Peripheral blood mononuclear cell cryopreservation protective agent and cryopreservation method

Abstract

The invention discloses a peripheral blood mononuclear cell cryopreservation protective agent and a cryopreservation method.The cryopreservation protective agent is prepared from, by volume, 5% of dimethyl sulfoxide (DMSO), 5% of dextran, 45% of autologous plasma and 45% of a serum-free culture medium. The method comprises the following steps: centrifuging a negative peripheral blood sample for infectious disease detection, counting mononuclear cells, preparing cryopreserved cell sap, carrying out programmed cooling, and transferring to low-temperature storage. The cryopreservation liquid has the advantages that the cryopreservation liquid can protect cells, and the effectiveness and the stability of the motility rate of the cryopreserved cells after recovery each time are ensured. The cell viability after recovery is high and can reach 95% or above, no heterologous animal component exists, and high-quality cryopreserved peripheral blood mononuclear cells are provided for recovery culture of immune cells later.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell cryopreservation, in particular to a cell cryopreservation protective agent and a cryopreservation method. BACKGROUND

[0002] Peripheral blood mononuclear cells (PBMC) refer to cells with single nuclei in peripheral blood, including lymphocytes, monocytes, dendritic cells and hematopoietic stem cells, etc. PBMC can be induced to differentiate into various immune cells in vitro, such as cytokine-induced killer cells (CIK), natural killer cells (NK), natural killer T cells (NKT), which have great use in anti-tumor, anti-infection treatment, etc. However, it is difficult to guarantee sufficient number and high recovery rate of PBMC in the process of clinical application, and the influencing factors include not only the acquisition method, but also the cryopreservation method.

[0003] The commonly used cell cryopreservation protective agent components currently mainly include isotonic reagents such as glycerol and dimethyl sulfoxide (DMSO), and non-isotonic reagents such as hydroxyethyl starch (HES), albumin and polyethylene glycol (PEG). Of course, there are also other cell cryopreservation protective agents with inconsistent components. Different peripheral blood mononuclear cell cryopreservation protective agent components are various, but the effectiveness and stability of the cell recovery rate after each cryopreservation cannot be guaranteed. It is difficult to avoid adverse reactions caused by heterologous animal components, and the cell viability and functionality after cryopreservation will be affected to a certain extent. SUMMARY

[0004] The present application aims to provide a peripheral blood mononuclear cell cryopreservation protective agent and a cryopreservation method, so as to solve the problem of lack of cryopreservation protective agent and cryopreservation method which can effectively guarantee the cell viability and functionality stability after cryopreservation in the prior art.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0006] A peripheral blood mononuclear cell cryopreservation protective agent, by volume fraction, comprising 5% dimethyl sulfoxide, 5% dextran, 45% autologous plasma and 45% serum-free medium.

[0007] The present application also provides a peripheral blood mononuclear cell cryopreservation method using the peripheral blood mononuclear cell cryopreservation protective agent according to claim 1, comprising the following steps:

[0008] (1) centrifuging peripheral blood samples, sucking out the upper plasma for standby, adding the lower blood sample after the plasma is taken to sample gradient separation liquid, centrifuging, taking white membrane layer cells, washing, supplementing to the target volume, centrifuging, to obtain peripheral blood mononuclear cells;

[0009] (2) Counting peripheral blood mononuclear cells and preparing for freezing;

[0010] (3) Mixing dimethyl sulfoxide and dextran together, pre-cooling, suspending peripheral blood mononuclear cells obtained in step (1) with autologous plasma obtained in step (1), adding serum-free medium to mix into a cell suspension, and adding the pre-cooled dimethyl sulfoxide and dextran mixture drop by drop into the cell suspension to mix thoroughly, thereby obtaining a freezing cell solution;

[0011] (4) Programmedly cooling the freezing cell solution;

[0012] (5) Transferring the programmedly cooled freezing cell solution into a low-temperature storage.

