vascular tissue, and the damage of ice crystals to the tissue cell structure is reduced; the cooling rate and the resuscitation temperature rise rate are optimized in the freezing process, the formation of ice crystals of the vascular tissue in the freezing storage and resuscitation processes can be greatly reduced, compared with the conventional vascular tissue freezing storage technology, the vascular tissue freezing storage technology has the advantages that the vascular tissue space structure is more effectively protected, the transplantation success rate and the function maintenance after transplantation are improved, the long-term patency rate after vascular transplantation is further improved, and the vascular storage effect is further improved.
(3) The recovery process gradually reduces the content of the cryo-free blood vessel preservation solution, and fully reduces the toxic effect on the blood vessel.
Drawings
FIG. 1 is a microscopic image of the size and position of ice crystals of rabbit carotid artery sections frozen by conventional freezing and no ice crystal;
FIG. 2 comparison of the contractile response of fresh blood vessels, blood vessels preserved by conventional freezing method and blood vessels preserved without ice crystals to a panel of stimulant drugs (norepinephrine, phenylephrine);
figure 3 dose/contraction response curves of fresh, cryopreserved and non-cryopreserved vessels against a panel of stimulant drugs (norepinephrine, phenylephrine).
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to illustrate the present invention and not to limit the scope of the present invention, and all simple modifications of the preparation method of the present invention based on the idea of the present invention are within the scope of the present invention. The following examples are experimental methods without specifying specific conditions, and generally follow the methods known in the art. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The specific experimental method is explained in the effect experiment.
Example 1:
1. 3.10mol of glycerol, 3.70mol of formamide, 0.5mol of polyvinylpyrrolidone, 0.5mg/L of nitroglycerin, 30mg/L of CGS26303 and 40 mu mol/L Z-DEVD are mixed uniformly and added with Euro-Collin solution to reach the constant volume of 1L.
2. Taking a proper amount of the solution obtained in the step 1, and cooling to 2-4 ℃. Adding the cooled solution into human vascular tissue to be cryopreserved at 2-4 deg.C by 6 steps, and completing introduction within 90min (adding cryo-free cryopreservation solution into vascular tissue by 6 steps at 2-4 deg.C for 15 min).
3. Cooling the human vascular tissue which is introduced into the frozen stock solution in the previous step to-100 ℃ at the speed of-43 ℃/min, then cooling the sample to-135 ℃ at the speed of-3 +/-0.2 ℃/min, and storing for at least 24h at the temperature; thereafter the samples can be stored in an environment of-135 ℃ for a long period of time.
4. Taking out a blood vessel sample to be resuscitated, recovering to-70 ℃ at the speed of 30 +/-0.2 ℃/minute, and recovering to 4 ℃ at the speed of 225 +/-15 ℃/minute;
5. gradually reducing and removing the non-ice crystal frozen stock solution from the blood vessel tissue by 6 steps at 2-4 deg.C for 5min, and replacing and eluting the non-ice crystal frozen stock solution within 30min each time for 5 min.
Example 2:
1. 3.10mol of glycerol, 3.70mol of formamide, 0.5mol of polyvinylpyrrolidone and 0.5mg/L of nitroglycerin are taken, mixed uniformly and added with Euro-Collin solution to be constant volume to 1L.
2. Taking a proper amount of the solution obtained in the step 1, and cooling to 2-4 ℃. Adding the cooled solution into human vascular tissue to be cryopreserved at 2-4 deg.C by 6 steps, and completing introduction within 90min (adding cryo-free cryopreservation solution into vascular tissue by 6 steps at 2-4 deg.C for 15 min).
3. Cooling the human vascular tissue in the last step to-100 ℃ at the speed of-43 ℃/min, cooling the sample to-135 ℃ at the speed of-3 +/-0.2 ℃/min, and storing at the temperature for at least 24 h; thereafter the samples can be stored in an environment of-135 ℃ for a long period of time.
4. The vascular sample to be resuscitated is removed and returned to-70 ℃ at a rate of 30 +/-0.2 ℃/min and then returned to 4 ℃ at a rate of 225 +/-15 ℃/min.
5. Gradually reducing and removing the non-ice crystal frozen stock solution from the blood vessel tissue by 6 steps at 2-4 deg.C for 5min, and replacing and eluting the non-ice crystal frozen stock solution within 30min each time for 5 min.
