Culture medium and culture method for persistent DC-CIK cells and application of culture medium and culture method

Abstract

The invention relates to a culture medium and a culture method of persistent DC-CIK cells and application of the culture medium and the culture method, and belongs to the technical field of cell culture. The invention provides a culture medium for persistent DC-CIK cells, which takes a serum-free culture medium as a solvent and comprises a recombinant human granulocyte macrophage colony stimulating factor, a recombinant human interleukin-12, a recombinant human tumor necrosis factor-alpha, a recombinant human interferon-gamma, L-cysteine, glutathione, ginseng polysaccharide and streptomycin sulfate. According to the culture medium and the culture method of the persistent DC-CIK cells, proliferation and growth of the DC-CIK cells can be promoted, and very high tumor killing activity of the DC-CIK cells can be maintained for a long time.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a culture medium and a culture method for persistent DC-CIK cells and applications thereof. Background Art

[0002] Cytokine-induced killer (CIK) cells are CD3 + Cells, CD56 + The effector cell group mainly composed of T cells can show the anti-tumor activity of T lymphocytes and the non-MHC restricted tumor killing activity of NK cells, and has the characteristics of rapid proliferation, high killing activity, broad tumor killing spectrum and small side effects. Dendritic cells (DC) are special cells with powerful antigen presentation function, which can efficiently absorb, process and present antigens, thereby activating initial T lymphocytes and triggering specific immune responses against tumor cells.

[0003] DC-CIK cells are a cell population obtained by co-culturing DC cells induced to mature by tumor antigens with CIK cells. Studies have found that co-culturing DC cells with CIK cells can not only promote the maturation of DC cells, but also enhance the cytotoxic T cell content of CIK cells, thereby exerting a specific killing effect on tumor cells, effectively eliminating residual lesions in the patient's body, and inducing immune memory in the patient to maintain a lasting anti-tumor effect.

[0004] However, in the existing DC-CIK cell co-culture method, the survival period of DC cells is short, while the culture time of CIK cells is long, which makes the culture time of DC cells and CIK cells asynchronous, and the culture environment of CIK cells is not conducive to the proliferation and growth of DC cells. Therefore, the effective cell content in the cultured DC-CIK cells is too small and the killing activity is low, and the tumor killing activity of DC-CIK cells cannot be maintained for a long time. Summary of the invention

[0005] The object of the present invention is to provide a culture medium and a culture method for persistent DC-CIK cells and their application, so as to solve the problems of low effective cell content, insufficient tumor killing activity and short effectiveness maintenance time of DC-CIK cells in the prior art.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a culture medium for persistent DC-CIK cells. The culture medium uses serum-free culture medium as solvent and comprises components with the following concentrations: 150-270 μg / L of recombinant human granulocyte macrophage colony stimulating factor, 1.2-2.8 μg / L of recombinant human interleukin-1, 65-105 μg / L of recombinant human tumor necrosis factor-α, 1200-1500 IU / mL of recombinant human interferon-γ, 180-360 μmol / L of L-cysteine, 80-200 μmol / L of glutathione, 12-20 mg / L of ginseng polysaccharide and 25-75 mg / L of streptomycin sulfate.

[0008] Preferably, the serum-free culture medium includes X-VIVO15 culture medium and GT-T551 H3 culture medium;

[0009] The volume ratio of the X-VIVO15 culture medium to the GT-T551 H3 culture medium is 1:2-5.

[0010] The present invention also provides application of the culture medium in culturing persistent DC-CIK cells.

[0011] The present invention also provides a method for culturing persistent DC-CIK cells, comprising the following steps:

[0012] (1) DC cells and CIK cells were inoculated into culture medium and cultured for 12 to 36 hours to obtain cell fluid;

[0013] (2) adding recombinant human granulocyte macrophage colony stimulating factor to the cell fluid described in step (1), culturing for 10 to 15 days, and obtaining persistent DC-CIK cells;

[0014] The culture medium described in step (1) is the culture medium of the persistent DC-CIK cells.

[0015] Preferably, the inoculation amount of DC cells in the culture medium in step (1) is 2.0×10 3 ~5.0×10 3 Pieces / mL;

[0016] The inoculation amount of CIK cells in the culture medium was 2.0×10 5 ~5.0×10 5 Pieces / mL.

[0017] Preferably, the mass volume ratio of recombinant human granulocyte macrophage colony stimulating factor and cell fluid added in step (2) is 7-12 mg:1L.

[0018] Preferably, the culture temperature in steps (1) to (2) is 35 to 38° C., and the CO 2 concentration in the culture is 4% to 8%.

