A long-term red blood cell storage solution and its use in storing and transporting red blood cells

Abstract

The application provides a red blood cell long-term storage solution and application thereof in storage and transportation of red blood cells, and belongs to the technical field of cell storage. The red blood cell long-term storage solution provided by the application takes 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. The application lowers the freezing point of the storage solution system to below-10 DEG C through effective combination of antifreeze components, ensures that the mixed red blood cell suspension still maintains a liquid state under the condition of-6 DEG C to-10 DEG C, avoids the damage of red blood cells caused by "ice crystal damage" in the traditional storage mode, improves the red blood cell yield, and maintains the activity of red blood cells.

Description

TECHNICAL FIELD

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

[0002] With the advent of cell culture technology in 1907, the technology of cryopreservation and recovery of cells has also emerged and gained increasing attention. Cryopreservation and cold storage are one of the most commonly used methods for cell preservation, and have been widely used in cell storage and transportation. The current common method for red blood cell preservation is to use a preservation solution to store at 4℃, with a storage period of one month. Another way of red blood cell preservation is to use high-concentration glycerol for cryopreservation in liquid nitrogen to achieve long-term preservation of red blood cells.

[0003] Red blood cells are mainly preserved at 4℃ by adding anticoagulants and energy metabolism substrates to maintain the basic life activities of red blood cells, while red blood cells are cryopreserved in liquid nitrogen using glycerol as a high-concentration osmotic protective agent for cryopreservation protection. Glycerol can pass through the cell membrane and combine with intracellular water, reducing the number of ice crystals generated during freezing and reducing the generation of needle-shaped ice crystals, thereby reducing the probability of damage to the cell membrane and organelle membrane caused by ice crystals to a certain extent.

[0004] However, the traditional red blood cell preservation method has many problems, such as the 4℃ preservation method with a time limit of one month, which is relatively short and causes a huge waste of red blood cell resources. Cryopreservation method will inevitably cause hemolysis due to freezing damage, and because of the problems of cytotoxicity, human toxicity and high osmotic pressure caused by high-concentration glycerol, it cannot be directly used for reinfusion, and requires a cumbersome elution operation, and the content of residual glycerol needs to be controlled, which greatly limits the application of frozen red blood cells.

[0005] Therefore, both the traditional 4℃ preservation method and the cryopreservation method have unavoidable problems that limit the preservation and use of red blood cells. SUMMARY

[0006] The purpose of the present application is to provide a long-term preservation solution for red blood cells and its application in preserving and transporting red blood cells. The present application lowers the freezing point of the preservation solution system to below -10℃ by effective combination of antifreeze components, ensuring that the mixed red blood cell suspension remains in a liquid state at -6 to -10℃, avoiding the damage to red blood cells caused by "ice crystal damage" in traditional preservation methods, improving the yield of red blood cells, and maintaining the activity of red blood cells.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The application provides a long-term red blood cell storage solution, which is a water-based solution 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.

[0009] Preferably, the long-term red blood cell storage solution is a water-based solution and comprises the following components 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.

[0010] Preferably, the long-term red blood cell storage solution is a water-based solution and comprises the following components 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] The application also provides a use of the long-term red blood cell storage solution in low-temperature non-freezing storage and transportation of red blood cells.

[0012] Preferably, the use method of the long-term red blood cell storage solution comprises the following steps: mixing red blood cells with the long-term red blood cell storage solution to obtain a cell suspension; and storing the cell suspension at-6 to-10 ℃.

[0013] Preferably, the density of the red blood cells in the cell suspension is (1-70)×10 8 cells / mL.

[0014] Preferably, the red blood cells are rewarmed at 18-26 ℃.

[0015] The application provides a long-term red blood cell storage solution and its use in storage and transportation of red blood cells. The long-term red blood cell storage solution can realize long-term storage of red blood cells at-6 to-10 ℃ under low-temperature non-freezing conditions. Through effective combination of anti-freezing components, the freezing point of the storage system is reduced to below-10 ℃, so that the mixed cell suspension still maintains a liquid state at-6 to-10 ℃ and no ice crystals are generated due to freezing, thereby avoiding the damage of red blood cells caused by "ice crystal damage" in the traditional storage method and improving the yield of red blood cells. Meanwhile, the storage temperature condition of-6 to-10 ℃ effectively reduces the metabolic rate of red blood cells.

