DC-CIK cell preparation, preparation method thereof and application of DC-CIK cell preparation in preparation of anti-cancer drugs

Abstract

The invention provides a DC-CIK cell preparation, a preparation method of the DC-CIK cell preparation and application of the DC-CIK cell preparation in preparation of anti-cancer drugs. The DC-CIK cell preparation comprises DC-CIK cells, 25-30% of human serum albumin and 1-2% of normal saline. The concentration of the DC-CIK cells is (3-6) * 10 < 7 > cells per mL of cells; the volume of the human serum albumin is 5-10 mL; the volume of the normal saline is 500 to 700 mL. DC and CIK culture solutions are optimized and modified, so that the cell amplification efficiency is obviously improved, the number of effective cells is obviously increased, the killing activity of the cells is improved, and the DC and CIK culture solutions are better applied to preparation of anti-cancer drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell preparation, and particularly relates to a DC-CIK cell preparation, a preparation method thereof and application of the DC-CIK cell preparation in preparation of an anticancer drug. BACKGROUND

[0002] With the development of science and technology, non-specific immune cells that can kill cancer cells, prevent recurrence and metastasis of cancer cells have become the research object of people. At present, the most commonly used non-specific immune cell treatment method is CIK cell and DC-CIK cell treatment.

[0003] Dendritic cells, namely DC cells, are widely distributed in blood, liver, spleen, lymph nodes and other non-immune organs and tissues, are the most powerful professional antigen presenting cells known at present, and play a key role in immune response. DC cells have a close relationship with the occurrence and development of tumors, can efficiently uptake, process and present antigens, and immature DC cells have strong migration ability; and mature DC cells can effectively activate initial T cells, and are in the central link of starting, regulating and maintaining immune response.

[0004] CIK is a group of heterogeneous cells obtained by culturing mononuclear cells under the action of anti-CD3 monoclonal antibody and various cytokines, in which CD3+, CD56+ lymphocytes are main effector cells. CIK can directly kill tumor cells and virus-infected cells, induce tumor cell apoptosis, and inhibit or kill tumor cells by releasing a large amount of inflammatory cytokines. CIK cells have the characteristics of fast proliferation speed, high tumor killing activity, wide tumor killing spectrum, sensitivity to multiple drug-resistant tumor cells, and small cytotoxicity to normal bone marrow hematopoietic precursor cells, are an ideal effector cell with strong tumor cell killing activity and suitable for clinical application, but such effector cells are extremely rare in normal peripheral blood, only accounting for 1-5%.

[0005] At present, the DC-CIK cell culture method is to add separated peripheral blood mononuclear cells into a culture solution, stimulate and induce by adding cell growth factors, and finally obtain a certain number of cells, but due to different culture solution components and culture time, the effective cell number and killing activity obtained are not ideal. SUMMARY

[0006] The purpose of the present application is to provide a DC-CIK cell preparation and a preparation method thereof, which significantly improve cell expansion efficiency, effective cell number and cell killing activity by optimizing and modifying the DC and CIK culture solution, and are better applied in preparation of an anticancer drug.

[0007] In order to achieve the above purpose, the present application provides the following technical solutions: A DC-CIK cell preparation, comprising DC-CIK cells, 25-30% human serum albumin and 1-2% physiological saline.

[0008] Further, the concentration of the DC-CIK cells is (3-6)×10 7 cells / mL; the human serum albumin is 5-10 mL; and the physiological saline is 500-700 mL.

[0009] A preparation method of a DC-CIK cell preparation, comprising the following steps: S1: separating mononuclear cells from peripheral blood, and dividing the cells into two parts; S2: culturing DC cells by adding half of the mononuclear cells separated in step S1 into a DC culture medium; S3: inducing CIK cells by adding the remaining cells in step S2 into a CIK culture medium; S4: mixing the DC cells and CIK cells obtained in step S2, washing with physiological saline, centrifuging and discarding the supernatant to obtain DC-CIK cells; S5: mixing the DC-CIK cells obtained in step S4 with 5-10 mL of 25-30% human serum albumin, stirring at a rate of 800-1000 r / min for 10-20 min; then adding 500-700 mL of 1-2% physiological saline, and continuing to mix for 5-10 min to obtain a DC-CIK cell preparation.

