Enhanced CIK cell as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cell culture, in particular to an enhanced CIK cell as well as a preparation method and application thereof. The preparation method of the enhanced CIK cells comprises the following steps: (1) mixing mononuclear cells with a serum-free culture medium, and culturing until the cell density is (2-6) * 10 < 6 > cells / mL; (2) transferring the cell suspension to a special culture medium for CIK cells, culturing for 5-7 days, adding a taxus chinensis extract and ginsenoside, and acting on the cells for 2-3 days; and (3) collecting the cells to obtain the enhanced CIK cells. The enhanced CIK cell prepared by using the method disclosed by the invention is high in proliferation speed and high in tumor killing capability, and a basis is provided for development of tumor drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to an enhanced CIK cell and a preparation method and application thereof. Background Art

[0002] In the process of tumor treatment, immune cell therapy has significantly changed the treatment of cancer as a revolutionary treatment method. It is a professional immunotherapy that uses living immune cells as biological agents to treat cancer.

[0003] CIK cells are heterogeneous immune effector cells with mixed T cell and NK cell-like phenotypes. During in vitro culture, CIK cells can secrete various cytokines, activate the cytotoxic activity of macrophages, NK cells, and CD8+T cells, thereby inhibiting the growth of tumor cells and promoting indirect killing effects. CIK cells also induce the expression of apoptosis genes and anti-tumor genes, carry out cytotoxic activity and promote tumor cell apoptosis. It can be seen that CIK cells play an important role in immune regulation and tumor immunotherapy.

[0004] In the process of preparing CIK cells in the prior art, due to the unreasonable addition of reagents, the proliferation efficiency and toxicity of the CIK cells obtained by culture need to be improved, and there are potential safety hazards.

[0005] Based on this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an enhanced CIK cell with fast proliferation efficiency and strong tumor killing efficiency, and in particular to an enhanced CIK cell and a preparation method and application thereof.

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

[0008] The present invention provides a method for preparing enhanced CIK cells, comprising the following steps:

[0009] (1) Mix mononuclear cells with serum-free medium and culture until the cell density is 2-6×10 6 Pieces / mL;

[0010] (2) Transfer the cell suspension to a CIK cell-specific culture medium. After culturing for 5 to 7 days, add yew extract and ginsenosides to the cells and allow them to act for 2 to 3 days.

[0011] (3) collecting cells to obtain enhanced CIK cells;

[0012] The CIK cell-specific culture medium in step (2) is based on GT-T1551 cell serum-free culture medium and also includes the following components at the following concentrations:

[0013] Anti-CD3 monoclonal antibody 160-200 ng / mL, anti-CD28 monoclonal antibody 50-80 ng / mL, interleukin-21 200-1400 IU / mL, interleukin-5 50-60 IU / mL, interleukin-1α 50-60 IU / mL, recombinant human granulocyte colony stimulating factor injection 3-5 μg / mL.

[0014] Preferably, the serum-free medium is based on serum-free RPMI-1640 medium and further includes components in the following concentrations:

[0015] Interferon-γ 1500-2000 IU / mL, human serum albumin 12-16 mg / mL, 6-8% autologous serum, creatine phosphate 0.2-0.5 mg / mL.

[0016] Preferably, the culture temperature in step (1) is 23-25°C.

[0017] Preferably, the culture temperature in step (2) is 36-38° C., the concentration of carbon dioxide introduced during culture is 4-6%, and the relative humidity during culture is 92-95%.

[0018] Preferably, the final concentration of the yew extract added in step (2) is 0.1 to 0.3 mg / mL;

[0019] The final concentration of ginsenoside added was 0.2-0.4 mg / mL.

[0020] Preferably, the method for preparing the mononuclear cells is: collecting them using a blood cell separator.

[0021] The present invention also provides enhanced CIK cells prepared by the preparation method.

[0022] The present invention also provides enhanced CIK cells prepared by the preparation method or use of the enhanced CIK cells in preparing a preparation for treating tumors.

[0023] The present invention also provides a preparation for treating tumors, comprising the enhanced CIK cells prepared by the preparation method or the enhanced CIK cells.

