CIK cell culture fluid suitable for CIK cell culture and method for culturing CIK cells

Abstract

The invention discloses a CIK cell culture fluid suitable for CIK cell culture and a method for culturing CIK cells, and relates to the technical field of immune cells. The CIK cell culture fluid suitable for CIK cell culture comprises a T cell amplification serum-free culture medium and a T cell serum-free culture medium additive component, wherein the T cell serum-free culture medium additive component comprises DPBS and sterile water, and the T cell serum-free culture medium additive component further comprises at least three of anti-human CD3 monoclonal antibody, anti-human CD28 antibody, IFN-γ, GM-CSF, IL-2, IL-15, IL-7, IL-21 and Nicotinamide; the volume ratio of the T cell amplification serum-free culture medium and the T cell serum-free culture medium additive component is 125:1; the cell phenotype of the CIK cells amplified and cultured by the CIK cells is relatively stable, the amplification multiple is large, the viability is high, the CIK cells are conducive to the culture and application of the CIK cells, and the CIK cells have a high amplification multiple, a long passage time and a high viability.

Description

Technical Field

[0001] The present invention relates to the field of immune cells, and in particular to a CIK cell culture fluid suitable for CIK cell culture and a method for culturing CIK cells. Background Art

[0002] CIK cells are the abbreviation of "Cytokine-Induced Killer Cells" and their full Chinese name is "cytokine-induced killer cells". CIK is a heterogeneous cell population with CD3+CD56+ cells as the main effector cells obtained by culturing mononuclear cells under the action of CD3 monoclonal antibodies and multiple cytokines (including IFN-g, IL-2, etc.). It has both the powerful anti-tumor activity of T lymphocytes and the non-MHC (major histocompatibility antigen) restricted tumor killing ability of NK cells (natural killer cells). CIK cells have the characteristics of high tumor killing activity, broad tumor killing spectrum, low toxicity to normal tissues, and high in vitro expansion. They are currently widely used adoptive immunotherapy cells in clinical practice and are the first line of defense for the body against pathogens and tumor cells.

[0003] The current CIK cell culture medium is usually prepared by adjusting various nutrients on the basis of the immune cell therapy culture medium, and cannot specifically improve the expansion effect of CIK cells. Various studies on CIK cells have shown that CIK cell culture cannot be separated from various amino acids and factor stimulations. Therefore, the present invention selects the OptiVitro® T cell expansion serum-free culture medium rich in amino acids suitable for CIK cell growth. At the same time, three of anti-human CD3 monoclonal antibodies, anti-human CD28 antibodies, IFN-γ, GM-CSF, IL-2, IL-15, IL-7, IL-21, and Nicotinamide are added to provide nutrition and induction factors for CIK cells, so that the number of CIK cells induced by the culture medium is effectively expanded.

[0004] Currently, most of the conventional serum-free culture media for immune cell therapy are supplemented with multiple growth factors to achieve large-scale culture of CIK cells. However, the addition of multiple growth factors and manual operation have potential risks in clinical applications and also increase the probability of cell contamination. Therefore, it is particularly important to provide a CIK cell culture fluid suitable for CIK cell culture that is animal-free, has simple ingredients, defined components, and a high cell proliferation rate, and a method for culturing CIK cells to reduce the clinical application risk of CIK immune cells and improve the proliferation efficiency of CIK immune cells. 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 CIK cell culture fluid suitable for CIK cell culture and a method for culturing CIK cells, wherein the cell phenotype of the CIK cells amplified and cultured is relatively stable, the amplification multiple is large, and the viability is high, which is beneficial to the culture and application of CIK cells; it has a high amplification multiple, and the use of the CIK cell culture fluid suitable for CIK cell culture and the method for culturing CIK cells provided in the present application can make the cell amplification multiple reach 280±100 times; it has a longer passage time and a higher viability.

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

[0007] A CIK cell culture fluid suitable for CIK cell culture comprises a T cell expansion serum-free culture medium and a T cell serum-free culture medium additive component, wherein the T cell serum-free culture medium additive component comprises DPBS and sterile water, and the T cell serum-free culture medium additive component further comprises at least three of anti-human CD3 monoclonal antibody, anti-human CD28 antibody, IFN-γ, GM-CSF, IL-2, IL-15, IL-7, IL-21 and Nicotinamide; the volume ratio of the T cell expansion serum-free culture medium and the T cell serum-free culture medium additive component is 125:1.