[0019] The present invention also provides application of the culture method in culturing persistent DC-CIK cells.

[0020] The present invention also provides persistent DC-CIK cells cultured by the culture method.

[0021] The present invention also provides the use of the persistent DC-CIK cells in preparing drugs for preventing and / or treating tumors.

[0022] The present invention has the following technical effects and advantages:

[0023] In the culture medium of the persistent DC-CIK cells prepared by the present invention, X-VIVO15 culture medium and GT-T551 The serum-free medium obtained by mixing H3 culture medium has higher types and contents of nutrients, which is more conducive to the culture of DC-CIK cells; recombinant human granulocyte macrophage colony stimulating factor can promote the survival of DC cells and enhance the antigen presentation function of cells; recombinant human interleukin-12 can induce cells to secrete GM-CSF, IFN-γ, TNF-α and other cytokines, start CIK cell culture, promote CIK cell proliferation, and help to improve the toxic effect of CIK cells; recombinant human tumor necrosis factor-α can promote immature DC cells to differentiate into mature DC cells, and significantly enhance the antigen presentation ability of cells; recombinant human interferon-γ can promote CIK cell maturation, enhance the antigen presentation function of cells, and mediate Th1 immune response; L-cysteine ​​can stimulate and promote the proliferation and growth of DC-CIK cells; glutathione can protect DC-CIK cells from oxidative stress and maintain normal tumor killing function; ginseng polysaccharide can significantly enhance and maintain the tumor killing function of DC-CIK cells. The culture medium for persistent DC-CIK cells prepared by the present invention can be used for co-culture of DC cells and CIK cells, and can also promote the proliferation and growth of DC-CIK cells, and maintain the high tumor killing activity of DC-CIK cells for a long time;

[0024] Compared with the prior art, the culture method of the persistent DC-CIK cells of the present invention has simple culture steps and a shorter culture time, and can increase the proportion of NKT cells and CTL cells with tumor killing activity in DC-CIK cells. DETAILED DESCRIPTION

[0025] The present invention provides a culture medium for persistent DC-CIK cells, wherein the culture medium uses a serum-free culture medium as a solvent and comprises the following components at the following concentrations: 150 to 270 μg / L of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF), preferably 180 to 240 μg / L, and more preferably 220 μg / L; 1.2 to 2.8 μg / L of recombinant human interleukin-12 (rhIL-12), preferably 1.8 to 2.5 μg / L, and more preferably 2 μg / L; 65 to 105 μg / L of recombinant human tumor necrosis factor-α (rhTNF-α), preferably 80 to 100 μg / L, and more preferably 90 μg / L; and 150 to 270 μg / L of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF), preferably 180 to 240 μg / L, and more preferably 220 μg / L. -γ) 1200~1500IU / mL, preferably 1250~1450IU / mL, more preferably 1300IU / mL; L-cysteine ​​180~360μmol / L, preferably 200~300μmol / L, more preferably 250μmol / L; glutathione (GSH) 80~200μmol / L, preferably 120~180μmol / L, more preferably 150μmol / L; ginseng polysaccharide 12~20mg / L, preferably 14~18mg / L, more preferably 16mg / L; streptomycin sulfate (Strep) 25~75mg / L, preferably 30~60mg / L, more preferably 50mg / L.

[0026] In the present invention, the serum-free culture medium includes X-VIVO15 culture medium and GT-T551 H3 culture medium;

[0027] The volume ratio of the X-VIVO15 culture medium to the GT-T551 H3 culture medium is 1:2 to 5, preferably 1:3.

[0028] The present invention also provides application of the culture medium in culturing persistent DC-CIK cells.

[0029] The present invention also provides a method for culturing persistent DC-CIK cells, comprising the following steps:

[0030] (1) inoculating DC cells and CIK cells in a culture medium and culturing for 12 to 36 hours, preferably 24 hours, to obtain a cell fluid;

[0031] (2) adding rhGM-CSF to the cell solution described in step (1), and culturing for 10 to 15 days, preferably 14 days, to obtain persistent DC-CIK cells;

[0032] The culture medium described in step (1) is the culture medium of the persistent DC-CIK cells.

[0033] In the present invention, the inoculation amount of DC cells in the culture medium in step (1) is 2.0×10 3 ~5.0×10 3 / mL, preferably 4.0×10 3 Pieces / mL;

[0034] The inoculation amount of CIK cells in the culture medium was 2.0×10 5 ~5.0×10 5 / mL, preferably 4.0×10 5 Pieces / mL.

[0035] In the present invention, the mass volume ratio of rhGM-CSF and cell fluid added in step (2) is 7-12 mg:1L, preferably 10 mg:1L.

