Preparation method suitable for cord blood mononuclear cell cryopreservation liquid

Abstract

The invention discloses a preparation method suitable for a cord blood mononuclear cell cryopreservation solution, and relates to the technical field of cord blood mononuclear cell cryopreservation solution preparation.The preparation method suitable for the cord blood mononuclear cell cryopreservation solution comprises the following steps that cord blood plasma is prepared, DMSO is added into the plasma which is detected to be qualified according to the proportion, and the cord blood plasma is obtained; uniformly mixing to obtain a cord blood mononuclear cell cryopreservation solution, wherein the cell cryopreservation solution contains a human mesenchymal stem cell serum-free culture medium, DMSO and human serum albumin; the cryopreservation liquid can improve the motility rate of cells and increase the recovery yield after the cells are unfrozen, and can effectively protect the cells and reduce the cell loss when the mononuclear cells of umbilical cord blood are recovered at low temperature by adding corresponding additives; according to the umbilical cord blood mononuclear cell cryopreservation method, under the condition that the prepared cryopreservation liquid is used, the survival rate of resuscitated cells reaches 90% or above, the biological characteristics of the cells are not changed, and the biological activity of the umbilical cord blood mononuclear cells is guaranteed.

Description

Technical Field

[0001] The invention relates to the field of preparation technology of cord blood mononuclear cell cryopreservation solution, in particular to a preparation method suitable for cord blood mononuclear cell cryopreservation solution. Background Art

[0002] Umbilical cord blood refers to the blood remaining in the placenta and umbilical cord after the fetus is delivered, the umbilical cord is tied and severed. Umbilical cord blood is rich in sources and easy to collect. It has the advantages of low matching requirements, low incidence of immune rejection, low transplantation costs and high transplantation success rate. It has been widely used in the treatment of leukemia and non-malignant blood diseases, congenital immunodeficiency and other diseases. Therefore, umbilical cord blood has become the third largest source of hematopoietic stem cells after bone marrow and peripheral blood. With the application of hematopoietic stem cells derived from umbilical cord blood, the storage problem has become a hot topic. Therefore, it is crucial to prepare different cryopreservation solutions to cryopreserve mononuclear cells derived from umbilical cord blood to establish a safe and stable umbilical cord blood bank.

[0003] Although there are finished cord blood cryopreservation solutions on the market, their DMSO concentration is relatively high and they contain xenobiotic ingredients, which are harmful to cells and the human body, greatly limiting their application in laboratories and clinics:

[0004] ① Different finished cord blood cryopreservation solutions have various ingredients, but lack a cryopreservation solution with added additives to protect the revived cells to ensure the effectiveness and stability of the viability after each cryopreserved cell resuscitation; ② The viability of cells after thawing is relatively low, and cell activity cannot be guaranteed; ③ Adverse reactions caused by heterologous animal components cannot be avoided. Therefore, the existing preparation methods of cord blood cryopreservation solutions still need to be improved. Summary of the invention

[0005] In view of this, the present invention aims at the deficiencies in the prior art, and its main purpose is to provide a method for preparing a cryopreservation solution for cord blood mononuclear cells, which can improve the cell viability and increase the recovery rate after the cells are thawed, and by adding corresponding additives, can effectively protect the cells, reduce cell loss, and maintain cell activity during low-temperature recovery of cord blood mononuclear cells; the cord blood mononuclear cell cryopreservation method of the present invention, when using this prepared cryopreservation solution, makes the survival rate of the recovered cells reach more than 90%, the biological characteristics of the cells do not change, and the biological activity of the cord blood mononuclear cells is guaranteed.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A method for preparing a cord blood mononuclear cell cryopreservation solution comprises the following steps: preparing cord blood plasma, adding DMSO to the plasma that has passed the test according to a proportion, and mixing to obtain the cord blood mononuclear cell cryopreservation solution, wherein the cell cryopreservation solution is a cell cryopreservation solution that does not contain a heterologous animal and other multiple components, and the cell cryopreservation solution contains a human mesenchymal stem cell serum-free culture medium, DMSO and human albumin; a method for freezing cells using the cord blood mononuclear cell cryopreservation solution is as follows: mixing the cord blood mononuclear cell suspension with the cell cryopreservation solution, wherein the volume of the cryopreserved cell suspension is equal to the total number of WBCs / cell cryopreservation density; performing a programmed cooling process of 70 to 90°C, and then transferring to liquid nitrogen at -200°C to -100°C for cryopreservation; and resuscitating the frozen cells.