[0013] Further, the detailed process of step (1) is as follows: the peripheral blood sample is distributed into a centrifuge tube, centrifuged, and after the end, the upper plasma is sucked out with a pipette into a new centrifuge tube for standby, the sample gradient separation liquid is carefully taken out and placed into a new centrifuge tube, the lower blood sample after the plasma is taken out is sucked with a pipette, the blood sample is slowly added vertically into the sample gradient separation liquid, and then the mixture of the blood sample and the sample gradient separation liquid is centrifuged, the white membrane layer cells are taken in the centrifuge tube, physiological saline is used for washing, the physiological saline is supplemented to the target volume, centrifuged, and peripheral blood mononuclear cells are obtained.

[0014] Further, the detailed process of step (1) is as follows: the peripheral blood sample is distributed into a 50 mL centrifuge tube, centrifuged under the condition of 2500 rpm, 10 min, 8 up and 8 down, after the end, the upper plasma is sucked out with a pipette into a new centrifuge tube for standby, the sample gradient separation liquid is carefully taken out and placed into a new centrifuge tube, the lower blood sample after the plasma is taken out is sucked with a pipette, the blood sample is slowly added vertically into the sample gradient separation liquid, and then the mixture of the blood sample and the sample gradient separation liquid is centrifuged under the condition of 20 ℃, 2100 rpm, 20 min, 4 up and 4 down, the white membrane layer cells are taken in the 50 mL centrifuge tube, physiological saline is used for washing, the physiological saline is supplemented to 45 mL, centrifuged under the condition of room temperature, 2000 rpm, 7 min, 8 up and 8 down, and peripheral blood mononuclear cells are obtained.

[0015] Further, the sample gradient separation liquid is a lymphocyte separation liquid.

[0016] Further, the peripheral blood sample is negative for infectious disease detection.

[0017] Further, in step (2), the peripheral blood mononuclear cells are prepared for freezing at 1.0×10 7 cells / mL.

[0018] Further, the detailed process of step (3) is as follows: after mixing dimethyl sulfoxide and dextran, place it in a 4°C refrigerator to pre-cool to 4°C, use the autologous plasma obtained in step (1) to suspend the peripheral blood mononuclear cells obtained in step (1), add serum-free culture medium and mix well to form a cell suspension, take out the 4°C dimethyl sulfoxide and dextran mixture and add it dropwise to the cell suspension, so that it is fully mixed with the cell suspension to obtain frozen cell solution.

[0019] Further, the detailed process of step (4) is as follows: the cryopreserved cell solution is cooled according to the following schedule: 4℃~-20℃, -0.5℃ / min; -20℃~-40℃, -0.8℃ / min; -40℃~-80℃, -5℃ / min; -80℃, maintain for 10 min.

[0020] Furthermore, step (5) involves transferring the cryopreserved cell fluid after the programmed cooling process into a liquid nitrogen biological container for liquid nitrogen storage.

[0021] The advantages of this invention are: the cryopreservation solution can protect cells, ensuring the effectiveness and stability of cell viability after each cryopreservation and thawing. The cell viability after thawing is high, reaching over 95%, and it contains no xenogeneic animal components, providing high-quality cryopreserved peripheral blood mononuclear cells for subsequent thawing and culture of immune cells. Attached Figure Description

[0022] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0023] Figure 1 This is a diagram illustrating the recovery process of a frozen peripheral blood mononuclear cell sample from Example 1.

[0024] Figure 2 This is a statistical chart of the results of sample 1 in Example 1 of CD3-&CD56+ flow cytometry detection;

[0025] Figure 3 This is a statistical chart of the results of sample 2 in Example 1 of CD3-&CD56+ flow cytometry detection;

[0026] Figure 4 This is a statistical chart of the results of sample 3 in Example 1 of CD3-&CD56+ flow cytometry detection;

[0027] Figure 5 This is a statistical chart of the results of sample 4 in Example 1 of CD3-&CD56+ flow cytometry detection;

[0028] Figure 6 This is a statistical chart of the results of a comparative sample for CD3- & CD56+ flow cytometry detection;

[0029] Figure 7This is a statistical chart of the results of flow cytometry analysis of the two comparative samples of CD3- & CD56+.

