CN112616827A - Erythrocyte cryopreservation method and cryopreserved erythrocytes - Google Patents
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Abstract
The invention relates to the technical field of erythrocyte preservation, in particular to a erythrocyte cryopreservation method and cryopreserved erythrocytes. The erythrocyte cryoprotectant used in the invention is an impermeable protectant, and under the protection of the protective protectant, erythrocytes are wrapped in a gel shell structure formed by crosslinking sodium alginate, for example, to form erythrocyte-containing fibers. Compared with the existing erythrocyte cryopreservation technology, the erythrocyte cryopreservation scheme provided by the invention saves the fussy processes of adding glycerol and removing glycerolysis, not only can ensure that the survival rate of the frozen erythrocyte reaches more than 90% in the glycerol-free rapid cryopreservation, but also can keep good microscopic morphology.
Description
Technical Field
The invention relates to the technical field of erythrocyte preservation, in particular to a erythrocyte cryopreservation method and cryopreserved erythrocytes.
Background
The cryopreservation of blood is the most widely and most important part in the preservation of all biological materials, and has important significance for the research of blood preservation in the aspects of military medicine, clinical treatment and the like, particularly has great significance for the long-term cryopreservation of human erythrocytes in human society, and has great demands for the preservation of human erythrocytes in the aspects of natural disaster emergency blood transfusion, rare blood type blood transfusion, research, military storage and the like. Therefore, it is of great significance to research how to efficiently preserve red blood cells for a long time.
Currently, the most common clinical preservation methods for red blood cells mainly include: 1) short-term storage at 4 deg.C; 2) slow freezing preservation under high concentration glycerol. The former has short shelf life, can be stored for only 35-42 days generally, and is very easy to be polluted, which causes red blood cell storage lesion. Although the latter method can prolong the preservation time of erythrocytes to several years or even several decades by using a high glycerol slow freezing method, the concentration of glycerol is as high as 40%, and the glycerol has toxic effect on cells, and a complex de-glycerolysis washing process is required before the use. The process of adding and removing glycerin wastes a great deal of time and energy, and it is highly likely to delay the treatment of the human body in an emergency.
In addition to these two methods commonly used in clinical practice, the low-concentration glycerol flash freezing method and the freeze-drying method are hot spots in the current research on preservation of red blood cells. Compared with slow freezing, the rapid freezing method utilizes less than 20% of glycerol to pretreat the red blood cells, and then directly throws the red blood cell suspension into liquid nitrogen at the temperature of-196 ℃ for low-temperature storage, so that the operation is not closed, and bacterial pollution is easily caused. And the red blood cells need to be rapidly rewarming when being thawed and rewarmed, and the red blood cells are easy to damage after being rewarmed for a long time. The red blood cells preserved by the method can be preserved for 5-10 years and even longer, and the morphology and the function of the red blood cells are well preserved. Nevertheless, the erythrocytes frozen by the low-concentration glycerol rapid cryopreservation method cannot be directly returned, and the problem that the glycerol removal operation is complicated and difficult before use still exists. In addition, in the existing research, the survival rate of the frozen and dried red blood cells is still low, the technology is not mature enough, and great difficulties are still faced in practice.
Disclosure of Invention
In view of the above, the present invention provides a method for cryopreserving red blood cells and cryopreserved red blood cells, so as to improve the recovery rate of red blood cells.
The cryopreservation method of the red blood cells provided by the invention comprises the following steps:
preparing a sodium alginate solution as a shell solution; preparing a calcium chloride solution as a cross-linking agent;
mixing the leukocyte-removed suspension with normal saline to obtain a erythrocyte solution;
mixing the erythrocyte solution with the cryoprotectant solution to prepare a nuclear solution;
and allowing the shell solution and the core solution to enter a cross-linking agent through a double-channel coaxial needle, and freezing and storing the obtained fiber liquid nitrogen after solidification.