[0008] As a preferred embodiment: the DMSO is dimethyl sulfoxide, a GMP grade solvent.

[0009] As a preferred embodiment, the cryopreservation density of the umbilical cord blood mononuclear cells used for programmed cooling is 3.6-4.4×10 7 Pieces / mL.

[0010] As a preferred embodiment: the total number of WBCs is the total number of mononuclear leukocytes in umbilical cord blood.

[0011] As a preferred embodiment: the human mesenchymal stem cell serum-free culture medium is a serum-free basic culture medium without animal-derived components and is a commercial product.

[0012] As a preferred embodiment: the cell freezing solution contains 5%-10% human serum albumin, 5%-10% DMSO and 80%-90% human mesenchymal stem cell serum-free culture medium.

[0013] As a preferred embodiment, the plasma accounts for 90-95% by volume, and the DMSO accounts for 5-10% by volume.

[0014] As a preferred embodiment, the cord blood mononuclear cell suspension is mixed with the cell freezing solution and transferred to liquid nitrogen for cryopreservation at a temperature of -196°C.

[0015] As a preferred solution, the umbilical cord blood mononuclear cells frozen for 15 days are revived in a water bath at 37° C., and the survival rate of the revived cells reaches more than 90%.

[0016] As a preferred embodiment: the human serum albumin is albumin.

[0017] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical scheme that the preparation method of the cord blood mononuclear cell cryopreservation solution is suitable for improving the cell viability and the recovery rate after the cells are thawed. By adding corresponding additives, the cells can be effectively protected, the cell loss can be reduced, and the cell activity can be maintained when the cord blood mononuclear cells are cryopreserved at low temperature; the cord blood mononuclear cell cryopreservation method of the present invention, when using this prepared cryopreservation solution, makes the survival rate of the recovered cells reach more than 90%, the cell biological characteristics do not change, and the biological activity of the cord blood mononuclear cells is guaranteed; the cord blood mononuclear cell cryopreservation solution of the present invention does not contain animal serum, etc., so no exogenous protein will be introduced, reducing the possibility of animal pathogen contamination; by adding corresponding additives, the cells can be effectively protected, the cell loss can be reduced, and the cell activity can be maintained when the cord blood mononuclear cells are cryopreserved at low temperature. Albumin, as a stabilizer, can effectively protect the cell phenotype and significantly improve the activity of mononuclear cells under cryopreservation conditions, thereby maintaining the physiological function and biological function of the cells after recovery.

[0018] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with specific embodiments. DETAILED DESCRIPTION

[0019] A method for preparing a cord blood mononuclear cell cryopreservation solution comprises the following steps: preparing cord blood plasma, adding DMSO to the plasma that has passed the test according to a proportion, and mixing to obtain the cord blood mononuclear cell cryopreservation solution, wherein the cell cryopreservation solution is a cell cryopreservation solution that does not contain a heterologous animal and other multiple components, and the cell cryopreservation solution contains a human mesenchymal stem cell serum-free culture medium, DMSO and human albumin; a method for freezing cells using the cord blood mononuclear cell cryopreservation solution is as follows: mixing the cord blood mononuclear cell suspension with the cell cryopreservation solution, wherein the volume of the cryopreserved cell suspension is equal to the total number of WBCs / cell cryopreservation density; performing a programmed cooling process of 70 to 90°C, and then transferring to liquid nitrogen at -200°C to -100°C for cryopreservation; and resuscitating the frozen cells.

[0020] The DMSO is dimethyl sulfoxide, a GMP grade solvent.

[0021] The cryopreservation density of the umbilical cord blood mononuclear cells used for programmed cooling is 3.6-4.4×10 7 Pieces / mL.

[0022] The total WBC count is the total number of mononuclear leukocytes in cord blood.

[0023] The human mesenchymal stem cell serum-free culture medium is a serum-free basic culture medium free of animal-derived components and is a commercial product.

[0024] The cell freezing solution contains 5%-10% human serum albumin, 5%-10% DMSO and 80%-90% human mesenchymal stem cell serum-free culture medium.