[0030] Figure 8 This is a statistical chart of the results of three samples in the CD3- & CD56+ flow cytometry comparison example;

[0031] Figure 9 This is a statistical chart of the results of flow cytometry analysis of four samples for comparison of CD3- and CD56+. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0033] In Example 1 and Comparative Examples 1-4, the peripheral blood mononuclear cells were all derived from umbilical cord blood samples. Unless otherwise specified, the techniques used are those that can be achieved through conventional laboratory techniques in this technical field.

[0034] Example 1

[0035] 1. Instruments

[0036] High-speed refrigerated centrifuges, biosafety cabinets, pipettes, programmed cooling systems, liquid nitrogen biological containers, and fully automated blood analyzers.

[0037] 2. Cryopreservation of peripheral blood mononuclear cells

[0038] (1) The collected peripheral blood samples were tested for four infectious diseases (AIDS, syphilis, hepatitis B, and hepatitis C). After the infectious disease test was negative, the peripheral blood samples were received. All peripheral blood samples were aspirated from the transfusion bag and evenly distributed into 50 mL centrifuge tubes. The samples were centrifuged at 2500 rpm for 10 min under the 8°C / ... White membrane cells were collected into a 50 mL centrifuge tube, washed with physiological saline, and the saline was added to a final volume of 45 mL. The tube was then centrifuged at room temperature, 2000 rpm, for 7 min, under the intensified and detensified conditions to obtain peripheral blood mononuclear cells.

[0039] (2) Peripheral blood mononuclear cell count: Peripheral blood mononuclear cell count was performed using a fully automated blood analyzer, at a rate of 1.0 × 10⁻⁶. 7 Prepare for cryopreservation at 100 cells / mL.

[0040] (3) By volume fraction, the cryoprotectant consists of 5% dimethyl sulfoxide (DMSO), 5% dextran, 45% autologous plasma, and 45% serum-free culture medium. After mixing DMSO and dextran, the mixture is pre-cooled to 4°C in a 4°C freezer. The peripheral blood mononuclear cells obtained in step (1) are suspended in the autologous plasma obtained in step (1), and then serum-free culture medium is added and mixed to form a cell suspension. The DMSO and dextran mixture at 4°C is added dropwise to the cell suspension and mixed thoroughly to obtain the cryopreserved cell solution.

[0041] (4) Cool the cryopreserved cell solution according to the following program: 4℃~-20℃, -0.5℃ / min; -20℃~-40℃, -0.8℃ / min; -40℃~-80℃, -5℃ / min; -80℃, maintain for 10 min.

[0042] (5) Transfer the frozen cell solution after the procedure cooling into a liquid nitrogen biological container for liquid nitrogen storage.

[0043] Four peripheral blood mononuclear cell samples were frozen according to steps (1)-(5), and numbered as Samples 1-4 of Example 1.

[0044] Comparative Example 1

[0045] This comparative example uses a commercially available peripheral blood mononuclear cell cryopreservation agent a, which, by volume percentage, consists of 10% DMSO and 90% fetal bovine serum.

[0046] Peripheral blood mononuclear cells were mixed with peripheral blood mononuclear cell cryoprotectant a, and then the peripheral blood mononuclear cell samples were cryopreserved using a cryopreservation box deep low temperature freezer method.

[0047] Comparative Example 2

[0048] This comparative example uses a commercially available peripheral blood mononuclear cell cryopreservation agent b, which, by volume percentage, consists of: 10% DMSO, 10% β-glucan, and 80% autologous plasma.

[0049] Peripheral blood mononuclear cells were mixed with peripheral blood mononuclear cell cryoprotectant b, and the peripheral blood mononuclear cell samples were cryopreserved using a cryopreservation box deep low temperature freezer method.

[0050] Comparative Example 3

[0051] This comparative example uses a commercially available peripheral blood mononuclear cell cryopreservation agent c, which, by volume percentage, consists of: 10% DMSO, 10% dextran, and 80% autologous plasma.

[0052] Peripheral blood mononuclear cells were mixed with peripheral blood mononuclear cell cryoprotectant c, and the peripheral blood mononuclear cell samples were cryopreserved using a cryopreservation box deep low temperature freezer method.