Example 3:
1. 3.10mol of glycerol, 3.70mol of formamide, 0.5mol of polyvinylpyrrolidone and 30mg/L of CGS26303 are uniformly mixed, and Euro-Collin solution is added to the mixture to be constant volume to 1L.
2. Taking a proper amount of the solution obtained in the step 1, and cooling to 2-4 ℃. Adding the cooled solution into human vascular tissue to be cryopreserved at 2-4 deg.C by 6 steps, and completing introduction within 90min (adding cryo-free cryopreservation solution into vascular tissue by 6 steps at 2-4 deg.C for 15 min).
3. Cooling the human vascular tissue which is introduced into the frozen stock solution in the previous step to-100 ℃ at the speed of-43 ℃/min, then cooling the sample to-135 ℃ at the speed of-3 +/-0.2 ℃/min, and storing for at least 24h at the temperature; thereafter the samples can be stored in an environment of-135 ℃ for a long period of time.
4. The vascular sample to be resuscitated is removed and returned to-70 ℃ at a rate of 30 +/-0.2 ℃/min and then returned to 4 ℃ at a rate of 225 +/-15 ℃/min.
5. Gradually reducing and removing the non-ice crystal frozen stock solution from the blood vessel tissue by 6 steps at 2-4 deg.C for 5min, and replacing and eluting the non-ice crystal frozen stock solution within 30min each time for 5 min.
Example 4:
1. 3.10mol of glycerol, 3.70mol of formamide, 0.5mol of polyvinylpyrrolidone and 40 mu mol/L Z-DEVD are taken, mixed uniformly and added with Euro-Collin solution to reach the constant volume of 1L.
2. Taking a proper amount of the solution obtained in the step 1, and cooling to 2-4 ℃. Adding the cooled solution into human vascular tissue to be cryopreserved at 2-4 deg.C by 6 steps, and completing introduction within 90min (adding cryo-free cryopreservation solution into vascular tissue by 6 steps at 2-4 deg.C for 15 min).
3. Cooling the human vascular tissue which is introduced into the frozen stock solution in the previous step to-100 ℃ at the speed of-43 ℃/min, then cooling the sample to-135 ℃ at the speed of-3 +/-0.2 ℃/min, and storing for at least 24h at the temperature; thereafter the samples can be stored in an environment of-135 ℃ for a long period of time.
4. Taking out a blood vessel sample to be resuscitated, recovering to-70 ℃ at the speed of 30 +/-0.2 ℃/minute, and recovering to 4 ℃ at the speed of 225 +/-15 ℃/minute; displacing and eluting the ice-free frozen stock solution within 90min at 2-4 ℃.
5. Gradually reducing and removing the non-ice crystal frozen stock solution from the blood vessel tissue by 6 steps at 2-4 deg.C for 5min, and replacing and eluting the non-ice crystal frozen stock solution within 30min each time for 5 min.
Control group 1: a conventional cryoprotectant solution is provided which contains 1.0MDMSO by volume percent.
At room temperature, the blood vessel cryoprotectant is added into a blood vessel to be cryopreserved, then the blood vessel is transplanted into a blood vessel containing 1.0MDMSO for balance for 1 minute, and then the blood vessel is put into a programmed cooling instrument, and is cooled to-90 ℃ at the speed of-1 ℃/min, and then the blood vessel is transferred into liquid nitrogen at the temperature of-196 ℃ for long-term preservation.
Control group 2: fresh blood vessels without freezing treatment are obtained and put in Euro-Collin solution and stored in environment of 2-10 ℃ for no more than 12 hours.
Effect experiment:
effect test 1 Effect test of vascular function and vascular transplantation
After the blood vessels were treated in the examples and the control group, experiments on the functions of the blood vessels and the effects of blood vessel transplantation were conducted. The vasoconstrictor and vasodilator functions are the tests of the response of blood vessels to different doses of systolic and diastolic drugs, as well as the sensitivity of blood vessels to these drugs. The recovery of vascular function was based on the recovery of vasoconstrictor function as the main index, vasodilation and sensitivity to drugs, and the recovery (%) of vascular function was the ratio of the vasoconstrictor power of the working group to the fresh vasoconstrictor power of the control group 2. The blood vessel transplantation is the carotid artery autograft of a new Zealand rabbit, and the carotid artery on one side is transplanted to the same part on the other side after being treated by the embodiment and the control group. The survival rate after the blood vessel transplantation refers to the patency of the blood vessel after 3 months of the blood vessel transplantation as an index. The recovery of vascular function and survival rate after vascular transplantation are shown in table 1.