[0010] Further, the DC culture medium in step S2 comprises a basic culture medium, 30-50 ng / mL FBS, 50-100 ng / mL GM-CSF, 10-20 ng / mL TNF-α, 40-60 ng / mL ginseng peptide and 35-55 ng / mL IL-6.

[0011] Further, the DC culture medium in step S2 further comprises 1-3 ng / mL amphotericin B; and the basic culture medium is RPMI 1640.

[0012] Further, the culturing process of the DC in step S2 is as follows: inoculating the single cells into RPMI 1640 culture medium, culturing in a 37°C, 5% CO2 incubator for 3-6 h; removing the RPMI 1640 culture medium, adding a DC culture solution into the culture bottle, and culturing in a 37°C, 5% CO2 incubator for 7-10 days to obtain a DC cell culture solution; centrifuging the cell solution at a centrifugal force of 300-500 g at room temperature for 10-20 min, discarding the supernatant, and obtaining DC cells.

[0013] Further, the culture medium of the CIK in step S3 comprises: a basic culture medium, 1-5 μg / mL CD3 monoclonal antibody, 800-1200 IU / mL IFN-γ, 600-800 IU / mL IL-2, 80-100 U / mL penicillin and 1-3 μg / mL recombinant human albumin.

[0014] Further, the culture medium of the CIK in step S3 further comprises: 30-50 U / mL IL-1β, 10-20 U / mL IL-6 and 10-20 μg / mL HEPES buffer.

[0015] Further, the culture process of the CIK in step S3 is as follows: the single cells are inoculated in the IMDM culture medium and cultured at 37°C in a 5% CO2 incubator for 2-3 hours; the IMDM culture medium is removed and the CIK cells are cultured at 37°C in a 5% CO2 incubator for 10-14 days to obtain a CIK cell culture solution; the CIK cell solution is centrifuged at a centrifugal force of 350-550 g at room temperature for 5-10 minutes, and the supernatant is discarded to obtain the CIK cells.

[0016] The DC-CIK cell preparation can be in the form of a suspension, an emulsion, a tablet, a capsule, a granule, an oral liquid or an injection.

[0017] The DC-CIK cell preparation prepared by any one of the above can be used in the preparation of an anticancer drug.

[0018] Compared with the prior art, the DC-CIK cell preparation has the following advantages and beneficial effects: The basic culture medium, FBS, GM-CSF, TNF-α, ginseng peptide, IL-6, amphotericin B and the like are used as the DC culture medium, and the CD3 monoclonal antibody, IFN-γ, IL-2, penicillin, recombinant human albumin, IL-1β, IL-6 and HEPES buffer are used as the CIK culture medium, and under the synergistic action of the components, the DC precursor cells can be promoted to mature, the cell activity can be enhanced, the CIK cells can be promoted to differentiate and proliferate, and the killing activity of the DC-CIK cells can be improved. The DC-CIK cell preparation obtained by the method has a large number of effective cells, high activity and strong killing effect on tumor cells. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0020] The raw materials used in the present application are as follows: Human serum albumin (Shanghai Yuan Ye Biotechnology Co., Ltd., Item No. S12018); FBS (Aibisheng (Shanghai) Biotechnology Co., Ltd., Item No. abs983); GM-CSF (Wuhan Enji Life Science and Technology Co., Ltd., Item No. PCH90015); TNF-α (Shanghai Genechem Biotech Co., Ltd., Item No. 313); Ginseng peptide (Fufeng Snort Biotechnology Co., Ltd., Item No. 121); IL-6 (Fuainde Technology (Wuhan) Co., Ltd., Item No. 1812); Amphotericin B (Shanghai Yuan Ye Biotechnology Co., Ltd., Item No. S17017); RPMI 1640 (Topvivo Biotechnology Co., Ltd., Item No. 6); CD3 monoclonal antibody (Jiangsu Eastland Biopharmaceutical Technology Co., Ltd., Item No. 97); IFN-γ (Guangzhou Aolida Biotechnology Co., Ltd., Item No. 4335); IL-2 (Hunan Ruwei Biological Technology Co., Ltd., Item No. 2774); Penicillin (Hubei Widel Chemicals Co., Ltd., Item No. HBWS-2059); Recombinant human albumin (Qingdao Jetseikang Biotechnology Co., Ltd., Item No. USP1012595); IL-1β (Shanghai Xitang Biotechnology Co., Ltd., Item No. F01220); HEPES buffer (Guangdong Huan Kai Microbial Science and Technology Co., Ltd., Item No. 794).

