Concentration gradient rhIL-2 dependent iNKT cell amplification method and application thereof

A technology of concentration gradient and cells, which is applied in the field of tumor cell immunotherapy, can solve the problems of low culture efficiency, achieve high amplification efficiency, enhance the immune enhancement of in vitro expanded iNKT cells and the effect of tumor immune monitoring

Inactive Publication Date: 2017-04-19
闾军 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of the present invention aims at the problems of low efficiency of iNKT in vitro expansion and culture and no selectivity

Method used

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  • Concentration gradient rhIL-2 dependent iNKT cell amplification method and application thereof
  • Concentration gradient rhIL-2 dependent iNKT cell amplification method and application thereof
  • Concentration gradient rhIL-2 dependent iNKT cell amplification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Peripheral Blood Mononuclear Cells (PBMC) Extraction

[0040] (1) Take 20ml of anticoagulated venous blood in the morning.

[0041] (2) Add 15ml Lymphoprep separation solution into a 50ml centrifuge tube.

[0042] (3) Slowly add an equal volume of 0.9% sterile normal saline to the anticoagulated venous blood and gently invert three times to obtain diluted blood.

[0043] (4) Use a 2ml sterile dropper to slowly add the diluted blood to the surface layer of the Lymphoprep separation solution, and do not shake or turn it upside down after all is transferred.

[0044] (5) Balance the centrifuge tube, and centrifuge at room temperature for 30 minutes with a horizontal centrifuge (horizontal rotor) at 1700 rpm, ace / brake: 4 / 0.

[0045] (6) Aspirate excess plasma from the uppermost layer. Gently aspirate the lymphocyte layer with a 2ml sterile pipette, transfer to a new 50ml centrifuge tube, and aspirate the lymphocyte layer in all tubes into the same 50ml centrif...

Embodiment 2

[0050] Example 2 Primary Expansion of iNKT Cells

[0051] (1) On the day of PBMC extraction, a final concentration of 100ng / ml α-GalCer was added and cultured for 48 hours.

[0052] (2) The concentration of rhIL-2 was gradually increased in the culture medium, and the concentration of rhIL-2 increased in an equiproportional or equidifferential manner from the time the cells were isolated to the 11th day of culture.

[0053] (3) On the 11th day of culture, the concentration of rhIL-2 increased to 400IU / ml.

[0054] Depend on figure 1 As shown in A, the concentration of rhIL-2 increased in a proportional manner every 48 hours (except the 24-hour interval from the 10th day to the 11th day) from the time of cell isolation.

[0055] Depend on figure 1 As shown in B, the concentration of rhIL-2 increased in an arithmetic manner every 72 hours (except for the 48-hour interval from the 10th day to the 11th day) from the cell isolation.

Embodiment 3

[0056] Example 3 Induction and culture of dendritic cells

[0057] (1) Resuspend the PBMC obtained by density gradient centrifugation in AIM V medium, add it to a cell culture dish, and store at 37°C in 5% CO 2 to cultivate.

[0058] (2) After 3 hours, take out the culture dish, shake it gently, and suck out the suspended cells.

[0059] (3) Add AIM V medium containing 500IU / ml rhIL-4 and 500IU / ml rhGM-CSF to the culture dish.

[0060] (4) On the 3rd day and the 5th day after culture, the medium was changed in half, and fresh AIM V medium containing 500IU / ml rhIL-4 and 500IU / ml rhGM-CSF was added.

[0061] (5) Add rhTNF-α at a final concentration of 10ng / ml on the 6th day of culture.

[0062] (6) Mature dendritic cells were harvested on day 7.

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Abstract

The invention discloses a concentration gradient rhIL-2 dependent iNKT cell amplification method and an application thereof. The method comprises the following steps: (1) extracting peripheral blood mononuclear cells PBMC; (2) adding [alpha]-GalCer, which is 100ng/ml in final concentration, to the PBMC cells which are extracted in the step (1), and conducting cultivation for 48h; and (3) gradually increasing an rhIL-2 concentration in a culture medium in the cells which are cultivated in the step (2) in a gradient mode. According to the cell amplification method provided by the invention, when the iNKT cells are collected on the 21st day of cultivation, the quantity of the iNKT cells is increased by 100,000 times, including more than 90% of CD4-iNKT cells which have effects of up-regulating immune reaction and directly killing tumors. The iNKT cells, which are activated and amplified by virtue of the method provided by the invention, are higher in amplification efficiency, and the method can selectively amplify the Th1-like iNKT cells and enhance functions of in vitro amplification iNKT cell immunity as well as tumor immunity surveillance and killing.

Description

technical field [0001] The invention belongs to the field of tumor cell immunotherapy, and more specifically, the invention relates to a method and application for rapid and massive expansion of Th1-like iNKT cells with tumor killing effect. Background technique [0002] Invariant natural killer T (invariant nature killer T, iNKT) cells are a type of naturally occurring immune cells that mediate innate immunity and acquired immunity. The innate immune function that stimulates the rapid production of cytokines and chemokines to regulate the immune response also produces specific responses to antigens through the T cell receptor (TCR). Different from the TCR of traditional T lymphocytes, iNKT cells have a highly conserved TCRα chain. The human TCRα chain is composed of Vα24-Jα28 and can react with various glycolipid antigens. In addition, unlike traditional T cells that recognize antigen presenting cells (antigen presenting cells, APCs) class I or class II major histocompatib...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/0784A61K35/17A61P35/00
CPCA61K35/17C12N5/0639C12N5/0646C12N2501/22C12N2501/2302C12N2501/2304C12N2501/2307C12N2501/2315C12N2501/25C12N2501/51C12N2501/515C12N2501/999C12N2502/1121C12N2533/50
Inventor 闾军孙文峰陈辉
Owner 闾军
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