[0053] Comparative Example 4

[0054] This comparative example uses a commercially available cryopreservation protectant for peripheral blood mononuclear cells, which, by volume percentage, consists of: 5% DMSO, 5% human serum albumin, and 90% serum-free culture medium.

[0055] Peripheral blood mononuclear cells were mixed with peripheral blood mononuclear cell cryoprotectant d, and then the peripheral blood mononuclear cell samples were cryopreserved using a cryopreservation box deep low temperature freezer method.

[0056] recovery

[0057] The following resuscitation method was used to resuscitate four frozen peripheral blood mononuclear cell samples from Example 1 and the frozen peripheral blood mononuclear cell samples from Comparative Examples 1-4: Peripheral blood mononuclear cell samples frozen for 72 hours were quickly placed in a 37°C water bath for 3-5 minutes for cell resuscitation, shaken until completely thawed, centrifuged at 2000 rpm for 8 minutes, washed with 4°C saline, and their resuscitation viability was measured. The resuscitation process of the frozen peripheral blood mononuclear cell samples from Example 1 is as follows: Figure 1 As shown.

[0058] Resuscitation survival rate testing

[0059] For the trypan blue staining method, take an appropriate amount of cell suspension, dilute it a certain factor, add 20 µl of 0.4% trypan blue to 180 µl of the diluted cell suspension, mix well, stain for 3 minutes, and then detect viability under a biological microscope. The results are shown in Table 1.

[0060] In Table 1, the cryopreservation quantity refers to the number of peripheral blood mononuclear cells (PBMCs) frozen in each umbilical cord blood sample; the thawing quantity refers to the number of thawed PBMCs; and the thawing volume refers to the volume of each thawed PBMC sample. White blood cell (WBC) concentration refers to the concentration of WBCs in PBMCs after thawing; WBC recovery count refers to the number of WBCs after thawing; WBC yield is the number of WBCs after thawing divided by the number of WBCs at cryopreservation; and thawing viability is the thawing viability of PBMCs.

[0061] Table 1. Cell data after thawing after 72 hours of cryopreservation.

[0062]

[0063] As shown in Table 1, the resuscitation survival rates of samples 1-4 in Example 1 were all higher than those of Comparative Examples 1-4.

[0064] NK cells were obtained by in vitro induction and expansion culture of leukocytes (WBCs) obtained from the four frozen peripheral blood mononuclear cell samples in Example 1 and the frozen peripheral blood mononuclear cell samples in Comparative Examples 1 to 4.

[0065] Detection of NK cell killing rate against K562 cells

[0066] NK cells and CFSE-stained K562 cells were co-cultured at a ratio of 10:1 for 4 hours. Cells were then collected for 7-AAD staining, and NK cell cytotoxic activity was detected using a Beckman DxFLEX flow cytometer. Results are shown in Table 2. Figures 2-9 As shown, the proportion of CD3- and CD56+ NK cells obtained by induced differentiation of leukocytes from samples 1-4 in Example 1 was higher than that of NK cells obtained by induced differentiation of leukocytes from samples 1-4 in Comparative Examples. The killing rate of NK cells against K562 by induced differentiation of leukocytes from samples 1-4 in Example 1 was also higher than that of NK cells against K562 by induced differentiation of leukocytes from samples 1-4 in Comparative Examples.

[0067] Table 2. Proportion of CD3- & CD56+ and K562 Kill Rate Data

[0068]

[0069] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A cryopreservation protectant for peripheral blood mononuclear cells, characterized in that, By volume fraction, it includes 5% dimethyl sulfoxide, 5% dextran, 45% autologous plasma, and 45% serum-free culture medium.