TABLE 1 vascular functional recovery and survival rates after vascular transplantation
Take an example of a set of 2 vessels: these two groups were control group 1 (conventional cell cryopreservation protectant (1.0mol DMSO) cryopreservation) and example 1 non-crystal cryopreservation solution group, respectively.
Healthy New Zealand rabbits weighing 2kg were selected, and the allogeneic blood vessels to be transplanted were taken from the carotid artery of the healthy New Zealand rabbits, and the carotid artery was perfused with the cryo-preservation solution without ice crystal prepared in example 1 or the conventional cell cryopreservation protective agent in control group 1. After the step-by-step introduction of the freezing agent is completed by respectively using the ice-crystal-free cryopreservation liquid and the traditional cell cryopreservation protective agent (1.0mol of DMSO) according to the method described in the embodiment 1, programmed cooling is carried out, samples of rabbit artery segments without ice crystals and frozen conventionally are selected at-90 ℃ for microscopic slice observation, as shown in fig. 1, ice crystal regions (B) with different sizes distributed on extracellular matrix of a vascular wall are obviously visible in a control group 1, white irregular-sized gaps (thin arrows) are left when the ice crystals occupy the gaps, from an endothelial layer at the innermost layer to a smooth muscle layer at the middle layer, so that the outer layer of the outermost blood vessel is fully covered with the ice crystals (thin arrows), the structure of the frozen blood vessel is obviously changed, and the ice crystals are more loose and more large in the outer layer, and the vascular endothelial layer and the muscle layer are less compact in the blood vessel. However, the formation of ice crystals in the gaps between the layers (thick arrows) destroys the overall function of the blood vessel, and particularly, the formation under the endothelium (thick arrows) is significant for maintaining the integrity of the endothelial layer after the blood vessel is transplanted, and if the endothelium is not complete, red blood cells, platelets and blood vessel blockage can be caused. Whereas example 1 frozen vessels without ice crystals had no ice crystals (a). The integrity of the vascular tissue preserved by using the ice-crystal-free cryopreservation liquid is higher than that of the vascular tissue preserved by using the traditional cell cryopreservation protective agent, and each cell and tissue in the blood vessel preserved by using the ice-crystal-free cryopreservation liquid is intact in shape and low in damage degree, so that the blood vessel of the control group 1 can obviously see ice crystal areas with different sizes distributed on extracellular matrix of the blood vessel wall, the structure of the frozen blood vessel is obviously distorted, while the ice-crystal-free cryopreserved blood vessel of the embodiment 1 has no ice crystal, and the effect is obviously improved.
Take an example of 3 sets of blood vessels to illustrate: the three groups are respectively blood vessels frozen and preserved by the recovered traditional cell freezing protective agent (a control group 1), blood vessels frozen and preserved without crystal ice in the example 1 (hereinafter, the embodiment 1) and fresh blood vessels (a control group 2) to be mechanically detected together.
1000ml of Krebs Henseleit Buffer (KHB) solution was injected into the isolated vascular tissue perfusion system for experiments. The constant-temperature perfusion is adjusted to 37 ℃, and the tension sensor is calibrated and zeroed. The perfusion tank is filled with 10ml of KHB solution and is continuously filled with 5 percent of O2And CO2. The vascular ring is sleeved between two probes connected with the sensor gently, the distance is adjusted to enable the initial tension to be 4g, the balance is carried out for 1h, the distance between the probes is continuously adjusted to enable the initial tension to be 4g, and the KHB solution in the tank is replaced once every 15 minutes. 75ul of 10% KCL solution was added sequentially to a final concentration of 10-90mmol/L, and the maximum contractile force after each addition was recorded separately. The KHB solution was changed twice, and 10ul of norepinephrine or phenylephrine (10 concentration each) was added sequentially-10、10-9、10-8、10-7、10-6、10-5、10-4mol/L), the maximum shrink force is recorded for each time. Finally, 10ul of 10 is added after the maximum contractile force is continuously and stably balanced-4mol/L of calcium ionophore A23187 or 10-4Sodium nitroprusside in mol/L, maximum post-diastolic blood vessel tension was recorded.
