Composition for stimulating and inducing single karyocyte to be amplified to gamma deltaT cell and application of composition

A nuclear cell and composition technology, applied in the direction of animal cells, vertebrate cells, cell culture active agents, etc., can solve the problems of low amplification factor, low cell purity, low content of γδT cells, etc., and achieve high proliferation ability, The effect of high purity and high cytotoxic activity

Active Publication Date: 2018-06-29
安徽瑞达健康产业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the extremely low content of γδT cells in peripheral blood, the clinical application of γδT cells as adoptive immune cells is greatly limited.
At present, γδT cells are expanded from peripheral blood mononuclear cells, the expansion factor is low, and the cell purity is not high

Method used

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  • Composition for stimulating and inducing single karyocyte to be amplified to gamma deltaT cell and application of composition
  • Composition for stimulating and inducing single karyocyte to be amplified to gamma deltaT cell and application of composition
  • Composition for stimulating and inducing single karyocyte to be amplified to gamma deltaT cell and application of composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Induction of γδT cells

[0031] Isolation of mononuclear cells (PBMCs) from peripheral blood and expansion of γδ T cells:

[0032] ① Turn on the biological safety cabinet 30 minutes before use;

[0033] ②Take D-PBS out of the refrigerator before use, and let it stand at room temperature for 30 minutes;

[0034] ③Transfer 30ml of peripheral blood samples (heparin anticoagulant) to two sterile 50ml centrifuge tubes, 15ml in each tube, then add 22.5ml of sterile D-PBS to each tube, invert the centrifuge tubes repeatedly, and mix well;

[0035] ④ Take two 50ml sterile centrifuge tubes, add 15ml Ficoll-Paque Plus solution respectively, and then slowly add 24ml blood diluted in step 3 (drawn from the two sterile tubes in step 3) to form layers , 20°C, 400×g, centrifuge for 30 minutes;

[0036] ⑤Put the two 50ml centrifuge tubes in step 4 into a biological safety cabinet, then use a 10ml pipette to suck off the 15ml serum layer, put it into a new sterile 50ml cen...

Embodiment 2

[0045] Embodiment 2: culture medium change liquid

[0046] ①Put the mixed culture medium in a 37°C water bath to warm it up, or put it at room temperature to equilibrate for 1 hour to room temperature;

[0047] ②Put the cell culture bottle into the biosafety cabinet, resuspend the cells, blow evenly with a 25ml pipette, and take 10μl of the cell suspension for counting;

[0048] ③ On day 4, transfer all the cells into a 50ml sterile centrifuge tube and replace the expansion medium. The specific replacement operation is as follows:

[0049] ④According to the total amount of cells, adjust the final cell concentration to 1×10 6 cells / ml. Among them, the improved method is: take half of the new mixed culture composition containing the expansion factors (anti-human CD3Ab, anti-human CD28Ab, IL-15, IL-21, and IL-2) required for amplification by the improved method The base is added to an empty cell culture bottle; the routine culture method is: take half of the new RPMI 1640 medi...

Embodiment 3

[0060] Example 3: Phenotype detection of γδT cells expanded and cultured by improved culture method

[0061] ①Take the cells cultured on the 21st day and put them into two 1.5mL EP tubes, each with 1 × 10 6 cells, centrifuge at 400×g for 5 minutes, and discard the supernatant;

[0062] ②Add 1 mL of LPBS to wash again, centrifuge at 400×g for 5 minutes, and remove the supernatant;

[0063] ③ Add 100 μL of PBS, and then add control antibodies Mouse IgG1-PerCP, Mouse IgG1-FITC ( Figure 4 Middle Ctrl) and detection antibody mouse anti-human CD3-PerCP, anti-human γδTCR-FITC fluorescent antibody ( Figure 4 Medium CD3 / γδ-TCR), placed at 4°C for 30 minutes;

[0064] ④Wash twice with PBS, discard the supernatant, and finally resuspend the cells with 200 μL PBS.

[0065] Use the AECA Novocyte flow cytometer to detect the cultured γδT cells, such as Figure 4 It was shown that the purity of γδT cells can reach 97.8% after culturing the cells for 21 days.

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Abstract

The invention relates to a composition for stimulating and inducing a single aryocyte to be amplified to a gamma delta T cell. The composition is prepared from zoledronic acid, anti-human CD3Ab, anti-human CD28Ab, IL-15, IL-21 and IL-2. The invention also provides a method for the in-vitro mass amplification of gamma deltaT cells by utilizing the composition, and particularly relates to a method for in-vitro inducing the mass amplification of gamma deltaT cells by utilizing PBMCs. The zoledronic acid is used for induction and activation, the anti-human CD3Ab, the anti-human CD28Ab, IL-15, IL-21 and IL-2 are used for inducing and activating the proliferation, and various amplification factor combinations to jointly stimulate and induce the amplification of gamma deltaT cells, and the obtained gamma deltaT cell has the characteristics of large quantity, high purity, high cell toxicity and the like and has good clinical application value.

Description

technical field [0001] The invention relates to a composition capable of stimulating mononuclear cells to induce expansion into γδT cells, and a method for using the composition to expand γδT cells in large quantities in vitro from mononuclear cells (PBMCs). Background technique [0002] Tumor is one of the major diseases threatening human health, with high mortality and morbidity. Tumor immunotherapy is the fourth cancer treatment method after surgery, radiotherapy and chemotherapy, and it is also the therapy that has received the most attention and has the best therapeutic effect. Mucosa (epidermal) tissue acts as a physical barrier containing a wide range of cell types including nonlymphoid and lymphoid immune cells, especially T cells. [0003] Studies have shown that T cells, especially T cells expressing γδ receptors, play a key role in maintaining mucosal tissue balance and preventing epidermal tissue pressure such as pathogenic microbial infection and malignant canc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2501/06C12N2501/2302C12N2501/2315C12N2501/2321C12N2501/51C12N2501/515
Inventor 赵报刘丹吴疆邓蒙蒙程箫
Owner 安徽瑞达健康产业有限公司
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