[0024] The present invention provides an enhanced CIK cell and a preparation method and application thereof. The preparation method of the enhanced CIK cell of the present invention comprises the following steps: (1) mixing mononuclear cells with serum-free culture medium and culturing the cells to a cell density of 2 to 6×10 6 / mL; (2) transfer the cell suspension to a CIK cell-specific culture medium, culture for 5 to 7 days, add yew extract and ginsenosides, and act on the cells for 2 to 3 days; (3) collect the cells to obtain enhanced CIK cells; the CIK cell-specific culture medium in step (2) is based on GT-T1551 cell serum-free culture medium and also includes the following concentrations of components: anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, interleukin-2, interleukin-5, interleukin-1α, and recombinant human granulocyte colony stimulating factor injection. Anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody are added to the special culture medium of the present invention. The anti-CD3 monoclonal antibody is used as the first signal to determine the specificity of the immune response, and the anti-CD28 monoclonal antibody is used as the second signal to determine the amount of the immune response and its spatiotemporal effect. The two work together to stimulate T cells to produce a series of physiological and biochemical reactions, thereby enhancing the tumor killing ability of CIK cells. The addition of recombinant human granulocyte colony stimulating factor injection synergizes with interleukin-2, interleukin-5, and interleukin-1α to enhance the phagocytic and killing activity mediated by neutrophils. Taxus extract is paclitaxel, which has anti-tumor effects. Ginseng saponins also have anti-tumor effects. The synergistic effect of the two on CIK cells will increase the tumor killing ability of the cells.

[0025] The enhanced CIK cells prepared by the method of the present invention have a fast proliferation rate and a strong ability to kill tumors. DETAILED DESCRIPTION

[0026] The present invention provides a method for preparing enhanced CIK cells, comprising the following steps:

[0027] (1) Mix mononuclear cells with serum-free medium and culture until the cell density is 2-6×10 6 Pieces / mL;

[0028] (2) Transfer the cell suspension to a CIK cell-specific culture medium. After culturing for 5 to 7 days, add yew extract and ginsenosides to the cells and allow them to act for 2 to 3 days.

[0029] (3) collecting cells to obtain enhanced CIK cells;

[0030] The CIK cell-specific culture medium in step (2) is based on GT-T1551 cell serum-free culture medium and also includes the following components at the following concentrations:

[0031] Anti-CD3 monoclonal antibody 160-200 ng / mL, preferably 180 ng / mL; anti-CD28 monoclonal antibody 50-80 ng / mL, preferably 65 ng / mL; interleukin-21 200-1400 IU / mL, preferably 1300 IU / mL; interleukin-5 50-60 IU / mL, preferably 55 IU / mL; interleukin-1α 50-60 IU / mL, preferably 55 IU / mL; recombinant human granulocyte colony stimulating factor injection 3-5 μg / mL, preferably 4 μg / mL.

[0032] In the present invention, the serum-free medium is based on serum-free RPMI-1640 medium and further includes the following components at the following concentrations:

[0033] γ-interferon 1500-2000 IU / mL, preferably 1800 IU / mL; human serum albumin 12-16 mg / mL, preferably 14 mg / mL; 6-8% autologous serum, preferably 7%; creatine phosphate 0.2-0.5 mg / mL, preferably 0.4 mg / mL.

[0034] In the present invention, the culture temperature in step (1) is 23-25°C, preferably 24°C; in the present invention, the cell density in step (1) is preferably 4×10 6 Pieces / mL.

[0035] In the present invention, the culture temperature in step (2) is 36-38°C, preferably 37°C; the concentration of carbon dioxide introduced during culture is 4-6%, preferably 5%; the relative humidity during culture is 92-95%, preferably 93%.

[0036] In the present invention, the final concentration of the yew extract added in step (2) is 0.1-0.3 mg / mL, preferably 0.2 mg / mL; the final concentration of ginsenoside added is 0.2-0.4 mg / mL, preferably 0.3 mg / mL.

[0037] In the present invention, the mononuclear cells are prepared by collecting the mononuclear cells using a blood cell separator. The present invention also provides enhanced CIK cells prepared by the preparation method.

[0038] The present invention also provides enhanced CIK cells prepared by the preparation method or use of the enhanced CIK cells in preparing a preparation for treating tumors.

[0039] The present invention also provides a preparation for treating tumors, comprising the enhanced CIK cells prepared by the preparation method or the enhanced CIK cells.