[0036] In the present invention, the culture temperature in steps (1) to (2) is 35 to 38°C, preferably 37°C; the CO2 concentration in the culture is 4% to 8%, preferably 7.5%.

[0037] The present invention also provides application of the culture method in culturing persistent DC-CIK cells.

[0038] The present invention also provides persistent DC-CIK cells cultured by the culture method.

[0039] The present invention also provides the use of the persistent DC-CIK cells in preparing drugs for preventing and / or treating tumors.

[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] In the present invention, X-VIVO15 culture medium was purchased from Beijing Bitbo Biotechnology Co., Ltd., GT-T551H3 culture medium was purchased from Shanghai Huaya Sichuang Biotechnology Co., Ltd., rhGM-CSF and rhTNF-α were purchased from Pepro Tech, USA, rhIL-12 was purchased from Jiangsu Dongkang Biopharmaceutical Technology Co., Ltd., rhIFN-γ was purchased from Shanghai Kaimao Biopharmaceutical Co., Ltd., L-cysteine ​​and Strep were purchased from Shanghai Yuanye Biotechnology Co., Ltd., GSH was purchased from Xi'an Zebang Biotechnology Co., Ltd., and ginseng polysaccharide was purchased from Shaanxi Ciyuan Biotechnology Co., Ltd.;

[0042] In the present invention, DC cells and CIK cells were purchased from Shanghai Kanglang Biotechnology Co., Ltd.; human renal cell adenocarcinoma cell line (ACHN) and human hepatoma cell line (Huh-7) were purchased from Wuhan Pronocell Life Science Co., Ltd.

[0043] Example 1: Culture medium for persistent DC-CIK cells

[0044] Referring to the instructions for use of X-VIVO15 medium and GT-T551 H3 medium, 300 mL of X-VIVO15 medium and 900 mL of GT-T551 H3 medium were prepared respectively, and then mixed evenly to obtain serum-free medium; 220 μg rhGM-CSF, 2 μg rhIL-12, 90 μg rhTNF-α, 1300000 IU rhIFN-γ, 250 μmol L-cysteine, 150 μmol GSH, 16 mg ginseng polysaccharide, and 50 mg Strep were added to a fixed volume of 1 L serum-free medium, filtered, and obtained a culture medium for persistent DC-CIK cells.

[0045] Example 2: Culture medium for persistent DC-CIK cells

[0046] Referring to the instructions for use of X-VIVO15 medium and GT-T551 H3 medium, 200 mL of X-VIVO15 medium and 1000 mL of GT-T551 H3 medium were prepared respectively, and then mixed evenly to obtain serum-free medium; 180 μg rhGM-CSF, 2.8 μg rhIL-12, 105 μg rhTNF-α, 1250000 IU rhIFN-γ, 300 μmol L-cysteine, 120 μmol GSH, 20 mg ginseng polysaccharide, and 60 mg Strep were fixed to 1 L of serum-free medium and filtered to obtain a culture medium for persistent DC-CIK cells.

[0047] Example 3: Culture medium for persistent DC-CIK cells

[0048] Referring to the instructions for use of X-VIVO15 medium and GT-T551 H3 medium, 400 mL of X-VIVO15 medium and 800 mL of GT-T551 H3 medium were prepared respectively, and then mixed evenly to obtain serum-free medium; 270 μg rhGM-CSF, 1.8 μg rhIL-12, 80 μg rhTNF-α, 1500000 IU rhIFN-γ, 180 μmol L-cysteine, 200 μmol GSH, 18 mg ginseng polysaccharide, and 75 mg Strep were added to a fixed volume of 1 L serum-free medium, filtered, and obtained a culture medium for persistent DC-CIK cells.

[0049] Comparative Example 1: Culture medium of DC-CIK cells

[0050] Referring to the instructions for use of X-VIVO15 medium and GT-T551 H3 medium, 300 mL of X-VIVO15 medium and 900 mL of GT-T551 H3 medium were prepared respectively, and then mixed evenly to obtain serum-free medium; 220 μg rhGM-CSF, 2 μg rhIL-12, 90 μg rhTNF-α, 1300000 IU rhIFN-γ, 150 μmol GSH, 16 mg ginseng polysaccharide, and 50 mg Strep were added to a fixed volume of 1 L serum-free medium and filtered to obtain a culture medium for DC-CIK cells.

