Cell culture method for improving DC-CIK (dendritic cell-cytokine-induced killer) cell killing ability

A technology of DC-CIK and cell culture, which is applied in the direction of cell culture active agent, co-culture, tissue culture, etc., can solve the problems of restricting technology development, DC-CIK cell lethality not being achieved, reducing DC-CIK cell lethality, etc. , to achieve the effect of stimulating lethality and improving the effect

Inactive Publication Date: 2017-01-25
青海七彩花生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, in practice, the lethality of DC-CIK cells is far from what researchers expected, which limits the development of this technology.
During the applicant's research, it was found that this was mainly due to the mutual inhibition of DC and CIK after co-culture. Although this inhibition was not obvious, it was this inhibition that reduced the lethality that DC-CIK cells should have.

Method used

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  • Cell culture method for improving DC-CIK (dendritic cell-cytokine-induced killer) cell killing ability
  • Cell culture method for improving DC-CIK (dendritic cell-cytokine-induced killer) cell killing ability
  • Cell culture method for improving DC-CIK (dendritic cell-cytokine-induced killer) cell killing ability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Culture of DC-CIK cells

[0022] Methods for isolation and culture of DC cells and CIK cells:

[0023] Collect 20mL of peripheral blood from healthy volunteers, dilute it with pre-cooled PBS 1:1, slowly add the upper layer of lymphocyte separation medium, centrifuge at 650g, 4°C for 20min, collect the white cell layer, separate mononuclear cells, and resuspend the cells in RPMI 1640 medium Place at 37°C, 5% CO 2 Incubate for 2 hours in the incubator. Culture of DC cells: Collect adherent cells after 2 hours of culture, add 1000U / mL GM-CSF and 1000U / mL IL-4 to RPMI1640 medium containing 10% fetal bovine serum, and store at 37°C, 5% CO 2 Cultured in an incubator, supplemented with GM-CSF and IL-4 after changing the medium, continued to culture for 6 days, and added 100ng / mL TNF-α to the medium on 5 days. Cultivation of CIK cells: collect the suspension cells in monocytes after 2 hours of culture, add 1000U / mL INF-γ to RPMI 1640 medium containing 10% fetal bo...

Embodiment 2

[0026] Embodiment 2: the contrast of embodiment 1, do not add eliciting peptide during mixed culture

[0027] Methods for isolation and culture of DC cells and CIK cells:

[0028] Collect 20mL of peripheral blood from healthy volunteers, dilute it with pre-cooled PBS 1:1, slowly add the upper layer of lymphocyte separation medium, centrifuge at 650g, 4°C for 20min, collect the white cell layer, separate mononuclear cells, and resuspend the cells in RPMI 1640 medium Place at 37°C, 5% CO 2 Incubate for 2 hours in the incubator. Culture of DC cells: Collect adherent cells after 2 hours of culture, add 1000U / mL GM-CSF and 1000U / mL IL-4 to RPMI1640 medium containing 10% fetal bovine serum, and store at 37°C, 5% CO 2 Cultured in an incubator, supplemented with GM-CSF and IL-4 after changing the medium, continued to culture for 6 days, and added 100ng / mL TNF-α to the medium on 5 days. Cultivation of CIK cells: collect the suspension cells in monocytes after 2 hours of culture, add...

Embodiment 3

[0031] Example 3: Cytotoxicity

[0032] The cytotoxicity of the DC-CIK cells prepared in Examples 1 and 2 were tested respectively. Using K562 cells as target cells, use complete medium (RPMI 1640 containing 10% fetal bovine serum) to adjust the cell concentration to 10 5 cells / mL, using the DC-CIK cells prepared in Examples 1 and 2 as effector cells, adding effector cells and target cells into a 96-well plate according to the effect target ratio of 10:1, at 37°C, 5% CO 2 Cultured in the incubator for 48h. Cell viability was detected by MTT detection kit.

[0033] Killing activity (%)=[1-(mean value of test wells-mean value of effect control wells) / mean value of control wells of target cells]×100%.

[0034] The results are shown in Table 1 and figure 1 .

[0035] Table 1 DC-CIK cell killing rate (%) to K562 target cell

[0036] Example 1 Example 2 Kill rate (%) 68.55±9.45 53.42±8.34

[0037] The results show that the method of the present inventio...

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Abstract

The invention discloses a cell culture method for improving DC-CIK (dendritic cell-cytokine-induced killer) cell killing ability. The cell culture method includes subjecting cultured DCs and CIKs to mixed culture in mixed culture liquid, wherein stimulating peptides shown in SEQ ID NO.1 are added into the mixed culture liquid and used for mixed culture of the DCs and the CIKs so as to stimulate to form DC-CIK cells. By changing the component of a culture system, the cell culture method changes the inhibiting effect between the DCs and the CIKs into the stimulating effect, so that the ability of the DC-CIK cells to kill target cells after co-culture can be improved, and improvement in immunological therapy effects is benefited.

Description

technical field [0001] The invention belongs to the biological field and relates to immune cell culture, in particular to a cell culture method for improving the lethality of DC-CIK cells. Background technique [0002] The incidence of hematological malignancies is increasing year by year, seriously threatening human health. At present, the treatment of tumors mainly includes surgical resection, physical radiation therapy, and chemical drug therapy. For hematologic malignancies, chemical drug therapy and bone marrow stem cell transplantation are currently the main treatments. However, the limited source of bone marrow stem cells and strict implementation conditions prevent it from being used as a treatment for hematological malignancies. At the same time, multidrug resistance (MDR) leads to leukemia relapse and chemotherapy failure has become the bottleneck of leukemia treatment. A novel treatment method against cancer through autoimmunity - tumor biotherapy is the fourth l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0784
CPCC12N5/0639C12N5/0638C12N2501/2301C12N2501/2302C12N2501/2304C12N2501/24C12N2501/515C12N2501/998C12N2502/00
Inventor 杨廷伟陈子扬朱荣富陈四海朱晓翔邓凯旋
Owner 青海七彩花生物科技有限公司
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