Method for separating and activating human peripheral blood T cells

A technology of human peripheral blood and peripheral blood, which is applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., to achieve the effect of simple and easy method, guaranteed treatment effect and good repeatability

Inactive Publication Date: 2017-05-31
刘晓明
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Whether the magnetically separated T cells have been activated, and whether positively selected T cells bound to microbeads affect their functional detection are still controversial

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  • Method for separating and activating human peripheral blood T cells

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Embodiment Construction

[0020] The technical solution of the present invention will be further described below, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered by the protection scope of the present invention middle.

[0021] The method for the separation and activation of human peripheral blood T cells of the present invention comprises the following steps:

[0022] (1) Peripheral blood was diluted 5X with pH 7.2 phosphate buffer;

[0023] (2) Add 15 mL of peripheral blood mononuclear cell separation medium Ficoll-Paque into a 50 mL centrifuge tube;

[0024] (3) Slowly add the diluted peripheral blood to the Ficoll-Paque layer by layer, and centrifuge at 400g for 30min at room temperature;

[0025] (4) Carefully suck off the upper layer of plasma, taking care not to stir the middle mononuclear cell layer;

[0...

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Abstract

The invention discloses a method for separating and activating human peripheral blood T cells. According to the method, the human peripheral blood T cells are separated with CD4 and CD8 antibody magnetic beads, then the obtained T cells are activated with a T cell activation reagent for preparation of conversion of slow viruses. The method comprises steps as follows: a peripheral blood mononuclear cell is separated with a peripheral blood mononuclear cell separating medium; the peripheral blood mononuclear cell is incubated and combined with CD4 and CD8 antibody magnetic beads; the incubated T cells are separated by separation columns arranged in a magnetic field, and the human peripheral blood T cells are obtained; the human peripheral blood T cells obtained through separation are suspended in a T cell culture medium, and the T cell activation reagent is added for activation. The method is simple, easy to implement and good in repeatability, CD4 and CD8 T cells with the purity higher than 90% can be obtained simultaneously, and the treatment effect is effectively guaranteed when the T cells expressing CD19 and CD20 antibody gene chimeric antibody receptors is used for treatment.

Description

[0001] Technical field: [0002] The invention relates to the technical field of cell sorting and cell activation in vitro, in particular to a method for separating and activating human peripheral blood T cells. [0003] Background technique: [0004] T lymphocytes are key members involved in the body's immune response, and in-depth research on the phenotypic characteristics and functions of T lymphocytes is of great significance in both basic theory and clinical practice. Stable and efficient isolation of high-purity T lymphocytes with unaffected physiological function and structure is the premise and basis for a series of immunological research. [0005] Julius et al first established a method for rapid separation of T cells: the surface of T cells is smooth, while the surface of B cells has more villi. When the mixed lymphocyte suspension passes through the nylon hair column, B cells, plasma cells, monocytes And some auxiliary cells are selectively adhered to the nylon wool...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 朱分禄刘晓明
Owner 刘晓明
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