Red blood cell long-term preservation solution, and use thereof in preservation and transportation of red blood cell

Abstract

The present invention belongs to the technical field of cell preservation. Provided are a red blood cell long-term preservation solution, and the use thereof in preservation and transportation of red blood cells. The red blood cell long-term preservation solution in the present invention uses water as a solvent and comprises the following components in concentration: 10-100 mg / ml of dextran, 10-50 mg / ml of glucose, 1-10 mg / ml of polyvinylpyrrolidone and 0.1-0.5 mg / ml of adenosine triphosphate. The present invention lowers the freezing point of the preservation solution system to below -10℃ by means of the effective combination of anti-freezing components, thereby ensuring that the mixed red blood cell suspension still remains in a liquid state even under the condition of -6 to -10℃, avoiding damage to red blood cells caused by "ice crystal damage" from traditional preservation modes, increasing the yield of red blood cells, and maintaining the activity of red blood cells.

Description

A long-term red blood cell storage solution and its application in storage and transportation of red blood cells Technical Field

[0001] The present invention relates to the technical field of cell preservation, in particular to a long-term red blood cell preservation solution and its application in preserving and transporting red blood cells. Background Art

[0002] With the advent of cell culture technology in 1907, cell cryopreservation and thawing technologies emerged and gained increasing attention. Cryopreservation and refrigeration are among the most commonly used methods for cell preservation and are widely used in cell storage and transportation. Currently, the most common method for preserving red blood cells is to store them in a preservation solution at 4°C, with a shelf life of one month. Another method for preserving red blood cells is to cryopreserve them in liquid nitrogen at high concentrations of glycerol for long-term storage.

[0003] The storage of red blood cells at 4°C is mainly achieved by adding anticoagulants and energy metabolism substrates to maintain the basic life activities of red blood cells, while the deep-cryopreservation of red blood cells in liquid nitrogen uses a high-concentration permeable protective agent such as glycerol to protect the cells. Glycerol can penetrate the cell membrane and combine with the intracellular water of red blood cells, reducing the number of ice crystals generated during the freezing process, and at the same time reducing the formation of needle-shaped ice crystals, thereby reducing the probability of cell membranes and organelle membranes being punctured and damaged by ice crystals to a certain extent.

[0004] However, traditional red blood cell preservation methods have many problems. For example, the storage time limit at 4°C is only one month, which is too short and results in a huge waste of red blood cell resources. The frozen storage method inevitably causes hemolysis due to freezing damage. At the same time, due to the cytotoxicity, human toxicity and high osmotic pressure caused by high concentrations of glycerol, it cannot be directly re-infused and requires cumbersome elution operations. At the same time, the residual glycerol content needs to be controlled. These problems also greatly limit the application of frozen red blood cells.

[0005] Therefore, whether it is the traditional method of storage at 4°C or the frozen storage method, there are inevitable problems that limit the storage and use of red blood cells.

[0006] Summary of the Invention

[0007] The present invention aims to provide a long-term red blood cell (RBC) storage solution and its use in the storage and transportation of RBCs. Through an effective combination of antifreeze components, the present invention lowers the freezing point of the storage solution system to below -10°C, ensuring that the mixed RBC suspension remains liquid at temperatures between -6°C and -10°C. This avoids the "ice crystal damage" associated with traditional storage methods, which can lead to RBC breakage, improves RBC yield, and maintains RBC activity.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a long-term red blood cell preservation solution, which uses water as a solvent and comprises the following components in the following concentrations: 10-100 mg / mL dextran, 10-50 mg / mL glucose, 1-10 mg / mL polyvinylpyrrolidone, and 0.1-0.5 mg / mL adenosine triphosphate.

[0010] Preferably, water is used as the solvent, and the components are included in the following concentrations: 30-70 mg / mL dextran, 20-40 mg / mL glucose, 3-7 mg / mL polyvinylpyrrolidone, and 0.2-0.4 mg / mL adenosine triphosphate.

[0011] Preferably, water is used as the solvent, and the components are included in the following concentrations: 70 mg / mL dextran, 40 mg / mL glucose, 7 mg / mL polyvinylpyrrolidone, and 0.4 mg / mL adenosine triphosphate.

[0012] The present invention also provides an application of the above-mentioned long-term red blood cell preservation solution in low-temperature non-freezing storage and transportation of red blood cells.

[0013] Preferably, the method for using the long-term red blood cell preservation solution is to mix red blood cells with the long-term red blood cell preservation solution to obtain a cell suspension; and store the cell suspension at -6 to -10°C.

