Culture method of DC-CIK (Dendritic Cell-Cytokine-Induced Killer) cells loaded with tumor cell exosomes

A technology of DC-CIK and tumor cells, applied in animal cells, nervous system cells, vertebrate cells, etc., can solve the problems of complex protein components and inability to play a specific killing effect, achieve high content and enhance cytotoxic activity , the effect of high purity

Inactive Publication Date: 2019-03-12
见多视光(北京)科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

The extracted protein components are complex and contain the required intracellular antigens. After the DCs are activated, the DCs enter th

Method used

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  • Culture method of DC-CIK (Dendritic Cell-Cytokine-Induced Killer) cells loaded with tumor cell exosomes
  • Culture method of DC-CIK (Dendritic Cell-Cytokine-Induced Killer) cells loaded with tumor cell exosomes
  • Culture method of DC-CIK (Dendritic Cell-Cytokine-Induced Killer) cells loaded with tumor cell exosomes

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Experimental program
Comparison scheme
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Embodiment 1

[0101] Preparation of tumor cell exosomes

[0102] 1. Medium

[0103] The components of the normal complete medium for tumor cells are: RPMI-1640 culture medium and FBS, and the amount of FBS added is 10% of the volume of the RPMI-1640 culture medium.

[0104] The components of the complete culture medium for exosomes are: phenol red-free RPMI-1640 culture medium and exosome-free FBS, and the amount of exosome-free FBS is 10% of the volume of the phenol red-free RPMI-1640 culture medium.

[0105] 2. Preparation of exosome-free FBS

[0106] Pour FBS into special ultracentrifuge tubes under sterile conditions, 38.6mL per tube, 4 tubes, after confirming the balance, put it into an ultracentrifuge at 120000g, and centrifuge overnight.

[0107] The next day, carefully draw the upper layer of clarified serum into a new sterile centrifuge tube, filter it with a 0.22 μm filter membrane into another centrifuge tube to ensure the sterility of the serum, seal it, and set it aside.

[...

Embodiment 2

[0120] A method for culturing peripheral blood DC cells loaded with tumor cell exosomes, comprising the following content:

[0121] 1. Peripheral blood DC cell culture medium

[0122] 1) DC medium

[0123] Based on the AIM-V medium, the following components were added according to the following contents: autologous plasma volume percentage was 1%±0.2%, GM-CSF concentration was 50ng / mL, and IL-4 concentration was 50ng / mL.

[0124] 2) DC maturation medium

[0125] Based on the AIM-V medium, add the following components according to the following content: the volume percentage of autologous plasma is 1%±0.2%, the concentration of GM-CSF is 50ng / mL, the concentration of IL-4 is 50ng / mL, and the concentration of TNF-α is 10ng / mL mL, PGE-2 concentration 1μg / mL.

[0126] 2. Peripheral blood DC cell culture method

[0127] On the 0th day of culture, take a T175 culture flask, add the obtained PBMC cells into the culture flask, and the number of cells in each flask is 1.5×10 8 , a...

Embodiment 3

[0141] 1. Solution

[0142] 1. Coating solution: D-PBS, CD3 monoclonal antibody concentration 500ng / mL, CD28 concentration 500ng / mL.

[0143] 2. CIK initial medium: VIVO medium (containing 5% autologous plasma).

[0144] 3. CIK medium: GT-T581 medium (containing 5% autologous plasma), IL-2 concentration 1000IU / mL.

[0145] 2. CIK cell culture method

[0146] 1) On the 0th day of culture, take a T25 culture bottle, add 3 mL of coating solution, place it in an incubator for coating for 2 hours, and set aside. Take out the coated T25 culture bottle, pour off the coating solution, and take the prepared PBMC1.0×10 7 , inoculate into a T25 culture flask, add 10mL CIK initial medium, add IFN-γ to a final concentration of 1000U / mL, place at 37°C, 5% CO 2 cultured in an incubator.

[0147] 2) On the first day of culture, take out the culture bottle, add IL-2 to make the final concentration 1000U / mL, add IL-12 to make the final concentration 2.5ng / mL, add IL-15 to make the final co...

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Abstract

The invention relates to the field of DC-CIK (Dendritic Cell-Cytokine-Induced Killer) cell culture and particularly relates to a culture method of DC-CIK cells loaded with tumor cell exosomes. The culture method comprises the following steps of: commonly culturing CIK cells and peripheral blood DC cells loaded with the tumor cell exosomes, after culturing for 65-75 hours, adding IL-2 with the amount of 1000-1500 IU/mL by volume of culture solution, and continuously culturing for 65-75 hours to obtain the DC-CIK cells; culturing by a CIK culture medium comprising the components of GT-T581 culture medium, autologous plasma and IL-2, wherein the autologous plasma is 5%+/-0.5% of the volume of the GT-T581 culture medium and the concentration of the IL-2 is 1000+/-100 IU/mL. The culture methodhas the beneficial effects that by culturing the CIK cells and the peripheral blood DC cells loaded with the tumor cell exosomes in a certain mode, the obtained DC-CIK cells loaded with the tumor cellexosomes have good killing capability.

Description

technical field [0001] The present invention relates to the field of DC-CIK cell culture, in particular to a method for culturing DC-CIK cells loaded with tumor cell exosomes. Background technique [0002] Cytokine-induced killer cells (CIK) are a group of CD3-derived cells induced by IL-1α, IL-2, IFN-γ and anti-CD3 monoclonal antibody in vitro. + CD56 + CD4 + and CD8 + The effector T cell population has both the strong tumor killing activity of T cells and the advantages of non-major histocompatibility complex (MHC)-restricted tumor killing of NK cells. [0003] Compared with other adoptive immunotherapy cells, CIK has the characteristics of fast proliferation, high killing activity, broad tumor killing spectrum and small side effects, and has little effect on normal bone marrow hematopoietic function. It is clinically considered to be an effective means of tumor immunotherapy . It is widely used in the treatment of various malignant tumors such as kidney cancer, melan...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/0784C12N5/079
Inventor 张权
Owner 见多视光(北京)科技有限公司
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