[0025] The plasma accounts for 90-95% by volume, and the DMSO accounts for 5-10% by volume.

[0026] The cord blood mononuclear cell suspension is mixed with the cell freezing solution and transferred to liquid nitrogen for freezing and preservation at a temperature of -196°C.

[0027] The umbilical cord blood mononuclear cells that were frozen for 15 days were revived in a water bath at 37°C, and the survival rate of the revived cells reached more than 90%.

[0028] The human serum albumin is albumin.

[0029] Example 1: Preparation method for cryopreservation solution of cord blood mononuclear cells

[0030] Use a 5mL pipette to add 0.5mL human albumin and 9mL human mesenchymal stem cell serum-free medium to a 50mL centrifuge tube, then use a pipette to mix, then slowly add 0.5mL DMSO to the 50mL centrifuge tube, and after mixing, you can get cord blood mononuclear cell cryopreservation solution, which has 5% by volume of DMSO, 5% by volume of human albumin, and 90% by volume of human mesenchymal stem cell serum-free medium. Store at 4°C, and then use the cord blood mononuclear cell cryopreservation solution to cryopreserve cells.

[0031] Example 2: Preparation method for cryopreservation solution of cord blood mononuclear cells

[0032] Use a 5mL pipette to add 1mL human albumin and 8.5mL human mesenchymal stem cell serum-free medium to a 50mL centrifuge tube, then use a pipette to mix, then slowly add 0.5mL DMSO to the 50mL centrifuge tube, and after mixing, you will get the cord blood mononuclear cell cryopreservation solution, which has a volume percentage of 5% DMSO, a volume percentage of 10% human albumin, and a volume percentage of 85% human mesenchymal stem cell serum-free medium. Store at 4°C, and then use the cord blood mononuclear cell cryopreservation solution to cryopreserve the cells.

[0033] Example 3: Preparation method for cryopreservation solution of cord blood mononuclear cells

[0034] Use a 5mL pipette to add 0.5mL human albumin and 8.5mL human mesenchymal stem cell serum-free medium to a 50mL centrifuge tube, then use a pipette to mix, then slowly add 1mL DMSO to the 50mL centrifuge tube, and mix to obtain cord blood mononuclear cell cryopreservation solution, which contains 10% DMSO by volume, 5% human albumin by volume, and 85% human mesenchymal stem cell serum-free medium by volume. Store at 4°C, and then use the cord blood mononuclear cell cryopreservation solution to cryopreserve cells.

[0035] Example 4: Preparation method for cryopreservation solution of cord blood mononuclear cells

[0036] Use a 5mL pipette to add 1mL human albumin and 8mL human mesenchymal stem cell serum-free medium to a 50mL centrifuge tube, then use a pipette to mix, then slowly add 1mL DMSO to the 50mL centrifuge tube, and mix to obtain cord blood mononuclear cell cryopreservation solution, which contains 10% DMSO, 10% human albumin, and 80% human mesenchymal stem cell serum-free medium by volume. Store at 4°C, and then use the cord blood mononuclear cell cryopreservation solution to cryopreserve cells.

[0037] Example 5: Cell cryopreservation

[0038] Cryopreservation steps: Umbilical cord blood is centrifuged at 2000g for 10 minutes. After centrifugation, the upper plasma is removed. The lower whole blood is diluted 1-2 times with saline and then coated with lymphocyte separation solution at a 1:1 ratio, so that the whole blood floats on the upper layer of the separation solution. Then, it is centrifuged at 600g for 20 minutes using a Ficoll density gradient. The upper waste liquid is removed and mononuclear cells are aspirated from the lymphocyte layer. Subsequently, the cells are centrifuged three times at 300g for 10 minutes, and then the cells are seeded in bottles. After culture, umbilical cord blood mononuclear cells are obtained. Mix the umbilical cord blood mononuclear cells with the freezing solution and put them into a 4.5mL cryopreservation tube. Put the cryopreservation tube into a program cooling box, then put it into an 80°C deep low temperature refrigerator for 24 hours, and then put it into a liquid nitrogen tank (196°C) for storage.

