CN112616827B - Erythrocyte cryopreservation method and cryopreserved erythrocytes - Google Patents

Abstract

The invention relates to the technical field of erythrocyte preservation, in particular to an 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

Red blood cell cryopreservation method and cryopreserved red blood cells

technical field

The present invention relates to the technical field of erythrocyte preservation, in particular to a method for cryopreservation of erythrocytes and cryopreserved erythrocytes.

Background technique

The cryopreservation of blood is the most widely used and most important part of the preservation of all biological materials. The research on blood preservation is of great significance to military medicine and clinical treatment, especially the long-term cryopreservation of human red blood cells is of great importance to human society. Significance, such as natural disaster emergency blood transfusion, rare blood type blood transfusion and research, military reserves, etc., all have a great demand for the preservation of human red blood cells. Therefore, it is of great significance to study how to efficiently preserve red blood cells for a long time.

At present, the most commonly used red blood cell preservation methods in clinic mainly include: 1) short-term preservation at 4°C; 2) slow cryopreservation at high concentration of glycerol. The former has a short shelf life, usually only 35 to 42 days, and is highly susceptible to contamination, causing red blood cell storage lesions. The latter method can prolong the storage time of red blood cells to several years or even decades by using high glycerol slow freezing method, but the concentration of glycerol is as high as 40%, and it has toxic effects on cells, and requires a tedious deglycerolization washing process before use. The process of adding and removing glycerin wastes a lot of time and effort, potentially delaying treatment of the body in emergency situations.

In addition to these two methods commonly used in clinical practice, low-concentration glycerol rapid freezing method and freeze-drying method are also hotspots in the current research on red blood cell preservation. Compared with slow freezing, the rapid freezing method uses less than 20% glycerol to pretreat red blood cells, and then directly throws the red blood cell suspension into liquid nitrogen at -196°C for cryopreservation. Moreover, rapid rewarming is required when red blood cells are thawed and rewarmed, and long rewarming time is likely to cause damage. The red blood cells preserved by this method can be stored for 5 to 10 years, or even longer, and the morphology and function of the red blood cells are well preserved. Despite this, the red blood cells cryopreserved by the low-concentration glycerol rapid cryopreservation method still cannot be directly reinfused, and the glycerol removal operation is still complicated and difficult before use. In addition, in the current research, the survival rate of red blood cells after freeze-drying is still relatively low, the technology is not mature enough, and there are still huge difficulties in practice.

SUMMARY OF THE INVENTION

In view of this, the technical problem to be solved by the present invention is to provide a method for cryopreserving erythrocytes and cryopreserved erythrocytes, so as to improve the recovery rate of erythrocytes.

The cryopreservation method of red blood cells provided by the present invention comprises:

Prepare sodium alginate solution as shell solution; prepare calcium chloride solution as crosslinking agent;

The leukocyte-depleted suspension is mixed with normal saline to obtain a red blood cell solution;

Mix the red blood cell solution with the cryoprotectant solution to prepare the nuclear solution;

The shell solution and the core solution were made to enter the cross-linking agent through a double-channel coaxial needle, and the fibers obtained after coagulation were frozen in liquid nitrogen and stored.

In the scheme of the present invention, the cryoprotectant can reduce the toxicity of the solution, prevent the osmotic pressure and ice crystals from causing damage to the red blood cells. However, in the present invention, only a single cryoprotectant can be used to achieve good protection of red blood cells. The red blood cells are human red blood cells.

In the present invention, the cryoprotectant is a non-permeable protective agent. In the embodiments of the present invention, the cryoprotectant is trehalose or glycerol; in some embodiments, the cryoprotectant is trehalose, and the concentration of trehalose in the cryoprotectant solution is 5% (w/v) ~30% (w/v); in some specific embodiments, the concentration of trehalose in the cryoprotectant solution is 1.2 mol/L.

In some embodiments, the cryoprotectant is glycerol, and in the cryoprotectant solution, the volume fraction of glycerol is 5% to 30% (w/v); in some specific embodiments, the cryoprotectant solution is , the volume fraction of glycerol is 10%.

According to the experimental results of the present invention, using glycerol as a cryoprotectant is less effective than using trehalose. Experiments show that using trehalose as a cryoprotectant can achieve better protection of red blood cells and obtain a higher cell recovery rate.

