CN109535241B - DC-CIK (dendritic cell-cytokine induced killer) co-culture cell, preparation method thereof, sensitizing antigen and application - Google Patents

Abstract

The invention discloses a DC-CIK co-cultured cell, a preparation method thereof, a sensitizing antigen and application. The DC-CIK co-culture cell is obtained by respectively inducing and culturing separated mononuclear cells into a DC cell and a CIK cell, wherein the DC cell is added with a self-constructed polypeptide sensitizing antigen which is verified to be widely expressed in various tumor cells in the culture process for inducing stimulation, so that the tumor killing effect can be improved. The DC-CIK co-cultured cell obtained by the method has a good killing effect on various tumor cells, and the cultured DC-CIK cell has high activity and strong amplification capability, releases more cytokines such as IFN-gamma and the like, and has strong killing power. The co-cultured cell can be widely used for treating various cancers, including renal cancer, prostatic cancer, breast cancer, bladder cancer, melanoma, colon cancer, ovarian cancer, bone cancer, lung cancer, neuroblastoma and the like, and has wide application range.

Description

DC-CIK (dendritic cell-cytokine induced killer) co-culture cell, preparation method thereof, sensitizing antigen and application

Technical Field

The invention relates to the field of immune cells, in particular to a DC-CIK co-cultured cell and a preparation method, a sensitizing antigen and application thereof.

Background

The DC-CIK cell therapy is a mixed therapy of killer cells and dendritic cells activated by cytokines, and comprises two cells of killer immune cells and dendritic cells. During tumor treatment, DC cells recognize antigens first, and then activate the adaptive immune system; the activated CIK cells can kill tumor cells efficiently by exerting self cytotoxicity and secreting cell factors.

Dendritic Cells (DCs) are the most powerful professional antigen presenting cells, so called because they mature with a dendritic-like or pseudopodoid membranous protrusion extending from the cell surface. DCs originate from bone marrow CD34+ HSC precursor cells and are normally distributed in small but widespread amounts throughout the body in various organs other than the brain, such as cord blood, human bone marrow, and peripheral blood. The precursor cells can be induced to mature DCs in vitro by the addition of a series of cytokines GM-CSF, IL-4, TNF-alpha, IL-1 beta, and the like. Mature DC cells present antigen and can activate naive T cells, which trigger the body to generate anti-tumor response.

CIK cells are a heterogeneous cell group mainly comprising CD3+ CD56, which is cultured by in vitro cytokine induction of human peripheral blood mononuclear cells, are also called NK cell-like T lymphocytes and have the advantages of anti-tumor activity of the T lymphocytes and non-MIH-limited tumor killing of the NK cells. CIK cells can directly kill tumor cells and cells infected by viruses by releasing cytoplasmic toxic particles (granzyme or perforin), and simultaneously the CIK cells generate a large amount of inflammatory cytokines (such as IFN-gamma, TNF-alpha, IL-6 and the like), so that the cytotoxicity of immune effector cells is improved or the sensitivity of the tumor cells to CIK is regulated, and the tumor growth and virus replication are inhibited.

DCs play an important role in activation and differentiation of T cells as powerful professional antigen-presenting cells. The CIK cells and the DC are combined to treat the tumor, so that the immunity incapability phenomenon of T cells on partial tumor cells is avoided, and the synergistic anti-tumor effect can be exerted. After the DC is combined with the CIK, the cell proliferation activity, the tumor killing activity and the release amount of cytokines of the CIK are obviously improved and increased. And the antigen-loaded DC can stimulate CIK cells to be further activated, release more cytokines and have higher tumoricidal activity. The antigen used by the existing in vitro sensitization DC is mainly known specific tumor-related antigen, tumor cell strain lysate or patient tumor specimen lysate. The tumor cell line lysate or the lysate of a patient tumor specimen has higher risk and the antigen components in the lysate are undefined; the known DC-CIK cells sensitized by specific tumor-associated antigens aim at specific tumors, cannot play a strong killing role on various tumors, and limit the in-vivo anti-tumor effect of the DC-CIK cells sensitized by the tumor antigens.

Disclosure of Invention

Based on the above, a DC-CIK co-cultured cell, a preparation method thereof, a sensitizing antigen and an application thereof are needed to be provided, so that the problems of poor anti-tumor killing effect and large limitation of the traditional DC-CIK co-cultured cell are solved.