In the scheme of the invention, the cryoprotectant can reduce the toxicity of the solution and prevent the damage of the erythrocyte caused by osmotic pressure and ice crystal. In the present invention, good protection of erythrocytes can be achieved by using only a single cryoprotectant. The red blood cells are human red blood cells.
In the invention, the low-temperature protective agent is an impermeable protective agent. In the embodiment of the invention, the cryoprotectant is trehalose or glycerol; in some embodiments, the cryoprotectant is trehalose, and the concentration of trehalose in the cryoprotectant solution is from 5% (w/v) to 30% (w/v); in some embodiments, the concentration of trehalose in the cryoprotectant solution is 1.2 mol/L.
In some embodiments, the cryoprotectant is glycerol, and the volume fraction of glycerol in the cryoprotectant solution is 5% to 30% (w/v); in some embodiments, the volume fraction of glycerol in the cryoprotectant solution is 10%.
According to the experimental results of the present invention, glycerol as a cryoprotectant was less effective than trehalose. Experiments show that the trehalose serving as the low-temperature protective agent can realize better protection on red blood cells and obtain higher cell recovery rate.
The invention designs a scheme for rapid cryopreservation of hydrogel fiber encapsulated human erythrocytes with a 'core-shell' structure based on a hydrogel encapsulation technology and a trehalose pre-dehydration technology. The core solution and the shell solution are respectively transmitted into a double-channel coaxial needle through two liquid propelling pumps, and are crosslinked at the outlet of the needle immersed in the crosslinking agent solution to form the core-shell hydrogel fiber encapsulating the red blood cells.
In the present invention, the concentration of sodium alginate in the shell solution is 0.5% to 3% (w/v); the concentration of calcium chloride in the cross-linking agent is 0.01-0.5 mol/L. In some embodiments, the concentration of sodium alginate in the shell solution is 1.5% (w/v); the concentration of calcium chloride in the cross-linking agent is 0.15 mol/L.
In the invention, the leukocyte-removed suspension is mixed with normal saline until the hematocrit is 75-85%. In some embodiments, the mixing is performed until the hematocrit is 80%. The volume ratio of the mixed erythrocyte solution and cryoprotectant solution is 1: 1. After mixing, the hematocrit in the nuclear solution was 40%. The packed volume of the red blood cells in the nuclear solution is proper, so that the red blood cells do not need to be diluted and then stored.
In the present invention, the method for preparing the red blood cell solution comprises: adding physiological saline into the suspension without the white blood cells, shaking uniformly, centrifuging for 5min at 2000g, removing supernatant, repeating the steps for 3 times to obtain a red blood cell solution with a volume of about 80%, and placing the red blood cell solution in a refrigerator at 4 ℃ for later use.
In the invention, the preparation method of the nuclear solution comprises the steps of uniformly mixing the erythrocyte solution with the volume of about 80% and the equal volume of the cryoprotectant solution to obtain the nuclear solution, and standing for 5 minutes at room temperature for later use.
In the invention, the flow rate of the shell solution passing through the double-channel coaxial needle head is 10-1000 mu L/min; the flow speed of the nuclear solution passing through the double-channel coaxial needle is 10-1000 mu L/min. In the embodiment of the invention, the flow rate of the shell solution passing through the double-channel coaxial needle head is 200-800 mu L/min; the flow speed of the nuclear solution passing through the double-channel coaxial needle is 200-800 mu L/min.
Experiments show that the flow rates of the core solution and the shell solution have obvious influence on the freezing effect. The flow rate of the core solution is greater than that of the shell solution, so that the volume ratio of the red blood cells in the cryopreserved fiber can be increased, and the recovery rate of the red blood cells after cryopreservation can be increased. In some embodiments, the shell solution is passed through the dual channel coaxial needle at a flow rate of 200-500 μ L/min; the flow rate of the nuclear solution passing through the double-channel coaxial needle is 500-800 mu L/min. In some embodiments, the shell solution is passed through the dual channel coaxial needle at a flow rate of 500 μ L/min; the flow rate of the nuclear solution through the double-channel coaxial needle is 800 muL/min. In other embodiments, the shell solution is passed through the dual channel coaxial needle at a flow rate of 200 μ L/min; the flow rate of the nuclear solution through the double-channel coaxial needle is 500 muL/min.