Based on the hydrogel encapsulation technology and the trehalose pre-dehydration technology, the present invention designs a solution for rapid freezing and preservation of human red blood cells encapsulated by hydrogel fibers with a "core-shell" structure. The present invention uses a sodium alginate solution as the outer shell solution, The erythrocyte suspension containing the cryoprotectant was used as the nucleus solution, and the calcium chloride solution was used as the crosslinker solution. The core and shell solutions were respectively transferred into a dual-channel coaxial needle through two liquid propelling pumps, and cross-linked at the exit of the needle immersed in the cross-linking agent solution to form core-shell hydrogel fibers encapsulating red blood cells.

In the present invention, the concentration of sodium alginate in the shell solution is 0.5%-3% (w/v); the concentration of calcium chloride in the cross-linking agent is 0.01-0.5mol/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 present invention, the leukocyte-depleted suspension is mixed with physiological saline until the hematocrit is 75%-85%. In some embodiments, the mixing is to a hematocrit of 80%. The volume ratio of the red blood cell solution and the cryoprotectant solution is 1:1. After mixing, the hematocrit in the nuclear solution was 40%. The hematocrit in the nuclear solution is appropriate, so there is no need to dilute the red blood cells for storage.

In the present invention, the preparation method of the red blood cell solution includes: adding the white red blood cell suspension, adding physiological saline, shaking well, centrifuging at 2000g for 5 minutes, removing the supernatant, and repeating the above steps 3 times to obtain a red blood cell solution with a volume of about 80% , placed in a 4°C refrigerator for later use.

In the present invention, the preparation method of the nuclear solution includes: mixing the red blood cell solution with a volume of about 80% and an equal volume of the cryoprotectant solution as the nuclear solution, and standing at room temperature for 5 minutes for use.

In the present invention, the flow rate of the shell solution through the dual-channel coaxial needle is 10-1000 μL/min; the flow rate of the core solution through the dual-channel coaxial needle is 10-1000 μL/min. In the embodiment of the present invention, the flow rate of the shell solution through the dual-channel coaxial needle is 200-800 μL/min; the flow rate of the core solution through the dual-channel coaxial needle is 200-800 μL/min.

Experiments show that the flow rates of core solution and shell solution have a significant effect on the cryopreservation effect. The flow rate of the core solution is greater than that of the shell solution, which can not only increase the volume ratio of erythrocytes in cryopreserved fibers, but also improve the recovery rate of erythrocytes after cryopreservation. In some embodiments, the flow rate of the shell solution through the dual-channel coaxial needle is 200-500 μL/min; the flow rate of the core solution through the dual-channel coaxial needle is 500-800 μL/min. In some specific embodiments, the flow rate of the shell solution through the dual-channel coaxial needle is 500 μL/min; the flow rate of the core solution through the dual-channel coaxial needle is 800 μL/min. In other specific embodiments, the flow rate of the shell solution through the dual-channel coaxial needle is 200 μL/min; the flow rate of the core solution through the dual-channel coaxial needle is 500 μL/min.

In the present invention, the diameter of the dual-channel coaxial needle is 1 mm, the diameter of the prepared fiber is 0.5-3 mm, and the freezing time in liquid nitrogen is not less than 20 minutes.

The cryopreservation method of the present invention protects the red blood cells with a cryoprotectant, and uses the gel formed by sodium alginate as a shell structure to encapsulate the red blood cells inside the fibers. A good cryopreservation effect was obtained, and the recovery rate of red blood cells was high after cryopreservation.

The present invention also provides red blood cell-containing fibers prepared by the cryopreservation method.

The fiber containing red blood cells of the present invention uses a gel formed by cross-linking of sodium alginate as a shell structure, and wraps the red blood cell solution containing a cryoprotectant.

The present invention also provides a method for thawing red blood cells frozen by the freezing method of the present invention. °C. During thawing, the concentration of the sodium citrate solution is 0.1 mol/L to 1 mol/L. The soaking time is 20-25 seconds. Preferably, after soaking in sodium citrate, the fibers are taken out from the sodium citrate solution and left at room temperature until the fibers are dissolved.