[0051] Comparative Example 2: Culture medium of DC-CIK cells

[0052] Referring to the instructions for use of X-VIVO15 medium and GT-T551 H3 medium, 300 mL of X-VIVO15 medium and 900 mL of GT-T551 H3 medium were prepared respectively, and then mixed evenly to obtain serum-free medium; 220 μg rhGM-CSF, 2 μg rhIL-12, 90 μg rhTNF-α, 1300000 IU rhIFN-γ, 250 μmol L-cysteine, 16 mg ginseng polysaccharide, and 50 mg Strep were added to a fixed volume of 1 L serum-free medium, filtered, and obtained a culture medium for DC-CIK cells.

[0053] Comparative Example 3: Culture medium of DC-CIK cells

[0054] Referring to the instructions for use of X-VIVO15 medium and GT-T551 H3 medium, 300 mL of X-VIVO15 medium and 900 mL of GT-T551 H3 medium were prepared respectively, and then mixed evenly to obtain serum-free medium; 220 μg rhGM-CSF, 2 μg rhIL-12, 90 μg rhTNF-α, 1300000 IU rhIFN-γ, 250 μmol L-cysteine, 150 μmol GSH, and 50 mg Strep were added to a fixed volume of 1 L serum-free medium and filtered to obtain a culture medium for DC-CIK cells.

[0055] Experimental Example 1: Preparation of DC-CIK cells

[0056] (1) DC cells and CIK cells that were revived and cultured for 7 days were co-inoculated in 50 mL of the culture medium of the persistent DC-CIK cells described in Examples 1 to 3 and the culture medium of the DC-CIK cells described in Comparative Examples 1 to 3 for mixed culture, wherein the inoculation amount of DC cells in each culture medium was 4.0×10 3 The inoculation amount of CIK cells in each culture medium was 4.0×105 / mL, and then cultured at 37℃, CO2 concentration of 7.5% for 24h to obtain cell fluid;

[0057] (2) Add 10 mg of rhGM-CSF to each liter of cell fluid, and culture at 37°C and 7.5% CO2 for 14 days to obtain DC-CIK cells.

[0058] Experimental Example 2: Cell Phenotype Detection of DC-CIK Cells

[0059] DC-CIK cells are mainly composed of two cell subsets with tumor killing activity, namely NKT cells and CTL cells. The cell phenotype of NKT cells is CD3 + CD56 + The cell phenotype of CTL cells is CD3 + CD8 + The DC-CIK cells cultured in Experimental Example 1 were adjusted into a single cell suspension, washed, and labeled with CD3, CD56, and CD8 flow cytometry antibodies. The percentage of surface markers of DC-CIK cells was detected by flow cytometry. The results are shown in Table 1.

[0060] The results showed that compared with Comparative Examples 1 to 3, the proportion of NKT cells and CTL cells with cytotoxic activity in DC-CIK cells cultured using the culture medium prepared in Examples 1 to 3 was higher, indicating that abandoning L-cysteine, glutathione, and ginseng polysaccharides respectively can reduce the effective cell content of DC-CIK cells to varying degrees.

[0061] Table 1 Cell phenotype of DC-CIK cells

[0062] Cell phenotype <![CDATA[CD3 + CD56 + ]]> <![CDATA[CD3 + CD8 + ]]> Example 1 72.19％ 98.22％ Example 2 67.33％ 94.45％ Example 3 69.26％ 96.18％ Comparative Example 1 49.82％ 82.33％ Comparative Example 2 35.26％ 74.95％ Comparative Example 3 36.74％ 65.29％

[0063] Experimental Example 3: Killing detection of DC-CIK cells

[0064] The DC-CIK cells cultured in Experimental Example 1 were used as effector cells, and human renal cell adenocarcinoma cell line (ACHN) and human hepatoma cell line (Huh-7) were used as target cells. The effector cells and target cells were plated in a 96-well plate at a seeding ratio of 5:1, and co-cultured for 24 h at 37 ° C and a CO2 concentration of 7.5%, wherein the culture wells in which only DC-CIK cells were added were used as negative controls, and the culture wells in which only ACHN or Huh-7 cell lines were added were used as positive controls; after the co-culture, 10 μL of CCK-8 reagent was added to each well for staining, and after incubation for 2 h at 37 ° C and a CO2 concentration of 7.5%, the OD of each well was measured. 450nm The killing rate of DC-CIK cells was calculated and the results are shown in Table 2.

[0065] The formula for calculating the kill rate is:

[0066]

[0067] Killing rate (%) = 1 - cell survival rate.