The experimental result shows that the contractility of the blood vessels of the control group 1 (conventional freezing) is obviously reduced to 23-26% after the blood vessels are subjected to freezing treatment. The example 1 (no ice crystal) group of vessels retained the contractile response of the control 2 (fresh), specifically as shown in fig. 2, the control 1 (conventional frozen) vessels had a significant decrease in contractile force to 23-26% after freezing treatment, while the example 1 (no ice crystal) vessels retained the contractile response of the control 2 vessels; blood vessel ratio of control 1 (regular frozen) blood vessels to control 2 (fresh), p < 0.0001; #: the control group 1 (conventional frozen) vessels had p <0.0001 compared to the example 1 (no ice crystal) group vessels. Noradrenaline group: control 2 blood vessels n-16; example 1 (ice crystal free) blood vessels n ═ 43; control 1 (conventional frozen) blood vessels n ═ 20; phenylephrine group: control 2 (fresh) vessels n-12; example 1 (no ice crystals) blood vessels n ═ 31; control 1 (conventional frozen) vessels n-20.
In addition, the blood vessel relaxation tension of the control group 1 is obviously reduced; the diastolic response in the example 1 group was weaker than that in the control 2 group, but better than that in the control 1 group, specifically shown in table 2, norepinephrine was preshrinkled, sodium nitroprusside (a non-vascular endothelial cell-relaxing factor, which acts directly on vascular smooth muscle cells) and calcium ionophore a23187 (a vascular endothelial cell-relaxing factor, which acts via vascular endothelial cells) were relaxed. Each drug was divided into three groups, control 2 vasoconstriction and relaxation (fresh preshrinkment/fresh relaxation), example 1 vasoconstriction and relaxation (no iced preshrinkment/no iced relaxation), control 1 vasoconstriction and relaxation (iced preshrinkment/iced relaxation). The vasodilation caused by the sodium nitroprusside effect is obviously reduced (9%) in the blood vessel relaxation tension of the control group 1, and the vasodilation response (35%) of the group in the example 1 is greatly better than that of the control group 1 and is the same as that of the control group 2 (37%). Calcium ionophore A23187 stimulated less relaxation, both example 1 (37%) and control 1 (28%) were lower than control 2 (68%). Indicating that hypothermia has an effect on the passage of vascular endothelial cells. Calcium ionophore a23187 group: control 2 blood vessels n-8; example 1 blood vessels n-33; control 1 with n-11; sodium nitroprusside group: control 2 blood vessels n-16; example 1 blood vessels n-33; control 1 blood vessels n-11.
TABLE 2 PRE-SHRINKAGE AND DESHRINKAGE OF BLOOD VESSELS IN COMPARATIVE GROUP 2, EXAMPLE 1 AND COMPARATIVE GROUP 1
General principles of pharmacology we use the drug concentration to reach half the maximal contractile force with pD2Expressed as the negative logarithm (-log) of the molar concentration producing 50% of the maximum effect, pD2Larger values indicate a more sensitive receptor to the drug. Control 2, example 1 and control 1 three groups of blood vessels sensitive to noradrenaline and phenylephrine to more accurately detect blood vessel transitDegree of damage after treatment in the same manner.
As shown in Table 3, the pD2 values of the three groups of blood vessels of control 2, example 1 and control 1 indicate the sensitivity of each of the three groups of blood vessels to the drug. The blood vessels of control 1 were significantly reduced in sensitivity after freezing treatment, while the blood vessels of the non-frozen group retained the same sensitivity as the blood vessels of control 2. @: control group 1 vessel to ice crystal free vessel ratio, p < 0.01; #: in control 1 vessels, p was <0.05 compared to example 1 vessels. Noradrenaline group: control 2 blood vessels n-16; example 1 blood vessel n-39; control group 1 angio n-20 phenylephrine group: control 2 with n-12; example 1 blood vessels n-27; control 1 blood vessels n-20.
