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Co-culture supernatant containing autologous CIK cells and application thereof

A technology of cell culture and supernatant, applied in the direction of cell culture active agents, animal cells, tissue culture, etc., to achieve the effect of improving anti-tumor effect, enhancing immune function, and high biological activity

Inactive Publication Date: 2018-03-09
广州市金航生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, it has not been reported that the co-culture supernatant containing autologous CIK cells is used to make cell suspension for anti-tumor effect

Method used

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  • Co-culture supernatant containing autologous CIK cells and application thereof
  • Co-culture supernatant containing autologous CIK cells and application thereof
  • Co-culture supernatant containing autologous CIK cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 The preparation method of the co-culture supernatant containing autologous CIK cells

[0018] 1. Pretreatment of culture flasks coated with anti-human CD3 monoclonal antibody

[0019] For the culture flask coated with anti-human CD3 monoclonal antibody, pour out the coating buffer in the culture flask, wash it once with sterile 0.9% physiological saline, and wash it once with RPMI-1640 medium for later use.

[0020] 2. Isolation, extraction and primary culture of peripheral blood mononuclear cells

[0021] Take 50ml of heparin-anticoagulated peripheral blood, slowly and evenly add it to the centrifuge tube that has been added with lymph separation fluid, and centrifuge for 20min in a low-speed horizontal centrifuge at a speed of 1500r / min. After separation, the plasma was collected and inactivated in a 56-degree water bath, and the mononuclear cell layer was extracted and washed twice with normal saline. Collect peripheral blood mononuclear cells in a centri...

Embodiment 2

[0031] Example 2 Quality Control of the Co-Culture Supernatant Containing Autologous CIK Cells

[0032] Bacteria detection

[0033] The whole process of autologous CIK cell culture was tracked for sterility monitoring. Samples were taken and inoculated on the sheep blood agar plate medium on the 0th, 7th, 9th, 11th, and 14th days for bacterial detection, and the colonies were observed every day. The results are shown in Table 1.

[0034] Table 1 Bacterial detection table of the co-culture supernatant containing autologous CIK cells

[0035] sample

[0036] Mold detection

[0037]The autologous CIK cells were cultured to the seventh day, and 6 samples were taken and inoculated on the amber plate medium respectively, and the cultured co-culture supernatant containing autologous CIK cells was tested for mold infection, and the mildew spots were observed every day, and the results are shown in the table 2.

[0038] Table 2 Mold detection table of co-culture supernata...

Embodiment 3

[0053] Example 3 Detection of cytokines secreted by co-culture supernatant containing autologous CIK cells

[0054] The autologous CIK cell culture supernatants on the 0th day, the 4th day, the 6th day, the 8th day, the 10th day, the 12th day, and the 14th day of the culture process were used to detect cytokines with ELISA kits, and the test results See Table 6.

[0055] Table 6 Cytokine detection table secreted by co-culture supernatant containing autologous CIK cells at different times

[0056] number of days

[0057] The present invention respectively analyzes the cytokines in the co-culture supernatant containing autologous CIK cells on the 0th day, the 4th day, the 6th day, the 8th day, the 10th day, the 12th day and the 14th day in the culture process The results in Table 6 show that the autologous CIK cells after induction and activation can mainly synthesize and secrete GM-CSF, IL-6, TNF-α, IFN-γ and other cytokines in large quantities. Compared with simila...

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Abstract

The invention discloses co-culture supernatant containing autologous CIK cells and application thereof, wherein the co-culture supernatant containing the autologous CIK cells is prepared by a following method. The specific method comprises the steps that: (1) the autogenous CIK cells are cultured until the fourteenth day, and concentration of the autologous CIK cells reaches 1-5*10<6> cells / ml, and after centrifugation, supernatant 1 and bottom cells 1 are obtained respectively; (2) the bottom cells 1 are washed with normal saline and centrifuged to obtain bottom cells 2; (3) the supernatant 1obtained in step (1) is further centrifuged, and bottom flocculent is discarded to obtain supernatant 2; (4) the supernatant 2 of the step (3) and the bottom cells 2 of the step (2) are mixed, addedwith the normal saline, human albumin, and gentamicin sulfate to prepare cell suspension, namely, the co-culture supernatant containing autologous CIK cells. Natural cytokines synthesized and secretedby the autologous CIK cells are more effective in enhancing immune function, resolving myelosuppression and immunosuppression, and improving anti-tumor effects of the autologous CIK cells.

Description

technical field [0001] The invention belongs to the biological field, in particular to a co-culture supernatant containing autologous CIK cells and application thereof. Background technique [0002] Advances in immune cell biology and immune molecular biology have advanced the application of immunology in tumor therapy. Nowadays, tumor immunotherapy has become one of the basic means of tumor treatment, among which cell-mediated adoptive immunotherapy is one of the current research hotspots. By infusing specific or non-specific immune effector cells that can inhibit the growth of tumor cells into tumor patients, it can directly kill tumor cells, or induce the body's immune response in patients to achieve the purpose of inhibiting and treating tumors. The current research and clinical preliminary data on tumor immune effector cells suggest that cytokine-induced killer (autologous CIK) cells are a new type of highly efficient MHC non-restricted immune effector with broad-spect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCA61K35/17C12N5/0638C12N2501/2302C12N2501/515
Inventor 何跃麦志国肖秀丽袁静娜何炳坤李维维李结珍来从显
Owner 广州市金航生物科技有限公司
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