Erythrocyte cryopreservation liquid of deep eutectic solvent system as well as preparation and application of erythrocyte cryopreservation liquid

Abstract

The invention relates to the technical field of low-temperature biomedicine, in particular to red blood cell cryopreservation liquid of a deep eutectic solvent system and preparation and application of the red blood cell cryopreservation liquid. The erythrocyte cryopreservation liquid of the eutectic solvent system comprises a hydrogen bond donor, a hydrogen bond receptor and a buffer solution, the hydrogen bond donor is glycerol. Based on the red blood cell cryopreservation liquid, the invention further provides a red blood cell cryopreservation method and a CPA elution method which are matched with the red blood cell cryopreservation liquid. After human red blood cells are quickly frozen in the red blood cell cryopreservation liquid provided by the invention, the survival rate after freezing can reach 97%, and the cryopreserved cells can maintain good morphological structures and functional states; the erythrocyte cryopreservation operation is simple and convenient, other special instruments and equipment are not needed, large-volume human erythrocytes can be cryopreserved through the ultralow-temperature cryopreservation bag, and efficient and rapid cryopreservation and rewarming washing processes are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cryobiology, in particular to a red blood cell cryopreservation solution based on deep eutectic solvents and its preparation and application. BACKGROUND

[0002] Currently, the cryopreservation of red blood cells mainly relies on high concentrations of glycerol (20-40%) as cryoprotective agents (CPA). However, high concentrations of glycerol can easily cause osmotic imbalance and toxic damage to cell membranes. In addition, it needs to go through multiple elution processes before it can be used for clinical transfusion. If the glycerolization is not complete, the red blood cells will swell and lyse during transfusion, leading to an increase in free hemoglobin concentration, which may cause kidney failure and other problems. Therefore, it is necessary to reduce the concentration of glycerol used in the cryopreservation of red blood cells or to develop new and safer cryoprotective agents for red blood cells.

[0003] Deep eutectic solvents (DESs) are a class of eutectic mixtures formed by hydrogen bond donors and hydrogen bond acceptors in a certain molar ratio, usually composed of two or three components. DESs are a kind of chemical solvents with high designability, which can be designed by combining different hydrogen bond donors and hydrogen bond acceptors to prepare specific DESs with specific physical and chemical properties (such as freezing point, viscosity, conductivity, and pH value, etc.). DESs were initially mainly used in green chemical industry and material science, and their application in low-temperature preservation, stabilization of protein, DNA and RNA structures, and other biomedical applications has only been explored recently.

[0004] Glycerol-based deep eutectic solvents are a class of eutectic systems composed of glycerol as a hydrogen bond donor and one or more hydrogen bond acceptors in a certain molar ratio. The hydrogen bond acceptors of sugars, amino acids and quaternary ammonium salts themselves have certain membrane stability and osmotic regulation ability. After they are combined with glycerol to form glycerol-based DESs, they not only maintain good biocompatibility, but also, due to their multiple hydrogen bond network structure, may play multiple roles such as osmotic regulation, inhibition of ice crystal formation and membrane protection during the freezing process. In addition, compared with traditional single-component CPA systems, glycerol-based DESs may produce synergistic effects among components, which are expected to reduce the concentration of each single component while providing equivalent or better cryoprotective effects, thereby simplifying the subsequent CPA elution process while significantly reducing osmotic stress and toxicity risk.

[0005] Therefore, it is essential to provide a new red blood cell cryopreservation system based on glycerol-based DESs to solve the many limitations of the current high-concentration glycerol solution, providing a safer, more efficient and convenient new path for clinical blood storage and emergency transfusion. SUMMARY

[0006] In order to solve the problems of high concentration of glycerol used in existing red blood cell cryopreservation and time-consuming and cumbersome elution process, the present application aims to provide a red blood cell cryopreservation solution of a deep eutectic solvent system and preparation and application thereof.

[0007] The object of the present application can be achieved by the following technical solutions.