The sensitizing antigen of the DC-CIK cocultured cell contains at least one polypeptide of a polypeptide 1 and a polypeptide 2, and the amino acid sequences of the polypeptide 1 and the polypeptide 2 are respectively shown as SEQ ID No.1 and SEQ ID No. 2.

In one embodiment, the sensitizing antigen contains the polypeptide 1 and the polypeptide 2 at the same time, and the molar ratio of the polypeptide 1 to the polypeptide 2 is (0.2-5): 1.

In one embodiment, the sensitizing antigen is used at a concentration of 2. mu.M to 20. mu.M.

In one embodiment, hemocyanin KLH is coupled to the polypeptide 1 and the polypeptide 2, and the coupling rate is not less than 90%.

A preparation method of DC-CIK co-cultured cells comprises the following steps:

collecting mononuclear cells, respectively inducing and culturing DC cells and CIK cells, and adding the sensitizing antigen described in any one of the embodiments in the induction culture process of the DC cells for stimulation induction;

and (3) co-culturing the DC cells after the stimulation and induction of the sensitizing antigen and the CIK cells.

In one embodiment, the number ratio of DC cells after the stimulation and induction of the sensitizing antigen to CIK cells in the co-culture is 1 (5-20).

In one embodiment, the mononuclear cells are collected from fresh peripheral blood or cord blood.

In one embodiment, the separately induced culturing of DC and CIK cells, the stimulation induction of the sensitizing antigen, and the co-culturing comprise the steps of:

according to 2-4 x 10⁶Inoculating the mononuclear cells into an adherent culture container at a cell/ml density, standing for a period of time, slightly shaking the culture container, collecting cells which do not adhere to the wall, adding the adherent cells into a DC culture medium for continuous culture, wherein the DC culture medium is an AIM-V culture medium added with 2-5% of autologous serum, 20-200 ng/ml of GM-CSF and 20-200 ng/ml of IL-4, the cells which do not adhere to the wall are cultured by using a CIK culture medium for induction culture, and the CIK culture medium is an AIM-V culture medium added with 2-5% of autologous serum, 500-2000 ng/ml of IFN-gamma and 500-2000 ng/ml of IL-2;

respectively adding a CD3 antibody and a CD28 antibody with the final concentration of 200-1000 ng/ml into a CIK cell culture solution on the 2 nd day for culture;

adding the sensitizing antigen into the DC cell culture solution on the 4 th day, and continuously culturing;

addition of pro-mature cytokines to the DC cell culture broth on day 6: continuing induction culture of TNF-alpha with the final concentration of 20-200 ng/ml, IL-1 beta with the final concentration of 5-50 ng/ml and PEG2 with the final concentration of 1-5 mu g/ml;

mature DC cells and CIK cells were harvested on day 8 and the DC cells were co-cultured with CIK cells.

A DC-CIK co-cultured cell prepared by the preparation method of any one of the above embodiments.

The DC-CIK co-cultured cell can be applied to the preparation of cell medicines for treating tumors.

The DC-CIK co-culture cell is obtained by respectively inducing and culturing separated mononuclear cells into DC cells and CIK cells, wherein the DC cells are induced and stimulated by adding self-constructed polypeptide sensitizing antigen which is verified to be widely expressed in various tumor cells in the culture process, so that the tumor killing effect can be improved. The DC-CIK co-cultured cell obtained by the method has a good killing effect on various tumor cells, and the cultured DC-CIK cell has high activity and strong amplification capability, releases more cytokines such as IFN-gamma and the like, and has strong killing power.

Furthermore, in the culture process, the mononuclear cells are induced to differentiate into DC and CIK cells by using various cytokines in vitro, and the antigen polypeptide mixture which is highly expressed in various cancer cells is added for stimulation, so that the tumor cell killing efficiency of the mononuclear cells is greatly improved; moreover, the DC-CIK co-cultured cell prepared by the preparation method can be widely used for treating various cancers, including renal cancer, prostatic cancer, breast cancer, bladder cancer, melanoma, colon cancer, ovarian cancer, bone cancer, lung cancer, neuroblastoma and the like, and has wide application range.

On the source of the mononuclear cells, fresh peripheral blood or cord blood can be used, and when the cord blood is used for culturing the DC-CIK co-culture cells activated by the antigen, the cell immunogenicity of the cord blood is low, so that the cord blood can be subjected to foreign body back transfusion treatment, the problem that part of patients are difficult to prepare qualified immune cells can be solved, and the clinical application is facilitated.