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] The yew extract described in the embodiment of the present invention is paclitaxel, purchased from Shanxi Tech Biotechnology Co., Ltd., with a purity of 99%;

[0042] The ginsenosides described in the examples of the present invention have a purity of 80% and are purchased from Shanxi Tech Biotechnology Co., Ltd.

[0043] The serum-free culture medium for GT-T1551 cells in the examples and comparative examples of the present invention was purchased from Shanghai Lianxun Biotechnology Co., Ltd.

[0044] The serum-free RPMI-1640 culture medium in the examples and comparative examples of the present invention was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.

[0045] The recombinant human granulocyte colony stimulating factor injection in the examples and comparative examples of the present invention was purchased from Chengdu Rongsheng Pharmaceutical Co., Ltd.

[0046] Example 1

[0047] Preparation method of serum-free culture medium: add 1600 IU / mL of γ-interferon, 16 mg / mL of human serum albumin, 6% of autologous serum, and 0.3 mg / mL of creatine phosphate to serum-free RPMI-1640 culture medium.

[0048] Preparation method of CIK cell-specific culture medium: add anti-CD3 monoclonal antibody (CD3-mAb) 180 ng / mL, anti-CD28 monoclonal antibody 80 ng / mL, interleukin-21 200 IU / mL, interleukin-5 60 IU / mL, interleukin-1α 55 IU / mL, and recombinant human granulocyte colony stimulating factor injection 4 μg / mL to the GT-T1551 cell serum-free culture medium.

[0049] Mononuclear cells (PBMC) were collected from the blood using a blood cell separator, and serum-free culture medium was added to the separated mononuclear cells. The cells were cultured at a temperature of 25°C and a humidity of 90% until the cell density reached 4×10 6 The cell suspension was transferred to a culture bottle and added to a CIK cell-specific culture medium. After culturing for 5 days at 37°C, 5% carbon dioxide concentration, and 92% humidity (the CIK cell-specific culture medium was supplemented once at 3 days), 0.2 mg / mL of paclitaxel and 0.3 mg / mL of ginsenoside were added, and the culture was continued for 2 days. The cells were collected to obtain enhanced CIK cells.

[0050] Example 2

[0051] Preparation method of serum-free culture medium: add 2000 IU / mL of γ-interferon, 12 mg / mL of human serum albumin, 7% of autologous serum, and 0.2 mg / mL of creatine phosphate to serum-free RPMI-1640 culture medium.

[0052] Preparation method of CIK cell-specific culture medium: add anti-CD3 monoclonal antibody (CD3-mAb) 160 ng / mL, anti-CD28 monoclonal antibody 70 ng / mL, interleukin-21 400 IU / mL, interleukin-5 50 IU / mL, interleukin-1α 50 IU / mL, and recombinant human granulocyte colony stimulating factor injection 5 μg / mL to the GT-T1551 cell serum-free culture medium.

[0053] Mononuclear cells (PBMC) were collected from the blood using a blood cell separator, and serum-free culture medium was added to the separated mononuclear cells. The cells were cultured at a temperature of 23°C and a humidity of 90% until the cell density reached 2×10 6 The cell suspension was transferred to a culture bottle and added to a CIK cell-specific culture medium. After culturing for 7 days at 37°C, 5% carbon dioxide concentration, and 95% humidity (CIK cell-specific culture medium was supplemented on the 3rd and 6th days of culture), 0.1 mg / mL of paclitaxel and 0.4 mg / mL of ginsenoside were added, and the culture was continued for 3 days. The cells were collected to obtain enhanced CIK cells.

[0054] Example 3

[0055] Preparation method of serum-free culture medium: add 1500 IU / mL of γ-interferon, 14 mg / mL of human serum albumin, 8% of autologous serum, and 0.5 mg / mL of creatine phosphate to serum-free RPMI-1640 culture medium.

[0056] Preparation method of CIK cell-specific culture medium: add anti-CD3 monoclonal antibody (CD3-mAb) 200 ng / mL, anti-CD28 monoclonal antibody 50 ng / mL, interleukin-21 300 IU / mL, interleukin-5 5 5 IU / mL, interleukin-1α 60 IU / mL, and recombinant human granulocyte colony stimulating factor injection 3 μg / mL to the GT-T1551 cell serum-free culture medium.