[0021] Example 1 The present embodiment provides a preparation method of DC-CIK cell preparation, comprising the following steps: S1: separating mononuclear cells from peripheral blood, and dividing the cells into two parts; S2: inoculating half of the mononuclear cells separated in step S1 in RPMI 1640 culture medium, and culturing in a 37℃, 5% CO2 incubator for 3h; removing the RPMI 1640 culture medium, adding DC culture solution (30ng / mL FBS, 50ng / mL GM-CSF, 10ng / mL TNF-α, 40ng / mL ginseng peptide, 35ng / mL IL-6, 1ng / mL amphotericin B) into the culture bottle, and culturing in a 37℃, 5% CO2 incubator for 7 days to obtain DC cell culture solution; centrifuging the cell solution at a centrifugal force of 300g at room temperature for 10min, discarding the supernatant, and obtaining DC cells; S3: the remaining cells in step S2 are inoculated in IMDM medium and cultured at 37℃ in a 5% CO2 incubator for 2h; the IMDM medium is removed and CIK culture solution (1 μg / mL CD3 monoclonal antibody, 800 IU / mL IFN-γ, 600 IU / mL IL-2, 80 U / mL penicillin, 1 μg / mL recombinant human albumin, 30 U / mL IL-1β, 10 U / mL IL-6 and 10 μg / mL HEPES buffer) is added to the culture bottle, which is cultured at 37℃ in a 5% CO2 incubator for 10 days to obtain CIK cell culture solution; the CIK cell solution is centrifuged at 350g at room temperature for 5min, and the supernatant is discarded to obtain CIK cells; S4: the DC cells obtained in step S2 and the CIK cells in step S3 are mixed, washed with normal saline, and the supernatant is discarded after centrifugation to obtain DC-CIK cells; S5: the DC-CIK cells obtained in step S4 are mixed with 5mL 25% human serum albumin, stirred at a rate of 800r / min for 10min; then 500mL of 1% normal saline is added and mixed for 5min to obtain a DC-CIK cell preparation.

[0022] Example 2 The present embodiment provides a preparation method of DC-CIK cell preparation, comprising the following steps: S1: single nucleated cells are isolated from peripheral blood, and the cells are divided into two parts; S2: half of the single nucleated cells isolated in step S1 are inoculated in RPMI 1640 medium and cultured at 37℃ in a 5% CO2 incubator for 6h; the RPMI 1640 medium is removed and DC culture solution (50ng / mL FBS, 100ng / mL GM-CSF, 20ng / mL TNF-α, 60ng / mL ginseng peptide, 55ng / mL IL-6 and 3ng / mL amphotericin B) is added to the culture bottle, which is cultured at 37℃ in a 5% CO2 incubator for 10 days to obtain DC cell culture solution; the cell solution is centrifuged at 500g at room temperature for 20min, and the supernatant is discarded to obtain DC cells; S3: the remaining cells in step S2 are inoculated in IMDM medium and cultured in a 37℃, 5% CO2 incubator for 3h; the IMDM medium is removed, and CIK culture solution (5 μg / mL CD3 monoclonal antibody, 1200 IU / mL IFN-γ, 800 IU / mL IL-2, 100 U / mL penicillin, 3 μg / mL recombinant human albumin, 50 U / mL IL-1β, 20 U / mL IL-6 and 20 μg / mL HEPES buffer) is added to the culture bottle, which is cultured in a 37℃, 5% CO2 incubator for 14 days to obtain CIK cell culture solution; the CIK cell solution is centrifuged at 550g for 10min at room temperature, and the supernatant is discarded to obtain CIK cells; S4: the DC cells obtained in step S2 and the CIK cells in step S3 are mixed, washed with normal saline, and the supernatant is discarded after centrifugation to obtain DC-CIK cells; S5: the DC-CIK cells obtained in step S4 are mixed with 10 mL of 30% human serum albumin, and stirred at a rate of 1000 r / min for 20min; then 700 mL of 2% normal saline is added and mixed for 10min to obtain a DC-CIK cell preparation.