[0008] As a preferred embodiment: the T cell serum-free culture medium supplemented components include at least one of anti-human CD3 monoclonal antibody, anti-human CD28 antibody, and IFN-γ; the T cell serum-free culture medium supplemented components include at least one of IL-2 and Nicotinamide; the T cell serum-free culture medium supplemented components include at least one of GM-CSF, IL-15, IL-7, and IL-21.

[0009] As a preferred embodiment: the CIK cell culture fluid includes T cell expansion serum-free medium, DPBS, sterile water, anti-human CD3 monoclonal antibody, anti-human CD28 antibody, IFN-γ, GM-CSF, IL-2, IL-15, IL-7 and IL-21; the mass ratio of the anti-human CD3 monoclonal antibody, anti-human CD28 antibody, IFN-γ, GM-CSF, IL-15, IL-7 and IL-21 is 14:14:14:4:8:1.6:7.8; the T cell expansion serum-free medium is OptiVitro® T cell expansion serum-free medium.

[0010] The method for culturing CIK cells with CIK cell culture fluid comprises culturing CIK cells by inducing and amplifying cryopreserved umbilical cord blood mononuclear cells with CIK cell culture fluid.

[0011] As a preferred embodiment, the method for culturing CIK cells, using the CIK cell culture medium to induce amplification of cryopreserved umbilical cord blood mononuclear cells, comprises the following steps:

[0012] S1. preparing CIK cell culture medium;

[0013] S2. Place the frozen umbilical cord blood mononuclear cells in a 35-40°C water bath to thaw for 1-3 minutes;

[0014] S3, washing the resuscitated umbilical cord blood mononuclear cells with CIK cell culture medium, and centrifuging at 300-500g for 8-10min;

[0015] S4, using CIK cell culture medium to resuspend the umbilical cord blood mononuclear cells centrifuged in step S3 and induce the seed bottle;

[0016] S5, adding CIK cell culture medium from the second to fourth day;

[0017] S6. Add CIK cell culture medium from the sixth to the eighth day and divide equally into another cell culture bottle;

[0018] S7, on the eleventh day, transfer to a culture bag for expansion culture and replenish with CIK cell culture medium;

[0019] S8, replenishing with CIK cell culture medium from the 14th to the 17th day;

[0020] S9. Collect the cells by centrifugation at 300-500 g for 8 min.

[0021] As a preferred embodiment: the umbilical cord blood mononuclear cells are prepared by the Ficoll method.

[0022] As a preferred solution: in step S4, the cell density of umbilical cord blood mononuclear cells needs to be adjusted to 4.0×10 6 Pieces / mL.

[0023] As a preferred solution: in step S5, before adding CIK cell culture medium, the cell density of umbilical cord blood mononuclear cells needs to be counted to reach 1.0×10 6 Pieces / mL.

[0024] As a preferred solution: in step S7, before changing the culture container and amplifying the culture, the cell density should be counted to reach 2.0×10 6 Pieces / mL.

[0025] As a preferred solution: before collecting cells in step S9, it is necessary to determine whether the cell number and cytotoxic activity meet the expected requirements, and then collect them after meeting the requirements.

[0026] 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 cell phenotype of the CIK cells amplified and cultured is relatively stable, the amplification multiple is large, and the viability is high, which is beneficial to the culture and application of CIK cells; it has a high amplification multiple. It can be seen from experimental data that the CIK cell culture medium suitable for CIK cell culture provided by the present application and the method for culturing CIK cells can make the cell amplification multiple reach 280±100 times; it has a long passage time, and the culture medium of the present invention still has a high proliferation amount on the 11th to 20th day; it has a high viability. The culture medium of the present invention is used throughout the entire culture cycle, and the viability reaches more than 90%; it has a relatively stable cell phenotype. The cell phenotype reaches the optimal state on the 11th to 20th day when the culture medium of the present invention is used, and the cells have the highest anti-tumor activity at this time. DETAILED DESCRIPTION

[0027] The invention provides a CIK cell culture fluid suitable for CIK cell culture. The CIK cell culture fluid comprises a T cell expansion serum-free culture medium and a T cell serum-free culture medium additive component. The T cell serum-free culture medium additive component comprises DPBS and sterile water. The T cell serum-free culture medium additive component further comprises at least three of anti-human CD3 monoclonal antibody, anti-human CD28 antibody, IFN-γ, GM-CSF, IL-2, IL-15, IL-7, IL-21 and Nicotinamide. The volume ratio of the T cell expansion serum-free culture medium and the T cell serum-free culture medium additive component is 125:1.