[0014] Preferably, the density of red blood cells in the cell suspension is (1-70)×10 8 cells / mL.

[0015] Preferably, when the red blood cells are used, the stored cell suspension is rewarmed at 18-26°C.

[0016] The present invention provides a long-term red blood cell preservation solution and its use in the preservation and transportation of red blood cells. The long-term red blood cell preservation solution of the present invention can achieve long-term non-frozen storage of red blood cells at low temperatures of -6 to -10°C. Through the effective combination of antifreeze components, the freezing point of the preservation system is reduced to below -10°C, ensuring that the suspension after mixing cells remains in a liquid state at -6 to -10°C. Ice crystals will not be generated due to freezing, avoiding the "ice crystal damage" caused by traditional preservation methods that causes damage to red blood cells and improving the red blood cell yield. At the same time, the storage temperature condition of -6 to -10°C effectively reduces the metabolic rate of red blood cells.

[0017] Dextran and polyvinylpyrrolidone, as non-permeable cell protectants, can effectively protect the stability of cell membranes. At the same time, as a blood volume expander, dextran and polyvinylpyrrolidone can effectively increase plasma colloidal osmotic pressure and maintain blood pressure after red blood cell transfusion, further promoting the effect of red blood cell transfusion therapy. Crucially, polyvinylpyrrolidone has the effect of lowering the freezing point of the preservation system, so that the preservation system remains liquid at -6 to -10°C without freezing to produce ice crystals. Glucose, as a nutrient source, is an important substance for cells to maintain life activities. At the same time, glucose in the present invention also plays a role in regulating the osmotic pressure of the preservation system. Adenosine triphosphate (ATP) plays an important role in cells. It is the main source of energy supply in cells and participates in cell biochemical reactions, signal transduction, ion balance and regulation of multiple cell activities. ATP is not only the basis of cell life activities, but also the guarantee for maintaining cell function.

[0018] Compared with the traditional 4°C storage method, the method of the present invention ensures that red blood cells remain in a liquid, non-frozen state under low temperature conditions of -6 to -10°C. At the same time, under relatively low temperature conditions, the metabolic rate of cells is further reduced, the consumption rate of energy substances such as adenosine triphosphate is reduced, and the life of red blood cells themselves is extended. Therefore, the storage period of red blood cells is extended from the current one month to more than four months, which greatly expands the storage time of red blood cells and breaks the problems of resource waste and application restrictions caused by time limits. At the same time, the appropriate component concentration combination in the present invention can effectively reduce the risk of red blood cell damage caused by fluid shear force during transportation compared to the 4°C storage method, avoid allergies and other problems caused by the release of red blood cell contents during clinical transfusion, and further improve the safety of clinical application.

[0019] Compared with the high-concentration glycerol cryopreservation method, the components of the cell preservation solution of the present invention are all of pharmaceutical grade, and the components dextran and polyvinylpyrrolidone themselves have the function of replacing plasma. Therefore, the cell preservation solution of the present invention can be directly re-infused as a whole for treatment after preserving red blood cells. The method is safer and more efficient, and does not require the addition of toxic high-concentration osmotic protective agents, nor does it require tedious elution operations during use. It avoids harsh storage and transportation conditions and reduces costs. At the same time, due to the design of lowering the freezing point of the preservation system, red blood cells will not be affected by ice crystal formation under low temperature conditions, avoiding ice crystals piercing the cell membrane and causing cell damage, effectively maintaining the activity of red blood cells and ensuring red blood cell function. DETAILED DESCRIPTION

[0020] The present invention provides a long-term red blood cell preservation solution, which uses water as a solvent and comprises the following components in the following concentrations: 10-100 mg / mL dextran, 10-50 mg / mL glucose, 1-10 mg / mL polyvinylpyrrolidone, and 0.1-0.5 mg / mL adenosine triphosphate.

[0021] In the present invention, the water is preferably water for injection.

[0022] In the present invention, the concentration of the dextran is preferably 10 to 100 mg / mL, more preferably 30 to 70 mg / mL, and even more preferably 70 mg / mL.

[0023] In the present invention, the concentration of glucose is preferably 10 to 50 mg / mL, more preferably 20 to 40 mg / mL, and even more preferably 40 mg / mL.

[0024] In the present invention, the concentration of polyvinyl pyrrolidone is preferably 1 to 10 mg / mL, more preferably 3 to 7 mg / mL, and even more preferably 7 mg / mL.