[0008] The first object of the present application is to provide a red blood cell cryopreservation solution of a deep eutectic solvent system, comprising a hydrogen bond donor, a hydrogen bond acceptor and a buffer solution.

[0009] The hydrogen bond donor is glycerol.

[0010] In an embodiment of the present application, the hydrogen bond acceptor is selected from one of glucose, trehalose, fructose, sucrose, alanine, glutamic acid, glycine, proline, betaine or choline chloride.

[0011] The buffer solution is selected from one of PBS buffer solution, DPBS buffer solution, HEPES buffer solution, HBSS buffer solution or EBSS buffer solution; preferably, the buffer solution is PBS buffer solution.

[0012] The second object of the present application is to provide a preparation method of a red blood cell cryopreservation solution of a deep eutectic solvent system, comprising the following steps.

[0013] The hydrogen bond acceptor and the hydrogen bond donor are heated and uniformly mixed to obtain a deep eutectic solvent.

[0014] The deep eutectic solvent is uniformly mixed with the buffer solution to obtain the red blood cell cryopreservation solution of the deep eutectic solvent system.

[0015] In an embodiment of the present application, during the heating and uniform mixing process, the temperature is 50-70℃, the time is 4-24h and the rotation speed is 200-700rpm.

[0016] The third object of the present application is to provide the application of the red blood cell cryopreservation solution of the deep eutectic solvent system in cryopreservation of red blood cells.

[0017] The fourth object of the present application is to provide a method for cryopreservation of red blood cells, comprising the following steps.

[0018] The red blood cell suspension is uniformly mixed with the red blood cell cryopreservation solution of the deep eutectic solvent system to obtain a mixed solution.

[0019] The mixed solution is post-processed and cryopreserved in liquid nitrogen to complete the cryopreservation of red blood cells.

[0020] In an embodiment of the present application, in the mixed solution, the concentration of the red blood cell cryopreservation solution of the deep eutectic solvent system is 5%v / v to 20%v / v.

[0021] Preferably, in the mixture, the concentration of the erythrocyte cryopreservation solution in the eutectic solvent system is 10% v / v;

[0022] The red blood cell suspension is a mixture of red blood cells and PBS buffer solution;

[0023] In the erythrocyte suspension, the hematocrit of erythrocytes is 20-60%;

[0024] Preferably, the hematocrit of erythrocytes is 20%.

[0025] In one embodiment of the present invention, the mixture is post-treated, placed in an ultra-low temperature freezing bag, and then frozen in liquid nitrogen.

[0026] In one embodiment of the present invention, the post-processing is to perform equilibration treatment on the mixture, or to perform loading treatment on the mixture;

[0027] During the equilibration process, the temperature is room temperature and the time is 5 to 15 minutes; preferably, the time is 10 minutes.

[0028] During the loading process, the temperature is 4–37°C and the time is 10 min–3 h; preferably, the temperature is 37°C and the time is 2 h.

[0029] The fifth objective of this invention is to provide a CPA elution method for cryopreserved red blood cells, comprising the following steps:

[0030] (S1) After the frozen red blood cells obtained by the above method are taken out, they are thawed and warmed, and then centrifuged to remove the supernatant.

[0031] (S2) Mix the lower layer of concentrated red blood cells with the washing solution, centrifuge again and remove the supernatant to complete the CPA elution of frozen red blood cells.

[0032] In one embodiment of the present invention, in step (S1), the temperature during the thawing and rewarming process is 37-45°C; preferably, the temperature is 37°C.

[0033] Step (S2) is repeated 2-3 times, with the washing buffer being the same volume as the thawed and rewarmed red blood cell suspension;

[0034] The washing solution is a 0.9-3.5% w / v sodium chloride solution.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The erythrocyte cryopreservation solution of the eutectic solvent system provided by the present invention is simple to prepare and can be prepared by heating and stirring. Human erythrocytes can achieve a 97% post-freezing survival rate after rapid freezing in the erythrocyte cryopreservation solution. The cryopreserved cells can maintain good morphological structure and functional state.

