NK cell serum-free culture medium and preparation method thereof

A technology of serum-free medium and NK cells, which is applied in the field of cell culture, can solve the problems of low expansion multiple, complicated operation, and easy pollution, and achieve the effect of increasing the multiple of proliferation

Inactive Publication Date: 2016-12-14
英普乐孚生物技术(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The existing NK cell expansion and activation culture system generally overactivates the killing activity of NK cells while expanding. This culture system is likely to cause premature senescence of NK cells, which is manifested as shortened survival time and increased multiples of expansion. not tall
In addition, in the process of cultivating NK cells using the existing medium, a variety of stimulatory factors and other substances need to be added, which is complicated and easy to cause pollution

Method used

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  • NK cell serum-free culture medium and preparation method thereof
  • NK cell serum-free culture medium and preparation method thereof
  • NK cell serum-free culture medium and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Step 1: Prepare the basic components of NK cell serum-free medium.

[0029] DME F12 medium powder (purchased from SIGMA company D8900) and 1.2 g of sodium bicarbonate were weighed to prepare DME F12 medium solution with water for injection.

[0030] According to each liter of DME F12 medium solution, add albumin solution (containing 0.0375mmol albumin) respectively; Then in each part, continue to add the following substances: recombinant human transferrin 50mg (T3705 product purchased from SIGMA company), Recombinant human insulin 10mg (purchased from Jiangsu Wanbang Biochemical Pharmaceutical Co., Ltd. insulin injection product), cholesterol 2mg (purchased from SIGMA company's C1231 product), ethanolamine 2mg (purchased from SIGMA company's E0135 product), ascorbic acid 2.5mg (purchased from A4544 product from SIGMA Company), gentamicin sulfate 10000IU (purchased from the gentamicin sulfate injection product of Shanghai First Biochemical Pharmaceutical Co., Ltd.), copp...

Embodiment 2

[0037] The operation of preparing medium is the same as in Example 1, only the final concentration of the added substance is different:

[0038] The concentration of albumin is 0.001mmol / L, the concentration of recombinant human transferrin is 0.1mg / L, the concentration of recombinant human insulin is 0.1mg / L, the concentration of cholesterol is 0.1mg / L, and the concentration of ethanolamine is 0.1mg / L L, the concentration of ascorbic acid is 0.1mg / L, the concentration of gentamicin sulfate is 1×10 3 IU / L, the concentration of copper sulfate is 0.0001mg / L, the concentration of zinc sulfate is 0.01mg / L, and the concentration of manganese chloride is 0.0001-0.001mg / L.

[0039] Preferably, the concentration of CD2 monoclonal antibody in the induction medium components is 1 μg / L, the concentration of CD16 monoclonal antibody is 1 μg / L, the concentration of CD335 monoclonal antibody is 1 μg / L, and the concentration of IL-2 is 1×10 4 IU / L, the concentration of IL-12 is 0.01 μg / L, t...

Embodiment 3

[0042] The operation of preparing medium is the same as in Example 1, only the final concentration of the added substance is different:

[0043] The concentration of albumin is 0.5mmol / L, the concentration of recombinant human transferrin is 500mg / L, the concentration of recombinant human insulin is 100mg / L, the concentration of cholesterol is 20mg / L, the concentration of ethanolamine is 20mg / L, and the concentration of ascorbic acid The concentration is 100mg / L, the concentration of gentamicin sulfate is 1×10 6 IU / L, the concentration of copper sulfate is 0.01mg / L, the concentration of zinc sulfate is 10mg / L, and the concentration of manganese chloride is 0.001mg / L.

[0044] Preferably, the concentration of CD2 monoclonal antibody in the induction medium components is 100 μg / L, the concentration of CD16 monoclonal antibody is 100 μg / L, the concentration of CD335 monoclonal antibody is 100 μg / L, and the concentration of IL-2 is 1×10 6 IU / L, the concentration of IL-12 was 100 ...

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Abstract

The invention provides an NK cell serum-free culture medium. The culture medium comprises two independently packaged components which are used in different stages of cell culture, so the NK cell culture process is divided into an induction stage and a proliferation process, the culture effect is improved, and the cell induction and culture process is simplified, that is, other extra cell induction factors do not need to be added in the culture process. Polyinosinic cells are added into the NK cell serum-free culture medium and are more suitable for NK cell efficient culture, and the proliferation rate is increased.

Description

technical field [0001] The invention relates to a NK cell serum-free medium and a preparation method thereof, belonging to the technical field of cell culture. Background technique [0002] Serum-free medium refers to a synthetic medium that can maintain the growth and reproduction of cells in vitro for a long time without adding serum, and can be used to cultivate immune cells, such as lymphocytes (for example, T lymphocytes, B cells, NK cells, etc.) , dendritic cells (also known as DC cells), monocytes / macrophages, etc. [0003] Natural killer cells (NK) are important immune cells of the body, which play an important role in the body's anti-tumor and anti-virus processes. NK cells recognize target cells non-specifically, and kill target cells by secreting granzymes, perforin, cytokines and other ways. At the same time, NK cells express FcγRⅢA, which binds to the Fc segment of antibody molecules, and achieves specific recognition and killing of target cells through antibo...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 武宁谢志明
Owner 英普乐孚生物技术(上海)有限公司
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