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Method for preparing high-purity and high-cytotoxin-activity gamma delta T cells

A cytotoxic and high-purity technology, applied in the field of preparation of γδT cells, can solve the problems of low cytotoxic activity and low purity, and achieve the effect of solving low cytotoxic activity and enhancing anti-tumor function.

Inactive Publication Date: 2011-08-17
郑骏年
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Overcome the technical problems of low purity and low cytotoxic activity in existing methods for preparing γδT cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Preparation of γδT cells

[0016] 1) Preparation of peripheral blood mononuclear cells (PBMC)

[0017] Use a blood cell separator to collect anticoagulant blood from the patient, centrifuge at 1500rpm / min for 10 minutes, absorb the upper layer of plasma, inactivate at 56°C for 30 minutes, and then centrifuge for use. Dilute the blood cell pellet with normal saline. The human lymphocyte separation medium with a specific gravity of 1.077 is mixed with Diluted blood was added to a centrifuge tube at a ratio of 1:1.5, centrifuged at 2000rpm / min for 15 minutes, the buffy coat was carefully extracted, washed twice with normal saline, and peripheral blood mononuclear cells (PBMC) were obtained after low-speed centrifugation.

[0018] 2) Forward sorting of γδT cells by Mini MACS indirect immunomagnetic beads

[0019] Resuspend peripheral blood mononuclear cells with RPMI1640 containing 10% (V / V) autoplasma, and adjust the cell concentration to 10 8 / ml, add 10 μl a...

Embodiment 2

[0023] 1. Morphological observation of cultured γδT cells

[0024] After 24 hours of cell culture, γδT cells can be seen to adhere to the wall, and each colony has about 3-6 cells. After 48 hours, the colony begins to grow larger. After 10 days of culture, large colonies and a very small number of single adherent cells can be seen. The single cell is in the shape of a shuttle. The cell smears cultured for 10 days were stained with Reggie's, and it was found that most of the cells were large in size, oval or irregular in shape, most of the nuclei were oval or round in shape, the chromatin was loose, and the nuclear membrane was irregular with bulges. A small nucleolus can be seen in the cell, which is dark blue; the cytoplasm is abundant, stained gray-blue, irregular in shape, with pseudopods, lightly stained near the nucleus, and also irregular in shape.

[0025] Transmission electron microscopy shows that the cells are round, with many slender and protruding microvilli on the...

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Abstract

The invention discloses a method for preparing high-purity and high-cytotoxin-activity gamma delta T cells and belongs to the field of in-vitro cell culture. The method comprises: collecting and separating peripheral blood mononuclear cells (PBMC) of a patient, screening gamma delta T positive cells by Mini MACS, activating by a phospho-antigen (E)-4-hydroxy-3-methylbut-2-enyl-pyrophophate (HDMAPP) (3 mu M), adding a gene recombinant human cell factor interleukin (IL)-2 (100IU / ml) and IL-21 (10ng / ml), placing at 37 DEG C, incubating in a culture box containing 5 of percent CO2, supplying a IL-2-containing culture medium after 4 days to continue culturing for 7 to 14 days, and thus, obtaining the high-purity and high-cytotoxin-activity gamma delta T cells. In the invention, a magnetic activated cell sorting technique is used to obtain the high-purity gamma delta T-Cell-Receptor (TCR) positive cells, and the IL-2 and IL-21 are used together to stimulate the cultured gamma delta T cells to strengthen the antitumor function of the cultured gamma delta T cells; and the problems of low purity and low cytotoxin activity of gamma delta T cells amplified in vitro in the prior art are solved.

Description

technical field [0001] The invention belongs to the field of in vitro cell culture, in particular to a method for preparing gamma delta T cells with high purity and high cytotoxic activity. Background of the invention [0002] γδT cells are a special type of immune cells between acquired immunity and innate immunity, with antigen-specific recognition function without MHC restriction. The γδT cells currently used in anti-tumor research mainly refer to Vγ9Vδ2T cells, which account for about 50-95% of peripheral blood γδT cells. [0003] Human peripheral blood Vγ9Vδ2 T cells recognize non-peptide phosphorylated antigens through cell surface TCR, and do not require the restriction and presentation of MHC molecules during the antigen recognition process. Currently, non-peptide phosphorylated antigens commonly used to activate Vγ9Vδ2T cells include: isoprenyl pyrophosphate (IPP), nitrogen-containing bisphosphonates (N-BPs) and alkylamines. Among them, IPP is an intermediate prod...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/14A61P35/00A61P31/12A61K35/17
Inventor 郑骏年李慧中李连涛刘俊杰徐为陈菲菲程乾章龙珍裴冬生
Owner 郑骏年
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