[0039] Example 6: Determination of resuscitation rate and quantity

[0040] The cells frozen in the freezing solution of Example 1 were taken out and quickly placed in a 37°C water bath for 12 minutes, shaken until the cell suspension was completely thawed, centrifuged at 300g for 8 minutes, washed with pre-cooled saline, and the recovery activity and number were measured.

[0041] Example 7: Determination of resuscitation rate and quantity

[0042] The cells frozen with the freezing solution of Example 2 were taken out and quickly placed in a 37°C water bath for 12 minutes, shaken until the cell suspension was completely thawed, centrifuged at 300g for 8 minutes, washed with pre-cooled saline, and the recovery activity and number were measured.

[0043] Example 8: Determination of resuscitation rate and quantity

[0044] The cells frozen with the freezing solution of Example 3 were taken out and quickly placed in a 37°C water bath for 12 minutes, shaken until the cell suspension was completely thawed, centrifuged at 300g for 8 minutes, washed with pre-cooled saline, and the recovery activity and number were measured.

[0045] Example 9: Determination of resuscitation rate and quantity

[0046] The cells frozen with the freezing solution of Example 4 were taken out and quickly placed in a 37°C water bath for 12 minutes, shaken until the cell suspension was completely thawed, centrifuged at 300g for 8 minutes, washed with pre-cooled saline, and the recovery activity and number were measured.

[0047] Comparative Example 1: A commercial cord blood mononuclear finished cell cryopreservation solution, with a concentration of 10% DMSO by volume, is used for cell cryopreservation.

[0048] Comparative Example 2: A commercial cord blood mononuclear finished cell cryopreservation solution, with a concentration of 10% DMSO by volume, is used for cell cryopreservation.

[0049] Comparative Example 3: A commercial cord blood mononuclear finished cell freezing solution, with a concentration of 10% DMSO by volume, is used for cell freezing.

[0050] Comparative Example 4: The cells frozen with the freezing solution of Comparative Example 1 were taken out and quickly placed in a 37°C water bath for 12 minutes for cell recovery. The cells were shaken until the cell suspension was completely melted, centrifuged at 300g for 8 minutes, washed with pre-cooled saline, and the recovery activity was measured.

[0051] Comparative Example 5: Take out the cells frozen with the freezing solution of Comparative Example 2 and quickly place them in a 37°C water bath for 12 minutes for cell recovery. Shake until the cell suspension is completely melted, centrifuge at 300g for 8min, wash with pre-cooled saline, and measure the recovery activity and quantity.

[0052] Comparative Example 6: Take out the cells frozen with the freezing solution of Comparative Example 3 and quickly place them in a 37°C water bath for 12 minutes for cell recovery. Shake until the cell suspension is completely melted, centrifuge at 300g for 8min, wash with pre-cooled saline, and measure the recovery activity and quantity.

[0053] Total number of WBC recovered after resuscitation = volume × WBC concentration; WBC recovery rate = total number of WBC recovered after resuscitation / total number of frozen storage.

[0054] Table 1: Umbilical cord blood mononuclear cells after thawing at 37°C

[0055]

[0056] The results show that:

[0057] 1. The data in Table 1 show that in the self-prepared cryopreservation solution used in Examples 6-9, the cell viability can reach 92% or more under 37°C water bath conditions, while the cell viability revived by the finished cryopreservation solution used in Comparative Examples 4-6 is significantly lower than that in the examples, and its resuscitation viability under 37°C water bath conditions is only 90%.

[0058] 2. The highest viability in Example 7 is 95.6%, and the cryopreservation solution used is prepared with 5% by volume of DMSO, 10% by volume of human albumin, and 85% by volume of human mesenchymal stem cell serum-free medium. This shows that when the DMSO concentration is 5% by volume, the effect on cell viability is the lowest, and human albumin can effectively protect cells and has a certain effect on the activity after recovery.

[0059] From the experimental results, the cryopreservation solution containing human albumin, human mesenchymal stem cell serum-free medium and DMSO significantly improves the viability and recovery rate of stem cells after resuscitation. Moreover, under the condition of 37°C water bath, the cell viability and recovery rate of the cryopreservation solution with a volume ratio of 5% DMSO and a volume percentage of 10% human albumin reached 95.6% and 72.47%, indicating that the cryopreservation solution prepared in Example 2 is more preferred for resuscitation under the condition of 37°C water bath. The cryopreservation solution can improve the cell viability and recovery rate after the cells are thawed. By adding corresponding additives, it can effectively protect the cells, reduce cell loss and maintain cell activity during the low-temperature resuscitation of umbilical cord blood mononuclear cells.