In the invention, the diameter of the double-channel coaxial needle is 1mm, the diameter of the prepared fiber is 0.5-3 mm, and the time for freezing by liquid nitrogen is not less than 20 min.
The cryopreservation method provided by the invention protects erythrocytes by using a low-temperature protective agent, and encapsulates erythrocytes in fibers by using gel formed by sodium alginate as a shell structure. Good cryopreservation effect is obtained, and the recovery rate of the red blood cells is higher after cryopreservation.
The invention also provides the fiber containing the red blood cells prepared by the cryopreservation method.
The fiber containing the red blood cells uses gel formed by crosslinking sodium alginate as a shell structure to wrap red blood cell solution containing a low-temperature protective agent.
The invention also provides a thawing method of the erythrocyte cryopreserved by the cryopreservation method, which comprises the step of soaking the fiber containing the erythrocyte in a sodium citrate solution, wherein the temperature of the sodium citrate solution is 37-45 ℃. And during thawing, the concentration of the sodium citrate solution is 0.1-1 mol/L. The soaking time is 20-25 seconds. Preferably, after the sodium citrate is soaked, the fiber is taken out of the sodium citrate solution and is kept standing at room temperature until the fiber is dissolved.
The invention provides a method for rapidly freezing and storing human red blood cells packaged by core-shell fibers. The erythrocyte cryoprotectant used in the invention is an impermeable protectant, and under the protection of the protective protectant, erythrocytes are wrapped in a gel shell structure formed by crosslinking sodium alginate, for example, to form erythrocyte-containing fibers. Compared with the existing erythrocyte cryopreservation technology, the erythrocyte cryopreservation scheme provided by the invention saves the fussy processes of adding glycerol and removing glycerolysis, not only can ensure that the survival rate of the frozen erythrocyte reaches more than 90% in the glycerol-free rapid cryopreservation, but also can keep good microscopic morphology.
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FIG. 1 is a schematic diagram of the structure of a nucleocapsid fiber encapsulating red blood cells prepared in example 1 of the present invention;
FIG. 2 is an optical microscope photograph of the erythrocyte encapsulated nucleocapsid fiber prepared in example 1 of the present invention.
Detailed Description
The invention provides a method for freezing and preserving red blood cells and the frozen and preserved red blood cells, and the technical personnel can appropriately improve the technological parameters by taking the contents into consideration. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1: trehalose is used as a low-temperature protective agent
1. The implementation method comprises the following steps:
1) solution preparation: the concentration of sodium alginate solution is 1.5% (w/v), calcium chloride solution is 0.15M, and sodium citrate solution is 0.8M. The formula of the low-temperature protective agent is as follows: 1.2M trehalose solution.
2) Pretreatment of red blood cells: taking appropriate amount of the suspension (stored in refrigerator at 4 deg.C for less than 15 days), adding 0.9% NaCl solution, centrifuging to remove supernatant, and repeating the above steps for 3 times to obtain erythrocyte solution with a volume of about 80%. An appropriate amount of 0.9% NaCl solution was added to obtain 25mL of a 40% packed red blood cell solution, which was placed in a refrigerator at 4 ℃ for further use.
3) Adding a low-temperature protective agent: adding 25ml of the pretreated red blood cells into a freezing tube, adding an isovolumetric cryoprotectant, uniformly mixing, and standing at room temperature for 5 min.
