Method for easily and efficiently preparing human gamma/deltaT cells

A cell and high-efficiency technology, applied in the field of cellular immunology, can solve the problems of γδT cells in human peripheral blood that have not been seen in practical and high-efficiency, and achieve the effects of reduced culture costs, large quantities, and low numbers of solutions

Active Publication Date: 2013-09-18
SHENZHEN HORNETCORN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After extensive review of domestic and foreign patent documents and various publications, so far, there has been no invention or report on the practical, efficient, and rapid expansion of human peripheral blood γδT cells in large numbers

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] In Example 1, cultured Mycobacterium tuberculosis was used to prepare Mycobacterium tuberculosis heat-resistant antigen. Include the following steps:

[0011] 1. Inoculate Mycobacterium tuberculosis seeds with a wet weight of 50-100 grams into 200ml Sutong-style liquid medium, and culture them statically in a 37°C incubator for 2 weeks, and then divide them to contain 10%-30% newborn In 250ml Sutong type liquid medium of bovine serum, 50ml per bottle, divided into 4 bottles, and then continue to culture in a 37°C incubator for 4 weeks, collect the cultured bacterial suspension, centrifuge at 4000 rpm 30 minutes to harvest Mycobacterium tuberculosis cells.

[0012] 2. Wash the collected bacteria twice with normal saline, resuspend the bacteria with 2 times the volume of distilled water, treat with high-pressure steam at 115°C for 20 minutes, centrifuge at 4000 rpm for 30 minutes, and collect the supernatant, which is Mtb -HAg.

Embodiment 2

[0014] In Example 2, the prepared Mtb-HAg was used to stimulate PBMCs to obtain a large number of γδT cells with high purity and high cytotoxic activity. The specific operation includes the following steps:

[0015] 1. Collect peripheral venous blood from the patient with a blood cell separator or a 50ml syringe under sterile conditions, and obtain mononuclear cells by density gradient centrifugation of Ficoll-diatrizoate glucosamine. The specific steps are: 1500 rpm, centrifuge for 10 minutes, absorb the upper plasma layer, inactivate at 56°C for 30 minutes and centrifuge for later use, dilute the precipitated blood cells with normal saline, and divide human lymphocyte separation medium and diluted blood at a ratio of 1:2 The proportion of the mixture was added to the centrifuge tube, 2000 rpm, centrifuged for 20 minutes, carefully sucked the buffy coat layer, washed twice with normal saline, the speeds were 1600 rpm, 1300 rpm, and centrifuged for 7 minutes to obtain the peri...

Embodiment 3

[0018] In Example 3, the morphology, purity, immunophenotype and cytotoxic activity of the above-mentioned cultured γδT cells were detected. The specific operation includes the following steps:

[0019] 1. After 24 hours of cell culture, γδT cells can be seen sinking to the bottom of the culture vessel and tend to colonize. After 48 hours, the colonies begin to grow larger. After 6 to 10 days of culture, large colonies and irregular mononuclear cells can be seen. Reggie's staining was carried out on the cells cultured for 10 days, and it was found that most of the cells were enlarged in size, oval or irregular in shape, most of the nuclei were oval or round, the nuclear staining was loose, the nuclear membrane was irregular, and there were protrusions. One small nucleolus can be seen in each cell, which is dark blue; the cytoplasm is abundant, stained gray-blue, irregular in shape, with pseudopodia, light staining near the nucleus, and irregular in shape. Take 100 μl of cells...

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PUM

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Abstract

The invention belongs to the technical field of cellular immunology, in particular to a method for amplifying human peripheral blood gamma / deltaT cells massively and preferentially in vitro. The method comprises the following steps of: (1) preparing tubercle bacillus heat stable antigen by using the cultured tubercle bacillus; (2) stimulating to amplify the gamma / deltaT cells by combining the prepared tubercle bacillus heat stable antigen and cell factors rhIL-2 and rhIL-21; and (3) performing morphologic observation through a microscope, performing purity and immunophenotyping analysis by using a flow cytometry, and performing cytotoxicity detection through MTT assay. The method has the advantages that: culture conditions and operation are simple, a large number of gamma / deltaT cells with high purity and high cytotoxicity can be prepared in short time, and the cost is low.

Description

technical field [0001] The invention belongs to the technical field of cellular immunology, and specifically relates to a method for amplifying human peripheral blood γδT cells in large quantities in vitro. Background technique [0002] T lymphocytes are divided into two types according to the expression of T cell antigen receptor (TCR): TCRαβT cells and TCRγδT cells, referred to as αβT cells and γδT cells, respectively. Among them, γδT cells are a special type of cells between specific immunity and nonspecific immunity. Most of them are double-negative for CD4 and CD8 molecules, and a few can express CD8 molecules. γδT cells are mainly distributed in skin and mucosal tissues, and generally do not exceed 5% of the total number of T cells. γδT cells have the ability to specifically recognize antigens without MHC restriction, have anti-infection, anti-tumor and immune regulation functions, and are the first line of defense for the body's immune surveillance. However, because...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783
Inventor 马飞王宇环罗晓玲
Owner SHENZHEN HORNETCORN BIOTECH
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