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Production method for cell population including nk cells

A manufacturing method and technology of NK cells, which can be applied in the direction of biological preparations, animal cells, embryonic cells to remove bad cells, etc., can solve the problem of low proliferation and achieve the effect of improving the proliferation rate

Pending Publication Date: 2021-11-05
GAIA BIOMEDICINE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for NK cells, the number obtained from peripheral blood etc. is small, and the proliferative property in vitro is low

Method used

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  • Production method for cell population including nk cells
  • Production method for cell population including nk cells
  • Production method for cell population including nk cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] [Example 1: Mixed culture 1 of NK cells obtained from fresh peripheral blood]

[0117] Blood was collected from healthy human volunteers, and peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll (GE Healthcare, 17144002). The isolated peripheral blood mononuclear cells were mixed in substantially the same ratio, and CD3 beads were added. ※1 and suspended, after 15 minutes of incubation at 4°C, 1 mL of separation buffer was added. ※2 Suspended well and centrifuged at 300 x g for 10 minutes. The supernatant was removed, suspended in 0.5 mL of separation buffer, added to an LD column (Miltenyi Biotech, 130-042-901) wetted by adding 2 mL of separation buffer in advance, and the eluate from the LD column was recovered . Further, 1 mL of separation buffer was added to the LD column, and the eluate was recovered. After that, the column was washed with 1 mL of separation buffer, the number of cells in the recovered liquid was coun...

Embodiment 2

[0124] [Example 2: Mixed culture 2 of NK cells obtained from fresh peripheral blood]

[0125] By the method described in Example 1, the data of the single-donor-derived culture (n=17) and the mixed culture of 4 or more people (n=10) in T-75 flasks were totaled and calculated for The cell proliferation rate of the obtained NK cell numbers was analyzed statistically. For the analysis software, the Wilcoxon rank sum test was performed using JMP Pro13.

[0126] show the result in figure 2 . In the case of culturing cells derived from a single donor, the proliferation rate (total number of cells after proliferation / total number of cells before proliferation) was 3.10±0.56, whereas in the case of mixed culture, the proliferation rate was 6.56 ±1.24, the proliferation rate of the mixed culture was statistically higher (P<0.01).

Embodiment 3

[0127] [Example 3: Tumor cytotoxic activity test]

[0128] "Manufacture of NK cells"

[0129] The mixed cultured NK cells of 4 people obtained by the method described in Example 1 from healthy volunteers, and the cultured NK cells from individual donors (1 out of 4 people) were collected, washed, and suspended in RPMI1640 medium (Wako Pure Chemical Industries, 189-02025) containing 10% FBS (Nichirei Bioscience, 171012-500ML) and 100 units of penicillin, 100 μg / mL of streptomycin (Nacalai Tesque, 26253-84) (described below) as 10% FBS / RPMI1640), prepared with the same medium as above to a concentration of 1x10 6 cells / mL.

[0130] "The Making of SKOV3"

[0131] SKOV3 cells (human ovarian cancer cell line) were prepared at a concentration of 1x10 in RPMI1640 medium (Wako Pure Chemical Industries, 189-02025) without serum components 6 cells / mL. The prepared SKOV3 cells were stained with PKH26 Red FluorescentCell Linker kit (Sigma, PKH26GL-1KT) and finally prepared as 2x10 wi...

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Abstract

The present invention addresses the problems of providing an effective method for producing a population of NK cells for cell therapy, improving NK cell expansion efficiency in vitro, and flexibly increasing the signalling necessary for NK cell licensing. Specifically provided is a production method for a cell population including NK cells by preparing a monocyte population that is derived from multiple donors and includes NK cells, incubating and culturing the prepared monocyte population under conditions effective for the processing and proliferation of NK cells, and inducing the proliferation of the NK cells.

Description

technical field [0001] The present invention relates to a method for the manufacture of a cell population comprising natural killer cells (NK cells) and uses thereof. Background technique [0002] Malignant tumors are the number one cause of death in Japan, and countermeasures against them are the top priority. In particular, it is extremely important and meaningful to develop new treatments for advanced refractory malignancies that are resistant to existing treatments including surgery, radiation therapy, and chemotherapy. In recent years, immunotherapy using immune checkpoint inhibitors and chimeric antigen receptor (CAR) gene-modified T cells (CAR-T therapy) has attracted attention as the fourth treatment method. However, most of these therapies use T cells activated by recognizing antigens as effectors, so there is a fundamental barrier to the restriction of specific antigens. [0003] As an immunotherapy using NK cells that function as major factors of innate immunity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P31/04A61P35/00C12N5/0783A61K35/17
CPCC12N5/0646C12N2506/115A61P31/04A61P35/00A61K39/4613A61K39/4644C12N5/0081C12N2506/02C12N2506/45
Inventor 米满吉和原田结
Owner GAIA BIOMEDICINE INC
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