[0028] The T cell serum-free culture medium additive component contains at least one of anti-human CD3 monoclonal antibody, anti-human CD28 antibody, and IFN-γ; the T cell serum-free culture medium additive component contains at least one of IL-2 and Nicotinamide; the T cell serum-free culture medium additive component contains at least one of GM-CSF, IL-15, IL-7, and IL-21.

[0029] The CIK cell culture fluid includes T cell expansion serum-free culture medium, DPBS, sterile water, anti-human CD3 monoclonal antibody, anti-human CD28 antibody, IFN-γ, GM-CSF, IL-2, IL-15, IL-7 and IL-21; the mass ratio of the anti-human CD3 monoclonal antibody, anti-human CD28 antibody, IFN-γ, GM-CSF, IL-15, IL-7 and IL-21 is 14:14:14:4:8:1.6:7.8; the T cell expansion serum-free culture medium is OptiVitro® T cell expansion serum-free culture medium.

[0030] The method for culturing CIK cells with a CIK cell culture fluid comprises culturing CIK cells by inducing and amplifying cryopreserved umbilical cord blood mononuclear cells with the CIK cell culture fluid.

[0031] The method for culturing CIK cells uses the CIK cell culture fluid to induce and amplify cryopreserved umbilical cord blood mononuclear cells, and comprises the following steps:

[0032] S1. preparing CIK cell culture medium;

[0033] S2. Place the frozen umbilical cord blood mononuclear cells in a 35-40°C water bath to thaw for 1-3 minutes;

[0034] S3, washing the resuscitated umbilical cord blood mononuclear cells with CIK cell culture medium, and centrifuging at 300-500g for 8-10min;

[0035] S4, using 17-20 mL CIK cell culture medium to resuspend the umbilical cord blood mononuclear cells centrifuged in step S3 and induce the seed bottle;

[0036] S5. Add CIK cell culture medium from the second to fourth day to 40-60 mL;

[0037] S6. Add CIK cell culture medium on the sixth to eighth day, add constant volume to 300-400 mL, and divide equally into another cell culture bottle;

[0038] S7. On the eleventh day, transfer to a culture bag for expansion culture and replenish the fluid to 600 mL-800 mL with CIK cell culture medium;

[0039] S8. On the 14th to 17th day, the volume of the cells was replenished with CIK cell culture medium to 2000 mL-3000 mL;

[0040] S9. Collect the cells by centrifugation at 300-500 g for 8 min.

[0041] The umbilical cord blood mononuclear cells are prepared by the Ficoll method.

[0042] In step S4, the cell density of umbilical cord blood mononuclear cells needs to be adjusted to 4.0×10 6 Pieces / mL.

[0043] In step S5, before adding CIK cell culture medium, the cell density of umbilical cord blood mononuclear cells should be counted to reach 1.0×10 6 Pieces / mL.

[0044] In step S7, the cell density should be counted to reach 2.0×10 before changing the culture container and amplifying the culture. 6 Pieces / mL.

[0045] Before collecting cells in step S9, it is necessary to determine whether the cell number and cytotoxic activity meet the expected requirements, and then collect the cells after meeting the requirements.

[0046] Example 1: CIK cell culture medium suitable for CIK cell culture

[0047] The CIK cell culture fluid comprises a T cell expansion serum-free culture medium and a T cell serum-free culture medium additive component. The T cell serum-free culture medium additive component comprises DPBS and sterile water. The T cell serum-free culture medium additive component also comprises IFN-γ: 1.4 μg; IL-2: 348,000 IU and IL-15: 0.8 μg.

[0048] Example 2: CIK cell culture medium suitable for CIK cell culture

[0049] The CIK cell culture fluid comprises a T cell expansion serum-free culture medium and a T cell serum-free culture medium supplement component, wherein the T cell serum-free culture medium supplement component comprises DPBS and sterilized water, and the T cell serum-free culture medium supplement component also comprises anti-human CD3 monoclonal antibody: 1.4 μg; anti-human CD28 antibody: 1.4 μg; IFN-γ: 1.4 μg; GM-CSF: 0.4 μg; IL-2: 348,000 IU; IL-15: 0.8 μg; IL-7: 0.16 μg and IL-21: 0.78 μg.