Furthermore, the invention uses the autologous serum to induce and expand the cells, reduces the possible immunogenicity risk of the allogenic serum, and improves the applicability of the DC-CIK co-cultured cells.

Drawings

FIG. 1 is an under-the-lens morphology (10X 10) of harvested DC mature cells, showing that the DC cells are more pseudopodic in extension with burr-like dendrites, as indicated by the black arrows.

FIG. 2 is a histogram of the phenotype CD80/CD86/CD83/HLA-DR flow of DC cells.

FIG. 3 shows the phenotype of CIK cells.

FIG. 4 is a graph showing the killing efficiency of sensitized antigen-activated DC-CIK co-cultured cells against HepG2 liver cancer cells; wherein the abscissa is the ratio of CIK cells to HepG2 cells, and the ordinate represents the killing efficiency.

FIG. 5 is a graph showing IFN-. gamma.release upon killing HepG2 liver cancer cells; the abscissa is the ratio of CIK cells to HepG2 cells and the ordinate represents the concentration (in pg/mL).

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention provides a sensitizing antigen of DC-CIK co-cultured cells, which contains at least one polypeptide of polypeptide 1 and polypeptide 2. The polypeptide 1 and the polypeptide 2 are broad-spectrum polypeptides which are widely expressed in various tumor cells, such as cancer cells of kidney cancer, prostate cancer, breast cancer, bladder cancer, melanoma, colon cancer, ovarian cancer, bone cancer, lung cancer, neuroblastoma and the like. The amino acid sequence of the polypeptide 1 is VLFIAKITPNNNGTYACFVSNLAT (SEQ ID NO.1), and the amino acid sequence of the polypeptide 2 is SAENFTVLIKNNIDFPGHNYTTRNILP (SEQ ID NO. 2).

The polypeptide 1 and the polypeptide 2 can be synthesized by SPSS (solid phase polypeptide synthesis method) or genetically engineered for transgenic expression. The purity requirement of the synthesized or expressed purified polypeptide 1 and polypeptide 2 is not less than 98%. The synthesized polypeptide can be coupled with hemocyanin KLH (polypeptide 1: VLFIAKITPNNNGTYACFVSNLAT-KLH and polypeptide 2: SAENFTVLIKNNIDFPGHNYTTRNILP-KLH) by adopting an SMCC method, and the coupling rate needs to be more than 90%.

In a specific example, the two polypeptides are mixed at a ratio of (0.2-5): 1, such as 0.2:1, 0.5:1, 0.8:1, 1:1, 1.2:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1, preferably at a ratio of 1:1, to form the sensitizing antigen of the antigen-polypeptide mixture.

More specifically, in one example, the sensitizing antigen is used at a concentration of 2. mu.M to 20. mu.M, such as 2. mu.M, 5. mu.M, 8. mu.M, 9. mu.M, 9.5. mu.M, 10. mu.M, 10.5. mu.M, 12. mu.M, 15. mu.M, 18. mu.M, 19. mu.M, or 20. mu.M.

The invention also provides a preparation method of the DC-CIK co-culture cell, which comprises the following steps:

collecting mononuclear cells, respectively carrying out induction culture on DC cells and CIK cells, and adding the sensitizing antigen to the induction culture process of the DC cells for stimulation induction;

and (3) co-culturing the DC cells after the stimulation and induction of the sensitizing antigen and the CIK cells.

In one particular example, the collection of mononuclear cells is from fresh peripheral blood or cord blood. Preferably, collecting fresh peripheral blood or cord blood, adding anticoagulant, avoiding freezing or refrigerating in the transportation process, and performing cell separation within 2h after collection; during separation, peripheral blood or cord blood is added into 50ml of human lymphocyte separation tubes, 20ml of the human lymphocyte separation tubes are added into each separation tube, the setting parameters are 20-25 ℃, the speed of acceleration and deceleration are respectively 9 and 0, and the separation tubes are centrifuged for 20min at 800-1000 g; after the centrifugation is finished, respectively collecting the upper part of plasma and the middle white membrane layer by using a Pasteur pipette, and transferring the plasma and the middle white membrane layer into a 50ml centrifuge tube; adding part of the plasma into normal saline to obtain a buffer solution with the final volume concentration of the normal saline and the plasma being 2%, inactivating the rest plasma for 30min at 56 ℃, then centrifuging, setting the parameters to be 20-25 ℃, increasing and decreasing the speed to 9, centrifuging for 5min at 500g, and removing the precipitate after centrifuging to obtain the autoserum; washing the leucocyte layer with a buffer solution with the final volume concentration of 2% of physiological saline and plasma, centrifuging at 20-25 ℃ at the speed of 9 and 1 respectively and 300g for 5min, removing the supernatant after centrifugation, and repeating the centrifugation step for washing once again to obtain the mononuclear cell.