[0025] In the present invention, the concentration of adenosine triphosphate is preferably 0.1 to 0.5 mg / mL, more preferably 0.2 to 0.4 mg / mL, and even more preferably 0.4 mg / mL.

[0026] In the present invention, the pH of the long-term red blood cell storage solution is preferably 6.8 to 7.8, more preferably 7.2.

[0027] In the present invention, the long-term red blood cell storage solution is preferably sterilized by filtration using a 0.22 μm sterile filter.

[0028] The present invention also provides an application of the above-mentioned long-term red blood cell preservation solution in low-temperature non-freezing storage and transportation of red blood cells.

[0029] In the present invention, the method for using the long-term red blood cell preservation solution is preferably as follows: mixing red blood cells with the long-term red blood cell preservation solution to obtain a cell suspension; and storing the cell suspension at -6 to -10°C.

[0030] In the present invention, the density of red blood cells in the cell suspension is preferably (1-70)×10 8 cells / mL, more preferably (50-70)×10 8 cells / mL, and further preferably 59×10 8 cells / mL.

[0031] In the present invention, the cell suspension is preferably stored at -6 to -10°C, more preferably at -8 to -10°C, and even more preferably at -10°C.

[0032] In the present invention, when red blood cells are used, the stored cell suspension is preferably rewarmed at 18 to 26°C, more preferably at 25°C.

[0033] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] This embodiment provides a long-term red blood cell storage solution and a method for storing red blood cells, as follows:

[0036] Preparation of long-term red blood cell storage solution: Dissolve dextran, polyvinylpyrrolidone, glucose, and ATP in water for injection to a concentration of 10 mg / mL dextran, 1 mg / mL polyvinylpyrrolidone, 10 mg / mL glucose, and 0.1 mg / mL ATP. Sterilize the solution through a 0.22 μm pore size sterile filter before use.

[0037] Storage method: The red blood cells collected by centrifugation were mixed with the cell long-term storage solution to obtain a cell suspension, and the density of the red blood cells in the cell suspension was 56×10 8 cells / mL. After mixing thoroughly, transfer the cell suspension to -10°C and store for 4 months. After 4 months, remove the cell suspension and rewarm it at 25°C for testing.

[0038] Example 2

[0039] This embodiment provides a long-term red blood cell storage solution and a method for storing red blood cells, as follows:

[0040] Preparation of long-term red blood cell storage solution: Dissolve dextran, polyvinylpyrrolidone, glucose, and ATP in water for injection to a concentration of 30 mg / mL dextran, 3 mg / mL polyvinylpyrrolidone, 20 mg / mL glucose, and 0.2 mg / mL ATP. Sterilize the solution through a 0.22 μm pore size sterile filter before use.

[0041] Storage method: The red blood cells collected by centrifugation were mixed with the cell long-term storage solution to obtain a cell suspension, and the density of the red blood cells in the cell suspension was 53×10 8 cells / mL. After mixing thoroughly, transfer the cell suspension to -10°C and store for 4 months. After 4 months, remove the cell suspension and rewarm it at 25°C for testing.

[0042] Example 3

[0043] This embodiment provides a long-term red blood cell storage solution and a method for storing red blood cells, as follows:

[0044] Preparation of long-term red blood cell storage solution: Dissolve dextran, polyvinylpyrrolidone, glucose, and ATP in water for injection to a concentration of 50 mg / mL dextran, 5 mg / mL polyvinylpyrrolidone, 30 mg / mL glucose, and 0.3 mg / mL ATP. Sterilize the solution through a 0.22 μm pore size sterile filter before use.

[0045] Storage method: The red blood cells collected by centrifugation were mixed with the cell long-term storage solution to obtain a cell suspension, and the density of the red blood cells in the cell suspension was 61×10 8 cells / mL. After mixing thoroughly, transfer the cell suspension to -10°C and store for 4 months. After 4 months, remove the cell suspension and rewarm it at 25°C for testing.

[0046] Example 4

[0047] This embodiment provides a long-term red blood cell storage solution and a method for storing red blood cells, as follows:

[0048] Preparation of long-term red blood cell storage solution: Dissolve dextran, polyvinylpyrrolidone, glucose, and ATP in water for injection to a concentration of 70 mg / mL dextran, 7 mg / mL polyvinylpyrrolidone, 40 mg / mL glucose, and 0.4 mg / mL ATP. Sterilize the solution through a 0.22 μm pore size sterile filter before use.

