A kind of NK cell culture fluid and culture method

A technology of NK cells and culture methods, applied in the direction of cell culture active agents, animal cells, culture process, etc., can solve problems such as inability to carry out quality control, safety hazards, etc., and achieve the effects of avoiding safety hazards, increasing magnification, and high killing activity

Active Publication Date: 2022-05-13
广州航华生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention provides a NK cell culture medium and a culture method, which solves the problem of the need to add inactivated K562 tumor cell lines and peripheral blood mononuclear cells for mixed culture to induce expansion in the existing process of using trophoblast cells to expand NK cells. The increase of NK cells makes it impossible to carry out quality control in the later stage and introduces the problem of potential safety hazards

Method used

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  • A kind of NK cell culture fluid and culture method
  • A kind of NK cell culture fluid and culture method
  • A kind of NK cell culture fluid and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] This embodiment is the cultivation of NK cells

[0068] (1) Separation of mononuclear cells from peripheral blood:

[0069] (1.1) Whole blood separation and plasma processing

[0070] Collect 5ml of human peripheral venous blood, transfer it to a centrifuge tube and centrifuge at 2500rpm for 10min, transfer the upper layer of plasma to a new centrifuge tube, heat inactivate it in a water bath at 56.0°C for 30min, and store it in a refrigerator at 4°C for later use;

[0071] (1.2) Separation of peripheral blood mononuclear cells: add an equal volume of cleaning solution to the lower cell layer separated in (1.1) to dilute, and blow evenly. After adding 5ml Ficoll to the 15ml centrifuge tube, tilt the centrifuge tube at 45°, add the diluted cells to the upper layer of Ficoll along the tube wall at a uniform and slow speed, centrifuge at 2500rpm for 20min; suck out most of the cleaning solution and discard the residual plasma mixed layer. Absorb and transfer the remainin...

Embodiment 2

[0092] This embodiment is the cultivation of NK cells

[0093] The difference between this embodiment and embodiment 1 is: (2.4) regular bottle rotation and rehydration steps:

[0094] Add IL-2 and IL-15 to the lymphocyte serum-free medium, mix well to obtain culture medium C, wherein the final concentrations of IL-2 and IL-15 in culture medium C are 1000IU / ml, 50μg / ml respectively .

[0095] Add IL-2 to the lymphocyte serum-free medium, and mix well to obtain culture medium D, wherein the final concentration of IL-2 in culture medium D is 1000 IU / ml.

[0096] Day2: Supplement 5ml culture medium C;

[0097] Day4: Transfer all the cells in the T25 flask to a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, and wash twice with washing solution. Resuspend the cells with culture medium D, transfer them all to a new T75 bottle, and supplement culture medium D to a total volume of 40ml, and 1ml (1.1) of heat-inactivated plasma after centrifugation; ...

Embodiment 3

[0101] This embodiment is the cultivation of NK cells

[0102] The difference between this embodiment and embodiment 1 is: (2.4) regularly turn the bottle and rehydration:

[0103] Add IL-2 to the lymphocyte serum-free medium, and mix well to obtain culture medium D, wherein the final concentration of IL-2 in culture medium D is 1000 IU / ml.

[0104]Day2: Supplement 5ml culture medium D;

[0105] Day4: Transfer all the cells in the T25 bottle to a new T75 bottle, and supplement the culture medium D to a total volume of 40ml, and 1ml (1.1) heat-inactivated plasma after centrifugation;

[0106] Day8: Transfer all the cells in the T75 bottle to a new T175 bottle, and supplement the culture medium D to a total volume of 100ml, and heat-inactivated plasma after centrifugation in 0.5ml (1.1);

[0107] Day11: Supplement 50ml of culture solution D.

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Abstract

The invention relates to the technical field of cell culture, in particular to an NK cell culture medium and a culture method. The invention discloses a NK cell culture medium, which is compounded with IFN-γ and IL-15, IL-2, IL-1α, and CD3 monoclonal antibody, and the combined effect is beneficial to stimulate peripheral blood The efficient conversion of mononuclear cells to NK cells increases the magnification of NK cells and obtains more NK cells, and the content of CD3CD56+ and CD3+CD56+ is high, and the killing activity is high, indicating that the NK cell culture medium provided by the present invention is increasing While increasing the number of NK cells, it significantly increased the cytotoxic activity.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to an NK cell culture medium and a culture method. Background technique [0002] NK cells are important immune cells involved in the body's resistance to tumors, anti-viral infections, and immune regulation. They have no MHC restriction in the process of killing target cells, do not rely on antibodies, and effectively kill a variety of tumor cells through direct and indirect ways. However, the number of NK cells in normal human peripheral blood is relatively small, accounting for only 10% to 20% of the total number of peripheral blood mononuclear cells. In recent years, breakthroughs have been made in cellular immunotherapy, and NK cell therapy has also achieved certain clinical curative effects. At present, how to obtain a large number of high-quality and high-purity NK cells is one of the most critical issues in its clinical application. [0003] In addition to adding cytok...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/24C12N2501/2315C12N2501/2302C12N2501/2301C12N2501/515C12N2501/51C12N2500/90
Inventor 马玉国
Owner 广州航华生物医药科技有限公司
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