Method for preparing DC-CIK immune cells with efficient tumor killing efficiency and prepared DC-CIK immune cells

A DC-CIK, immune cell technology, applied in blood/immune system cells, biochemical equipment and methods, animal cells, etc., can solve problems such as inability to break through and limited effect, and achieve the effect of improving tumor killing activity

Inactive Publication Date: 2016-06-22
TIANJIN PURUI SAIER BIOLOGICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Dendritic cells do not directly kill cells, they are just an immune presenting cell that presents signals to killing immune cells. In theory, DC-CIK combined with immune cells should have a stronger ability to kill cancer cells, but clinically DC-CIK The therapeutic effect is also very limited, because it cannot break through the bottleneck of DC-CIK therapy: cancer immunosuppression

Method used

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  • Method for preparing DC-CIK immune cells with efficient tumor killing efficiency and prepared DC-CIK immune cells
  • Method for preparing DC-CIK immune cells with efficient tumor killing efficiency and prepared DC-CIK immune cells
  • Method for preparing DC-CIK immune cells with efficient tumor killing efficiency and prepared DC-CIK immune cells

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preparation example Construction

[0027] The method for preparing DC-CIK immune cells with high-efficiency tumor killing of the present invention uses peripheral blood as a raw material, and obtains PBMC and upper plasma by Ficoll density gradient centrifugation, and the upper plasma is inactivated and freeze-thawed to obtain autologous serum. Autologous serum combined with immune cell culture medium and various cytokines, separate PBMCs to induce DC and CIK respectively, CIK cells are expanded and cultured every 2 days, DC cells are expanded and cultured on the 4th day, and then mixed culture is carried out on the 7th day. DC cells were stimulated with TNF-α on day 5 to promote their differentiation and maturation, then Anti-PDL-1MAb was added to block DC cells, and after blocking for 12-24 hours, all DC cells were recovered and added to the CIK cell induction system for co-culture ; Add CD28 and CD3 monoclonal antibodies to CIK cells on the 7th day to improve the expansion efficiency of CIK, use Anti-PD-1 MAb...

Embodiment 1

[0047] 1. Collect 120ml of peripheral blood, dilute it with 240ml of normal saline, and use Ficoll density gradient centrifugation to efficiently separate mononuclear cells.

[0048] 2. After separation, transfer the upper plasma layer to a 500ml centrifuge cup to obtain a total of 300ml diluted plasma, and inactivate it in a water bath at 56°C for 30 minutes. After inactivation, centrifuge at 12,000rpm for 10min, and filter the supernatant with a 0.4um filter membrane to obtain nearly 290ml of autologous serum, which is divided into 40ml / tubes for later use.

[0049] 3. Prepare cell induction medium for use:

[0050] DC cell induction medium: the immune cell culture medium contains autologous serum prepared in step 2 with a volume concentration of 8%, IL-4 of 150IU / ml and GM-CSF of 1000IU / ml;

[0051] CIK cell induction medium: the immune cell medium contains autologous serum prepared in step 2 with a volume concentration of 6% and IL-2 of 1000IU / ml;

[0052] 4. The isolate...

Embodiment 2

[0064] 1. Collect 120ml of umbilical cord blood, dilute it with 240ml of normal saline, and use Ficoll density gradient centrifugation to efficiently separate mononuclear cells.

[0065] 2. After separation, transfer the upper plasma layer to a 500ml centrifuge cup to obtain a total of 315ml diluted plasma, and inactivate it in a water bath at 56°C for 30 minutes. After inactivation, centrifuge at 12,000rpm for 10min, filter the supernatant with a 0.4um filter to obtain nearly 285ml of autologous serum, and divide into 40ml / tubes for later use.

[0066] 3. Prepare cell induction medium for use:

[0067] DC cell induction medium: the immune cell culture medium contains autologous serum prepared in step 2 with a volume concentration of 8%, IL-4 of 150IU / ml and GM-CSF of 1000IU / ml;

[0068] CIK cell induction medium: the immune cell medium contains autologous serum prepared in step 2 with a volume concentration of 6% and IL-2 of 1000IU / ml;

[0069] 4. The isolated PBMCs were wa...

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Abstract

The invention discloses a method for preparing high-efficiency tumor-killing DC-CIK immune cells and the prepared DC-CIK immune cells, in particular, adding After Anti-PD-1MAb and Anti-PDL-1MAb, their tumor killing activity was significantly improved. In the present invention, TexMACS immune cell culture medium produced by Miltenyi Company, autologous serum, various cytokines and joint culture technology are used to induce and culture DC and CIK, respectively, and at an appropriate time point, DC and CIK-related PD- 1 site is blocked, and then the cells are mixed cultured and applied at a certain time point, and finally the tumor killing activity of the mixed cells is greatly improved and has higher clinical application value.

Description

technical field [0001] The invention relates to a method for preparing DC-CIK immune cells, in particular to a method for preparing highly efficient tumor-killing DC-CIK immune cells and the prepared DC-CIK immune cells. Background technique [0002] DC-CIK combined with immune cells, the full name is cytokine-activated killer cells-dendritic cell mixed therapy. It contains both killer immune cells and dendritic cells. Dendritic cells, named for their resemblance to tree branches, are an important part of the immune system. Dendritic cells do not directly kill cells, they are just an immune presenting cell that presents signals to killing immune cells. In theory, DC-CIK combined with immune cells should have a stronger ability to kill cancer cells, but clinically DC-CIK The therapeutic effect is also very limited, because it cannot break through the bottleneck of DC-CIK therapy: cancer immunosuppression. Contents of the invention [0003] The problem to be solved by the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0784
CPCC12N5/0638C12N5/0639C12N2501/22C12N2501/2302C12N2501/2304C12N2501/24C12N2501/25C12N2501/51C12N2501/515C12N2502/1114C12N2502/1121
Inventor 张冰晶鲁振宇韩洪起刘俊江秦臻
Owner TIANJIN PURUI SAIER BIOLOGICAL TECH CO LTD
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