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Culture method for DC-CTL cells loaded with tumor cell exosomes

A DC-CTL, tumor cell technology, applied in the field of DC-CTL cell culture, can solve the problems of complex protein composition, unable to play a specific killing effect, etc., and achieve the effects of high content, simple method and high purity

Inactive Publication Date: 2019-03-01
见多视光(北京)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 for DC-CTL cells loaded with tumor cell exosomes
  • Culture method for DC-CTL cells loaded with tumor cell exosomes
  • Culture method for DC-CTL cells loaded with tumor cell exosomes

Examples

Experimental program
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Example Embodiment

[0099] Example 1

[0100] Preparation of tumor cell exosomes

[0101] 1. Medium

[0102] 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.

[0103]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.

[0104] 2. Preparation of exosome-free FBS

[0105] 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.

[0106] 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...

Example Embodiment

[0118] Example 2

[0119] A method for culturing peripheral blood DC cells loaded with tumor cell exosomes, comprising the following content:

[0120] 1. Peripheral blood DC cell culture medium

[0121] 1) DC medium

[0122] 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.

[0123] 2) DC maturation medium

[0124] 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.

[0125] 2. Peripheral blood DC cell culture method

[0126] 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 fl...

Example Embodiment

[0139] Example 3

[0140] The cells harvested in Example 2 were centrifuged, the medium was removed, and the peripheral blood DC cells loaded with tumor cell exosomes were co-cultured to obtain DC-CTL cells loaded with tumor cell exosomes. The specific steps were as follows:

[0141] 1. Solution

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

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

[0144] 3. CTL medium: AIM-V medium (containing 5% autologous plasma), IL-2 concentration 1000IU / mL.

[0145] 2. Peripheral blood DC-CTL 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 15mL of CTL initial medium, a...

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Abstract

The invention relates to the field of DC-CTL cell culture, in particular to a culture method for DC-CTL cells loaded with tumor cell exosomes. The culture method for the DC-CTL cells loaded with tumorcell exosomes comprises the following step: carrying out co-culture on CTL cells and peripheral blood DC cells loaded with the tumor cell exosomes to obtain DC-CTL cells loaded with the tumor cell exosomes. A culture medium is a CTL culture medium, the CTL culture medium comprises the following components: an AIM-V culture medium, autologous plasma and IL-2, the content of the autologous plasma is 5%+/- 0.5% of the volume of the AIM-V culture medium, and the final concentration of the IL-2 is 1000+/- 100 IU/mL. Components of the culture medium are optimized, and the obtained DC-CTL cells loaded with the tumor cell exosomes have good killability.

Description

technical field [0001] The present invention relates to the field of DC-CTL cell culture, in particular to a method for culturing DC-CTL cells loaded with tumor cell exosomes. Background technique [0002] Cytotoxic T lymphocytes (cytotoxic lymphocyte, CTL) is a specific, T cells with killing function, specialized in secreting various cytokines to participate in immune function. It has a killing effect on antigenic substances such as certain viruses and tumor cells, and forms an important line of defense for the body's anti-virus and anti-tumor immunity with natural killer cells. [0003] Activated CTLs can produce specific tumor-killing activity, and this cytotoxic effect mainly includes (1) perforin-granzyme pathway; (2) activated CTLs highly express FaSL, which can bind to the FaS antigen on the target cell surface Induce target cell apoptosis; (3) secrete TNF and other cytokines to directly kill target cells; (4) produce a variety of chemokines to attract natural immune...

Claims

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Application Information

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