TABLE 3 pD of blood vessels of control 2, example 1 and control 12Value of
| | 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 maximal contraction and relaxation of blood vessels represents only the maximal limit that blood vessels can reach after being preserved by different treatments, but do not respond to the sensitivity of drugs. For example, for a drug, the amount of concentration at which the blood vessel responds most, in other words, if the blood vessel is damaged, a higher drug concentration may be required to achieve the effect that would be achieved at a lower drug concentration if the blood vessel were undamaged. Sensitivity to drug response tests for cellular receptor function, smooth muscle contraction and relaxation sensitivity, and the degree of integrity or impairment of vascular global function. We divided the contractile force for each drug dose by the maximal contractile force to obtain a percentage, and generated a drug dose/contractile response curve with the results shown in fig. 3: control 2 (fresh) vessels, control 1 vessels and example 1 (no ice crystal) vessels dose-contraction response curves to a panel of stimulant drugs (norepinephrine, phenylephrine). The drug dose is 10 from the minimum dose-10Molar to maximum dose 10-4Molar, the corresponding shrink force is a percentage of the upper maximum shrink force. It is evident from the dose-contraction response curve that after treatment of the control 1 (conventional frozen) vessels, the curve has moved significantly to the right, the sensitivity of the vessels to the drug has decreased, and a larger dose is required to achieve the contractile force of the control 2 (fresh) vessels and the example 1 (no ice crystal) vessels. The contractile response of the blood vessels of the example 1 (without ice crystals) group was similar to that of the control 2 (fresh) vessels, indicating that the sensitivity to the drug was not affected. Noradrenaline group: control 2 (fresh) vessels n ═ 16; example 1 (ice crystal free) blood vessels n ═ 43; control group 1 (conventional frozen) angion-20 phenylephrine group: control 2 (fresh) vessels n-12; example 1 (ice crystal free) blood vessels n-27-39; control 1 (conventional frozen) vessels n-20.
Three groups of blood vessel specimens were fixed with 10% formalin, paraffin sections were prepared, and pathological changes of blood vessels were observed by Hematoxylin Eosin (HE) staining. The results show that:
(1) control group 2: the endothelial cells have good continuity and regular and ordered arrangement, and can be found that very individual endothelial cells fall off, the nucleus of the medial smooth muscle cell is clear, and the structures of the collagen fiber and the elastic fiber are normal.
(2) Control group 1: the endothelial cells are seriously fallen off, the arrangement sequence of the endothelial cells is irregular, and the middle smooth muscle, the collagen fiber and the elastic fiber are partially broken.
(3) Example 1 group: endothelial cells slightly drop off, a small amount of gaps are visible between the endothelial layer and the endothelial lower layer, and the structures of the endothelial lower layer, the internal elastic membrane and smooth muscle cells are good.
Effect experiment 4 autoblood vessel transplantation rabbit model test of blood vessel patency after blood vessel transplantation
Take an example of 3 groups of vascular grafts: the three groups are respectively a control group 2, a control group 1 (traditional cell cryopreservation protective agent (1.0mol DMSO) cryopreservation) and an ice crystal-free cryopreservation solution group of example 1, and 9-12 operations are respectively completed in each group by using the middle section of the rabbit neck total artery as an anastomosis part of the autologous artery. Patency rate of blood vessels (table 3) three groups of transplanted blood vessels were probed with an ultrasonic doppler probe 2 weeks and 4 weeks after surgery for patency of blood flow. At 12 weeks post-surgery, the specimens were formalin-fixed in 10% for graft vessels. The fixed blood vessel specimen is made into a paraffin section, Hematoxylin Eosin (HE) staining is carried out to observe the pathological change of the transplanted blood vessel, and the change of the intima, the media and the adventitia of the transplanted artery and the infiltration condition of mononuclear/lymphocyte in the blood vessel wall are observed after 3 months.
TABLE 4 comparison of vascular patency after 2 weeks to 3 months of vascular implantation in control 2, control 1 and example 1
The results are shown in table 4, and the vascular graft model shows that the patency of the vascular tissue cryopreserved using the ice-free cryopreservation solution after transplantation is close to that of fresh vascular tissue, which indicates that the patency of the ice-free cryopreserved blood vessel can be increased by adding A, B, C components in the present invention.
After 3 months of operation, each group is observed by transplanting blood vessels, and each treatment group has luminal hyperplasia and inflammatory reaction with different degrees. The major tissue components of intimal hyperplasia are proliferating vascular smooth muscle cells and stroma.
Control group 2: the artery wall is slightly thicker than before transplantation, the smooth geometric shape of the lumen is basically regular, the lumen is narrow, the hyperplasia degree of the wall is the lowest, and the proliferation of the smooth muscle of the blood vessel and the matrix deposition are less. The patency after vascular tissue transplantation is 90%.