[0050] Example 3: CIK cell culture medium suitable for CIK cell culture

[0051] The CIK cell culture fluid comprises a T cell expansion serum-free culture medium and a T cell serum-free culture medium supplement component, wherein the T cell serum-free culture medium supplement component comprises DPBS and sterilized water, and the T cell serum-free culture medium supplement component further comprises anti-human CD3 monoclonal antibody: 1.4 μg; anti-human CD28 antibody: 1.4 μg; IFN-γ: 1.4 μg; GM-CSF: 0.4 μg; IL-2: 348,000 IU; IL-15: 0.8 μg; IL-7: 0.16 μg; IL-21: 0.78 μg and Nicotinamide: 14 mL.

[0052] Examples 1 to 3 all used homologous umbilical cord blood mononuclear cells.

[0053] Verification Example 1: Method for culturing CIK cells

[0054] Prepare CIK cell culture medium by culturing; revive frozen cord blood mononuclear cells in a 35-40°C water bath for 1-3 minutes; wash the revive cord blood mononuclear cells with CIK cell culture medium, and centrifuge at 300g for 8 minutes; resuspend them with 20mL CIK cell culture medium and induce seed bottles; add 20mL and 40mL CIK cell culture medium on the second and fourth days, respectively; add CIK cell culture medium on the sixth and eighth days, and make up to 300 and 400mL, respectively, and divide equally into another cell culture bottle on the eighth day; transfer to a culture bag for expansion culture on the eleventh day, and make up to 800mL with CIK cell culture medium; make up to 2000mL and 3000mL with CIK cell culture medium on the fourteenth and seventeenth days, respectively; and collect cells by centrifugation at 500g for 8 minutes.

[0055] Samples were collected for counting and viability at D0, D8, D11, D14, D17, and D20. Samples were collected for flow cytometry at D0, D11, and D20. Samples were collected at D20 for tumoricidal activity and abnormal toxicity testing; the results are shown in Tables 1 to 4.

[0056] Table 1: Changes in cell number from Example 1 to Example 3

[0057]

[0058] Table 2: Changes in cell viability from Example 1 to Example 3

[0059]

[0060] Table 3: Flow cytometry changes of cells from Example 1 to Example 3

[0061]

[0062] Table 4: Tumoricidal activity and abnormal toxicity detection of Examples 1 to 3

[0063]

[0064] It can be seen from Table 1 to Table 4 that: Examples 1 to 3 all have certain tumoricidal activity, among which Example 2 is better; there are no abnormalities in abnormal toxicity detection; Flow cytometry detection of Examples 1 to 3 can detect the corresponding CIK indicators, among which Example 2 is better; the number of cells in Examples 1 to 3 increases with time, among which Example 2 grows the fastest and the final harvest number is the largest, followed by Example 3; the cell viability of Examples 1 to 3 is higher than 90% at D0 time except that Example 2 is lower than 90%. Under the same source of umbilical cord blood mononuclear cells, different factor ratios also have different effects, among which Example 2 has the best effect, and the subsequent examples all prepare CIK cell culture fluid according to the factor ratio of Example 2.

[0065] Examples 4 to 13: CIK cell culture fluid suitable for CIK cell culture

[0066] The CIK cell culture medium of Examples 4 to 13 is the same as that of Example 2; it contains anti-human CD3 monoclonal antibody: 1.4 μg, anti-human CD28 antibody: 1.4 μg, IFN-γ: 1.4 μg, GM-CSF: 0.4 μg, IL-2: 348,000 IU, IL-15: 0.8 μg, IL-7: 0.16 μg and IL-21: 0.78 μg.

[0067] That is, the CIK cell culture fluid includes T cell expansion serum-free medium and T cell serum-free medium supplement components, the T cell serum-free medium supplement components include DPBS and sterile water, and the T cell serum-free medium supplement components also include anti-human CD3 monoclonal antibody: 1.4μg; anti-human CD28 antibody: 1.4μg; IFN-γ: 1.4μg; GM-CSF: 0.4μg; IL-2: 348,000 IU; IL-15: 0.8μg; IL-7: 0.16μg and IL-21: 0.78μg.

[0068] Examples 4 to 13 used umbilical cord blood mononuclear cells from five sources, of which Examples 4 to 6 were from the same source, Examples 7 and 8 were from the same source, Examples 9 to 11 were from the same source, Example 12 was from one source, and Example 13 was from one source.

