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Method for producing natural killer cells

A technology of cells and nuclear cells, applied in the field of producing natural killer cells

Pending Publication Date: 2020-11-06
GREEN CROSS LAB CELL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method is limited to obtain a large number of high-purity NK cells

Method used

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  • Method for producing natural killer cells
  • Method for producing natural killer cells
  • Method for producing natural killer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1. Study of NK cell growth by suspension culture

[0088] (1) Culture of NK cells

[0089] To deplete CD3-positive cells and obtain feeder cells (PBMCs) in peripheral blood mononuclear cells (PBMCs) collected from healthy donors, transfer the cells to new 50 mL tubes, 5x10 7 cells. Then, the cells were centrifuged at 1,200 rpm and 4°C for 10 minutes. After centrifugation, the recovered feeder cells (1x10 8 cells) into vials and frozen in liquid nitrogen tanks. Thaw frozen cells before use.

[0090] To obtain PBMCs from which CD3-positive cells were removed, 400 μL of MACS running buffer (Miltenyi Biotech, Korea) and 100 μL of CD3 magnetic beads (Miltenyi Biotech, Korea) were added to a mixture consisting of 5x10 7 A cell pellet consisting of two cells was added and allowed to react at 4°C for 20 minutes. Then, the cells were centrifuged with 20 mL of MACS running buffer at 1,200 rpm and 4° C. for 10 minutes, and then resuspended in 2 mL of MACS running buf...

Embodiment 2

[0153] Example 2. Study of NK cell growth according to the time of repeated stimulation (re-stimulation) using feeder cells

[0154] To investigate when to initiate restimulation with feeder cells during NK cell culture, restimulation with feeder cells was performed at the time when a specific accumulation population doubling level was reached. As in Example 1(1), at 37°C and 5% CO 2 The culture was carried out in the reactor under the conditions of 2-3, 3-4, 4-5 and 5-6, respectively, instead of 7 days after the start of culture. Cells were restimulated (Table 4).

[0155] Culture was performed as follows.

[0156] After thawing frozen feeder cells and frozen PBMC from which CD3-positive cells were removed and inactivating the feeder cells by irradiating at about 2000 cGy using a gamma irradiator, 500-1,000 IU of IL-2 and 10 ng / mL of OKT-3 was added to the tube containing the feeder cells for co-culture of the inactivated feeder cells and the PBMC from which the CD3-posit...

Embodiment 3

[0164] Example 3. Development and Characterization of Agitated Bioreactor Process

[0165] In this example, a bioreactor process capable of culturing NK cells in large quantities under a uniform environment was developed and its characteristics were analyzed.

[0166] (1) To study the growth of NK cells according to the agitation speed of the agitated bioreactor

[0167] NK cells cultured for about 12 days were inoculated into a stirred bioreactor, and then cultured by a fed-batch method for up to 21 days.

[0168] Culture was performed as follows.

[0169] The frozen feeder cells and the frozen PBMC from which the CD3-positive cells were removed were thawed, and then cultured statically for 5 days. Then, the cell culture was switched to suspension culture (stirring speed 160 rpm). Cells were cultured for a total of 12 days.

[0170] After thawing frozen feeder cells and frozen PBMC from which CD3-positive cells were removed and inactivating the feeder cells by irradiating...

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PUM

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Abstract

The present invention relates to a method for producing natural killer (NK) cells. More specifically, the present invention relates to a method for producing NK cells, characterized in that peripheralblood mononuclear cells from which CD3-positive cells are removed are proliferated together with supporting cells, and the peripheral blood mononuclear cells are re-stimulated with supporting cells at the time of reaching a specific number of cumulative division times. The present invention also relates to a method for producing NK cells, characterized in that NK cells are cultured under appropriate culture conditions by using a bioreactor. The production method according to the present invention has an advantage that NK cells having a high cell killing ability and cell survival rate can be produced with high purity and at high efficiency in a short period of time by a clinically friendly method as compared with existing methods, thereby increasing the productivity of an NK cell therapy agent.

Description

technical field [0001] The present application relates to a method for producing natural killer (NK) cells, more specifically to a method for producing NK cells, characterized in that peripheral blood mononuclear cells from which CD3-positive cells are removed are proliferated together with feeder cells, And the peripheral blood mononuclear cells are re-stimulated with feeder cells at a time when a specified accumulation population doubling level is reached. [0002] The present application also relates to a method for producing NK cells, characterized by culturing the NK cells under appropriate culture conditions by using a bioreactor. Background technique [0003] Natural killer (NK) cells are lymphoid cells that make up approximately 10% of blood cells and play an important role in the immune response. NK cells have many functions. Specifically, they are used to remove abnormal cells that have undergone or are undergoing tumorization because NK cells have the ability to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0087C12N5/0646C12N2501/515C12N2501/2302C12N2501/2312C12N2501/2315C12N2501/2318C12N2501/2321C12N2500/60C12N2523/00C12N2527/00C12N2500/02C12N2501/998C12N2506/11
Inventor 黄琉炅白相勋韩升烈李尚贤南亨辰金周映M·R·韩鲁东一
Owner GREEN CROSS LAB CELL CORP
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