In a specific example, the number ratio of DC cells to CIK cells after induction of sensitization antigen stimulation is 1 (5-20), such as 1:5, 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, 1:19 or 1:20, preferably the ratio of 1: 10.

In one specific example, separately inducing culture of DC and CIK cells, priming antigen for stimulation induction, and co-culture comprise the steps of:

according to 2-4 x 10⁶Inoculating mononuclear cells in an adherent culture container at the cell/ml density, standing for a period of time, slightly shaking the culture container, collecting cells which do not adhere to the wall, adding the adherent cells into a DC culture medium for continuous culture, wherein the DC culture medium is an AIM-V culture medium added with 2-5% of autologous serum, 20-200 ng/ml of GM-CSF and 20-200 ng/ml of IL-4, culturing the cells which do not adhere to the wall by using a CIK culture medium for induction culture, and the CIK culture medium is an AIM-V culture medium added with 2-5% of autologous serum, 500-2000 ng/ml of IFN-gamma and 500-2000 ng/ml of IL-2;

respectively adding a CD3 antibody and a CD28 antibody with the final concentration of 200-1000 ng/ml into a CIK cell culture solution on the 2 nd day for culture;

adding sensitizing antigen into the DC cell culture solution on day 4, and continuously culturing;

addition of pro-mature cytokines to the DC cell culture broth on day 6: continuing induction culture of TNF-alpha with the final concentration of 20-200 ng/ml, IL-1 beta with the final concentration of 5-50 ng/ml and PEG2 with the final concentration of 1-5 mu g/ml;

mature DC cells and CIK cells were harvested on day 8 and the DC cells were co-cultured with CIK cells.

Generally, the killing performance of the DC-CIK co-cultured cells on related tumor cells can be detected when the cells are co-cultured to about day 14.

The DC-CIK co-culture cell is obtained by respectively inducing and culturing separated mononuclear cells into DC cells and CIK cells, wherein the DC cells are induced and stimulated by adding self-constructed polypeptide sensitizing antigen which is verified to be widely expressed in various tumor cells in the culture process, so that the tumor killing effect can be improved. The DC-CIK co-cultured cell obtained by the method has a good killing effect on various tumor cells, and the cultured DC-CIK cell has high activity and strong amplification capability, releases more cytokines such as IFN-gamma and the like, and has strong killing power. The DC-CIK co-cultured cell can be widely used for various cancers, including renal cancer, prostatic cancer, breast cancer, bladder cancer, melanoma, colon cancer, ovarian cancer, bone cancer, lung cancer, neuroblastoma and the like, and has wide adaptation range.

The following are specific examples.

Synthesis of polypeptide chains of first, sensitizing antigens

The sensitizing antigen of the embodiment is designed with two polypeptides, namely polypeptide 1 and polypeptide 2, and the amino acid sequences are respectively:

polypeptide 1: VLFIAKITPNNNGTYACFVSNLAT-KLH;

polypeptide 2: SAENFTVLIKNNIDFPGHNYTTRNILP-KLH.

The polypeptide 1 and the polypeptide 2 are both synthesized by Shanghai bioengineering GmbH, and the purity is more than 98%; the polypeptide is coupled with KLH by adopting an SMCC method, and the coupling rate reaches more than 90%.

Mixing the two polypeptides according to the ratio of 1:1 to form the sensitizing antigen of the antigen polypeptide mixture.