Control group 1: the vascular wall is obviously thickened, the vascular intimal hyperplasia is obvious, a large amount of smooth muscle cells are proliferated, the matrix is deposited, the lumen is irregularly narrowed, and endothelial cell necrosis with different degrees can be seen. Patency 33% after vascular tissue transplantation.
Example 1: the wall of the artery is slightly thicker than that before transplantation, the lumen is smooth and has regular geometric shape, the degree of the stenosis and the hyperplasia of the wall is lighter than that of the former two groups, and the proliferation of the smooth muscle of the blood vessel and the deposition of the stroma are less. The endothelial sublayer, the internal elastic membrane and the smooth muscle cells were structurally sound. The patency after vascular tissue transplantation reached 84%, which was close to 90% of the patency of the blood vessels of control group 2.
Claims (10)
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; preferably, the composition comprises 2.0-5.0mol/L of glycerol, 2.5-5.5mol/L of formamide, 0.1-1.0mol/L of polyvinylpyrrolidone and the 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.
2. The cryo-preservation solution for blood vessels according to claim 1, wherein the component X comprises the components A, B and C.
3. The vascular ice-crystal-free cryopreservation liquid according to claim 1, wherein the component A is one or more of 0.05-2mg/L nitroglycerin, 0.1-5mg/L isosorbide dinitrate, 0.5-10mg/L molsidomine or 0.3-7mg/L nicorandil; preferably 0.05-1mg/L nitroglycerin; 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; preferably 20-40mg/ml of ET-1 convertase inhibitor CGS 26303; the component C is one or more of Caspase inhibitor Z-VAD of 5-100 mu mol/L and Z-DEVD of 1-50 mu mol/L; preferably 30-50. mu. mol/L of Z-DEVD.
4. 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; preferably, the other components of the ice crystal-free frozen storage solution are solvents; further preferably, the solvent is a solution of Euro-Collin.
5. 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 ℃; the preferable step-by-step mode is specifically that the ice-free crystal frozen preservation solution is added into the vascular tissue in 6 steps by 15min step by step under the condition of 2-4 ℃, and the ice-free crystal frozen preservation solution is introduced into the vascular tissue every 15 min.
6. The method for preserving blood vessels according to claim 5, wherein after introducing the cryo-preservation solution of claim 1 in a stepwise manner, the cryo-preservation solution is first cooled to-80 ℃ to-120 ℃ at a rate of-20 ℃ to-70 ℃/min, and then cooled to-120 ℃ to-180 ℃ at a rate of-2 ℃ to-40 ℃/min, and preserved at the temperature; preferably, the temperature is reduced to-90-110 ℃ at the rate of-38-50 ℃/min for the first time; further preferably, the temperature is reduced to-100 ℃ at the speed of-43 +/-2 ℃/min; preferably, the second temperature reduction rate is-2 ℃ to-10 ℃/minute for the second temperature reduction to-120 ℃ to-150 ℃; further preferably, the temperature is reduced to-135 ℃ at a rate of-3 +/-0.2 ℃/min.
7. 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 ℃; preferably, the step-by-step mode is specifically that the ice-free crystal frozen preservation solution is reduced and removed from the vascular tissue in 6 steps by 5min step by step at the temperature of 2-4 ℃, and each time is 5 min.
8. The vascular resuscitation method according to claim 8, wherein during resuscitation, after the cryopreserved sample is taken out of the storage environment, the temperature is raised to-70 ℃ to-120 ℃ for the first time at a speed of 10-50 ℃/min, then raised to 1-10 ℃ for the second time at a speed of 140-350 ℃/min, and then the non-crystal cryopreservation liquid according to claim 1 is eluted from the vascular tissue in a step-by-step manner; preferably, the first temperature rise is to rise to-100 ℃ at the rate of 30 +/-0.2 ℃/minute; preferably, the second temperature rise is a rapid rewarming to 4 ℃ at a rate of 225 +/-15 ℃/min.
9. A method of vascular preservation and resuscitation which is a combination of the method of vascular preservation according to claim 5 and the method of resuscitation according to claim 7.
10. 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 ℃; preferably, the step-by-step mode is specifically that the ice crystal-free frozen preservation solution is added into the vascular tissue in 6 steps in 15min step by step at the temperature of 2-4 ℃, and the ice crystal-free frozen preservation solution is introduced into the vascular tissue every 15 min.
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