[0068] The results show that the killing rates of DC-CIK cells cultured in the culture medium prepared in Examples 1 to 3 against ACHN and Huh-7 cell lines are all above 90%, while the killing rates of DC-CIK cells cultured in the culture medium in which L-cysteine, glutathione and ginseng polysaccharide are respectively discarded in Comparative Documents 1 to 3 against ACHN and Huh-7 cell lines are significantly reduced. This indicates that L-cysteine, glutathione and ginseng polysaccharide play an important role in promoting the proliferation and growth of DC-CIK cells and maintaining tumor killing activity.

[0069] Table 2 Cytotoxic activity of DC-CIK cells against ACHN and Huh-7 cell lines

[0070]

[0071]

[0072] Experimental Example 4: Persistence Detection of DC-CIK Cells

[0073] The DC-CIK cells cultured in Experimental Example 1 were cultured continuously for 14 days at 37°C and a CO2 concentration of 7.5%. DC-CIK cells were collected on the 7th and 14th days of continuous culture, and the CD3 expression of DC-CIK cells was detected according to the method described in Experimental Example 2. + CD56 + The results are shown in Table 3.

[0074] The results show that compared with comparative examples 1 to 3, after the DC-CIK cells obtained by culture in Examples 1 to 3 were continuously cultured for 14 days, the proportion of NKT cells and CTL cells in the DC-CIK cells was still greater than 80%; and abandoning L-cysteine, glutathione, and ginseng polysaccharide respectively can reduce the persistence of DC-CIK cells to varying degrees. It shows that the culture medium and culture method of the present invention can prolong the persistence of the killing activity of DC-CIK cells.

[0075] Table 3 Cell phenotypes of DC-CIK cells after continuous culture

[0076] Detection time No. 7d 14d Example 1 96.45％ 90.03％ Example 2 87.36％ 82.19％ Example 3 86.75％ 80.97％ Comparative Example 1 69.88％ 64.35％ Comparative Example 2 47.33％ 28.18％ Comparative Example 3 51.46％ 38.72％

[0077] As can be seen from the above examples, the present invention provides a culture medium and a culture method for persistent DC-CIK cells and their applications. The culture medium and culture method for persistent DC-CIK cells of the present invention can increase the ratio of NKT cells and CTL cells in DC-CIK cells, promote the proliferation and growth of DC-CIK cells, and maintain the high tumor killing activity of DC-CIK cells for a long time.

[0078] 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 culture medium for persistent DC-CIK cells, characterized in that: The culture medium uses serum-free culture medium as solvent and includes components with the following concentrations: recombinant human granulocyte macrophage colony stimulating factor 150-270 μg / L, recombinant human interleukin-1 21.2-2.8 μg / L, recombinant human tumor necrosis factor-α 65-105 μg / L, recombinant human interferon-γ 1200-1500 IU / mL, L-cysteine ​​180-360 μmol / L, glutathione 80-200 μmol / L, ginseng polysaccharide 12-20 mg / L, and streptomycin sulfate 25-75 mg / L.

2. The culture medium according to claim 1, characterized in that The serum-free culture medium includes X-VIVO15 culture medium and GT-T551 H3 culture medium; The volume ratio of the X-VIVO15 culture medium to the GT-T551 H3 culture medium is 1:2-5.

3. Use of the culture medium according to claim 1 or 2 in culturing persistent DC-CIK cells.

4. A method for culturing persistent DC-CIK cells, characterized in that: The steps include: (1) DC cells and CIK cells were inoculated into culture medium and cultured for 12 to 36 hours to obtain cell fluid; (2) adding recombinant human granulocyte macrophage colony stimulating factor to the cell fluid described in step (1), culturing for 10 to 15 days, and obtaining persistent DC-CIK cells; The culture medium described in step (1) is the culture medium of the persistent DC-CIK cells described in claim 1.

5. The culture method according to claim 4, characterized in that The inoculation amount of DC cells in the culture medium in step (1) is 2.0×10 3 ~5.0×10 3 Pieces / mL; The inoculation amount of CIK cells in the culture medium was 2.0×10 5 ~5.0×10 5 Pieces / mL.

6. The culture method according to claim 5, characterized in that: The mass volume ratio of the recombinant human granulocyte macrophage colony stimulating factor and the cell fluid added in step (2) is 7-12 mg:1L.

7. The culture method according to claim 6, characterized in that: The culture temperature in steps (1) to (2) is 35 to 38°C, and the CO2 concentration in the culture is 4% to 8%.

8. Use of the culture method according to any one of claims 4 to 7 in culturing persistent DC-CIK cells.

9. Persistent DC-CIK cells cultured by the culture method according to any one of claims 4 to 7.

10. Use of the persistent DC-CIK cells according to claim 9 in the preparation of drugs for preventing and / or treating tumors.

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