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Method for preparing NK (natural killer) cell

A technology of NK cells, cells

Inactive Publication Date: 2014-01-01
青岛麦迪赛斯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Subsequently, the researchers tried to stimulate NK cells with a combination of factors such as IL‐12, IL‐15, IL‐21, CD2 antibodies, etc., but the number and purity of NK cells obtained were limited

Method used

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  • Method for preparing NK (natural killer) cell
  • Method for preparing NK (natural killer) cell

Examples

Experimental program
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preparation example Construction

[0023] The efficient preparation method of NK cells of the present invention comprises the following steps:

[0024] 1) Construction of feeder cells: K562 cells in good condition were selected, and NCR3LG1 and mIL-15 gene nucleotide fragments were respectively inserted into the two multiple cloning sites of the lentiviral vector pEF1alpha-IRES Vector to construct the target gene vector; 24 hours will be 5 x 10 6 A Lenti-X293T packaging cell was inoculated into a 10 cm culture dish; the target gene carrier was mixed with 36 microliters of Lenti-X HTX Packaging Mix, 7.5 microliters of Xfect Polymer, and 1150 microliters of Xfect Reaction Buffer, and allowed to stand at room temperature for 10 minutes. Then add it to the petri dish; place the petri dish in a 37°C carbon dioxide incubator overnight; replace with a new medium; after 48 hours, collect the supernatant and pass it through a 0.45 micron sterile filter, the filtrate contains the target gene virus suspension; the detect...

experiment example 1

[0033] 1) Cell inoculation: collect 50 ml of venous peripheral blood, use lymphocyte separation medium to obtain PBMCs by centrifugation, wash twice with normal saline, resuspend with 60 ml of serum-free medium, sample and count, and the number of cells is 4.6×10 7 . Cells were seeded into culture flasks, and the same number of inactivated feeder cells was added, 360,000 units of IL-2, and 600 ng of IL-21 were added.

[0034] 2) Transfer culture: After inoculated cells were cultured for 4 days, they were transferred to a gas-permeable cell culture bag to continue culturing, adding serum-free medium to 300 ml, adding 15,000 units of IL-2, and 3,000 ng of IL-21.

[0035] 3) Secondary stimulation: when the culture was expanded to the 7th day, samples were counted, and the same amount of inactivated feeder cells were added for secondary stimulation, and IL-2 was added at a concentration of 500 units / ml, and IL‐2 was added at a concentration of 10 ng / ml. Concentrations of IL‐21 we...

experiment example 2

[0040] 1) Cell inoculation: 50 ml of venous peripheral blood was collected, and PBMCs were obtained by centrifugation using lymphocyte separation medium, washed twice with normal saline, and resuspended in 70 ml of serum-free medium. Sampling and counting, the number of cells is 5.1×10 7 . Cells were seeded into culture flasks, and the same number of inactivated feeder cells was added, 420,000 units of IL-2, and 700 ng of IL-21 were added.

[0041] 2) Transfer culture: After inoculated cells were cultured for 4 days, they were transferred to a gas-permeable cell culture bag to continue culturing, adding serum-free medium to 400 ml, adding 20,000 units of IL-2, and 4,000 ng of IL-21.

[0042] 3) Secondary stimulation: when the culture was expanded to the 7th day, samples were counted, and the same amount of inactivated feeder cells were added for secondary stimulation, and IL-2 was added at a concentration of 500 units / ml, and IL‐2 was added at a concentration of 10 ng / ml. Co...

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Abstract

The invention provides a method for efficiently preparing an NK cell, which can improve the multiplication rate and the purity of the NK cell through combination of stimulation effects of cell factors and feeder cells. The method is mainly characterized by comprising the steps as follows: NCR3LG1 and m IL-15 are transfected to a K562 cell simultaneously, m IL-15 can be used for adjusting activation and multiplication of the NK cell, NCR3LG1 serving as a ligand of NKp30 which is one of main activated receptors on the surface of the NK cell can effectively stimulate activation of the NK cell, and NCR3LG1 and m IL-15 have a synergistic effect; and PBMC(peripheral blood mononuclear cells) can be multiplied over 500 times in 21 cultivation days through stimulation of factors of freely added IL-2, IL-21 and the like, and a proportion of CD3-CD56+NK cell exceeds 70%; and up to now, a research report that NCR3LG1 and m IL-15 are transfected to the feeder cells simultaneously and jointly stimulate and activate the NK cell in combination of free cell factors is absent. The invention firstly provides a method for jointly cultivating and preparing the NK cell in combination of the feeder cells and the free factors.

Description

technical field [0001] The invention belongs to the field of cell culture, in particular to a natural killer cell (Natural Killer, NK) culture, that is, using transgenic feeder cells and cytokines to jointly stimulate peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cell, PBMC) to obtain a large number of high Purity of NK cells. Background technique [0002] NK cells are a group of larger granulated lymphocytes that express CD56 on the cell surface and lack CD3 expression, so the surface marker CD3 is usually used ‐ CD56 + to define NK cells. NK cells can account for about 5-15% of peripheral blood lymphocytes, and are distributed in the liver, viscera, bone marrow, and lymph nodes of the human body. [0003] NK cells are an important part of the innate immune system, and can kill tumor cells or infected cells in a variety of ways: 1. After contacting with target cells, they release perforin and granzyme; 2. Through their own expressed ligands, Stimulate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 徐矫健孙威
Owner 青岛麦迪赛斯生物科技有限公司
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