Method of expanding nk cell and composition for culturing

a composition and cell technology, applied in the field of enhancing natural killer cell (nk cell) expansion, can solve the problems of inability to maintain prone to apoptosis or cellular senescence, and inability to carry out long-term survival. or long-term survival of ex vivo-cultured immune cells, etc., to achieve the effect of increasing the number of cells

Inactive Publication Date: 2017-05-04
KOREA UNIV RES & BUSINESS FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0022]When natural killer (NK) cells are cultured by treating the cells with a reactive oxygen species (ROS) inhibitor and / or a p53 protein inhibitor according to the present invention, a higher cell number may be obtained than the conventional culture method without a change in the function of the cells, and thus the NK cells required for production of cell therapeutic agents may be more effectively obtained.

Problems solved by technology

A human immune system is regulated by complicated mechanisms, and when there is an imbalance of the immune system, a variety of intractable diseases such as cancer may occur.
However, ex vivo-cultured immune cells do not maintain their survival for long term.
In particular, as the cells proliferate, they are liable to undergo apoptosis or cellular senescence.
ROS rapidly increased under environmental stress and give rise to oxidative stress, which causes cellular apoptosis or senescence.
However, excessive ROS may damage a macromolecule such as DNA, a protein or a lipid, ultimately resulting in cellular senescence or apoptosis.
However, there have been no satisfactory results describing the relationship between the p53 gene and immune cells.

Method used

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  • Method of expanding nk cell and composition for culturing
  • Method of expanding nk cell and composition for culturing
  • Method of expanding nk cell and composition for culturing

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

Preparation and Culture of NK Cells

[0078]Human blood was prepared, and subjected to centrifugation at 2500 rpm for 30 minutes using Ficoll (Ficoll-Paque™ PLUS, GE Healthcare). Then, peripheral blood mononuclear cells were isolated from a buffy coat.

[0079]Afterward, a Jurkat cell line irradiated at 100 Gy and an EBV-LCL cell line were co-cultured with PBMCs: KL-1: EBV-LCL in a ratio of 1:0.5:0.5 in hRPMI medium prepared by adding 10% FBS and 1% penicillin / streptomycin to RPMI1640 medium in the presence of 500 U / ml IL-2, and the medium was exchanged with hRPMI medium supplemented with 500 U / ml IL-2 once for 2 to 3 days.

experimental example 1

Examination of Influence of ROS and / or p53 Inhibitor on Culture of NK Cells

[0080]An examination of a change of proteins expression of NK cells during the culture of the NK cell as in the Preparation Example 1 was conducted. A cell cycle-associated marker pRb, cellular senescence markers P53 and P21, ER stress markers Bip and CHOP, a DNA damage marker γH2AX, and apoptosis markers PARP and cl-caspase-7 were identified.

[0081]The results are shown in FIG. 2, confirming that most of the markers peaked at 13 to 16 days.

[0082]To examine the influence of an ROS inhibitor on NK cells, the NK cells were cultured for 10 days under the conditions of Preparation Example 1. Then, 400 μM of an apocynin (Calbiochem), 50 μM of trolox (Santa Cruz) and 1 μM of pifithrin-α (Santa Cruz) were treated individually, and 50 μM trolox and 500 nM pifithrin-α were treated in a combination. The inhibitor was treated at three-day intervals, and a cell number was assessed after culture for 2 weeks. Here, the medi...

experimental example 2

Measurement of Changes in Cancer Cell Killing Activity of NK Cells

[0085]An experiment was performed to examine cancer cell killing activity of NK cells treated with the inhibitor.

[0086]As target cells, K562 and A375 cells were prepared, labeled with chromium for 1 hour, co-cultured with the NK cells in a suitable ratio, and then a supernatant was taken from the cell culture after 4 hours so as to measure an isotopic value using a gamma counter. The experiment was performed on a control group which was not treated, and groups treated with apocynin, trolox, pifithrin-α and trolox+pifithrin-α, respectively, and the results are shown in FIG. 7.

[0087]As shown in FIG. 7, even when the cells were cultured with the ROS inhibitor and / or p53 inhibitor according to the present invention, the cancer cell killing activity was maintained at the same level as that in the non-treated control group.

[0088]Also, as target cells, K562 and A375 cells were prepared, and NK cells and the target cells were...

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Abstract

Provided are a method of ex-vivo culture of natural killer (NK) cells by treating the cells with a reactive oxygen species (ROS) inhibitor and/or a p53 protein inhibitor; and a composition comprising the cultured NK cells. By reducing the activity of ROS and p53 proteins during ex-vivo culture, NK cells may have achieved greater expansion efficiency without altering their anti-tumor cytotoxicity.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims priority to and the benefit of Korean Patent Application No. 2015-0098269 filed on Jul. 10, 2015, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND[0002]1. Field of the Invention[0003]The present invention relates to a method of enhancing natural killer cell (NK cell) expansion, and a composition for culturing NK cell.[0004]2. Discussion of Related Art[0005]A human immune system is regulated by complicated mechanisms, and when there is an imbalance of the immune system, a variety of intractable diseases such as cancer may occur. Therefore, the development of an immune cell therapeutic agent, which is a method for resolving the imbalance occurring in the immune system so as to recover and maintain a normal condition thereof, would be desirable.[0006]The human immune system is divided into an innate immune system and an adaptive immune system. The innate immune system consists of c...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/00C12N5/0783
CPCA61K39/0011C12N5/0646C12N2500/00A61K2039/585C12N2501/998A61K2039/5158A61K35/17C12N2501/999A61K31/045A61K31/05A61K31/428C07K14/4703
Inventor LEE, KYUNG-MISOHN, JEONGWONLIM, SEON AHLIM, JANG-MI
Owner KOREA UNIV RES & BUSINESS FOUND
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