2. A method for cryopreserving peripheral blood mononuclear cells using the cryopreservation protectant for peripheral blood mononuclear cells according to claim 1, characterized in that the steps include... include: (1) Centrifuge the peripheral blood sample, aspirate the upper plasma layer for later use, add the lower blood sample after plasma collection to the sample gradient separation solution, centrifuge, take the white membrane layer cells, wash, add to the target volume, centrifuge, and obtain peripheral blood mononuclear cells; (2) Count peripheral blood mononuclear cells and prepare for cryopreservation; (3) After mixing dimethyl sulfoxide and dextran, pre-cool the mixture, use the autologous plasma obtained in step (1) to suspend the peripheral blood mononuclear cells obtained in step (1), add serum-free culture medium and mix well to form a cell suspension, take out the pre-cooled dimethyl sulfoxide and dextran mixture and add it drop by drop to the cell suspension, mix it thoroughly with the cell suspension to obtain the frozen cell solution; (4) Procedurally cool the frozen cell solution; (5) Transfer the frozen cell solution after the program cooling to low temperature storage.

3. The method for cryopreserving peripheral blood mononuclear cells according to claim 2, characterized in that, The detailed process of step (1) is as follows: peripheral blood samples are allocated to centrifuge tubes and centrifuged. After centrifugation, the upper plasma layer is aspirated into a new centrifuge tube for later use. The sample gradient separation solution is carefully taken out and placed into a new centrifuge tube. The lower blood sample after the plasma is collected is aspirated with a pipette and added vertically and slowly into the sample gradient separation solution. The mixture of blood sample and sample gradient separation solution is then centrifuged. White membrane cells are taken into a centrifuge tube, washed with physiological saline, and physiological saline is added to the target volume. The mixture is then centrifuged to obtain peripheral blood mononuclear cells.

4. The method for cryopreserving peripheral blood mononuclear cells according to claim 3, characterized in that, The detailed process of step (1) is as follows: peripheral blood samples are allocated to 50 mL centrifuge tubes and centrifuged at 2500 rpm for 10 min under the conditions of rising 8 and falling 8. After centrifugation, the upper plasma layer is aspirated with a pipette and placed into a new centrifuge tube for later use. The sample gradient separation solution is carefully taken out and placed into a new centrifuge tube. The lower blood sample after the plasma is collected is aspirated with a pipette and the blood sample is slowly added vertically to the sample gradient separation solution. Then, the mixture of blood sample and sample gradient separation solution is centrifuged at 20 ℃, 2100 rpm for 20 min under the conditions of rising 4 and falling 4. The white membrane cells are taken into a 50 mL centrifuge tube, washed with physiological saline, and the physiological saline is added to 45 mL. The mixture is centrifuged at room temperature, 2000 rpm for 7 min under the conditions of rising 8 and falling 8 to obtain peripheral blood mononuclear cells.

5. The method for cryopreserving peripheral blood mononuclear cells according to any one of claims 1-3, characterized in that, The sample gradient separation solution is a lymphocyte separation solution.

6. The method for cryopreserving peripheral blood mononuclear cells according to claim 2, characterized in that, The peripheral blood sample tested negative for infectious diseases.

7. The method for cryopreservation of peripheral blood mononuclear cells according to claim 2, characterized in that, In step (2), the ratio is 1.0 × 10 7 Prepare for cryopreservation of cells / mL.

8. The method for cryopreserving peripheral blood mononuclear cells according to claim 2, characterized in that, The detailed process of step (3) is as follows: After mixing dimethyl sulfoxide and dextran, place them in a 4°C refrigerator to pre-cool to 4°C. Use the autologous plasma obtained in step (1) to suspend the peripheral blood mononuclear cells obtained in step (1), and then add serum-free culture medium to mix into a cell suspension. Take out the 4°C dimethyl sulfoxide and dextran mixture and add it dropwise to the cell suspension to mix it thoroughly with the cell suspension to obtain frozen cell solution.

9. The method for cryopreserving peripheral blood mononuclear cells according to claim 2, characterized in that, The detailed process of step (4) is as follows: the cryopreserved cell solution is cooled in the following manner: 4℃~-20℃, -0.5℃ / min; -20℃~-40℃, -0.8℃ / min; -40℃~-80℃, -5℃ / min; -80℃, maintain for 10 min.

10. The method for cryopreserving peripheral blood mononuclear cells according to claim 2, characterized in that, Step (5) involves transferring the cryopreserved cell fluid, after being cooled by the procedure, into a liquid nitrogen biological container for storage under liquid nitrogen.

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