[0057] Mononuclear cells (PBMC) were collected from the blood using a blood cell separator, and serum-free culture medium was added to the separated mononuclear cells. The cells were cultured at a temperature of 23°C and a humidity of 90% until the cell density reached 6×10 6The cell suspension was transferred to a culture flask and added to a CIK cell-specific culture medium. After culturing for 5 days at 37°C, 5% carbon dioxide concentration, and 93% humidity (CIK cell-specific culture medium was supplemented on the 3rd day of culture), 0.3 mg / mL of paclitaxel and 0.2 mg / mL of ginsenoside were added, and the culture was continued for 2 days. The cells were collected to obtain enhanced CIK cells.

[0058] Comparative Example 1

[0059] Comparative Example 1 was prepared according to the scheme of Example 1. The difference from Example 1 was that paclitaxel was not added when the enhanced CIK cells of Comparative Example 1 were cultured.

[0060] Comparative Example 2

[0061] Comparative Example 2 was prepared according to the scheme of Example 1. The difference from Example 1 was that ginsenoside was not added when the enhanced CIK cells of Comparative Example 2 were cultured.

[0062] Comparative Example 3

[0063] Comparative Example 3 was prepared according to the scheme of Example 1. The difference from Example 1 was that when the enhanced CIK cells of Comparative Example 3 were cultured, no recombinant human granulocyte colony stimulating factor injection was added to the CIK cell-specific culture medium.

[0064] Comparative Example 4

[0065] Mononuclear cells (PBMCs) were collected from the blood using a blood cell separator, and serum-free RPMI-1640 medium was added to the separated mononuclear cells. The cells were cultured at a temperature of 23°C and a humidity of 90% until the cell density reached 2×10 6 The cell suspension was transferred to a culture flask, and GT-T1551 cell serum-free culture medium was added, and cultured at 37°C, 5% carbon dioxide concentration, and 95% humidity for 10 days (the culture medium was supplemented on the 3rd, 6th, and 9th days of culture), and the cells were collected to obtain enhanced CIK cells.

[0066] Experimental Example 1

[0067] The enhanced CIK cells of Examples 1 to 3 and the enhanced CIK cells obtained by the methods of Comparative Examples 1 to 4 were respectively taken, and the cells of each group were counted using a cell counter. The results are shown in Table 1.

[0068] Table 1 Proliferation number of enhanced CIK cells obtained by different methods

[0069] Group Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Cell number / mL <![CDATA[2.89×10 9 ]]> <![CDATA[2.25×10 9 ]]> <![CDATA[3.25×10 9 ]]> <![CDATA[4.15×10 8 ]]> <![CDATA[5.18×10 8 ]]> <![CDATA[5.24×10 8 ]]> <![CDATA[1.87×10 8 ]]>

[0070] Table 1 shows that the number of enhanced CIK cells obtained by the methods of Examples 1 to 3 is higher than that of Comparative Examples 1 to 4, which indicates that the method of the present invention is suitable for the growth of CIK cells.

[0071] Experimental Example 2

[0072] The enhanced CIK cells of Examples 1 to 3 and Comparative Examples 1 to 4 were respectively taken to prepare cell suspensions, and the concentration of the cell suspensions was adjusted to 1×10 6 5 μL of anti-CD3, CD4, CD8, CD56 antibodies and isotype control IgG1 monoclonal antibody were added to the cell suspensions of different groups, incubated at 4°C in the dark for 30 minutes, and the percentage of cells in each group was detected by flow cytometry. The results are shown in Table 2. In the table, NKT cells are natural killer-like T cells, and Th is helper T cells.

[0073] Table 2 Expression of antibodies in enhanced CIK cells prepared by different methods

[0074]

[0075] As shown in Table 2, the NKT capacity of the enhanced CIK cells prepared by the scheme of the present invention can reach 59.7-63.4%, which can ensure the cytotoxicity of CIK cells.