[0037] (2) Compared with the 17.5% w / v compound glycerol system used in the traditional low-glycerol rapid freezing method, the concentration of glycerol used in the red blood cell cryopreservation solution of the present invention is reduced to about 10% w / v, which greatly shortens the deglycerol removal time. Human red blood cells cryopreserved with the red blood cell cryopreservation solution of the present invention only need two washing steps to meet clinical standards, greatly shortening the washing time, while the washing recovery rate reaches more than 85%.

[0038] (3) The red blood cell cryopreservation method provided by the present invention is simple to operate and does not require other special instruments and equipment. Large volumes of human red blood cells can be cryopreserved through ultra-low temperature cryopreservation bags, realizing efficient and rapid cryopreservation and thawing and washing of human red blood cells. Attached Figure Description

[0039] Figure 1 This is a schematic diagram showing the cryopreservation recovery rate of erythrocytes frozen at different concentrations of glycerol-based DESs in Example 6;

[0040] Figure 2 This is a schematic diagram illustrating the cryopreservation recovery rate of erythrocytes cryopreserved with glycerol-based DESs at different cryopreservation volumes in Example 8;

[0041] Figure 3 This is a flowchart illustrating the cryopreservation of red blood cells using glycerol-based DESs in a cryopreservation bag in Example 10.

[0042] Figure 4 Morphological image of thawed-washed red blood cells in Example 12 (scale bar is 5 μm);

[0043] Figure 5 This is a schematic diagram showing the ATP content of red blood cells after thawing and washing in Example 13;

[0044] Figure 6 This is a schematic diagram of the SOD activity of red blood cells after thawing and washing in Example 13;

[0045] Figure 7 This is a schematic diagram of the PS exposure rate of red blood cells after thawing and washing in Example 13. Detailed Implementation

[0046] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0047] Unless otherwise specified, all reagents used in the following embodiments are commercially available reagents, and all detection methods and techniques used are conventional detection methods and techniques in the art.

[0048] Example 1

[0049] This embodiment provides a method for preparing a erythrocyte cryopreservation solution using a eutectic solvent system: the method is as follows:

[0050] (1) Trehalose (as a hydrogen bond acceptor) and glycerol (the molar ratio of trehalose to glycerol is 1:30) are mixed to obtain a mixture; the mixture is stirred at 500 rpm at 60°C using a magnetic thermostatic stirrer until a uniform, transparent, viscous liquid is obtained, and then the air bubbles introduced during the stirring process are removed by ultrasonic treatment to obtain a eutectic solvent.

[0051] (2) Mix the eutectic solvent prepared in step (1) with PBS buffer (the volume ratio of eutectic solvent to PBS buffer is 1:9) to obtain a erythrocyte cryopreservation solution with glycerol-based eutectic solvent (TreGly) (eutectic solvent content is 10% v / v).

[0052] Mix the eutectic solvent prepared in step (1) with PBS buffer (the volume ratio of eutectic solvent to PBS buffer is 1:4) to obtain a erythrocyte cryopreservation solution with glycerol-based eutectic solvent (TreGly) (eutectic solvent content is 20% v / v).

[0053] The eutectic solvent prepared in step (1) was mixed with PBS buffer (the volume ratio of eutectic solvent to PBS buffer was 2:3) to obtain a erythrocyte cryopreservation solution with glycerol-based eutectic solvent (TreGly) (eutectic solvent content was 40% v / v).

[0054] Example 2

[0055] This embodiment provides a erythrocyte cryopreservation solution in a eutectic solvent system: the preparation method of the erythrocyte cryopreservation solution in a glycerol-based eutectic solvent is the same as in Example 1, except for the following conditions:

[0056] Glucose was used as the hydrogen bond acceptor, and the molar ratio of glucose to glycerol was 1:36.