4) Hydrogel fibers were generated to encapsulate red blood cells: the erythrocyte suspension added with the protective agent is taken as a core solution, a sodium alginate solution is taken as a shell solution, a calcium chloride solution is taken as a cross-linking agent, and the existing core-shell structure hydrogel fiber generation technology in the prior art is utilized to encapsulate the erythrocytes. Starting the shell solution injection pump to make the seaweedThe sodium solution is squeezed out, the nuclear solution injection pump is started a few seconds later, and the reactor is immersed in CaCl2Hydrogel fibers with a core-shell structure are formed at the needle point in the solution. The resulting hydrogel microfibrils encapsulating the core-shell structure of erythrocytes were collected in a beaker for subsequent experiments. The size of the generated core-shell fiber is adjusted by adjusting the flow rate of the propeller pump.
5) Cryopreservation of red blood cell-hydrogel fibers: the fibers were thrown directly into liquid nitrogen and frozen for at least 20 min. And then taking out the fiber from the liquid nitrogen, immediately putting the fiber into warm sodium citrate solution, taking out the fiber from the water bath after 20-25 seconds, standing the fiber for 5 minutes at room temperature until the fiber is completely dissolved, and centrifuging the obtained erythrocyte suspension for later use.
6) Calculating the recovery rate of the red blood cells after freezing: and (3) detecting the absorbance value A of the supernatant at 415nm after each group of erythrocyte suspension in the step 5) is centrifuged by using a spectrophotometer. Another group containing physiological saline as positive control group A0Assuming a positive control group with 0% hemolysis; taking a group added with pure water and repeatedly freezing and thawing for 3 times as a negative control group A1The negative control group was assumed to be 100% haemolytic. The freezing recovery rate of the red blood cells was calculated by the following formula:
wherein, the recovery rate after freezing (the shell solution flow rate is uniformly 500 muL/min) of the hydrogel fiber packaging red blood cells of the core-shell structure for quick freezing preservation at different core solution flow rates of 0.6M trehalose protectant is shown in table 1, and the recovery rate after freezing (the core rate is uniformly 500 muL/min) of the hydrogel fiber packaging red blood cells of the core-shell structure for quick freezing preservation at different shell solution flow rates of 0.6M trehalose protectant is shown in table 2. Each set of experiments was set to 5 replicates.
TABLE 1
TABLE 2
The result shows that the trehalose is used as the low-temperature protective agent to obtain good protection effect, wherein when the flow rate of the core solution is 500-800 mu L/min and the flow rate of the shell solution is 200-500 mu L/min, the highest recovery rate of the cryopreserved red blood cells can reach 90.43% -94.51%. When the flow rate of the core solution is 800 mu L/min and the flow rate of the shell solution is 500 mu L/min, the recovery rate of the red blood cells can reach 94.51 percent, which is obviously superior to the freezing effect of other groups of conditions, and p is less than 0.05.
Example 2: uses glycerin as low-temperature protective agent
1. The implementation method comprises the following steps:
1) solution preparation: the concentration of sodium alginate solution is 1.5% (w/v), calcium chloride solution is 0.15M, and sodium citrate solution is 0.8M. The formula of the low-temperature protective agent is as follows: 10% (w/v) glycerol.
2) Pretreatment of red blood cells: taking appropriate amount of the suspension (stored in refrigerator at 4 deg.C for less than 15 days), adding 0.9% NaCl solution, centrifuging to remove supernatant, and repeating the above steps for 3 times to obtain erythrocyte solution with a volume of about 80%. An appropriate amount of 0.9% NaCl solution was added to obtain 25mL of a 40% packed red blood cell solution, which was placed in a refrigerator at 4 ℃ for further use.
3) Adding a low-temperature protective agent: adding 25ml of the pretreated red blood cells into a freezing tube, adding the equal volume of the cryoprotectant, uniformly mixing, and standing at room temperature for 5 min.
