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NKT (Natural Killer T) cell preparation prepared by sensitization by using tumor cells and simulating factors and preparation method of NKT cell preparation

A technology of NKT cells and stimulating factors, which is applied in the field of biomedicine, can solve the problems of poor killing activity and insignificant amplification effect, and achieve the effect of enhancing the amplification effect and improving the killing effect

Inactive Publication Date: 2016-05-04
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, IL-2 is mainly used to promote the proliferation of NKT cells, the expansion effect is not significant, and the killing activity is relatively poor

Method used

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  • NKT (Natural Killer T) cell preparation prepared by sensitization by using tumor cells and simulating factors and preparation method of NKT cell preparation
  • NKT (Natural Killer T) cell preparation prepared by sensitization by using tumor cells and simulating factors and preparation method of NKT cell preparation
  • NKT (Natural Killer T) cell preparation prepared by sensitization by using tumor cells and simulating factors and preparation method of NKT cell preparation

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Experimental program
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Embodiment 1

[0027] This embodiment provides a method for preparing NKT cell preparations sensitized with tumor cells and stimulating factors, comprising the following steps:

[0028] 1) Collect 20 mL of the patient's own peripheral blood, and collect the plasma after centrifugation;

[0029] 2) Dilute the blood cells in the lower layer with normal saline, then add the diluted solution into a sepmate centrifuge tube with Ficoll separation solution, centrifuge at 300g for 10min, and the volume ratio of blood cells to normal saline is 1:2;

[0030] 3) Pour out the supernatant liquid after centrifugation in step 2) from the centrifuge tube, then add 15 mL of RPMI1640 medium to resuspend the cells, and centrifuge at 300 g for 10 min;

[0031] 4) Add X-VIVO-15 medium for culture, the cell density is 5×10 5 / mL; at the same time, add 100U / mL of IL-15, 100U / mL of IL-21, 50μg / mL of OKT-3, 100U / mL of IL-2 to the culture medium, and observe the morphology of NKT cells every day. , the morphology o...

Embodiment 2

[0041] This embodiment provides a method for preparing NKT cell preparations sensitized with tumor cells and stimulating factors, comprising the following steps:

[0042] 1) Collect 20 mL of the patient's own peripheral blood, and collect the plasma after centrifugation;

[0043] 2) Dilute the blood cells in the lower layer with normal saline, then add the diluted solution into a sepmate centrifuge tube with Ficoll separation solution, centrifuge at 400g for 15min, and the volume ratio of blood cells to normal saline is 1:1;

[0044] 3) Pour out the supernatant liquid after centrifugation in step 2) from the centrifuge tube, then add 20 mL of RPMI1640 medium to resuspend the cells, and centrifuge at 400 g for 15 min;

[0045] 4) Add X-VIVO-15 medium for culture, the cell density is 8×10 5 / mL; at the same time, add 500U / mL IL-15, 500U / mL IL-21, 300μg / mL OKT-3, 500U / mL IL-2 to the culture medium, and observe the morphology of NKT cells every day;

[0046] 5) Count the cells o...

Embodiment 3

[0052] This embodiment provides a method for preparing NKT cell preparations sensitized with tumor cells and stimulating factors, comprising the following steps:

[0053] 1) Collect 20 mL of the patient's own peripheral blood, and collect the plasma after centrifugation;

[0054] 2) Dilute the blood cells in the lower layer with normal saline, then add the diluted solution into a sepmate centrifuge tube with Ficoll separation solution, centrifuge at 500g for 20min, and the volume ratio of blood cells to normal saline is 2:1;

[0055] 3) Pour out the supernatant liquid after centrifugation in step 2) from the centrifuge tube, then add 25 mL of RPMI1640 medium to resuspend the cells, and centrifuge at 500 g for 20 min;

[0056] 4) Add X-VIVO-15 medium for culture, the cell density is 10×10 5 / mL; at the same time, add 1000U / mL IL-15, 1000U / mL IL-21, 500μg / mL OKT-3, 1000U / mL IL-2 to the medium, and observe the morphology of NKT cells every day;

[0057] 5) Count the cells once ...

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Abstract

The invention relates to the technical field of biomedicines, in particular to an NKT (Natural Killer T) cell preparation prepared by sensitization by using tumor cells and simulating factors and a preparation method of the NKT cell preparation. The NKT cell preparation is high in killing activity and can effectively kill remaining cancer cells, eliminate recurrence and metastasis factors, increase the curability, prolong the survival time of patients and improve the life quality by applying an immunotherapy. In the preparation process of the preparation, NKT cells are jointly cultured by adopting IL-2, IL-15 and IL-21 and sensitized by adopting the tumor cells and the stimulating factors, so that the expansion effects of the NKT cells are enhanced, and the killing activity of the NKT cell preparation is also greatly enhanced.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to an NKT cell preparation sensitized with tumor cells and stimulating factors and a preparation method thereof. Background technique [0002] Tumor cell immunotherapy is an emerging tumor treatment model with significant curative effect, and it is a new treatment method for autoimmune anti-cancer. It is a method of using biotechnology and biological agents to culture and amplify immune cells collected from patients in vitro and then reinfuse them back into the patient's body to stimulate and enhance the body's own immune function, so as to achieve the purpose of treating tumors, experimental and clinical All suggest that the immune system of the body has the effect of eliminating cancer. After the primary cancer is surgically removed or the local cancer is ablated by minimally invasive surgery such as argon-fluorine knife, immunotherapy can kill the remaining cancer cells and e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61P35/00C12N5/0783
CPCA61K39/0011A61K2039/5158C12N5/0646C12N2501/25C12N2501/998
Inventor 王一飞陈海佳葛啸虎罗二梅
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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