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CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture

A technology of cell culture and lentinan, applied in the biological field, can solve the problems of poor proliferation of CIK cells, achieve high cell surface antigen content, large amplification multiple, and promote proliferation

Inactive Publication Date: 2015-11-25
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when using this method to culture CIK cells, the proliferation effect of CIK cells is poor.

Method used

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  • CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture
  • CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1) Extract 40ml of peripheral blood, transfer the peripheral blood to a 50ml centrifuge tube, dilute and mix with 1 volume of normal saline, slowly add to Ficoll (Norway Axis-Shield company, product number: 1114547), centrifuge at 3500rpm for 30min ;

[0046] 2) After centrifugation, extract the PBMC band layer in the centrifuge tube, resuspend it with normal saline, and then centrifuge at 2000pm for 15min;

[0047] 3) After the centrifugation, remove the supernatant, resuspend the residue with normal saline again, and then centrifuge at 2000rpm for 15min;

[0048] 4) After centrifugation, remove the supernatant to obtain about 1.5×10 7 a PBMC;

[0049] 5) Use the culture medium to resuspend the PBMC obtained above to obtain a cell suspension; Company’s commercially available product), serum and RPMI-1640 medium, wherein, the content of IFN-γ is 1000U / ml, the content of CD3 stimulating monoclonal antibody is 80ng / ml, the content of IL-2 is 600U / ml, and the content of...

Embodiment 2

[0056] 1) Extract 40ml of peripheral blood, transfer the peripheral blood to a 50ml centrifuge tube, dilute and mix with 1 volume of normal saline, slowly add to Ficoll (Norway Axis-Shield company, product number: 1114547), centrifuge at 2800rpm for 20min ;

[0057] 2) After the centrifugation, extract the PBMC band layer in the centrifuge tube, resuspend with normal saline, and then centrifuge at 1800pm for 10min;

[0058] 3) After centrifugation, remove the supernatant, and resuspend the residue with normal saline again, then centrifuge at 1800rpm for 10min;

[0059] 4) After centrifugation, remove the supernatant to obtain 1.8×10 7 a PBMC;

[0060] 5) Use the culture medium to resuspend the PBMC obtained above to obtain a cell suspension; Company’s commercially available product), serum and RPMI-1640 medium, wherein, the content of IFN-γ is 500U / ml, the content of CD3 stimulating monoclonal antibody is 50ng / ml, the content of IL-2 is 500U / ml, and the content of lentinan ...

Embodiment 3

[0067]1) Extract 40ml of peripheral blood, transfer the peripheral blood to a 50ml centrifuge tube, dilute and mix with 1 volume of normal saline, slowly add to Ficoll (Norway Axis-Shield company, product number: 1114547), centrifuge at 2000rpm for 15min ;

[0068] 2) After centrifugation, extract the PBMC band layer in the centrifuge tube, resuspend it with normal saline, and then centrifuge at 1500pm for 5min;

[0069] 3) After the centrifugation, remove the supernatant, resuspend the residue with normal saline again, and then centrifuge at 1500rpm for 5min;

[0070] 4) After centrifugation, remove the supernatant to obtain 1.2×10 7 a PBMC;

[0071] 5) Use the culture medium to resuspend the PBMC obtained above to obtain a cell suspension; Company’s commercially available product), serum and RPMI-1640 medium, wherein, the content of IFN-γ is 200U / ml, the content of CD3 stimulating monoclonal antibody is 10ng / ml, the content of IL-2 is 100U / ml, and the content of lentinan ...

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Abstract

The invention relates to the biotechnology field, in particular to CIK cell culture fluid, a CIK cell culture method and application of lentinan in CIK cell culture. The CIK cell culture fluid comprises interferon-gamma, CD3 stimulated monoclonal antibodies, interleukin-2, the lentinan and serum-free basal culture media. When the CIK cell culture fluid with the lentinan is used for CIK cell culture, PBMCs (peripheral blood mononuclear cells) can generate lymphocyte activation factors and release helper T cell factors to promote CIK cell multiplication, so that obtained CIK cells have large amplification multiple, high killing activity and high cell surface antigen content. Experiment results show that after 14-day CIK cell culture by the CIK cell culture fluid, the amplification multiple of the CIK cells is larger than 300, in-vitro killing rate (effector-target ratio being 40:1) of the CIK cells is larger than (80+ / -2)%, and the number of CIK cell surface antigens (CD3+ and CD56+) is larger than 48%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a CIK cell culture solution, a CIK cell culture method and the application of lentinan in CIK cell culture. Background technique [0002] Autologous immune cell therapy is the most mature tumor biotherapy technology. It isolates mononuclear cells from the patient's autologous peripheral blood, activates, modifies, and expands them in an in vitro laboratory, and then infuses them back into the patient's body to regulate and enhance the patient's immune system. immune function, and directly kill tumor cells and virus-infected cells. Since its application in clinical tumor treatment in the 1980s, autologous immune cell therapy technology has become more and more mature, and has been more and more recognized by tumor patients and doctors. [0003] According to different methods, autologous immune cell therapy can be mainly divided into active immunotherapy and adoptive immunotherapy. A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 陈海佳王一飞葛啸虎李丽娟万桦
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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