[0023] Example 3 The present embodiment provides a preparation method of DC-CIK cell preparation, comprising the following steps: S1: single nucleated cells are separated from peripheral blood, and the cells are divided into two parts; S2: half of the single nucleated cells separated in step S1 are inoculated in RPMI 1640 medium and cultured in a 37℃, 5% CO2 incubator for 5.5h; the RPMI 1640 medium is removed, and DC culture solution (40 ng / mL FBS, 75 ng / mL GM-CSF, 15 ng / mL TNF-α, 55 ng / mL ginseng peptide, 50 ng / mL IL-6, 2 ng / mL amphotericin B) is added to the culture bottle, which is cultured in a 37℃, 5% CO2 incubator for 8 days to obtain DC cell culture solution; the cell solution is centrifuged at 400g for 16min at room temperature, and the supernatant is discarded to obtain DC cells; S3: the residual cells in step S2 are inoculated in IMDM culture medium and cultured at 37℃ in a 5% CO2 incubator for 2.5 hours; the IMDM culture medium is removed, and CIK culture solution (basic culture medium RPMI 1640, 4 μg / mL CD3 monoclonal antibody, 1000 IU / mL IFN-γ, 700 IU / mL IL-2, 95 U / mL penicillin, 2 μg / mL recombinant human albumin, 45 U / mL IL-1β, 15 U / mL IL-6 and 16 μg / mL HEPES buffer) is added to the culture bottle, which is cultured at 37℃ in a 5% CO2 incubator for 12 days to obtain CIK cell culture solution; the CIK cell solution is centrifuged at a centrifugal force of 400 g at room temperature for 7 min, and the supernatant is discarded to obtain CIK cells; S4: the DC cells obtained in step S2 and the CIK cells in step S3 are mixed, washed with normal saline, and centrifuged to discard the supernatant to obtain DC-CIK cells; S5: the DC-CIK cells obtained in step S4 are mixed with 8 mL of 28% human serum albumin, stirred at a rate of 900 r / min for 15 min; then 650 mL of 2% normal saline is added, and the mixing is continued for 8 min to obtain a DC-CIK cell preparation.

[0024] The DC-CIK cell preparation obtained in Example 3 is applied to the preparation of an anticancer drug. In a logarithmic growth period human colorectal cancer SW480 and HCT116 cell suspension with a concentration of 5×104 / ml, the DC-CIK cell preparation is added to make the final concentration reach 0.1 μmol / L, 0.2 μmol / L, 0.4 μmol / L, 1 μmol / L and 2 μmol / L, which are co-cultured for 12 hours, 24 hours and 48 hours under the condition of 5% CO2 and 37℃. The CCK8 is used to determine the inhibitory effect of the DC-CIK cell preparation on the proliferation of human colorectal cancer cells SW480 and HCT116 for different culture times. The results show that the DC-CIK cell preparation can induce the apoptosis of human colorectal cancer cells SW480 and HCT116 in a concentration-dependent manner, and can induce the cycle arrest of human colorectal cancer cells SW480 and HCT116.

[0025] Comparative Example 1 The difference from Example 1 is that the DC culture medium in step S2 does not contain ginseng peptide, and the other steps are unchanged.

[0026] Comparative Example 2 The difference from Example 1 is that the DC culture medium in step S2 does not contain amphotericin B, and the other steps are unchanged.

[0027] Comparative Example 3 The difference from Example 1 is that the CIK culture medium in step S3 does not contain IFN-γ and IL-2, and other steps are unchanged.

[0028] Comparative Example 4 The difference from Example 1 is that the CIK culture medium in step S3 does not contain IL-1β, IL-6 and HEPES buffer, and other steps are unchanged.

[0029] The mature DC-CIK cells collected in each example and comparative example are counted by using a cell counting plate and the viability is calculated by using trypan blue staining method. The results are shown in the following table.