[0057] Finally, the following red blood cell cryopreservation solutions were prepared: one with glycerol-based eutectic solvent (GluGly) (eutectic solvent content of 10% v / v), one with glycerol-based eutectic solvent (GluGly) (eutectic solvent content of 20% v / v), and one with glycerol-based eutectic solvent (GluGly) (eutectic solvent content of 40% v / v).

[0058] Example 3

[0059] This embodiment provides a erythrocyte cryopreservation solution in a eutectic solvent system: the preparation method of the erythrocyte cryopreservation solution in a glycerol-based eutectic solvent is the same as in Example 1, except for the following conditions:

[0060] L-proline was used as the hydrogen bond acceptor, and the molar ratio of L-proline to glycerol was 1:3.

[0061] Finally, the following red blood cell cryopreservation solutions were prepared: one with glycerol-based eutectic solvent (ProGly) (eutectic solvent content of 10% v / v), one with glycerol-based eutectic solvent (ProGly) (eutectic solvent content of 20% v / v), and one with glycerol-based eutectic solvent (ProGly) (eutectic solvent content of 40% v / v).

[0062] Example 4

[0063] This embodiment provides a erythrocyte cryopreservation solution in a eutectic solvent system: the preparation method of the erythrocyte cryopreservation solution in a glycerol-based eutectic solvent is the same as in Example 1, except for the following conditions:

[0064] Betaine was used as the hydrogen bond acceptor, and the molar ratio of betaine to glycerol was 1:2.

[0065] Finally, the following red blood cell cryopreservation solutions were prepared: one with glycerol-based eutectic solvent (BetGly) (eutectic solvent content of 10% v / v), one with glycerol-based eutectic solvent (BetGly) (eutectic solvent content of 20% v / v), and one with glycerol-based eutectic solvent (BetGly) (eutectic solvent content of 40% v / v).

[0066] Example 5

[0067] This embodiment provides the preparation of a red blood cell suspension, and the steps are as follows:

[0068] (1) Place the red blood cells in a centrifuge tube, add PBS solution and mix well. Centrifuge at 2780 rpm for 5 min and then remove the supernatant.

[0069] (2) Repeat step (1) until the supernatant is clear. After removing the supernatant, retain the lower layer of hematocrit red blood cells and adjust the hematocrit to 20% by adding PBS solution.

[0070] Example 6

[0071] This embodiment provides an effect test on the cryopreservation effect of eutectic solvent systems (DESs) on red blood cells. Red blood cell cryopreservation solutions prepared in Examples 1, 2, 3, and 4 were used as the cryopreservation solutions, with 35% w / v compound glycerol (Gly) as the control group, for rapid cryopreservation of red blood cells. The specific method is as follows:

[0072] Add 0.2 mL of the red blood cell suspension prepared in Example 5 to a 1.5 mL centrifuge tube, and then add an equal volume of red blood cell cryopreservation solution or compound glycerol to make a mixture of 0.4 mL. After mixing, the concentrations of DESs in the mixtures of each group are 5%, 10%, and 20% v / v, respectively, while the concentration of the control group is 17.5% w / v.

[0073] Each mixture was equilibrated at room temperature for 10 minutes, and then frozen in liquid nitrogen at -196°C for at least 30 minutes to complete the cryopreservation of red blood cells (hereinafter referred to as "cryopreserved samples").

[0074] After the frozen samples were removed, they were quickly placed in a 37°C water bath and gently thawed. The thawed red blood cells were collected and centrifuged. The absorbance (A) of the supernatant at 415 nm was measured using a microplate reader. A positive control group (0% hemolysis) was established, containing only physiological saline but not frozen, and its absorbance (A0) at 415 nm was measured. A negative control group (100% hemolysis) was established, containing pure water and subjected to three freeze-thaw cycles, and its absorbance (A1) at 415 nm was measured. The red blood cell recovery rate was calculated using the following formula:

[0075]

[0076] pass Figure 1 It was found that the recovery rate of erythrocytes after thawing showed a trend of first increasing and then decreasing in eutectic solvent systems (DESs) at different concentrations. The optimal concentration of DESs for rapid cryopreservation of erythrocytes was 10% v / v, with 10% v / v TreGly and 10% v / v GluGly showing the best results, with recovery rates of 97.24% and 97.26%, respectively, which were higher than the recovery rate (94.83%) of the control group of 17.5% w / v Gly.