[0060] The design focus of the present invention is a preparation method for a cryopreservation solution for cord blood mononuclear cells, which can improve the cell viability and recovery rate after the cells are thawed. By adding corresponding additives, the cells can be effectively protected, cell loss can be reduced, and cell activity can be maintained when the cord blood mononuclear cells are cryopreserved at low temperature; the cryopreservation method of the present invention, when using this prepared cryopreservation solution, makes the survival rate of the recovered cells reach more than 90%, the biological characteristics of the cells do not change, and the biological activity of the cord blood mononuclear cells is guaranteed; the cryopreservation solution of the present invention does not contain animal serum, etc., so no exogenous protein will be introduced, reducing the possibility of animal pathogen contamination; by adding corresponding additives, the cells can be effectively protected, cell loss can be reduced, and cell activity can be maintained when the cord blood mononuclear cells are cryopreserved at low temperature. Albumin, as a stabilizer, can effectively protect the cell phenotype and significantly improve the activity of mononuclear cells under cryopreservation conditions, thereby maintaining the physiological and biological functions of the cells after recovery.

[0061] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a cryopreservation solution of cord blood mononuclear cells, characterized in that: The method comprises the following steps: preparing umbilical cord blood plasma, adding DMSO to the plasma that has passed the test according to a proportion, and mixing to obtain umbilical cord blood mononuclear cell freezing solution, wherein the cell freezing solution is a cell freezing solution that does not contain heterologous animals and other multiple components, and the cell freezing solution contains human mesenchymal stem cell serum-free culture medium, DMSO and human albumin; a method for freezing cells using the umbilical cord blood mononuclear cell freezing solution is as follows: mixing the umbilical cord blood mononuclear cell suspension with the cell freezing solution, wherein the volume of the frozen cell suspension is equal to the total number of WBCs / cell freezing density; performing a programmed cooling process of 70 to 90°C, and then transferring to liquid nitrogen at -200°C to -100°C for cryopreservation; and resuscitating the frozen cells.

2. The method for preparing a cryopreservation solution for cord blood mononuclear cells according to claim 1, characterized in that: The DMSO is dimethyl sulfoxide, a GMP grade solvent.

3. The method for preparing a cryopreservation solution for cord blood mononuclear cells according to claim 1, characterized in that: The cryopreservation density of the umbilical cord blood mononuclear cells used for programmed cooling is 3.6-4.4×10 7 Pieces / mL.

4. The method for preparing a cryopreservation solution for cord blood mononuclear cells according to claim 1, characterized in that: The total number of WBCs is the total number of mononuclear leukocytes in cord blood.

5. The method for preparing a cryopreservation solution for cord blood mononuclear cells according to claim 1, characterized in that: The human mesenchymal stem cell serum-free culture medium is a serum-free basic culture medium without animal-derived components and is a commercial product.

6. The method for preparing a cryopreservation solution for cord blood mononuclear cells according to claim 1, characterized in that: The cell freezing solution contains 5%-10% human serum albumin, 5%-10% DMSO and 80%-90% human mesenchymal stem cell serum-free culture medium.

7. The method for preparing a cryopreservation solution for cord blood mononuclear cells according to claim 1, characterized in that: The plasma accounts for 90-95% by volume, and the DMSO accounts for 5-10% by volume.

8. The method for preparing a cryopreservation solution for cord blood mononuclear cells according to claim 1, characterized in that: The cord blood mononuclear cell suspension is mixed with the cell freezing solution and transferred to liquid nitrogen for cryopreservation at a temperature of -196°C.

9. The method for preparing a cryopreservation solution for cord blood mononuclear cells according to claim 1, characterized in that: The umbilical cord blood mononuclear cells that had been frozen for 15 days were revived in a water bath at 37°C, and the survival rate of the revived cells reached more than 90%.

10. The method for preparing a cryopreservation solution for cord blood mononuclear cells according to claim 1, characterized in that: The human serum albumin is albumin.

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