II, separation, induction culture and co-culture of DC cells and CIK cells

1. Collecting fresh cord blood, adding anticoagulant, mixing uniformly, adding the cord blood into 50ml of human lymphocyte separation tubes, adding 20ml of the blood into each separation tube, setting the parameters to be 20-25 ℃, increasing and decreasing the speed to be 9 and 0 respectively, and centrifuging for 20min at 800-1000 g;

2. after the centrifugation is finished, respectively collecting the upper part of plasma and the middle white membrane layer by using a Pasteur pipette, and transferring the plasma and the middle white membrane layer into a 50ml centrifuge tube;

3. adding part of the plasma into normal saline to obtain a buffer solution with the final volume concentration of the normal saline and the plasma being 2%, inactivating the rest plasma for 30min at 56 ℃, then centrifuging, setting the parameters to be 20-25 ℃, increasing and decreasing the speed to 9, centrifuging for 5min at 500g, and removing the precipitate after centrifuging to obtain the autoserum;

4. washing the leucocyte with a buffer solution with the final volume concentration of 2% of physiological saline and plasma, centrifuging for 5min at 20-25 ℃ with the speed increasing and decreasing of 9 and 1 respectively, removing supernatant after centrifugation, and repeating the previous centrifuging step to continuously wash once;

5. the cells were counted and then counted at 4X 10⁶Inoculating the cells/ml in an adherent culture bottle at a density, standing for 2h, then slightly shaking the culture bottle, collecting cells which are not attached to the wall, adding a DC culture medium into the adherent cells to continue culturing, wherein the DC culture medium is an AIM-V culture medium added with 5% of autologous serum in final volume percentage, 100ng/ml of GM-CSF in final concentration and 100ng/ml of IL-4, inducing and culturing the cells which are not attached to the wall by using a CIK culture medium, and the CIK culture medium is an AIM-V culture medium added with 5% of autologous serum in final volume percentage, 1000ng/ml of IFN-gamma and 1000ng/ml of IL-2 in final concentration;

6. adding 500ng/ml of CD3 and CD28 antibodies into the CIK culture medium on day 2 for culture;

7. adding the sensitizing antigen of the polypeptide mixture into the DC cells on the 4 th day until the final concentration is 10 mu M, and continuing to culture;

8. adding TNF-alpha which promotes mature cell factor to be 100ng/ml, IL-1 beta which promotes mature cell factor to be 20ng/ml and PEG2 which promotes mature cell factor to be 2 mu g/ml on the 6 th day, and continuing induction culture;

9. and (3) harvesting mature DC cells on the 8 th day, co-culturing the DC cells and the CIK cells according to the ratio of 1:10 to about 14 days, and then detecting the killing capacity and other capacities of the DC-CIK co-cultured cells on related tumor cells.

The results are shown in FIGS. 1-3, and it can be seen under the mirror that the DC protrudes more pseudopoda, the burr-like dendrites, the cytoplasm is in star-like bulge, the nuclei are extremely irregular, the expression of CD80 and CD86 is more than 99% as seen by flow type phenotypic analysis, and the expression of CD83 and HLA-DR is also more than 74%, which indicates that the growth state and phenotype of the DC are normal. The flow phenotype analysis of the CIK cells shows that 69% of the cells co-express CD3 and CD56, indicating that the CIK cells are normal in state.

Thirdly, detecting the tumor cell killing ability of the DC-CIK cells activated by the antigen polypeptide mixture

HepG2 tumor cells were harvested at log phase of growth and adjusted to a density of 10⁵Cells/ml; taking DC-stimulated CIK cells (namely DC-CIK co-culture cells), counting, and adjusting the concentration to 2 × 10⁶Cells/ml; inoculating the CIK cells and the HepG2 cells to a 96-well plate according to the proportion of 20 ten thousand: 1 ten thousand, 10 ten thousand: 1 ten thousand and 5 ten thousand: 1 ten thousand, uniformly mixing, and then putting the mixture into a 37 ℃ incubator for incubation for about 24 hours; and (3) taking out the 96-well plate after the incubation is finished, and detecting the killing effect of the DC-CIK cells stimulated by the antigen polypeptide on HepG2 tumor cells by using an LDH lactate dehydrogenase-cytotoxicity detection and analysis kit.

The control group cells are DC-CIK co-culture cells which are not stimulated by sensitizing antigen.

The results are shown in FIG. 4, and it is understood by comparison that the killing efficiency of HepG2 tumor cells is significantly improved by using the sensitizing antigen (Peptides group in the figure) compared with the Control group (Control in the figure).