[0077] Example 7

[0078] This embodiment demonstrates the effect of different loading times and temperatures on the cryopreservation of red blood cells using ProGly and BetGly.

[0079] Since the recoveries of both the 10% v / v BetGly and 10% v / v ProGly groups were below 88% after equilibration at room temperature for 10 min before cryopreservation, different CPA loading times and loading temperatures were set based on the 10% v / v BetGly and 10% v / v ProGly groups to further evaluate the effectiveness of glycerol-based DESs in cryopreserving erythrocytes. The specific experimental methods are as follows:

[0080] Take 0.2 mL of the red blood cell suspension prepared in Example 5 and add it to a 1.5 mL centrifuge tube. Then add equal volumes of 20% v / v ProGly and 20% v / v BetGly respectively, and mix them to form a 0.4 mL mixture. After mixing, the final concentration of DESs in each mixture is 10% v / v.

[0081] Centrifuge tubes containing red blood cell cryopreservation mixture were placed in a 37°C water bath for 10 min, 1 h, 2 h, and 3 h, respectively, and then frozen in liquid nitrogen. After 30 min, the tubes were removed and quickly placed in a 37°C water bath for gentle thawing. Finally, the supernatant was centrifuged and the cryopreservation recovery rate was measured (the results are shown in Table 1).

[0082] Table 1. Freezing recovery rate of red blood cells at different loading times

[0083]

[0084] As shown in Table 1, the erythrocyte recovery rate of the 10% v / v ProGly group significantly increased with the extension of loading time, reaching its highest value after 2 hours of loading. However, the recovery rate of the 10% v / v BetGly group decreased with prolonged loading time.

[0085] To further optimize the cryopreservation process of red blood cells using ProGly, red blood cells from the 10% v / v ProGly group were loaded at different loading temperatures for 2 hours, and the recovery rate of red blood cells after rewarming was shown in Table 2.

[0086] Table 2. Freezing recovery rate of erythrocytes in the 10% v / v ProGly group at different loading temperatures.

[0087]

[0088] Table 2 shows that 10% v / v ProGly achieves the best cryopreservation effect when loaded at 37°C for 2 hours.

[0089] Example 8

[0090] This example demonstrates the effect of different cryopreservation volumes on the cryopreservation of erythrocytes using glycerol-based DESs.

[0091] Three different cryopreservation containers were used to evaluate the effectiveness of DESs cryopreservation of red blood cells at different cryopreservation volumes. The three containers were 1.5 mL centrifuge tubes, 2 mL centrifuge tubes, and 10 mL cryopreservation tubes. The specific experimental methods are as follows:

[0092] Take 0.5 mL of the erythrocyte suspension prepared in Example 5 and add it to a 1.5 mL centrifuge tube; take 1 mL of the erythrocyte suspension prepared in Example 5 and add it to a 2 mL centrifuge tube; take 5 mL of the erythrocyte suspension prepared in Example 5 and add it to a 10 mL cryopreservation tube. Then add equal volumes of 20% v / v GluGly, 20% v / v TreGly, and 20% v / v ProGly, respectively, and mix to form 1 mL, 2 mL, and 10 mL mixtures, respectively. The final concentrations of DESs in the mixtures are 10% v / v GluGly, 10% v / v TreGly, and 10% v / v ProGly, respectively. The GluGly and TreGly groups were equilibrated at room temperature for 10 min and then frozen in liquid nitrogen. The ProGly group was incubated in a 37°C water bath for 2 h and then frozen in liquid nitrogen. After 30 min, the mixture was removed, quickly placed in a 37°C water bath, and gently thawed. After centrifugation, the supernatant was collected to determine the cryopreservation recovery rate (results are shown in Figure 1). Figure 2 (As shown).

