Activation method for enhanced NK (Natural Killer) cells and cell preparation method

A technology of NK cells and monocytes, which is applied in the field of cell preparation and enhanced NK cell activation, can solve the problems of not being suitable for large-scale applications, large demand for cytokines, and unsatisfactory purity, and achieve safe, simple and effective activation and proliferate the effect

Active Publication Date: 2015-09-23
BEIJING BIOHEALTHCARE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For many years, people have been trying to achieve large-scale expansion of NK cells in vitro. Traditional NK cell culture methods require the use of PHA and/or tumor cell lines (such as K562, K562-MB15-41BBL, HFWT) as stimulatory factors and trophoblasts cells to cultivate NK cells, in addition to the cumbersome cell culture steps, the use of PHA and tumor cell lines also brings safety issues in clinical applications
The current commonly used technology is mainly to add IL-2, IL-12, IL-

Method used

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  • Activation method for enhanced NK (Natural Killer) cells and cell preparation method
  • Activation method for enhanced NK (Natural Killer) cells and cell preparation method
  • Activation method for enhanced NK (Natural Killer) cells and cell preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Ex vivo culture of NK cells collected from peripheral blood

[0032] The method for culturing NK cells in vitro provided in this embodiment comprises the following steps:

[0033] 1) Collect mononuclear cells from isolated peripheral blood. After diluting 20ml of isolated venous blood with normal saline at 1:1, take 10ml of the diluted blood and slowly add it to a 15ml centrifuge tube, centrifuge at 1500rpm for 20min, take buffy coat cells and wash with normal saline, centrifuge at 1200rpm for 10min Resuspend with normal saline, and discard the supernatant after repeating once.

[0034] 2) resuspend the above-mentioned cells with the special medium for NK cells of the present invention, and the special medium for NK cells contains the following components in the serum-free medium: human albumin with a concentration of 10 g / L; a concentration of 20 ng IL-2 per ml; IL-15 at a concentration of 20 ng / ml; 2% volume percentage of isolated human plasma. And add hu...

Embodiment 2

[0036] Example 2 Ex vivo culture of NK cells collected from ascites

[0037] The method for culturing NK cells in vitro provided in this embodiment comprises the following steps:

[0038] 1) Collect mononuclear cells from ascites.

[0039] 2) resuspend the above cells with the special medium for NK cells of the present invention, the special medium for NK cells contains the following components in the serum-free medium: human albumin with a concentration of 5 g / L; a concentration of 30 ng IL-2 per ml; IL-15 at a concentration of 30 ng / ml; 1% volume percentage of isolated human plasma. And add human CD3 antibody (Orthoclone OKT-3; Ortho Biotech, Raritan, NJ) as CD3 agonist, the antibody final concentration is adjusted to 150ng / ml; TLR7 / 8 agonist (GDQ, Gardiquimod purchased from Invivo Gen company, use The endotoxin is dissolved in sterile water, the concentration is 1-2.5mg / ml, and the final concentration is adjusted to 5ug / ml when stored at -20°C for future use; the serum-fr...

Embodiment 3

[0041] Embodiment 3 Conventional methods cultivate NK cells

[0042] Mononuclear cells were collected from peripheral blood. After diluting 20ml of isolated venous blood with normal saline at 1:1, take 10ml of the diluted blood and slowly add it to a 15ml centrifuge tube, centrifuge at 1500rpm for 20min, take buffy coat cells and wash with normal saline, centrifuge at 1200rpm for 10min Resuspend with normal saline, and discard the supernatant after repeating once.

[0043] The above-mentioned cells were resuspended with NK cell medium, and the NK cell medium contained components in the following content range in CellGro SCGM serum-free medium (CellGenix, Freiburg, Germany; product number: YS-SO-0001334): the concentration was Human albumin at 50g / L; IL-2 at a concentration of 60ng / ml; IL-7 at 50ng / ml; IL-12 at 10ng / ml; IL-18 at 10ng / ml; TNF-α at 25ng / ml ; 8% volume percentage of isolated human plasma. After culturing to the fifth day, replace the fresh NK cell culture mediu...

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Abstract

The invention discloses a culture medium for activating, amplifying and culturing NK (Natural Killer) cells in vitro, a method for activating, amplifying and culturing the NK cells in vitro by utilizing the culture medium, and a biological agent containing the NK cells which are prepared through the method and are activated and amplified in vitro. Mononuclear cells are re-suspended by using the special culture medium for the NK cells, and a human CD3 antibody and a TLR7/8 agonist are added. The special culture medium for the NK cells is a serum-free medium containing human albumin, IL-2, IL-15 and in-vitro human plasma.

Description

Technical field: [0001] The invention relates to the field of in vitro culture of immune cells, in particular to an enhanced NK cell (natural killer cell) activation method, a NK cell proliferation method using the NK cell, and a cell preparation method. Background technique: [0002] As early as the 1970s, in the study of tumor immunity, it was discovered that lymphocytes from normal organisms could kill certain tumor cells, and it was later confirmed that the killing effect of these lymphocytes was natural, without the presence of antibodies or prior sensitization, so Name it natural killer cell (NK) cell. NK cells are important immunoregulatory cells for the body to resist malignant tumors or virus infections. They regulate their own activation through the specific binding of surface receptors and ligands on the surface of target cells, directly killing tumor infiltrating or virus-infected cells, or secreting cytokines. Induces inflammatory responses and regulates other ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 刘静维卢戌王跃杨照敏
Owner BEIJING BIOHEALTHCARE BIOTECH
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