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External amplification process of gamma delta T-lymphocyte

A lymphocyte, in vitro amplification technology, applied in the field of immunology, can solve problems such as high cost, loss of receptor library, and complicated procedures

Inactive Publication Date: 2007-03-21
北京佳德和细胞治疗技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned cells or molecules often only activate the Vγ9 / Vδ2 subpopulation in γδT cells, so this method will face the problem of loss of receptor pool
Anti-CD 3 After antibody activation of PBMC, αβT cells are removed by magnetic bead sorting, and the purity of γδT cells in the obtained cell population can reach 90%, but the procedure is complicated and the cost is relatively expensive

Method used

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  • External amplification process of gamma delta T-lymphocyte
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] (1) Antibody coating of cell culture plate

[0039] Add 500 μl of RPMI-1640 medium containing 1 μg / ml anti-TCRγδ monoclonal antibody to each well of a 24-well plastic culture plate, and place it in 37°C CO 2 Incubate in a saturated humid environment for 2 hours, and wash 3 times with RPMI1640 medium before use. This is a solid-phase antibody-coated culture plate, stored at 4°C for later use.

Embodiment 2

[0041] (2) Antibody coating of cell culture flask

[0042] At 25cm 2 2.5ml of RPMI1640 medium containing 1.0μg / ml anti-TCRγδ monoclonal antibody was added to the plastic culture flask for coating. Incubate at 37°C for 2 hours, and wash 3 times with RPMI1640 medium before use.

Embodiment 3

[0044] Peripheral blood mononuclear cell (PBMC) isolation

[0045] Collect 5ml of aseptic blood, dilute the peripheral blood sample with RPMI1640 medium 1:1, add it to a centrifuge tube containing lymphocyte separation solution (diluted venous blood and lymphocyte separation solution volume ratio 2:1), centrifuge at 500×g for 20 minutes. Aspirate the milky white mononuclear cell layer at the interface of the lymphocyte separation liquid, wash it twice with RPMI1640 medium, and centrifuge as above. Adjust the cell concentration to 10 with complete medium (RPMI1640 medium containing 10% fetal bovine serum) 6 Cells / ml are reserved.

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Abstract

The present invention relates to the field of immunology, and is especially external amplification process of gamma delta T-lymphocyte.

Description

Technical field [0001] The invention relates to the field of immunology. Specifically, the present invention relates to a method for amplifying γδ T lymphocytes in vitro. Background technique [0002] T lymphocytes can be divided into two types of T lymphocytes according to their surface antigen receptor (TCR) expression: TCRαβT lymphocytes and TCRγδT lymphocytes. Among them, the number of TCRγδT lymphocytes is small, the recognition antigen is extensive and there is no restriction of MHC molecules. Studies have shown that the growth of tumor cells can induce the accumulation of TCRγδ lymphocytes in situ, and TCRγδ lymphocytes are also abundant in tumor infiltrating lymphocytes. Under the action of IL-2, activated TCRγδ lymphocytes can multiply in large numbers, and show killing activity on tumor cells in in vitro experiments, but have no obvious killing effect on normal lymphocytes. Therefore, γδT cells as candidate cells for adoptive immunotherapy have attracted the attention o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00C12N5/08C12N5/06
Inventor 何维张素梅于松涛董小黎牛海涛陈娟宋卫华张建民胡愉
Owner 北京佳德和细胞治疗技术有限公司
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