[0093] pass Figure 2 It can be observed that when the cryopreservation volume of red blood cells is 1 mL, the cryopreservation recovery rate of both the 10% v / v GluGly group and the 10% v / v TreGly group reaches 96%; when the cryopreservation volume is further increased to 10 mL, the cryopreservation recovery rate of the 10% v / v GluGly group is still high, reaching 92.39%, which is significantly higher than other DESs groups.

[0094] Example 9

[0095] The 10 mL cryopreserved 10% v / v GluGly group from Example 8 was subjected to CPA elution, while the control group with the same cryopreserved volume of 17.5% w / v Gly was used. The washing steps after cell rewarming for both groups are as follows:

[0096] (1) For the DESs group: After rewarming and centrifugation to remove the supernatant, the first wash was performed, and 3.2% sodium chloride solution was added to the stratified red blood cells. After mixing, the supernatant was collected by centrifugation, and the hemolysis rate of the first wash was measured. After the second wash, the remaining supernatant was removed, and 0.9% sodium chloride solution was added to the stratified red blood cells. After mixing, the supernatant was collected by centrifugation, and the hemolysis rate of the second wash was measured.

[0097] (2) For the control group 17.5% w / v Gly: First wash, add 3.5% sodium chloride solution to the stratified red blood cells, mix well, centrifuge and collect the supernatant to measure the hemolysis rate of the first wash; Second wash, remove the remaining supernatant, add 0.9% sodium chloride solution to the stratified red blood cells, shake gently to mix well, centrifuge and collect the supernatant to measure the hemolysis rate of the second wash; Third wash, remove the remaining supernatant, add 0.9% sodium chloride solution to the stratified red blood cells, mix well, centrifuge and collect the supernatant to measure the hemolysis rate of the third wash.

[0098] The hemolysis rates at each step of the washing process in the two groups are shown in Table 3:

[0099] Table 3 Hemolysis rate of red blood cells during different washing processes

[0100]

[0101] The recovery rates after washing for the two groups are shown in Table 4:

[0102] Table 4. Recovery rate of red blood cells in the two groups

[0103]

[0104] Tables 3 and 4 show that the 10% v / v GluGly group experienced significantly less hemolysis of red blood cells during the washing process. It achieved the same effect as the traditional cryopreservation method of the 17.5% w / v Gly group after three washes with only two washes. The total recovery rate of the two cryopreservation-washing methods was not significantly different and could reach over 85%.

[0105] Example 10

[0106] The effect of different cryopreservation volumes in cryopreservation bags on the cryopreservation effect of glycerol-based DESs on red blood cells

[0107] Large-volume cryopreservation of red blood cells was conducted using 250 mL cryopreservation bags as freezing containers to evaluate the feasibility of cryopreservation of large-volume red blood cells using cryopreservation ethers (DESs). Three DESs with potential for cryopreservation of red blood cells—10% v / v GluGly, 10% v / v ProGly, and 10% v / v TreGly—were selected for rapid cryopreservation of red blood cells, with a control group of 17.5% w / v Gly. The specific experimental methods are as follows:

[0108] Take 10 mL, 20 mL, and 50 mL of the erythrocyte suspension prepared in Example 5, respectively, and add them to cryopreservation bags. Then, add equal volumes of 35% w / v Gly, 20% v / v TreGly, 20% v / v GluGly, and 20% v / v ProGly, respectively, to form 20 mL, 40 mL, and 100 mL mixtures with different cryoprotectants. The final concentrations of DESs in each mixture are 17.5% w / v Gly, 10% v / v TreGly, 10% v / v GluGly, and 10% v / v ProGly, respectively. After adding the cryoprotectants, vacuum seal the cryopreservation bags. The erythrocytes in the 10% v / v ProGly group need to be incubated in a 37°C water bath for 2 hours, while the other groups are equilibrated at room temperature for 10 minutes. To keep the bags flat and prevent deformation and curling after immersion in liquid nitrogen, the bags were placed in a self-made double-sided wire mesh for pressing and fixing, then immersed in liquid nitrogen for freezing. After 30 minutes, they were removed and quickly placed in a 37°C water bath for gentle thawing. After rewarming, the mixture was stirred, and samples were taken, centrifuged, and the supernatant was used to determine the cryogenic recovery rate. The operating procedure is as follows: Figure 3 As shown in Table 5, the results are as follows.

[0109] Table 5. Freezing recovery rate of red blood cells with different frozen volumes

[0110]

[0111] When the cryopreservation volume was 20 mL, the cryopreservation recovery rates of all three DESs groups were higher than those of the control group (17.5% w / v Gly). Among them, the 10% v / v TreGly and 10% v / v ProGly groups showed the best cryopreservation effects, especially the 10% v / v ProGly group, which had the highest cryopreservation recovery rate. When the cryopreservation volume of red blood cells reached 100 mL, the cryopreservation recovery rates of the 10% v / v TreGly and 10% v / v GluGly groups both reached over 90%, with no significant difference compared to the control group (17.5% w / v Gly). Among them, the 10% v / v GluGly group performed best, with the highest cryopreservation recovery rate.

[0112] Example 11

[0113] CPA elution was performed on the 10% v / v TreGly group, 10% v / v ProGly group, and the control group (17.5% w / v Gly) with a frozen volume of 20 mL in Example 10. The specific steps were the same as steps (1) and (2) in Example 6. The hemolysis rates at each step of the washing process are shown in Table 6.

[0114] Table 6. Hemolysis rate of red blood cells during different washing processes

[0115]

[0116] The recovery rates of each group after washing are shown in Table 7:

[0117] Table 7. Recovery rate of red blood cells in each group

[0118]

[0119] As can be seen from the table, the hemolysis rates of the 10% v / v TreGly group and the 10% v / v ProGly group were significantly lower than those of the control group (17.5% w / v Gly) in the first two washes, and the final total recovery rate of the freeze-wash was over 85%.

[0120] Example 12

[0121] Red blood cells from the 10% v / v TreGly and 10% v / v ProGly groups, after rewarming and washing in Example 10, were collected, and fresh red blood cells were prepared. The morphology of red blood cells in each group was observed under a scanning electron microscope (results are shown in Figure 10). Figure 4 (As shown). Through Figure 4 It can be observed that DESs-frozen red blood cells maintain a well-defined disc-shaped morphology with a central indentation.

[0122] Example 13

[0123] Red blood cells from the 10% v / v TreGly and 10% v / v ProGly groups, after rewarming and washing in Example 10, were collected, and fresh red blood cells were prepared. The ATP content, SOD activity, and PS exposure rate of each group of red blood cells were calculated. The specific methods are as follows:

[0124] Red blood cells were centrifuged to remove the supernatant, resulting in concentrated red blood cells. After mixing, the cells were placed on ice. The ATP content of each group was calculated using an ATP assay kit based on a standard curve prepared from the standards (results are shown below). Figure 5 (As shown). Through Figure 5 It can be observed that the TreGly group was able to maintain ATP content well after red blood cell cryopreservation, and its energy metabolism state was close to that of fresh red blood cells. The ATP content of the ProGly group decreased, but still retained more than 64%.

[0125] Red blood cells from each group were centrifuged to remove the supernatant. Three times the volume of physiological saline was added to the concentrated red blood cells in the lower layer. The SOD activity of the red blood cells in each group was measured using a total superoxide dismutase (T-SOD) assay kit (results are shown in the figure). Figure 6 (As shown). Through Figure 6 It was found that there was no significant difference in SOD activity between the 10% v / v TreGly group (5.06 U / g Hb) and the 10% v / v ProGly group (5.02 U / g Hb) compared with the fresh group (5.67 U / g Hb).