[0069] Verification Example 2: Method for culturing CIK cells

[0070] Prepare CIK cell culture medium by culturing; revive frozen cord blood mononuclear cells in a 35-40°C water bath for 1-3 minutes; wash the revive cord blood mononuclear cells with CIK cell culture medium, and centrifuge at 300g for 8 minutes; resuspend them with 20mL CIK cell culture medium and induce seed bottles; add 20mL and 40mL CIK cell culture medium on the second and fourth days, respectively; add CIK cell culture medium on the sixth and eighth days, and make up to 300 and 400mL, respectively, and divide equally into another cell culture bottle on the eighth day; transfer to a culture bag for expansion culture on the eleventh day, and make up to 800mL with CIK cell culture medium; make up to 2000mL and 3000mL with CIK cell culture medium on the fourteenth and seventeenth days, respectively; and collect cells by centrifugation at 500g for 8 minutes.

[0071] Sampling counting and viability were carried out at D0, D8, D11, D14, D17 and D20; the results are shown in Tables 5 and 6.

[0072] Table 5: Changes in cell number from Example 4 to Example 13

[0073]

[0074] Table 6: Changes in cell viability from Example 4 to Example 13

[0075]

[0076] It can be seen from Table 5 and Table 6 that: through the data of Examples 4 to 13, it can be analyzed that, regardless of whether the umbilical cord blood mononuclear cells are from the same source or not, after being expanded by the method provided by the present application, the viability can reach more than 90%. The growth curves of umbilical cord blood mononuclear cells from different sources are similar, and they can all reach more than 7 billion at the final harvest time.

[0077] The design focus of the present invention is that the cell phenotype of the CIK cells amplified and cultured is relatively stable, the amplification multiple is large, and the viability is high, which is beneficial to the culture and application of CIK cells; it has a high amplification multiple. It can be seen from experimental data that the CIK cell culture medium suitable for CIK cell culture and the method for culturing CIK cells provided by the present application can make the cell amplification multiple reach 280±100 times; it has a long passage time, and the culture medium of the present invention still has a high proliferation amount on the 11th to 20th day; it has a high viability. The culture medium of the present invention is used throughout the entire culture cycle, and the viability reaches more than 90%; it has a relatively stable cell phenotype. The cell phenotype reaches the optimal state on the 11th to 20th day when the culture medium of the present invention is used, and the cells have the highest anti-tumor activity at this time.

[0078] 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 culturing CIK cells using CIK cell culture medium, characterized in that: The CIK cell culture fluid comprises a T cell expansion serum-free medium and a T cell serum-free medium supplement component, wherein the T cell serum-free medium supplement component comprises DPBS and sterilized water, and the T cell serum-free medium supplement component further comprises anti-human CD3 monoclonal antibody: 1.4 μg; anti-human CD28 antibody: 1.4 μg; IFN-γ: 1.4 μg; GM-CSF: 0.4 μg; IL-2: 348,000 IU; IL-15: 0.8 μg; IL-7: 0.16 μg and IL-21: 0.78 μg; the volume ratio of the T cell expansion serum-free medium and the T cell serum-free medium supplement component is 125:1; the T cell expansion serum-free medium is OptiVitro® T cell expansion serum-free medium; A method for culturing CIK cells with CIK cell culture medium, wherein the CIK cells are cultured by inducing and amplifying cryopreserved umbilical cord blood mononuclear cells with CIK cell culture medium; The method of inducing and amplifying frozen umbilical cord blood mononuclear cells using the CIK cell culture medium comprises the following steps: The cryopreserved umbilical cord blood mononuclear cells were revived in a 35-40°C water bath for 1-3 minutes; the revived umbilical cord blood mononuclear cells were washed with CIK cell culture medium, and centrifuged at 300g for 8 minutes; they were resuspended with 20mL CIK cell culture medium and induced into seed bottles; 20mL and 40mL CIK cell culture medium were added on the second and fourth days, respectively; CIK cell culture medium was added on the sixth and eighth days, and the volume was fixed to 300 and 400mL, respectively, and evenly divided into another cell culture bottle on the eighth day; on the eleventh day, the cells were transferred to the culture bag for expansion culture, and the volume was supplemented to 800mL with CIK cell culture medium; on the fourteenth and seventeenth days, the volume was supplemented to 2000mL and 3000mL with CIK cell culture medium, respectively; the cells were collected by centrifugation at 500g for 8 minutes.

2. The method for culturing CIK cells using CIK cell culture medium according to claim 1, characterized in that: The umbilical cord blood mononuclear cells are prepared by the Ficoll method.

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