Peripheral blood memory T cell culture method

A cell culture and peripheral blood technology, applied in the field of peripheral blood memory T cell culture, can solve the problems of poor cell activity and low memory T cell magnification, and achieve the effect of increasing the ratio

Active Publication Date: 2019-08-06
新疆西部赛澳生物科技有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the existing methods for culturing memory T cells in vitro generally ha

Method used

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  • Peripheral blood memory T cell culture method

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preparation example Construction

[0048] In some embodiments, the preparation method of the autologous tumor antigen comprises:

[0049] The tumor tissue was mechanically crushed and resuspended in the antigen extract solution, then ultrasonically crushed at 280W-320W for 50s-70s, shaken in an ice bath, centrifuged, and the supernatant was dialyzed at low temperature.

[0050] In some embodiments, the working concentration of the following components of the antigen extraction solution in the aqueous solution is within the following range:

[0051] -Metal ion chelating agent 4mM~6mM,

[0052] -Iodoacetamide 2nM~3nM,

[0053] -Zwittergent 0.18g / mL%~0.28g / mL%, and

[0054] - buffer components;

[0055] In addition, the pH of the antigen extraction solution during the antigen extraction process is 8.2-8.6.

[0056] In some embodiments, the working concentration of the following components of the antigen extraction solution in the aqueous solution is within the following range:

[0057] - metal ion chelator ...

Embodiment 1

[0071] 1. Preparation of Tumor Antigens

[0072] 1) Under sterile conditions, the tumor tissue block was washed with an appropriate amount of normal saline to remove blood and other tissues.

[0073] 2) After being minced with scissors to the greatest extent, resuspended in antigen extraction solution (0.01M Tris-HCl, pH 8.4, containing 5mM EDTA, 2.5nM iodoacetamide and 0.2% Zwittergent 3-12).

[0074] 3) Ultrasonic wave at 300W for 1 min to further dissociate the small pieces of tissue, and shake in an ice bath at 4°C for 2 h.

[0075] 4) Centrifuge at 12000 rpm for 15 minutes, place the supernatant in a dialysis bag, and dialyze in distilled water overnight at 4°C.

[0076] 5) Tumor antigens are obtained after concentration.

[0077] 2. Peripheral Blood DC Cell Culture

[0078] 2.1. Peripheral blood DC cell culture medium

[0079] 1) DC culture: AIM-V medium (containing 1% autologous plasma), GM-CSF concentration 50 ng / mL, IL-4 concentration 50 ng / mL.

[0080] 2) DC mat...

Embodiment 2

[0103] 1. Preparation of Tumor Antigens

[0104] 1) Under sterile conditions, the tumor tissue block was washed with an appropriate amount of normal saline to remove blood and other tissues.

[0105] 2) After cutting to the maximum extent with scissors, resuspend in antigen extraction solution (0.012M Tris-HCl, pH8.2, containing 4mM EDTA, 3nM iodoacetamide and 0.18g / mL% Zwittergent 3-12).

[0106] 3) Ultrasonic wave 280W for 70 s to further dissociate small pieces of tissue, and shake in an ice bath at 4°C for 2 h.

[0107] 4) Centrifuge at 12000 rpm for 15 minutes, place the supernatant in a dialysis bag, and dialyze in distilled water overnight at 4°C.

[0108] 5) Tumor antigens are obtained after concentration.

[0109] 2. Peripheral Blood DC Cell Culture

[0110] With embodiment 1.

[0111] 3. Peripheral blood memory T cell culture

[0112] 3.1 Peripheral blood memory T cell culture medium

[0113] 1) Coating solution: D-PBS, CD3 monoclonal antibody concentration 450...

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Abstract

The invention relates to a peripheral blood memory T cell culture method. The method comprises: a) magnetically separating the PBMC isolated from the peripheral blood to obtain CD8<+> T cells; and inoculating the CD8<+> T cells in a culture container coated with a CD3 antibody and a CD28 antibody for culture; wherein a culture system contains autologous plasma, and the cytokine mainly includes IFN-gamma and IL-2; b) changing liquid for amplification culture, wherein a novel culture system contains autologous plasma and the cytokines mainly include IL-2, IL-1a, IL-7, and IL-15; and c) co-culturing the expanded T cells with DC cells; wherein the DC cells are previously co-cultured with an autologous tumor antigen. The method can effectively amplify memory T cells in vitro and effectively increase the proportion of active memory T cells.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing peripheral blood memory T cells. Background technique [0002] T cells can be divided into naive T cells, memory T cells, and effector T cells according to their degree of differentiation. Naive T cells mature in the thymus and migrate to peripheral lymphoid tissues (such as spleen and lymph nodes). Naive T cells in tumor patients contact tumor-associated antigens (TAA) on the surface of tumor cells, and under the recognition and presentation of APC cells, as well as a series of signaling pathways, they proliferate and differentiate into effector T cells and memory T cells. Effector T cells and memory T cells have different functions. The former secrete substances such as perforin, granzyme and granulysin, and directly kill tumor cells through the Fas ligand, but the survival time in the body is shorter (3 to 4 months ), and cannot continue to prolif...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2501/2302C12N2501/24C12N2502/1121
Inventor 张权
Owner 新疆西部赛澳生物科技有限责任公司
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