[0126] 50,000 to 100,000 resuspended red blood cells were collected from each group, centrifuged at 1000g for 5 minutes, and the supernatant was discarded. The PS exposure rate of each group was detected using the Annexin V-FITC apoptosis detection kit (results are shown in the figure). Figure 7 (As shown). Through Figure 7 It was found that the PS exposure rates of erythrocytes in the fresh group, the 10% v / v TreGly group, and the 10% v / v ProGly group were 0.47%, 2.01%, and 11.04%, respectively. Among them, the PS exposure rate of the 10% v / v TreGly group was lower, showing a better membrane protection effect.

[0127] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A eutectic solvent system for red blood cell cryopreservation, characterized in that, This includes hydrogen bond donors, hydrogen bond acceptors, and buffer solutions; The hydrogen bond donor is glycerol.

2. The erythrocyte cryopreservation solution in a eutectic solvent system according to claim 1, characterized in that, The hydrogen bond acceptor is selected from one of glucose, trehalose, fructose, sucrose, alanine, glutamic acid, glycine, proline, betaine, or choline chloride. The buffer solution is selected from one of PBS buffer, DPBS buffer, HEPES buffer, HBSS buffer, or EBSS buffer.

3. A method for preparing a erythrocyte cryopreservation solution using a eutectic solvent system as described in any one of claims 1 to 2, characterized in that, Includes the following steps: The hydrogen bond acceptor and hydrogen bond donor are heated and mixed to obtain a eutectic solvent; The eutectic solvent and buffer solution were mixed to obtain the eutectic solvent system for red blood cell cryopreservation.

4. The method for preparing a erythrocyte cryopreservation solution in a eutectic solvent system according to claim 3, characterized in that, During the heating and mixing process, the temperature is 50-70℃, the time is 4-24h, and the rotation speed is 200-700rpm.

5. The application of a erythrocyte cryopreservation solution of any one of claims 1 to 2 in cryopreserved erythrocytes.

6. A method for cryopreserving red blood cells, characterized in that, Includes the following steps: The red blood cell suspension is mixed with the red blood cell cryopreservation solution of the eutectic solvent system according to any one of claims 1 to 2 to obtain a mixture; After post-processing the mixture, it was frozen in liquid nitrogen to complete the cryopreservation of red blood cells.

7. A method for cryopreserving red blood cells according to claim 6, characterized in that, In the mixture, the concentration of the erythrocyte cryopreservation solution in the eutectic solvent system is 5% v / v to 20% v / v; The red blood cell suspension is a mixture of red blood cells and PBS buffer solution; In the erythrocyte suspension, the hematocrit of erythrocytes is 20-60%.

8. A method for cryopreserving red blood cells according to claim 6, characterized in that, The post-processing involves either balancing the mixture or loading it. During the equilibration process, the temperature was room temperature and the time was 5–15 min. During the loading process, the temperature is 4–37℃ and the time is 10 min–3 h.

9. A CPA elution method for cryopreserved red blood cells, characterized in that, Includes the following steps: (S1) The frozen red blood cells obtained by the method of claim 6 are taken out, thawed and warmed, and then centrifuged to remove the supernatant; (S2) Mix the lower layer of concentrated red blood cells with the washing solution, centrifuge again and remove the supernatant to complete the CPA elution of frozen red blood cells.

10. The CPA elution method for cryopreserved red blood cells according to claim 9, characterized in that, In step (S1), the temperature during the thawing and rewarming process is 37–45°C; Step (S2) is repeated 2-3 times, with the washing buffer being the same volume as the thawed and rewarmed red blood cell suspension; The washing solution is a 0.9-3.5% w / v sodium chloride solution.

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