[0076] Experimental Example 3

[0077] The drug-resistant lung adenocarcinoma cell line A549 / DDP in the logarithmic growth phase was used as the target cell. 3 Cells / well were plated in a 96-well plate, and different groups of effector cells (enhanced CIK cells prepared in Example 1), enhanced CIK cells prepared in Comparative Example 1, enhanced CIK cells prepared in Comparative Example 2, enhanced CIK cells prepared in Comparative Example 3, and enhanced CIK cells prepared in Comparative Example 4) were added according to the ratio of effector cells (enhanced CIK cells prepared by different methods): target cells of 20:1, with 4 replicates per well, and corresponding single target cells and single effector cell wells were set as controls for each group, and cultured in an incubator for 4 hours. After the culture was completed, 10 μL of CCK-8 solution was added to each well, cultured for 4 hours, the absorbance of each well at 450 nm was detected, and the killing rate was calculated according to the following formula. The results are shown in Table 3.

[0078] Killing rate = [1-(OD value of the experimental group-OD value of the effector cell group alone) / OD value of the target cell group alone] × 100%.

[0079] Table 3 Killing rate of enhanced CIK cells prepared by different methods

[0080] Group Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Kill rate% 92.4±2.6 71.3±1.6 69.5±1.2 65.4±1.1 54.7±0.9

[0081] As shown in Table 3, the enhanced CIK cells prepared by the embodiment of the present invention have a higher killing rate on tumor cells than those in Comparative Examples 1 to 4, and can reach a killing rate of 92.4%, indicating that the method of the present invention can cultivate CIK cells with a strong tumor killing rate.

[0082] It can be seen from the above embodiments that the present invention provides an enhanced CIK cell and a preparation method and application thereof. The preparation method of the enhanced CIK cell of the present invention comprises the following steps:

[0083] (1) Mix mononuclear cells with serum-free medium and culture until the cell density is 2-6×10 6 / mL; (2) transferring the cell suspension to a CIK cell-specific culture medium, culturing for 5 to 7 days, adding yew extract and ginsenosides, and acting on the cells for 2 to 3 days; (3) collecting the cells to obtain enhanced CIK cells. The enhanced CIK cells prepared by the method of the present invention have a fast proliferation rate and a strong ability to kill tumors.

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing enhanced CIK cells, It is characterized in that The steps include: (1) Mix mononuclear cells with serum-free medium and culture until the cell density is 2-6×10 6 Pieces / mL; (2) Transfer the cell suspension to a CIK cell-specific culture medium. After culturing for 5 to 7 days, add yew extract and ginsenosides to the cells and allow them to act for 2 to 3 days. (3) collecting cells to obtain enhanced CIK cells; The CIK cell-specific culture medium in step (2) is based on GT-T1551 cell serum-free culture medium and also includes the following components at the following concentrations: Anti-CD3 monoclonal antibody 160-200 ng / mL, anti-CD28 monoclonal antibody 50-80 ng / mL, interleukin-21 200-1400 IU / mL, interleukin-5 50-60 IU / mL, interleukin-1α 50-60 IU / mL, recombinant human granulocyte colony stimulating factor injection 3-5 μg / mL.

2. The preparation method according to claim 1, It is characterized in that The serum-free medium is based on serum-free RPMI-1640 medium and also includes the following components at the following concentrations: Interferon-γ 1500-2000 IU / mL, human serum albumin 12-16 mg / mL, 6-8% autologous serum, creatine phosphate 0.2-0.5 mg / mL.

3. The preparation method according to claim 2, It is characterized in that The culture temperature in step (1) is 23-25°C.

4. The preparation method according to claim 3, It is characterized in that The culture temperature in step (2) is 36-38° C., the concentration of carbon dioxide introduced during the culture is 4-6%, and the relative humidity during the culture is 92-95%.

5. The preparation method according to claim 4, It is characterized in that The final concentration of the yew extract added in step (2) is 0.1-0.3 mg / mL; The final concentration of ginsenoside added was 0.2-0.4 mg / mL.

6. The preparation method according to any one of claims 1 to 5, It is characterized in that The method for preparing the mononuclear cells is as follows: the mononuclear cells are collected by using a blood cell separator.

7. The enhanced CIK cell prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the enhanced CIK cells prepared by the preparation method according to any one of claims 1 to 6 or the enhanced CIK cells according to claim 7 in the preparation of a preparation for treating tumors.

9. A preparation for treating tumors, It is characterized in that The enhanced CIK cells include the enhanced CIK cells prepared by the preparation method according to any one of claims 1 to 6 or the enhanced CIK cells according to claim 7.

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