Addition agent of NKT cell induction culture and method of induction culture

A technology of NKT cells and additives, applied in the field of cells, can solve the problems of potential safety hazards, insufficient expansion of NKT cells, and affecting the effect of immunotherapy, etc., and achieve the effect of inducing and promoting proliferation, promoting large-scale proliferation, and good killing effect

Inactive Publication Date: 2015-11-11
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing method of inducing and culturing NKT cells does not expand the expansion factor of NKT cells large enough, and if it is used for immunotherapy or sub-health care, it will greatly affect the effect of immunotherapy
Moreover, the existing method of inducing and culturing NKT cells uses fetal bovine serum, which may introduce some mycoplasma and viruses, and there are certain potential safety hazards

Method used

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  • Addition agent of NKT cell induction culture and method of induction culture
  • Addition agent of NKT cell induction culture and method of induction culture
  • Addition agent of NKT cell induction culture and method of induction culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, peripheral blood separation obtains PBMC:

[0032] 1) Take 40mL of human peripheral blood into two 50mL centrifuge tubes, dilute the peripheral blood 1:1 with normal saline and mix well;

[0033] 2) Take two new 50mL centrifuge tubes, add 15mL of lymphocyte separation medium, and then according to the volume ratio of lymphocyte separation medium: blood dilution solution is 1:2, slowly add the mixed blood dilution solution along the tube wall to lymphocytes The upper layer of the separation liquid, be careful not to break through the separation liquid layer;

[0034] 3) Put it into a refrigerated centrifuge (Eppendorf), centrifuge at 3000rpm / min for 30min, and adjust the speed of lifting to 0;

[0035]4) After centrifugation, suck the mononuclear cells into a 15mL centrifuge tube with a Pasteur pipette, wash once with 10mL normal saline, and centrifuge at 400g for 5min.

Embodiment 2

[0036] Embodiment 2, the induction culture of NKT cell

[0037] 1. Resuspend the cell pellet obtained in Example 1 with 5mLX-VIVO15 medium and count them. According to the counting results, divide them into 3 groups for culture:

[0038] Group 1: press 1×10 with X-VIVO-15 medium 6 cell / mL was inoculated into a T75 culture flask, and 300U / mLIL-2, 30ng / mLIL-15, 50ng / mLIL-21, 50ng / mLIL-7 and 50ng / mLOKT-3 were added, placed at 37°C, 5% CO 2 NKT cells were induced and cultured in a constant temperature incubator.

[0039] Group 2: press 1×10 with X-VIVO-15 medium 6 cell / mL was inoculated into a T75 culture flask, and 300U / mLIL-2 and 30ng / mLIL-15 were added, placed at 37°C, 5% CO 2 NKT cells were induced and cultured in a constant temperature incubator.

[0040] Group 3: press 1×10 with RPMI1640 medium 6 cell / mL was inoculated into a T75 culture flask, and 300U / mLIL-2, 30ng / mLIL-15, 50ng / mLIL-21, 50ng / mLIL-7 and 50ng / mLOKT-3 were added, placed at 37°C, 5% CO 2 NKT cells were i...

Embodiment 3

[0045] Embodiment 3, cell viability detection

[0046] Get each group 8 * 10 in embodiment 2 respectively 6 The cells on the third day and the fourteenth day of cell culture were observed, and the results are shown in Figure 1-3 .

[0047] At the same time, the number of cells was counted, and cell viability was detected. The results are shown in Table 1 and Table 2.

[0048] Table 1 Cell number

[0049] cell number

[0050] Table 2 Cell viability

[0051] cell viability

[0052] Depend on Figure 1-3 The results showed that the cells of group 1 were better than those of group 2 on the third day and the fourteenth day of culture. From the results in Table 1 and Table 2, it can be seen that the cells of Group 1 on the 14th day of culture proliferated 45.5 times compared to the cells on the 3rd day of culture, which was much higher than that of Group 2 and Group 3, while the cell viability was comparable. It shows that the additive for inducing culture...

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PUM

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Abstract

The invention relates to the field of cells and discloses an addition agent of NKT cell induction culture and a method of induction culture. The addition agent of the NKT cell induction comprises growth factors IL-21, IL-7 and OKT-3. The growth factors IL-21, IL-7 and OKT-3 has a synergistic effect with the recognized growth factors IL-2 and IL-15, the multiplication of the NKT cells can be better induce and promoted, the mass multiplication of the NKT cells can be promoted, and the addition agent has a great killing effect to tumor cells. The method for inducing and culturing the NKT cells is simple in operation, capable of promoting the mass multiplication of the NKT cells and suitable for the scale production of the NKT cells.

Description

technical field [0001] The invention relates to the field of cells, in particular to an additive for inducing and culturing NKT cells and a method for inducing and culturing NKT cells. Background technique [0002] Natural killer T (natural killer T, NKT) cells are a group of special T cell subsets that have both T cell receptor TCR and NK cell receptor on the cell surface. NKT cells can express the T cells of NKT cell marker NKT1.1, and their function has dual characteristics of T cells and NKT cells. NKT cells, mediated by TCR and NKR, produce a large amount of IL-4 and INF-γ, which can kill tumor cells. [0003] NKT cells mainly include immune regulation and cytotoxicity. The antigen recognition of NKT cells is different from that of traditional T cells, and they cannot recognize lipid and protein antigens presented by classic MHC-I and II molecules. NKT cells are activated and rapidly produce IL-4, IFN-γ, IL-10, IL-13 and other cytokines, thus playing an important role...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 陈海佳王一飞葛啸虎曾维杰
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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