[0016] Dextran and polyvinylpyrrolidone as non-permeable cell protectants can effectively protect the stability of the cell membrane, at the same time, dextran and polyvinylpyrrolidone as a blood volume expander can effectively improve the plasma colloid osmotic pressure after reinfusion, maintain blood pressure, further promote the effect of red blood cell reinfusion therapy, and the key is that polyvinylpyrrolidone has the effect of reducing the freezing point of the preservation system, so that the preservation system still maintains a liquid state at-6~ -10℃ without freezing ice crystals. Glucose as a nutrient source is an important substance for cells to maintain life activities, and at the same time, glucose also plays a role in regulating the osmotic pressure of the preservation system. Adenine nucleotide triphosphate (ATP) plays an important role in cells, is the main energy source in cells, participates in biochemical reactions, signal transduction, ion balance and regulates and controls many cell activities, and ATP is not only the basis of cell life activities, but also the guarantee of cell function maintenance.

[0017] Compared with the traditional 4℃ preservation method, the method of the application ensures that the red blood cells are still in a liquid non-frozen state at a low temperature of-6~ -10℃, and at a relatively low temperature, the metabolic rate of the cells is further reduced, the consumption rate of energy substances such as adenosine triphosphate is reduced, and the life span of the red blood cells is prolonged, so that the preservation time of the red blood cells is prolonged from the existing one month to more than four months, which greatly expands the preservation time of the red blood cells and solves the problems of resource waste and application limitation caused by time limit; at the same time, compared with the 4℃ preservation method, the appropriate component concentration combination in the application can effectively reduce the risk of red blood cell damage caused by fluid shear force during transportation, avoid the problem of allergy caused by the release of red blood cell contents during clinical reinfusion, and further improve the safety of clinical application.

[0018] Compared with the high-concentration glycerol freezing preservation method, the components of the cell preservation solution of the application are all pharmaceutical grades, and the components dextran and polyvinylpyrrolidone also have the function of replacing plasma, so that the red blood cells preserved by the cell preservation solution of the application can be directly reinfused and treated as a whole, the method is safer and more efficient, no toxic high-concentration osmotic protectant needs to be added, and no complicated elution operation is needed during use, the strict storage and transportation conditions are avoided and the cost is reduced, at the same time, because of the design of reducing the freezing point of the preservation system, the red blood cells will not be affected by the generation of ice crystals under low temperature conditions, the cell membrane is avoided to be damaged by ice crystals, the activity of the red blood cells is effectively maintained, and the function of the red blood cells is guaranteed. DETAILED DESCRIPTION

[0019] The application provides a red blood cell long-term preservation solution, which takes water as a solvent and comprises the following components at 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 adenine nucleotide triphosphate.

[0020] In the present application, the water is preferably water for injection.

[0021] In the present application, the concentration of the dextran is preferably 10-100 mg / mL, further preferably 30-70 mg / mL, and still further preferably 70 mg / mL.

[0022] In the present application, the concentration of the glucose is preferably 10-50 mg / mL, further preferably 20-40 mg / mL, and still further preferably 40 mg / mL.

[0023] In the present application, the concentration of the polyvinylpyrrolidone is preferably 1-10 mg / mL, further preferably 3-7 mg / mL, and still further preferably 7 mg / mL.

[0024] In the present application, the concentration of the adenosine triphosphate is preferably 0.1-0.5 mg / mL, further preferably 0.2-0.4 mg / mL, and still further preferably 0.4 mg / mL.

[0025] In the present application, the pH of the long-term erythrocyte storage solution is preferably 6.8-7.8, and further preferably 7.2.

[0026] In the present application, the long-term erythrocyte storage solution is preferably sterilized by filtration using a 0.22 μm sterile filter.

[0027] The present application also provides a use of the above long-term erythrocyte storage solution in low-temperature non-frozen preservation and transportation of erythrocytes.