The present invention provides a method for rapid cryopreservation of human red blood cells encapsulated by core-shell fibers. The red blood cell cryoprotectant used in the present invention is a non-permeable protective agent. Under the protection, red blood cells are wrapped in a gel shell structure formed by cross-linking of sodium alginate to form fibers containing red blood cells. Compared with the existing erythrocyte cryopreservation technology, the erythrocyte cryopreservation scheme provided by the present invention saves the tedious process of adding glycerol and deglycerolization, and not only can make the survival rate of human erythrocytes reach 90% in glycerol-free rapid cryopreservation % or more, while red blood cells can maintain a good microscopic morphology.

Description of drawings

Fig. 1 is a schematic diagram of the structure of the core-shell fiber of the encapsulated erythrocyte prepared in Example 1 of the present invention;

FIG. 2 is an optical microscope image of the core-shell fibers encapsulating erythrocytes prepared in Example 1 of the present invention.

Detailed ways

The present invention provides a method for cryopreserving erythrocytes and the cryopreserved erythrocytes, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invention technology.

The test materials used in the present invention are all common commercial products and can be purchased in the market.

Below in conjunction with embodiment, the present invention is further elaborated:

Embodiment 1: take trehalose as cryoprotectant

1. Implementation method:

1) Solution preparation: the concentration of sodium alginate solution is 1.5% (w/v), the calcium chloride solution is 0.15M, and the sodium citrate solution is 0.8M. The formula of cryoprotectant is: 1.2M trehalose solution.

2) Red blood cell pretreatment: Take an appropriate amount of white red blood cell suspension (stored in a refrigerator at 4°C for less than 15 days), add 0.9% NaCl solution and centrifuge to remove the supernatant, repeat the above steps 3 times to obtain a volume of about 80%. Red blood cell solution. An appropriate amount of 0.9% NaCl solution was added to obtain 25 mL of red blood cell solution with a volume of 40%, which was placed in a 4°C refrigerator for later use.

3) Add cryoprotectant: add 25 ml of the above pretreated red blood cells into a cryopreservation tube, then add an equal volume of cryoprotectant to mix well, and let stand for 5 minutes at room temperature.

4) generate hydrogel fibers to encapsulate red blood cells: take the above-mentioned red blood cell suspension added with protective agent as core solution, sodium alginate solution as shell solution, and calcium chloride solution as cross-linking agent, utilizing existing in the prior art. Core-shell structure hydrogel fiber generation technology to encapsulate red blood cells. The shell solution injection pump was started first, so that the sodium alginate solution was squeezed out, and the core solution injection pump was started after a few seconds, and the core-shell structure hydrogel fibers were formed at the needle tip immersed in the _CaCl2 solution. The resulting hydrogel microfibrils encapsulating the core-shell structure of erythrocytes were collected into a beaker for subsequent experiments. The size of the generated core-shell fibers was adjusted by adjusting the flow rate of the propelling pump.

5) Cryopreservation of erythrocyte-hydrogel fibers: directly throw the fibers into liquid nitrogen and freeze for at least 20 minutes. Then take it out for thawing, take out the fiber from the liquid nitrogen and immediately put it into a warm sodium citrate solution, take it out from the water bath after 20 to 25 seconds, and let it stand for 5 minutes at room temperature until the fiber is completely dissolved, and the red blood cell suspension is obtained. Centrifuge the liquid for use.

6) Calculate the recovery rate of erythrocytes after freezing: use a spectrophotometer to detect the absorbance value A of the supernatant at 415 nm after centrifugation of the erythrocyte suspensions of each group in the above step 5). Another group added with only normal saline was taken as the positive control group A ₀ , and the positive control group was assumed to be 0% hemolyzed; another group added with pure water and repeated freezing and thawing 3 times was taken as the negative control group A ₁ . The negative control group was assumed to be 100% hemolyzed. Freeze recovery of red blood cells was calculated by the following formula:

Among them, the post-freezing recovery rates of the core-shell structure hydrogel fiber-encapsulated red blood cells under different core solution flow rates of 0.6M trehalose protective agent (the flow rate of the shell solution is uniformly 500 μL/min) are shown in Table 1, and 0.6M Table 2 shows the post-freezing recovery rates of the core-shell structure hydrogel fiber-encapsulated red blood cells for rapid cryopreservation under different shell solution flow rates of trehalose protective agent (the core rate is uniformly 500 μL/min). Each group of experiments was set up with 5 repetitions.