[0030] Table 1

[0031] From the performance test results, it can be seen that the DC-CIK cells prepared in Examples 1-3 have a large number and high viability. However, Comparative Examples 1-4 do not use the necessary technical solutions, resulting in a significant difference in the corresponding performance tests compared to the examples. The above experimental results further prove the importance of the technical solutions defined in the present application to its technical effects.

[0032] The above is the preferred embodiment of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made. These improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A DC-CIK cell preparation, characterized in that, The DC-CIK cells are mixed with 5-10 mL of 25-30% human serum albumin, stirred at a speed of 800-1000 r / min for 10-20 min, and then 500-700 mL of 1-2% physiological saline is added and mixed for 5-10 min to obtain the DC-CIK cell preparation. 2.The DC-CIK cell preparation of claim 1, characterized in that, The concentration of the DC-CIK cells is (3-6) x 10 7 cells / mL; the human serum albumin is 5-10 mL; and the physiological saline is 500-700 mL.

3. A method for preparing a DC-CIK cell preparation, characterized in that, The method comprises the following steps: S1: separating mononuclear cells from peripheral blood, and dividing the cells into two parts; S2: culturing the half of the mononuclear cells separated in step S1 in a DC culture medium to obtain DC cells; S3: culturing the remaining cells in step S2 in a CIK culture medium to induce CIK cells; S4: mixing the DC cells and the CIK cells obtained in step S2, washing with physiological saline, centrifuging and discarding the supernatant to obtain DC-CIK cells; S5: mixing the DC-CIK cells obtained in step S4 with 5-10 mL of 25-30% human serum albumin, stirring at a speed of 800-1000 r / min for 10-20 min, and then adding 500-700 mL of 1-2% physiological saline and mixing for 5-10 min to obtain the DC-CIK cell preparation. 4.The method for preparing DC-CIK cell preparation according to claim 3, characterized in that, The DC culture medium in step S2 comprises a basic culture medium, 30-50 ng / mL FBS, 50-100 ng / mL GM-CSF, 10-20 ng / mL TNF-α, 40-60 ng / mL ginseng peptide and 35-55 ng / mL IL-6.

5. The preparation method of DC-CIK cell preparation according to claim 3, characterized in that, The DC culture medium in step S2 further comprises 1-3 ng / mL amphotericin B, and the basic culture medium is RPMI 1640. 6.The preparation method of the DC-CIK cell preparation according to claim 3, characterized in that, The DC culture process in step S2 is as follows: the single cells are inoculated in the RPMI 1640 culture medium, and cultured in a 37°C, 5% CO2 incubator for 3-6 h; the RPMI 1640 culture medium is removed, the DC culture solution is added to the culture bottle, and the culture is carried out in a 37°C, 5% CO2 incubator for 7-10 days to obtain the DC cell culture solution; the cell solution is centrifuged at a centrifugal force of 300-500 g at room temperature for 10-20 min, and the supernatant is discarded to obtain the DC cells.

7. The preparation method of DC-CIK cell preparation according to claim 3, characterized in that, The CIK culture medium in step S3 comprises a basic culture medium, 1-5 μg / mL CD3 monoclonal antibody, 800-1200 IU / mL IFN-γ, 600-800 IU / mL IL-2, 80-100 U / mL penicillin and 1-3 μg / mL recombinant human albumin. 8.The preparation method of the DC-CIK cell preparation according to claim 3, characterized in that, The CIK culture medium in step S3 further comprises 30-50 U / mL IL-1β, 10-20 U / mL IL-6 and 10-20 μg / mL HEPES buffer. 9.The preparation method of the DC-CIK cell preparation according to claim 3, characterized in that, The CIK culture process in step S3 is as follows: the single cells are inoculated in the IMDM culture medium, and cultured in a 37°C, 5% CO2 incubator for 2-3 h; the IMDM culture medium is removed, and the culture is carried out in a 37°C, 5% CO2 incubator for 10-14 days to obtain the CIK cell culture solution; the CIK cell solution is centrifuged at a centrifugal force of 350-550 g at room temperature for 5-10 min, and the supernatant is discarded to obtain the CIK cells.

10. The use of the DC-CIK cell preparation prepared according to any one of claims 1-9 in the preparation of an anticancer drug.

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