[0028] In the present application, the method for using the long-term erythrocyte storage solution is preferably mixing erythrocytes with the long-term erythrocyte storage solution to obtain a cell suspension, and storing the cell suspension at -6 to -10°C.

[0029] In the present application, the density of erythrocytes in the cell suspension is preferably (1-70) x 10 8 cells / mL, further preferably (50-70) x 10 8 cells / mL, and still further preferably 59 x 10 8 cells / mL.

[0030] In the present application, the cell suspension is preferably stored at -6 to -10°C, further preferably at -8 to -10°C, and still further preferably at -10°C.

[0031] In the present application, when the erythrocytes are used, the stored cell suspension is preferably warmed at 18 to 26°C, and further preferably at 25°C.

[0032] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0033] Example 1

[0034] The present example provides a red blood cell long-term preservation solution and a red blood cell preservation method, which are as follows:

[0035] Preparation of the red blood cell long-term preservation solution: dissolve dextran, polyvinylpyrrolidone, glucose, and ATP in water for injection to make the concentration of dextran 10 mg / mL, the concentration of polyvinylpyrrolidone 1 mg / mL, the concentration of glucose 10 mg / mL, and the concentration of ATP 0.1 mg / mL. After sterilization by filtering with a sterile filter with a pore size of 0.22 μm, the solution is ready for use.

[0036] Preservation method: mix the centrifugally collected red blood cells with the cell long-term preservation solution to obtain a cell suspension, and make the density of red blood cells in the cell suspension 56 x 10 8 cells / mL. After mixing evenly, the cell suspension is transferred to -10℃ for preservation for 4 months. After 4 months, it is taken out and placed at 25℃ for rewarming detection.

[0037] Example 2

[0038] The present example provides a red blood cell long-term preservation solution and a red blood cell preservation method, which are as follows:

[0039] Preparation of the red blood cell long-term preservation solution: dissolve dextran, polyvinylpyrrolidone, glucose, and ATP in water for injection to make the concentration of dextran 30 mg / mL, the concentration of polyvinylpyrrolidone 3 mg / mL, the concentration of glucose 20 mg / mL, and the concentration of ATP 0.2 mg / mL. After sterilization by filtering with a sterile filter with a pore size of 0.22 μm, the solution is ready for use.

[0040] Preservation method: mix the centrifugally collected red blood cells with the cell long-term preservation solution to obtain a cell suspension, and make the density of red blood cells in the cell suspension 53 x 10 8 cells / mL. After mixing evenly, the cell suspension is transferred to -10℃ for preservation for 4 months. After 4 months, it is taken out and placed at 25℃ for rewarming detection.

[0041] Example 3

[0042] The present example provides a red blood cell long-term preservation solution and a red blood cell preservation method, which are as follows:

[0043] Preparation of the long-term storage solution for red blood cells: dissolve dextran, polyvinylpyrrolidone, glucose and ATP in water for injection to obtain a solution with a dextran concentration of 50 mg / mL, a polyvinylpyrrolidone concentration of 5 mg / mL, a glucose concentration of 30 mg / mL and an ATP concentration of 0.3 mg / mL. The solution is sterilized by filtration through a sterile filter with a pore size of 0.22 μm and stored for later use.

[0044] Storage method: the centrifugally collected red blood cells are mixed with the long-term storage solution for cells to obtain a cell suspension with a red blood cell density of 61 x 10 8 cells / mL. After mixing, the cell suspension is transferred to -10°C for storage for 4 months. After 4 months, the cell suspension is taken out and warmed at 25°C for testing.

[0045] Example 4

[0046] This example provides a long-term storage solution for red blood cells and a storage method for red blood cells, which are as follows:

[0047] Preparation of the long-term storage solution for red blood cells: dissolve dextran, polyvinylpyrrolidone, glucose and ATP in water for injection to obtain a solution with a dextran concentration of 70 mg / mL, a polyvinylpyrrolidone concentration of 7 mg / mL, a glucose concentration of 40 mg / mL and an ATP concentration of 0.4 mg / mL. The solution is sterilized by filtration through a sterile filter with a pore size of 0.22 μm and stored for later use.