Table 1

Table 2

The results showed that trehalose as a cryoprotectant could achieve good protective effect. When the flow rate of the core solution was 500-800 μL/min and the flow rate of the shell solution was 200-500 μL/min, the recovery rate of cryopreserved erythrocytes was the highest, up to 90.43%～94.51%. When the flow rate of the nucleus solution was 800μL/min and the flow rate of the shell solution was 500μL/min, the recovery rate of red blood cells could reach 94.51%, which was significantly better than that of the other groups, p<0.05.

Example 2: Using glycerol as cryoprotectant

1. Implementation method:

1) Solution preparation: the concentration of sodium alginate solution is 1.5% (w/v), the calcium chloride solution is 0.15M, and the sodium citrate solution is 0.8M. The cryoprotectant formulation is: 10% (w/v) glycerol.

2) Red blood cell pretreatment: Take an appropriate amount of white red blood cell suspension (stored in a refrigerator at 4°C for less than 15 days), add 0.9% NaCl solution and centrifuge to remove the supernatant, repeat the above steps 3 times to obtain a volume of about 80%. Red blood cell solution. An appropriate amount of 0.9% NaCl solution was added to obtain 25 mL of red blood cell solution with a volume of 40%, which was placed in a 4°C refrigerator for later use.

3) Add cryoprotectant: add 25 ml of the above pretreated red blood cells into a cryopreservation tube, add an equal volume of cryoprotectant, mix well, and let stand at room temperature for 5 minutes.

4) generate hydrogel fibers to encapsulate red blood cells: take the above-mentioned red blood cell suspension added with protective agent as core solution, sodium alginate solution as shell solution, and calcium chloride solution as cross-linking agent, utilizing existing in the prior art. Core-shell structure hydrogel fiber generation technology to encapsulate red blood cells. The shell solution injection pump was started first, so that the sodium alginate solution was squeezed out, and the core solution injection pump was started after a few seconds, and the core-shell structure hydrogel fibers were formed at the needle tip immersed in the _CaCl2 solution. The resulting hydrogel microfibrils encapsulating the core-shell structure of erythrocytes were collected into a beaker for subsequent experiments. The size of the generated core-shell fibers was adjusted by adjusting the flow rate of the propelling pump.

5) Cryopreservation of erythrocyte-hydrogel fibers: directly throw the fibers into liquid nitrogen and freeze for at least 20 minutes. Then take it out for thawing, take out the fiber from the liquid nitrogen and immediately put it into a warm sodium citrate solution, take it out from the water bath after 20 to 25 seconds, and let it stand for 5 minutes at room temperature until the fiber is completely dissolved.

6) Calculate the recovery rate of erythrocytes after freezing: use a spectrophotometer to detect the absorbance value A of the supernatant at 415 nm after centrifugation of the erythrocyte suspensions of each group in the above step 5). Another group added with only normal saline was taken as the positive control group A ₀ , and the positive control group was assumed to be 0% hemolyzed; another group added with pure water and repeated freezing and thawing 3 times was taken as the negative control group A ₁ . The negative control group was assumed to be 100% hemolyzed. Freeze recovery of red blood cells was calculated by the following formula:

Table 3 The recovery rate after freezing of the erythrocytes encapsulated by the core-shell structure hydrogel fibers under the condition of 5% glycerol for rapid cryopreservation (the flow rate of the shell solution is uniformly 500 μL/min)

The results showed that although the cryopreservation of erythrocytes could also be achieved with glycerol as a cryoprotectant, the recovery rate of erythrocytes after cryopreservation was low.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.

Claims (9)

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;

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;

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.

2. The cryopreservation method of claim 1, wherein the cryoprotectant is trehalose; the concentration of trehalose in the cryoprotectant solution 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 to a packed volume of erythrocytes of 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 diameter of the fiber is 0.5-3 mm, and the time for freezing with liquid nitrogen is not less than 20 min.

7. Erythrocyte-containing fiber obtained by the cryopreservation method according to any one of claims 1 to 6.

8. The method for thawing erythrocytes frozen according to any one of claims 1 to 6, which comprises immersing the fibres containing erythrocytes in a sodium citrate solution, the temperature of said sodium citrate solution being comprised between 37 ℃ and 45 ℃.

9. The thawing method according to claim 8, wherein the concentration of the sodium citrate solution is 0.1-1 mol/L.

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