[0049] Storage method: The red blood cells collected by centrifugation were mixed with the cell long-term storage solution to obtain a cell suspension, and the density of the red blood cells in the cell suspension was 59×10 8 cells / mL. After mixing thoroughly, transfer the cell suspension to -10°C and store for 4 months. After 4 months, remove the cell suspension and rewarm it at 25°C for testing.

[0050] Example 5

[0051] This embodiment provides a long-term red blood cell storage solution and a method for storing red blood cells, as follows:

[0052] Preparation of long-term red blood cell storage solution: Dissolve dextran, polyvinylpyrrolidone, glucose, and ATP in water for injection to a concentration of 100 mg / mL dextran, 10 mg / mL polyvinylpyrrolidone, 50 mg / mL glucose, and 0.5 mg / mL ATP. Sterilize the solution through a 0.22 μm pore size sterile filter before use.

[0053] Storage method: The red blood cells collected by centrifugation were mixed with the cell long-term storage solution to obtain a cell suspension, and the density of the red blood cells in the cell suspension was 57×10 8cells / mL. After mixing thoroughly, transfer the cell suspension to -10°C and store for 4 months. After 4 months, remove the cell suspension and rewarm it at 25°C for testing.

[0054] Comparative Example 1

[0055] The difference between this comparative example and Example 4 is that polyvinyl pyrrolidone is omitted. The details are as follows:

[0056] Preparation of long-term red blood cell storage solution: Dissolve dextran, glucose, and ATP in water for injection to a concentration of 70 mg / mL dextran, 40 mg / mL glucose, and 0.4 mg / mL ATP. Sterilize the solution by filtration through a 0.22 μm pore size sterile filter before use.

[0057] Storage method: The red blood cells collected by centrifugation were mixed with the cell long-term storage solution to obtain a cell suspension, and the density of the red blood cells in the cell suspension was 59×10 8 cells / mL. After mixing thoroughly, transfer the cell suspension to -10°C and store for 4 months. After 4 months, remove the cell suspension and rewarm it at 25°C for testing.

[0058] Comparative Example 2

[0059] In this comparative example, commercially available red blood cell preservation solution (Sichuan Nanger Biotechnology Co., Ltd.) was used to mix the red blood cell suspension with the red blood cell preservation solution in a ratio of 4:1 according to the instructions. The density of the red blood cells in the mixed solution was 47×10 8 cells / mL. Store red blood cells at 4°C ± 2°C for 4 months.

[0060] Comparative Example 3

[0061] In this comparative example, glycerol freezing method was used to preserve red blood cells. The final concentration of glycerol was 40% (v / v). The density of red blood cells in the preservation system was 51×10 8 cells / mL. The red blood cells were stored in liquid nitrogen (-196°C) for 4 months.

[0062] Test Example 1

[0063] This test was conducted on the red blood cells stored in Examples 1 to 5 and Comparative Examples 1 to 3 to test the red blood cell recovery rate, free hemoglobin content, red blood cell membrane tolerance, and red blood cell deformability to evaluate the effects of different preservation solutions and methods. The specific process is as follows:

[0064] 1. Red blood cell recovery rate

[0065] The red blood cells were counted before and after storage. The red blood cell recovery rate = the number of red blood cells before storage / the number of red blood cells after storage. The results are shown in Table 1.

[0066] Table 1 Red blood cell recovery results

[0067] 2. Free hemoglobin content

[0068] After rewarming, red blood cell suspension samples were collected from each group and centrifuged at 2000 rpm for 10 minutes. The supernatant was collected and the absorbance (OD) value was measured at a single wavelength of 405 nm using a hemoglobin detection kit (chromogenic method / Biyuntian) to calculate the free hemoglobin content. The results are shown in Table 2.

[0069] Table 2 Results of free hemoglobin content

[0070] 3. Red blood cell membrane tolerance

[0071] Tolerance of erythrocyte membranes to 0.45% (m / v) sodium chloride solution: Rewarmed cell suspension samples were taken from each group and added to 0.45% (m / v) sodium chloride solution, with a volume ratio of cell suspension to sodium chloride solution of 1:25. The samples were incubated at room temperature for 2 hours and centrifuged at 2000 rpm for 10 minutes. The supernatant was collected and the absorbance at a single wavelength of 405 nm was measured using a hemoglobin assay kit (chromogenic method, Biyuntian). The free hemoglobin content was calculated and used to evaluate erythrocyte membrane tolerance. The results are shown in Table 3.