[0048] Storage method: the centrifugally collected red blood cells are mixed with the long-term storage solution for cells to obtain a cell suspension with a red blood cell density of 59 x 10 8 cells / mL. After mixing, the cell suspension is transferred to -10°C for storage for 4 months. After 4 months, the cell suspension is taken out and warmed at 25°C for testing.

[0049] Example 5

[0050] This example provides a long-term storage solution for red blood cells and a storage method for red blood cells, which are as follows:

[0051] Preparation of the long-term storage solution for red blood cells: dissolve dextran, polyvinylpyrrolidone, glucose and ATP in water for injection to obtain a solution with a dextran concentration of 100 mg / mL, a polyvinylpyrrolidone concentration of 10 mg / mL, a glucose concentration of 50 mg / mL and an ATP concentration of 0.5 mg / mL. The solution is sterilized by filtration through a sterile filter with a pore size of 0.22 μm and stored for later use.

[0052] Storage method: the centrifugally collected red blood cells are mixed with the long-term storage solution for cells to obtain a cell suspension with a red blood cell density of 57 x 10 8cells / mL. After mixing well, the cell suspension was transferred to -10°C for 4 months. After 4 months, it was taken out and tested at 25°C.

[0053] Comparative Example 1

[0054] The difference between this comparative example and Example 4 is that polyvinylpyrrolidone is omitted. The details are as follows:

[0055] Preparation of the red blood cell long-term storage solution: dissolve dextran, glucose and ATP in water for injection to make the concentration of dextran 70 mg / mL, the concentration of glucose 40 mg / mL and the concentration of ATP 0.4 mg / mL. After sterilization by filtering through a sterile filter with a pore size of 0.22 μm, it is ready for use.

[0056] Storage method: mix the centrifugally collected red blood cells with the cell long-term storage solution to obtain a cell suspension, and make the density of red blood cells in the cell suspension 59 x 10 8 cells / mL. After mixing well, the cell suspension was transferred to -10°C for 4 months. After 4 months, it was taken out and tested at 25°C.

[0057] Comparative Example 2

[0058] In this comparative example, commercially available red blood cell storage solution (Sichuan Nager Biotechnology Co., Ltd.) was used to mix the red blood cell suspension with the red blood cell storage solution at a ratio of 4:1 according to the instructions, and the density of red blood cells in the mixed solution was 47 x 10 8 cells / mL. The red blood cells were stored at 4°C ± 2°C for 4 months.

[0059] Comparative Example 3

[0060] In this comparative example, the red blood cells were stored by glycerol freezing method, the final concentration of glycerol was 40% (v / v), and the density of red blood cells in the storage system was 51 x 10 8 cells / mL. The red blood cells were stored in liquid nitrogen (-196°C) for 4 months.

[0061] Test Example 1

[0062] In this test example, the red blood cells stored in Examples 1-5 and Comparative Examples 1-3 were tested for red blood cell recovery rate, free hemoglobin content, red blood cell membrane tolerance and red blood cell deformability to evaluate the effects of different storage solutions and storage methods. The specific process is as follows:

[0063] 1. Red blood cell recovery rate

[0064] The red blood cells before and after storage were counted respectively, and 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.

[0065] Table 1 Red blood cell recovery rate results

[0066]

[0067] 2. Free hemoglobin content

[0068] Each group took the rewarmed red blood cell suspension sample, centrifuged at 2000 r / min for 10 minutes, took the supernatant, used the hemoglobin detection kit (chromogenic method / Biyuntian) to detect the absorbance OD value at 405 nm single wavelength, calculated the free hemoglobin content. The results are shown in Table 2.

[0069] Table 2 Free hemoglobin content results

[0070]

[0071]

[0072] 3. Red blood cell membrane tolerance

[0073] Red blood cell membrane tolerance to 0.45% (m / v) sodium chloride solution: each group took the rewarmed cell suspension sample and added it to 0.45% (m / v) sodium chloride solution, the volume ratio of cell suspension to sodium chloride solution was 1:25. Place at room temperature for 2h, centrifuge at 2000 r / min for 10 minutes, take the supernatant, use the hemoglobin detection kit (chromogenic method / Biyuntian) to detect the absorbance OD value at 405 nm single wavelength, calculate the free hemoglobin content, and evaluate the red blood cell membrane tolerance according to the free hemoglobin content. The results are shown in Table 3.

