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Cell culture medium composition containing resveratrol and silk sericin

A technology of resveratrol and sericin, which is applied in the direction of cell culture medium, cell culture active agent, animal cells, etc., to achieve the effect of increasing expression level, improving in vitro amplification ability, and enhancing homing ability

Active Publication Date: 2016-08-17
BEIJING SHIHE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no report in the prior art of combining resveratrol and silk sericin and adding it to the natural killer cell culture system to amplify and cultivate it and further use it for treatment.

Method used

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  • Cell culture medium composition containing resveratrol and silk sericin
  • Cell culture medium composition containing resveratrol and silk sericin
  • Cell culture medium composition containing resveratrol and silk sericin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Preparation of stock solutions of resveratrol and sericin

[0052] Resveratrol powder (purchased from Sigma, USA) was dissolved in cell culture-grade DMSO to prepare a storage solution with a final concentration of 5 mM. The solution was sterilized by filtering through a 0.22 μm filter, and divided into 1 ml / bottle to avoid The light is stored at -80 degrees.

[0053] Silk sericin dry powder (purchased from Wako Company, Japan) was dissolved in PBS with a pH of 7.4 and prepared as a 10 mg / ml storage solution. The solution was sterilized by filtering through a 0.22 μm filter, divided into 1 ml / cartridge, and frozen in Save at -80 degrees.

[0054] When in use, take out the stock solutions of the two additives and melt them quickly at room temperature;

[0055] Mix with the culture medium according to the following ratio

[0056] Take 2ml of resveratrol storage solution, 1ml of sericin storage solution, 97ml of SCGM medium,

[0057] Mix well at room temperature, that ...

Embodiment 2

[0077] Example 2. Resveratrol combined with sericin to evaluate the proliferation ability of NK cells in vitro. Collect 30ml of peripheral blood from healthy people. According to the method reported in the literature, use human mononuclear cell separation medium (Beijing Dongfang Huahui Biopharmaceutical Co., Ltd. Technology Co., Ltd., Cat.No.25610) for the separation of PBMC. The separated PBMCs were counted by trypan blue exclusion method, the cell number was adjusted to 0.5x106 cells / ml, and resuspended with D-PBS.

[0078] According to the grouping method of the experimental design in Table 1., NK cells were cultured in vitro using different culture recipes. SCGM medium was purchased from CellGenix, humanized CD3 monoclonal antibody OKT-3 was purchased from Takara, recombinant human interleukin- 2 (IL-2) was purchased from Beijing Shuanglu Pharmaceutical.

[0079] The cultivation system is respectively,

[0080] Blank SCGM medium,

[0081] SCGM medium containing resvera...

Embodiment 3

[0089] Example 3. Effects of Resveratrol and Sericin on the Expression Levels of Different Molecular Receptors on the Surface of NK Cells

[0090] On the 14th day of culture, different groups of NK cells were collected, and the representative activating receptors, inhibitory receptors, chemotactic receptors and cytokine receptors expressed on the surface of NK were analyzed by flow cytometry. analyze. The operation process is briefly described as follows: the number of cells in each sample for flow cytometry is not less than 10 6 cell. The sample was centrifuged at 350g for 10 minutes at room temperature, the supernatant of the culture medium was discarded, and resuspended in cell preservation solution with a final volume of 200 μl. Add different specific antibodies according to the detected molecules, the detected molecules and the calculation method of the expression fold change of the corresponding molecules are shown in Table 2 below.

[0091] Table 2. NK cell surface m...

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Abstract

The invention relates to a cell culture medium composition containing resveratrol and silk sericin. In a culture system, the concentration of resveratrol is 1-100 micrometers, and the concentration of silk sericin is 1-100 micrograms per milliliter. By combining resveratrol and silk sericin and adding resveratrol and silk sericin into the natural killer cell culture system, it is accidentally found that the migration activity and killing activity of Natural killer cell (NK) for in-vitro expansion of a Peripheral blood monocyto (PBMC) source can be improved by the cell culture medium composition.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to a medium composition for cultivating natural killer cells, which contains resveratrol and silk sericin. Background technique [0002] Natural killer cells (Natural killer cells, NK) cells are a kind of lymphoid cells that widely exist in peripheral blood, spleen, lymph nodes and other tissues and organs, and contain large particles in the patina. The main effector cells of tumor immunity. NK cells exert immune activity mainly through direct and indirect ways. The direct way is mainly that NK cells distinguish "self" and "non-self" cells in the body through specific activating or / and inhibitory receptors on their surface, and after recognition, these surface receptors and corresponding molecules on the surface of target cells After specific binding, NK cells release perforin, granzyme, IL-2, IL-15, INF-γ and other cytokines to kill and clear target cells. The indirect method is al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C12N5/0783
CPCC12N5/0037C12N5/0646C12N2500/35C12N2501/2302C12N2501/998
Inventor 赵宇胡杨刘彩云李帅民王敏贾乐伟
Owner BEIJING SHIHE BIOTECH
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