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Method for preparing autologous natural killer cell in cocktail culture and and kit product

A technology for natural killer cells and cell culture, which is used in the preparation of autologous natural killer cell cocktail culture and the field of kit products, which can solve the problems of shortened telomere length, limited NK cells and long culture time, and low killing function of NK cells, etc. question

Inactive Publication Date: 2015-07-22
JIANGSU JIESHENG BIOSCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The number of NK cells and the killing function are directly related to the curative effect. The problems in the treatment are the limited number of NK cells and the long culture time
The traditionally used interleukin-2 activation growth factor is limited, the telomere length is shortened, and the long culture time causes the low killing function of NK cells

Method used

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  • Method for preparing autologous natural killer cell in cocktail culture and and kit product
  • Method for preparing autologous natural killer cell in cocktail culture and and kit product
  • Method for preparing autologous natural killer cell in cocktail culture and and kit product

Examples

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Effect test

preparation example Construction

[0027]The present invention finds a preparation method through cocktail culture, that is, in the mixture containing recombinant human interleukin 15, recombinant human interleukin 18, recombinant human interleukin 21, recombinant human interleukin 12, recombinant human interleukin 7 and recombinant human MHC-I chain-related molecule A Under the joint action of more than three cytokines, it can activate the massive proliferation of natural killer cells and enhance their killing activity; NK cells are derived from 30ml of autologous peripheral blood mononuclear cells of patients, and the NK cells are cultured in cocktail style until the cell proliferation multiple reaches more than 1000 times in 21 days. The number of cells reaches 30×10 9 The above meets the needs of clinical treatment, and the telomerase activity of the expanded NK cells is enhanced, the length of the telomere is increased, and the killing activity of a young state is maintained.

[0028] The preparation metho...

Embodiment 1

[0052] Cocktail culture of autologous natural killer cells

[0053] Collect 30ml of peripheral blood from patients with renal cell carcinoma, liver cancer, prostate cancer, breast cancer and glioblastoma (with informed consent signed), and obtain 36×10 6 For mononuclear cells, resuspend with 1.6ml of autologous plasma, add 32ml of CellGro SCGM cell culture medium (containing 50ng / ml recombinant human interleukin 15, 20ng / ml recombinant human interleukin 18, 20ng / ml recombinant human interleukin 21 and 50ng / ml recombinant human MHC-class I chain-related molecule A), mixed well and added to 12-well cell culture plate at 37°C, 5% CO 2 Continue culturing in the incubator for more than 5 days.

[0054] After 5 days of cocktail-type activation culture, the natural killer cells were pipetted with a 5 mL pipette, pipetted into a new cell culture bottle, and supplemented with 20 mL of fresh cell culture medium (containing 500 activity units / mL recombinant human interleukin-2), at 37...

Embodiment 2

[0059] Kit for Cocktail Culture of Autologous Natural Killer Cells

[0060] (1) 200ml lymphocyte separation fluid;

[0061] (2) 500ml lymphocyte serum-free medium;

[0062] (3) Cocktail culture reagent: 10ng-50ng / ml recombinant human interleukin 15, 10ng-50ng / ml recombinant human interleukin 18, 10ng-50ng / ml recombinant human interleukin 21, 10ng-50ng / ml recombinant human interleukin 12, 10ng- 50ng / ml recombinant human interleukin 7 and 10ng-50ng / ml recombinant human MHC-class I chain-related molecule A of three or more cytokines;

[0063] (4) 50-500 activity units / mL recombinant human interleukin-2;

[0064] (5) 100ml physiological saline;

[0065] (6) instruction manual;

[0066] Wherein, the instruction manual includes the method described in Example 1.

[0067] Packing is carried out after aliquoting to obtain a kit for cultivating autologous natural killer cells in a cocktail style.

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Abstract

The invention discloses a method for preparing autologous natural killer cell in cocktail culture and a kit product. The method is characterized in that proliferation of natural killer cells of peripheral blood mononuclear cells from a cancer patient is activated under the combined action of a recombinant human interleukin 15, a recombinant human interleukin 18, a recombinant human interleukin 21, a recombinant human interleukin 12, a recombinant human interleukin 7 and a recombinant human MHC-1 chain related molecule A, and the natural killer cell killing potential is reinforced. The invention also discloses a kit containing the autologous natural killer cell in cocktail culture prepared by the method. The kit can be used for clinically acquiring plenty of natural killer cells and performing antitumor and antivirus treatment.

Description

technical field [0001] The present invention relates to a method for preparing autologous natural killer cells in a cocktail style, which includes the following features: the peripheral blood mononuclear cells from cancer patients themselves are mixed with recombinant human interleukin 15, recombinant human interleukin 18, recombinant human Interleukin 21, recombinant human interleukin 12, recombinant human interleukin 7, and recombinant human MHC-I chain-related molecule A activate the proliferation of natural killer cells and enhance the killing potential of natural killer cells; The autologous natural killer cell cocktail culture kit obtained by the method can proliferate in large quantities in vitro to obtain natural killer cells with killing activity, and can be used for multiple courses of immunotherapy for various cancers including solid tumors and hematological tumors. Background technique [0002] Natural killer cells (NK) mainly express the CD16+ / CD56+ phenotype, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00A61P35/02
Inventor 王俊荣宋现让
Owner JIANGSU JIESHENG BIOSCI CO LTD
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