[0074] Table 3 Free hemoglobin content results

[0075]

[0076] 4. Red blood cell deformability

[0077] Red blood cell deformability through 5μm pore size: each group took the red blood cell suspension sample through the way of suction filtration to make the red blood cells pass through 5μm pore size and collect the suspension, centrifuged at 2000 r / min for 10 minutes, took the supernatant, used the hemoglobin detection kit (chromogenic method / Biyuntian) to detect the absorbance OD value at 405 nm single wavelength, calculated the free hemoglobin content, and evaluated the red blood cell deformability when passing through 5μm pore size according to the free hemoglobin content. The results are shown in Table 4.

[0078] Table 4 Free hemoglobin content results

[0079]

[0080] As can be seen from Tables 1 to 4, the red blood cell long-term preservation liquid and the preservation method of the present application can effectively maintain the activity of red blood cells.

[0081] The different concentrations of the protective component in each embodiment have different effects on the preservation of red blood cells: compared with each embodiment, the red blood cells preserved in Example 4 have the highest recovery rate and the lowest amount of free hemoglobin, indicating that the integrity of the red blood cells in Example 4 is best maintained; in the detection experiments of the tolerance of red blood cells to 0.45% sodium chloride solution and the deformability of red blood cells passing through a 5 μm pore size filter after preservation, the red blood cells in Example 4 release the lowest amount of free protein, indicating that the cell membranes of the red blood cells preserved in Example 4 have better toughness, can tolerate low osmotic pressure and maintain the integrity of the cell membranes, and will not deform and rupture after passing through a 5 μm filter.

[0082] Compared with each embodiment, the red blood cells in the comparative examples have significantly poorer preservation effects. Comparative Example 1 omits polyvinylpyrrolidone, and the cell suspension is frozen at -10°C, which cannot maintain a liquid state, and the red blood cells are severely dissolved and inactivated. The commercial red blood cell preservation liquid used in Comparative Example 2 preserves red blood cells at 4°C±2°C, and after 4 months of preservation of red blood cells, the recovery rate of red blood cells is much lower than that of the embodiments, and after 4 months of preservation of red blood cells, the red blood cells cannot maintain the integrity of the cell membranes, release a large amount of free hemoglobin, and also cannot maintain good cell membrane toughness and deformability, and cannot support long-term preservation of red blood cells. The glycerol freezing method of Comparative Example 3 preserves red blood cells, and although the long-term preservation effect is significantly improved compared with Comparative Example 2, the preservation effect is still not as good as most of the embodiments, and more importantly, the red blood cells preserved by the glycerol freezing method of Comparative Example 3 need to be further washed to remove high-concentration glycerol components when used, which not only increases the cumbersome washing operation, but also further affects the performance of the red blood cells during the washing process, limiting the clinical application.

[0083] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, such as using other protective agent combinations to lower the freezing point to achieve the purpose of preserving different types of cells in a low-temperature non-frozen state, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A long-term preservation solution for red blood cells, characterized in that, The water solvent is composed of the following components with 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; The red blood cell long-term storage solution can store red blood cells at-6 to-10℃.

2. The long-term preservation solution for red blood cells according to claim 1, characterized in that, The water solvent is composed of the following components with 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.

3. The long-term preservation solution for red blood cells according to claim 2, characterized in that, The water solvent is composed of the following components with 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.

4. The use of the red blood cell long-term storage solution according to any one of claims 1 to 3 for the low-temperature non-frozen storage and transport of red blood cells, characterized in that, The method for using the red blood cell long-term storage solution comprises the following steps: mixing red blood cells with the red blood cell long-term storage solution to obtain a cell suspension; and storing the cell suspension at 10°C. 6~ 10°C. The density of red blood cells in the cell suspension is (1-70) x 10 8 cells / mL; The red blood cell long-term storage solution can store red blood cells at-6 to-10℃. The red blood cell long-term storage solution can store red blood cells at-6 to-10℃.

Images