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NK cell in-vitro amplification culture medium combination and culture method

An in vitro expansion and NK cell technology, which is applied in the direction of cell culture active agents, tissue cell/virus culture devices, biochemical equipment and methods, etc., can solve unsafe and ethical, cytokine expression changes, and cell culture Safety and other issues, to achieve effective amplification and activation, high activity, and increased safety

Active Publication Date: 2017-05-24
浙江丹晖生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the composition of animal serum is relatively complex, which can easily bring unsafe factors to cell culture; improper addition of cytokines can easily cause changes in the expression of NK cell activation receptors and inhibitory receptors; at the same time, allogeneic cells are often medically unsafe and ethical issues

Method used

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  • NK cell in-vitro amplification culture medium combination and culture method
  • NK cell in-vitro amplification culture medium combination and culture method
  • NK cell in-vitro amplification culture medium combination and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Prepare basal medium, induction medium, proliferation medium and activation adaptation medium according to the medium composition given in Table 1.

[0091] Healthy volunteers collected 25mL of peripheral blood from the cubital vein on an empty stomach in the morning, added heparin for anticoagulation; used lymphocyte separation medium to perform density gradient centrifugation, collected mononuclear cell layers, and counted A after washing with PBS centrifugation. Mononuclear cells were resuspended, and the cell density was adjusted to 5×10 5 cells / mL, cell culture was carried out in the immune cell in vitro expansion device described in the present disclosure, firstly in 5% CO 2 , the basal culture was carried out for 1.5 days under the environment of 37° C. to obtain the basal culture cells. Collect the cells by centrifugation and adjust the cell density to 5×10 5 cells / mL, in 5% CO 2 , the induction culture was carried out for 3 days under the environment of 37° ...

Embodiment 2

[0098] The method of Example 1 was used to culture NK cells in vitro, the only difference being that the components of each culture medium used are shown in Table 2. The amplification factor and purity results of the detected cells are shown in Table 5 and Figure 4 , 5 As shown, the killing rate results of NK cells are shown in Table 6 and Image 6 shown.

[0099] Table 2

[0100]

Embodiment 3

[0102] The method of Example 1 was used to culture NK cells in vitro, the only difference being that the components of each culture medium used are shown in Table 3. The amplification factor and purity results of the detected cells are shown in Table 5 and Figure 4 , 5 As shown, the killing rate results of NK cells are shown in Table 6 and Image 6 shown.

[0103] table 3

[0104]

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Abstract

The invention relates to an NK cell in-vitro amplification culture medium combination and a culture method. The culture medium combination comprises a base culture medium, an induced culture medium, a proliferation culture medium and an activated adaptive culture medium, wherein the base culture medium is prepared from the following components: an RPMI-1640 (Roswell Park Memorial Institute-1640) culture medium, a GT-T551 culture medium, gentamicin and BSA (Bovine Serum Albumin); the induced culture medium is prepared from the following components: the RPMI-1640 culture medium, the GT-T551 culture medium, the BSA, lentinan, ganoderma lucidum polysaccharides, tea polyphenols, allicin and paclitaxel; the proliferation culture medium is prepared from the following components: the RPMI-1640 culture medium, the GT-T551 culture medium, the BSA and interleukins; and the activated adaptive culture medium is prepared from the following components: the RPMI-1640 culture medium, the GT-T551 culture medium, insulin, transferrin and glutamine. According to the technical scheme, the quantity of NK cells cultured by the NK cell in-vitro amplification culture medium combination and the method is more, and the NK cells also have higher activity in clinical treatment.

Description

technical field [0001] The present disclosure relates to the field of biotechnology, in particular, to a medium combination and a culture method for NK cell expansion in vitro. Background technique [0002] Natural killer cells (NK) are important immune cells in the body, mainly distributed in peripheral blood, and can participate in the physiological processes of anti-tumor, anti-virus infection and immune regulation. Existing studies have shown that NK cells are the cells with the strongest anti-cancer activity in the human body. They can release perforin to form perforations on the surface of target cells, and then enter target cells through granzyme to induce apoptosis of target cells, and can secrete a large amount of cytokines at the same time. For target cells, it can further activate other types of immune cells to attack target cells, and can express apoptosis-inducing proteins and tumor necrosis factor-related apoptosis-inducing ligands, so that programmed apoptosis...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12M3/02
CPCC12M23/14C12M23/56C12M27/02C12N5/0646C12N2500/24C12N2500/30C12N2500/32C12N2500/34C12N2501/2312C12N2501/2315C12N2501/2318C12N2501/2321C12N2501/33
Inventor 郭天欢杨小芳何淑婷
Owner 浙江丹晖生物科技有限公司
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