Culture system and culture method of NK cells

A technology of NK cells and culture system, applied in the field of cells, can solve the problems that tumor cells cannot have a good killing effect, affect the therapeutic effect, and weaken the therapeutic effect, so as to avoid toxic effects and the risk of serum-derived contamination, with a clear composition, Avoid the effect of quality variation

Active Publication Date: 2021-03-02
杭州中赢生物医疗科技有限公司
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Problems solved by technology

However, in the past ten years, NK cell therapy technology has not been widely used clinically. In some previous clinical trials, the curative effect of autologous NK cell therapy in clinical application was not obvious. NK immune cells affect the therapeutic effect of NK cells. It is conceivable that in patients with a large tumor burden, if more than 90% of the reinfused NK cells are in a silent state instead of an activated state, then it must not have a significant effect on tumor cells. Good killing effect, in addition, according to in vitro experiments, when the effective target ratio of NK cells and tumor cells is 10:1, the killing efficiency can reach more than 80%, but if the amount of NK cells reinfused is far less than the amount of tumor cells in the body , the therapeutic effect will be weakened

Method used

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  • Culture system and culture method of NK cells
  • Culture system and culture method of NK cells
  • Culture system and culture method of NK cells

Examples

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Effect test

Embodiment 1

[0064] Embodiment 1 culture system of the present invention

[0065] Component 1: NK serum-free medium and inactivated genetically engineered cells;

[0066] Among them, the NK serum-free medium contains KBM581 culture medium and 5v / v% albumin, 200U / ml IL2, 80U / ml gentamicin; the genetically engineered cells are K562 cells expressing IL21-CD4-CD137L, that is, IL21-CD4 - CD137L-K562 cells.

[0067] Preparation: take KBM581 culture solution and add IL2 and gentamicin, add albumin and inactivated genetically engineered cells when cultivating NK cells.

[0068] Component 2: NK cell serum-free medium;

[0069] Wherein, the NK serum-free medium comprises KBM581 culture solution and 5v / v% albumin, 200U / ml IL2 and 80U / ml gentamicin.

[0070] Preparation: take KBM581 culture solution and add IL2 and gentamicin, and then add albumin when culturing NK cells.

[0071] Example 2 Expansion of NK cells

[0072] (1) PBMC extraction: Centrifuge fresh blood at a suitable centrifugal speed ...

Embodiment 3

[0095] The influence of embodiment 3 different NK cell basal mediums on NK cell expansion

[0096] (1) Investigate the influence of interleukin 2 of different concentrations on NK cell expansion, culture method is the same as embodiment 2, culture time is 14 days, the results are shown in Table 1 and image 3 .

[0097] The influence of interleukin 2 of different concentrations in table 1 on NK cell culture method

[0098]

[0099]

[0100] The results showed that when the IL2 concentration was 200U / ml, the expansion multiple (quantity) and survival rate of NK cells were significantly higher than other concentrations, and the purity was slightly higher than other concentrations.

[0101] (2) To investigate the effects of different concentrations of albumin on the expansion of NK cells

[0102] Culture method is the same as embodiment 2, and culture time is 14 days, and the results are shown in Table 2 and Figure 4 .

[0103] Table 2 Effects of different albumin conc...

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Abstract

The invention relates to the technical field of cell culture and particularly relates to a culture system and a culture method of NK cells. The culture system provided by the invention comprises a component 1 and a component 2, wherein the component 1 comprises an NK cell serum-free culture medium and inactivated genetically engineered cells; and the component 2 is an NK cell serum-free culture medium. The culture system is used for culturing and amplifying the NK cells, more than 90% of effector cells can be obtained after 14 days, the amplification multiple is about 11000, and a foundation is laid for broad-spectrum application of adoptive immunotherapy of the NK cells.

Description

technical field [0001] The invention relates to the field of cell technology, in particular to a culture system and a culture method of NK cells. Background technique [0002] NK cells are currently the most important immune cells known to kill tumor cells undergoing tumor immunoediting, and are one of the important means for clinical tumor cell immunotherapy. Clinical studies have shown that allogeneic and autologous NK cell immunotherapy is safe. In 2009, the Ministry of Health issued the "Management Regulations for Autologous Immune Cell (T Cell, NK Cell) Therapy Technology (Draft for Comment)", which included NK cell immunotherapy technology as the third type of new medical technology in the application scope of clinical treatment . However, in the past ten years, NK cell therapy technology has not been widely used clinically. In some previous clinical trials, the curative effect of autologous NK cell therapy in clinical application was not obvious. NK immune cells af...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/10
CPCC12N5/0646C12N2500/90C12N2501/2302C12N2501/998C12N2502/99C12N2501/2321C12N2501/2312C12N2501/505C12N2501/599Y02A50/30
Inventor 俞英豪彭昉王冶陶
Owner 杭州中赢生物医疗科技有限公司
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