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Enhanced DCIK (dendritic cell activated cytokine-induced killer) cell preparation method and cell preparation

A cell preparation, enhanced technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of limited cell expansion ability, lack of antigen specificity, difficult source of TIL, etc., and improve the storage time. , safety guarantee, high reliability effect

Active Publication Date: 2014-08-06
武汉光谷高新科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this cell's attack on malignant cells is as antigen-specific as CIK cells
When the cells are co-cultured with tumor-infiltrating lymphocytes (TIL) after being pulsed with tumor-associated antigens, the induced effector cells have the ability to specifically attack malignant cells, but the ability to expand the immune effector cells is limited. And the source of TIL is difficult

Method used

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  • Enhanced DCIK (dendritic cell activated cytokine-induced killer) cell preparation method and cell preparation

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Embodiment 1

[0039] A method for preparing enhanced DCIK cells, comprising the following steps:

[0040] 1) Collect the patient’s anticoagulant blood under sterile conditions, centrifuge at 1500rpm / min for 6 minutes to absorb the upper plasma, inactivate at 56°C for 30 minutes and then centrifuge for use. Dilute the blood cell pellet with compound normal saline with the same volume as the blood cell pellet, with a specific gravity of 1.077 Human lymphocyte separation solution and diluted blood were added to the centrifuge tube at a ratio of 1:1.5, centrifuged at 2000rpm / min for 15 minutes, carefully extracted the white blood cell layer in the middle, washed twice with compound normal saline, and obtained peripheral blood mononuclear cells after low-speed centrifugation (PBMC);

[0041] 2) Peripheral blood mononuclear cells were isolated from peripheral blood collected from patients; they were placed in blood-free medium to adjust the cell concentration to 3×10 6 / ml of mononuclear cells, ...

Embodiment 2

[0061] A method for preparing enhanced DCIK cells, comprising the following steps:

[0062] 1) Collect the patient’s anticoagulant blood under sterile conditions, centrifuge at 1500rpm / min for 6 minutes to absorb the upper plasma, inactivate at 56°C for 30 minutes and then centrifuge for use. Dilute the blood cell pellet with compound normal saline with the same volume as the blood cell pellet, with a specific gravity of 1.077 Human lymphocyte separation solution and diluted blood were added to the centrifuge tube at a ratio of 1:1.5, centrifuged at 2000rpm / min for 15 minutes, carefully extracted the white blood cell layer in the middle, washed twice with compound normal saline, and obtained peripheral blood mononuclear cells after low-speed centrifugation (PBMC);

[0063] 2) Peripheral blood mononuclear cells were isolated from peripheral blood collected from patients; they were placed in blood-free medium to adjust the cell concentration to 3×10 6 / ml of mononuclear cells, ...

Embodiment 3

[0068] A method for preparing enhanced DCIK cells, comprising the following steps:

[0069]1) Collect the patient’s anticoagulant blood under sterile conditions, centrifuge at 1500rpm / min for 6 minutes to absorb the upper plasma, inactivate at 56°C for 30 minutes and then centrifuge for use. Dilute the blood cell pellet with compound normal saline with the same volume as the blood cell pellet, with a specific gravity of 1.077 Human lymphocyte separation solution and diluted blood were added to the centrifuge tube at a ratio of 1:1.5, centrifuged at 2000rpm / min for 15 minutes, carefully extracted the white blood cell layer in the middle, washed twice with compound normal saline, and obtained peripheral blood mononuclear cells after low-speed centrifugation (PBMC);

[0070] 2) Peripheral blood mononuclear cells were isolated from peripheral blood collected from patients; they were placed in blood-free medium to adjust the cell concentration to 3×10 6 / ml of mononuclear cells, t...

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Abstract

The invention discloses an enhanced DCIK (dendritic cell activated cytokine-induced killer) cellpreparation method and cell preparation. The method comprises the steps as follows: a PBMC (peripheral blood mononuclear cell) is separated from collectedperipheral blood of a patient; the PBMC is placed in a blood-free medium for culture to obtain nuclear cells with the concentration of 1-3*10<6> / ml, IFN-gamma (interferon-gamma), phytohemagglutinin and PGE2 (prostaglandin E2) are added, and the mixture is transferred into a culture flask or a culture bag to be cultured for 24 h; then a CD3 monoclonal antibody, IL-2 (interleukin-2), IL-1alpha, IL-4 and a GM-CSF (granulocyte-macrophage colony-stimulating factor) are added, the mixture is cultured for 3-5 days, the cell density is controlled in a range of 1-6*10<6> / ml, the mixture is cultured for 14-21 days continuously, and an enhanced CIK cell is obtained through centrifugal collection. The enhanced DCIK cell preparation comprises the enhanced DCIK cell, human blood albumin, the IL-2, amino acid, vitamins and inorganic salt. The preservation time of the DCIK cell preparation is substantially longer than those of preparations in previous patents and is prolonged to 36 hours from 6 hours under the room temperature condition. The preparation is higher in reliability in clinical applications, and the safety is effectively guaranteed.

Description

technical field [0001] The invention relates to the field of in vitro culture of immune cells, in particular to a method for preparing enhanced DCIK cells and a cell preparation. Background technique [0002] Adoptive cellular immunotherapy refers to a treatment method that directly kills tumor cells or viruses by infusing self or allogeneic specific and non-specific anti-tumor immune effector cells. Adoptive cellular immunotherapy has entered clinical application and has become the fourth major tumor treatment method after surgery, radiotherapy and chemotherapy. The types of such immune cells include lymphokine-activated killer cells (LAK), tumor infiltrating lymphocytes (TIL), cytotoxic lymphocytes (CTL), etc. Among many immune cells, dendritic cells (DC) The research and application of a new generation of tumor vaccines has become a hot spot in active immunotherapy research in recent years. In the body's immune response, DC is the most important antigen-presenting cell,...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/14A61P35/00
Inventor 许亮艾伟石纪刚柳海洋张辉
Owner 武汉光谷高新科技发展有限公司
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