In-vitro culture method for increasing human Vdelta2 T cell amplification efficiency and application thereof

A technology of expansion efficiency and in vitro culture, applied in the field of improvement and protocol optimization, can solve the problems of insufficient number of Vδ2T cells, limited expansion efficiency, low expansion efficiency, etc., and achieve a large number of induced cells, a small amount of peripheral blood, The effect of high amplification efficiency

Inactive Publication Date: 2015-06-17
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a high-efficiency in vitro method to overcome the shortcomings of limited expansion efficiency and large blood volume in the existing Vδ2 T cell in vitro expansion scheme. A method for massively expanding peripheral blood Vδ2 T cells, which overcomes the limitations of insufficient number of Vδ2 T cells, low expansion efficiency, and high cost

Method used

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  • In-vitro culture method for increasing human Vdelta2 T cell amplification efficiency and application thereof
  • In-vitro culture method for increasing human Vdelta2 T cell amplification efficiency and application thereof
  • In-vitro culture method for increasing human Vdelta2 T cell amplification efficiency and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: In vitro stimulation and expansion of Vδ2 T cells

[0037] 1. After the obtained 15ml heparin sodium anticoagulated peripheral blood was separated from the plasma at 1500rpm for 10min, use an equal amount of PBS buffer to fully mix the remaining venous blood; carefully add 4ml lymphocyte extract to the 15ml centrifuge tube (be careful not to stain the tube wall), then add the diluted human blood that has been mixed into 15ml centrifuge tubes, and add 6ml to each tube; room temperature, 600g, 30min, centrifuge (set the speed of the centrifuge to 6, and the speed to fall to 2); carefully draw Medium milky white mononuclear cell layer (usually called buffy coat); after resuspended in PBS, centrifuge at 2000rpm for 15min; discard the supernatant, resuspend in PBS and centrifuge again at 1500rpm for 10min, discard the supernatant. Adjust the cell density to 3-5 million cells / ml using AIMV+10% FBS medium. The cell suspension was added to a 24-well plate at an amou...

Embodiment 2

[0041] Example 2: Vδ2 T cells are stimulated and expanded in vitro (the concentration of the stimulator is different when cultured)

[0042] 1. After the obtained 15ml heparin sodium anticoagulated peripheral blood was separated from the plasma at 1500rpm for 10min, use an equal amount of PBS buffer to fully mix the remaining venous blood; carefully add 4ml lymphocyte extract to the 15ml centrifuge tube (be careful not to stain the tube wall), then add the diluted human blood that has been mixed into 15ml centrifuge tubes, and add 6ml to each tube; room temperature, 600g, 30min, centrifuge (set the speed of the centrifuge to 6, and the speed to fall to 2); carefully draw Medium milky white mononuclear cell layer; after resuspending in PBS, centrifuge at 2000rpm for 15min; discard the supernatant, resuspend in PBS and centrifuge again at 1500rpm for 10min, discard the supernatant. Adjust the cell density to 3-5 million cells / ml using AIMV+10% FBS medium. The cell suspension wa...

Embodiment 3

[0046] Example 3: Vδ2 T cells are stimulated and expanded in vitro (the concentration of the stimulator is different when cultured)

[0047] 1. After the obtained 15ml heparin sodium anticoagulated peripheral blood was separated from the plasma at 1500rpm for 10min, use an equal amount of PBS buffer to fully mix the remaining venous blood; carefully add 4ml lymphocyte extract to the 15ml centrifuge tube (be careful not to stain the tube wall), then add the diluted human blood that has been mixed into 15ml centrifuge tubes, and add 6ml to each tube; room temperature, 600g, 30min, centrifuge (set the speed of the centrifuge to 6, and the speed to fall to 2); carefully draw Medium milky white mononuclear cell layer; after resuspending in PBS, centrifuge at 2000rpm for 15min; discard the supernatant, resuspend in PBS and centrifuge again at 1500rpm for 10min, discard the supernatant. Adjust the cell density to 3-5 million cells / ml using AIMV+10% FBS medium. The cell suspension wa...

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Abstract

The invention relates to an in-vitro culture method for increasing human Vdelta2 T cell amplification efficiency and an application thereof. The method is characterized in that peripheral blood mononuclear cells are separated, a medium (AIMU+10%FBS)is used to prepare a cell suspension, zoledronate is added for in-vitro stimulation; after one-day stimulation of zoledronate, cytokine rhIL-2 is added every day; the human peripheral blood mononuclear cells are cultured for 72-96 hours, half amount or 2 / 3 mount of nutrient solution can be replaced when the nutrient solution turns yellow; half amount of nutrient solution can be replaced when the nutrient solution turns yellow after 96 hours; when culture is carried out in 11th-14th day, when the amplified Vdelta2 T cell frequency and absolute amount reach the requirements, the cells can be acquired. The Vdelta2 T cell purity can reach more than 90%. The method has the advantages of simple culture method, the required peripheral blood amount is little, induction cell quantity is more, and the amplification efficiency is high, Vdelta2 T cell is expected to increase curative effect of for liver cancer immunity treatment, and has good application potential.

Description

technical field [0001] The invention belongs to the technical field of cell biology. Specifically, the present invention relates to an increase in the number of innate immune cell Vδ2 T expansion cultures and optimization of the protocol. It is a method for inducing human Vδ2 T cells in combination with zoledronic acid and cytokines, which can be more effective for this type of cells. It is widely used in the clinical treatment of liver cancer cells to provide a solid foundation. Background technique [0002] γδT cells are a large class of T lymphocytes, which were first discovered by Brenner in 1986 in the antibody prepared by using the peptide encoded by the γ gene sequence of the T cell receptor. γδT cells are mainly distributed in the skin and mucosal tissues, and in the peripheral blood of healthy people, γδT cells account for 1% to 5% of lymphocytes, and the Vδ2 T cell subset mainly accounts for 50% to 90% of γδT cells (often paired with Vγ9). form Vγ9Vδ2 T cells), a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61P35/00
Inventor 武晓丽尹芝南赵立青吴震州王倩
Owner TIANJIN UNIV
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