Fourthly, detecting the release amount of IFN-gamma cell factors when the DC-CIK co-culture cells stimulated by the sensitizing antigen kill the tumor cells

HepG2 tumor cells were harvested at log phase of growth and adjusted to a density of 10⁵Cells/ml; taking DC stimulated CIK cells, counting, adjusting concentration to 2 × 10⁶Cells/ml; inoculating the CIK and the HepG2 cells to a 96-well plate according to the proportion of 20 ten thousand: 1 ten thousand, 10 ten thousand: 1 ten thousand and 5 ten thousand: 1 ten thousand, uniformly mixing, and then putting the mixture into an incubator at 37 ℃ for incubation for about 24 hours; after the incubation, the 96-well plate was removed, and 100. mu.l of the supernatant was collected, and the IFN-. gamma.release amount was measured using IFN-. gamma.ELISA kit according to the instructions. The control group was DC-CIK co-cultured cells that were not stimulated by the antigen-polypeptide mixture.

The results are shown in FIG. 4, and it is understood by comparison that IFN-. gamma.release amount was significantly increased in the Control group (Control) when the sensitizing antigen (Peptides group in the figure) was used.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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GUANHAO BIOTECH Co.,Ltd.

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Claims (8)

1. The sensitizing antigen of the DC-CIK cocultured cell is characterized by comprising a polypeptide 1 and a polypeptide 2, wherein the amino acid sequences of the polypeptide 1 and the polypeptide 2 are respectively shown as SEQ ID No.1 and SEQ ID No.2, and the molar ratio of the polypeptide 1 to the polypeptide 2 is 1: 1.

2. The sensitizing antigen of DC-CIK co-cultured cells of claim 1, wherein the sensitizing antigen is used at a concentration of 2 μ M to 20 μ M.

3. The sensitizing antigen for DC-CIK cocultured cells according to any one of claims 1-2, wherein hemocyanin KLH is coupled to the polypeptide 1 and the polypeptide 2, and the coupling rate is not less than 90%.

4. A preparation method of DC-CIK co-cultured cells is characterized by comprising the following steps:

collecting mononuclear cells, respectively inducing and culturing DC cells and CIK cells, and adding the sensitizing antigen of any one of claims 1-3 in the induction culture process of the DC cells for stimulation and induction;

and (3) co-culturing the DC cells after the stimulation and induction of the sensitizing antigen and the CIK cells.

5. The method for preparing DC-CIK co-cultured cells according to claim 4, wherein the number ratio of DC cells after stimulation and induction of the sensitizing antigen to CIK cells during co-culture is 1 (5-20).

6. The method of claim 4, wherein the ratio of the number of DC cells induced by the sensitizing antigen stimulation to the number of CIK cells in the co-culture is 1: 10.

7. The method for preparing DC-CIK co-cultured cells according to any one of claims 4 to 6, wherein the collection of mononuclear cells is from fresh peripheral blood or cord blood.

8. The method for preparing DC-CIK co-cultured cells according to any one of claims 4 to 6, wherein the separately induced culture of DC and CIK cells, the stimulation induction of the sensitizing antigen and the co-culture comprise the following steps:

according to 2-4 x 10⁶Inoculating the mononuclear cells into an adherent culture container at a cell/ml density, standing for a period of time, slightly shaking the culture container, collecting cells which do not adhere to the wall, adding the adherent cells into a DC culture medium for continuous culture, wherein the DC culture medium is an AIM-V culture medium added with 2-5% of autologous serum, 20-200 ng/ml of GM-CSF and 20-200 ng/ml of IL-4, the cells which do not adhere to the wall are cultured by using a CIK culture medium for induction culture, and the CIK culture medium is an AIM-V culture medium added with 2-5% of autologous serum, 500-2000 ng/ml of IFN-gamma and 500-2000 ng/ml of IL-2;

respectively adding a CD3 antibody and a CD28 antibody with the final concentration of 200-1000 ng/ml into a CIK cell culture solution on the 2 nd day for culture;

adding the sensitizing antigen into the DC cell culture solution on the 4 th day, and continuously culturing;

addition of pro-mature cytokines to the DC cell culture broth on day 6: continuing induction culture of TNF-alpha with the final concentration of 20-200 ng/ml, IL-1 beta with the final concentration of 5-50 ng/ml and PEG2 with the final concentration of 1-5 mu g/ml;

mature DC cells and CIK cells were harvested on day 8 and the DC cells were co-cultured with CIK cells.

Images