[0072] Table 3 Free hemoglobin content results

[0073] 4. Red blood cell deformability

[0074] Deformability of erythrocytes through a 5μm pore: Erythrocyte suspension samples were collected from each group and filtered through a 5μm pore. The suspension was then centrifuged at 2000 rpm for 10 minutes. The supernatant was collected and the absorbance (OD) was measured at a single wavelength of 405 nm using a hemoglobin assay kit (chromogenic method, Biyuntian). The free hemoglobin content was calculated and used to evaluate the deformability of erythrocytes through a 5μm pore. The results are shown in Table 4.

[0075] Table 4 Free hemoglobin content results

[0076] It can be seen from Tables 1 to 4 that the long-term red blood cell preservation solution and preservation method of the present invention can effectively maintain the activity of red blood cells.

[0077] There are differences in the preservation effects of red blood cells due to the distribution ratios of different concentrations of the protection groups in each embodiment: compared with the various embodiments, Example 4 has the highest recovery rate of preserved red blood cells and the lowest amount of free hemoglobin, indicating that the integrity of the red blood cells in Example 4 is best maintained; in the test experiments of the red blood cells in each embodiment after preservation to withstand 0.45% sodium chloride solution and pass through a 5μm pore size, the free protein content released by the red blood cells in Example 4 is the lowest, indicating that the cell membrane of the red blood cells preserved in Example 4 has better toughness, can withstand low osmotic pressure and maintain the integrity of the cell membrane, and will not deform or rupture after passing through a 5μm filter membrane.

[0078] Compared with the examples, the red blood cell preservation effect of the comparative examples is significantly worse. Comparative Example 1 omitted polyvinylpyrrolidone, and the cell suspension was frozen at -10°C, unable to maintain a liquid state. The red blood cells were severely lysed and a large number of them were inactivated. Comparative Example 2 used a commercial red blood cell preservation solution to store red blood cells at 4°C ± 2°C. After 4 months of storage, the red blood cell recovery rate was much lower than that of the examples. Moreover, after 4 months of storage, the red blood cells could not maintain the integrity of the cell membrane, releasing a large amount of free hemoglobin. At the same time, it was also unable to maintain good cell membrane toughness and deformability, and could not support the long-term storage of red blood cells. Comparative Example 3 used a glycerol freezing method to store red blood cells. Although the long-term storage effect of 4 months was significantly improved compared to Comparative Example 2, the preservation effect was still inferior to most of the examples. What is more unavoidable is that the red blood cells stored by the glycerol freezing method in Comparative Example 3 need to be further eluted to remove the high concentration of glycerol components when used. This not only increases the cumbersome elution operation, but the elution process also further affects the performance of the red blood cells, limiting clinical application.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, several improvements and modifications can be made. For example, other protective agent combinations can be used to lower the freezing point to achieve the purpose of preserving different types of cells in a low-temperature non-frozen state. These improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A long-term preservation solution for red blood cells, characterized in that, Using water as a solvent, it comprises components with the following concentrations: 10 - 100 mg / mL of dextran, 10 - 50 mg / mL of glucose, 1 - 10 mg / mL of polyvinylpyrrolidone, and 0.1 - 0.5 mg / mL of adenosine triphosphate.

2. The long-term preservation solution for red blood cells according to claim 1, characterized in that, Using water as a solvent, it comprises components with the following concentrations: 30 - 70 mg / mL of dextran, 20 - 40 mg / mL of glucose, 3 - 7 mg / mL of polyvinylpyrrolidone, and 0.2 - 0.4 mg / mL of adenosine triphosphate.

3. The long-term preservation solution for red blood cells according to claim 2, characterized in that, Using water as a solvent, it comprises components with the following concentrations: 70 mg / mL of dextran, 40 mg / mL of glucose, 7 mg / mL of polyvinylpyrrolidone, and 0.4 mg / mL of adenosine triphosphate.

4. Application of the long-term preservation solution for red blood cells according to any one of claims 1 to 3 in cryogenic non-freezing preservation and transportation of red blood cells.

5. The application according to claim 4, characterized in that, The method of using the long-term erythrocyte preservation solution is to mix erythrocytes with the long-term erythrocyte preservation solution to obtain a cell suspension; and preserve the cell suspension at -6 to -10 °C.

6. The application according to claim 5, characterized in that, The density of red blood cells in the cell suspension is (1-70)×10 8 cells / mL.

7. The application according to claim 6, characterized in that, When using erythrocytes, warm up the preserved cell suspension at 18 - 26 °C.

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