4) Hydrogel fibers were generated to encapsulate red blood cells: the erythrocyte suspension added with the protective agent is taken as a core solution, a sodium alginate solution is taken as a shell solution, a calcium chloride solution is taken as a cross-linking agent, and the existing core-shell structure hydrogel fiber generation technology in the prior art is utilized to encapsulate the erythrocytes. The shell solution injection pump is started first to ensure thatExtruding out sodium alginate solution, starting the injection pump of nuclear solution after several seconds, and immersing in CaCl2Hydrogel fibers with a core-shell structure are formed at the needle point in the solution. The resulting hydrogel microfibrils encapsulating the core-shell structure of erythrocytes were collected in a beaker for subsequent experiments. The size of the generated core-shell fiber is adjusted by adjusting the flow rate of the propeller pump.
5) Cryopreservation of red blood cell-hydrogel fibers: the fibers were thrown directly into liquid nitrogen and frozen for at least 20 min. And then taking out the fiber from the liquid nitrogen for thawing, immediately putting the fiber into warm sodium citrate solution, taking out the fiber from the water bath after 20-25 seconds, and standing for 5 minutes at room temperature until the fiber is completely dissolved.
6) Calculating the recovery rate of the red blood cells after freezing: and (3) detecting the absorbance value A of the supernatant at 415nm after each group of erythrocyte suspension in the step 5) is centrifuged by using a spectrophotometer. Another group containing physiological saline as positive control group A0Assuming a positive control group with 0% hemolysis; taking a group added with pure water and repeatedly freezing and thawing for 3 times as a negative control group A1The negative control group was assumed to be 100% haemolytic. The freezing recovery rate of the red blood cells was calculated by the following formula:
TABLE 35% Glycerol recovery after freezing for fast cryopreservation of core-shell structure hydrogel fiber-encapsulated erythrocytes (unified shell solution flow rate of 500. mu.L/min)
The results show that the recovery of erythrocytes after cryopreservation is low, although cryopreservation of erythrocytes can be achieved with glycerol as cryoprotectant.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. A method for cryopreserving red blood cells, comprising:
preparing a sodium alginate solution as a shell solution; preparing a calcium chloride solution as a cross-linking agent;
mixing the leukocyte-removed suspension with normal saline to obtain a erythrocyte solution;
mixing the erythrocyte solution with the cryoprotectant solution to prepare a nuclear solution;
and allowing the shell solution and the core solution to enter a cross-linking agent through a double-channel coaxial needle, and freezing and storing the obtained fiber liquid nitrogen after solidification.
2. The cryopreservation method of claim 1, wherein the cryoprotectant is trehalose or glycerol; the concentration of trehalose in the cryoprotectant solution is from 5% (w/v) to 30% (w/v); the volume fraction of glycerol in the solution in the cryoprotectant is 5% (w/v) to 30% (w/v).
3. The cryopreservation method of claim 1, wherein the concentration of sodium alginate in the shell solution is 0.5% to 3% (w/v); the concentration of calcium chloride in the cross-linking agent is 0.15 mol/L.
4. The cryopreservation method of claim 1, wherein the suspension of de-leukocytes is mixed with physiological saline until the hematocrit is 75% to 85%.
5. The cryopreservation method of claim 1, wherein the volume ratio of the red blood cell solution to the cryoprotectant solution is 1: 1.
6. The cryopreservation method of claim 1, wherein the flow rate of the shell solution through the double-channel coaxial needle is 10-1000 μ L/min; the flow speed of the nuclear solution passing through the double-channel coaxial needle is 10-1000 mu L/min.
7. The cryopreservation method of claim 1, wherein the diameter of the fiber is 0.5-3 mm, and the time for freezing with liquid nitrogen is not less than 20 min.
8. Erythrocyte-containing fiber obtained by the cryopreservation method according to any one of claims 1 to 7.
9. The method for thawing erythrocytes cryopreserved by the cryopreservation method according to any one of claims 1 to 7, which comprises immersing the erythrocyte-containing fiber in a sodium citrate solution at a temperature of 37 ℃ to 45 ℃.
10. The thawing method according to claim 9, wherein the concentration of the sodium citrate solution is 0.1 to 1 mol/L.
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