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Preparation method of CIK (cytokine-induced killer) cells

A nucleated cell and concentration technology, applied in the field of CIK preparation, can solve the problems of insufficient cell activity, increased cell expansion multiple, poor cell proliferation effect, etc., and achieves the effect of strong activity and increased expansion multiple.

Inactive Publication Date: 2015-12-16
SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the defects of poor cell proliferation effect and insufficient cell activity in the existing CIK preparation method, the present invention discloses a CIK preparation method, adding fibrin gel to the CIK culture system to better simulate in vivo cells The three-dimensional microenvironment for growth provides cells with sufficient growth space and a spatial structure that supports intercellular connections, significantly increases the expansion factor of cells, and obtains stronger cell activity

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  • Preparation method of CIK (cytokine-induced killer) cells
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  • Preparation method of CIK (cytokine-induced killer) cells

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preparation example Construction

[0025] Specifically, the preparation method of a kind of CIK provided by the present invention comprises the following steps:

[0026] Obtain mononuclear cells, fully mix mononuclear cells with uncoagulated or semi-coagulated fibrin gel, place at 10-40°C until the fibrin gel is completely solidified, and then induce and culture mononuclear cells into CIK . Among them, the fibrin gel is at least 1000mm 3 As a quantitative unit, mononuclear cells and fibrin gels are 1*10 2 -1*10 6 : mixed in a quantity ratio of 1; in the prior art, the method of inducing and culturing mononuclear cells into CIK by using cell growth factors and medium is applicable to the present invention.

[0027] Because fibrin gel is a three-dimensional cured product, and fibrin gel has good biocompatibility, it is a polymer network system with a three-dimensional pore structure. These pores not only allow cells to migrate and proliferate freely , It also provides the basis for the penetration of nutrient...

Embodiment 1

[0052] A kind of preparation method of CIK developed by the present invention comprises the following steps:

[0053] (1) separating mononuclear cells from blood;

[0054] (2) Mix the mononuclear cell suspension with 1000mm 3 Each uncoagulated fibrin gel takes 1*10 4 After mixing at a ratio of 1:1, place it in a 37°C incubator until the fibrin gel is completely solidified, and then place the solidified fibrin gel with mononuclear cells in 50ng / ml of CD3McAb and 10g / ml of RetroNectin. In the coated culture flask, and add the GTT551 medium containing the PPP of 1% volume fraction and the IFN-γ of 1000IU / ml concentration;

[0055] (3) On the second day, IL-1α and IL-2 were added, and their final concentrations in the GTT551 medium in step 2) were 100IU / ml and 300IU / ml respectively;

[0056] (4) On the 3rd day, supplement the GTT551 medium in step 2) to a volume of 100ml, add IL-2 at the same time, and maintain its concentration in the medium at 300IU / ml;

[0057] (5) Repeat s...

Embodiment 2

[0085] The preparation method of another kind of CIK provided by the present invention comprises the following steps:

[0086] (1) separating mononuclear cells from blood;

[0087] (2) Mix the mononuclear cell suspension with 1000mm 3 Each uncoagulated fibrin gel takes 1*10 2 After mixing at a ratio of 1:1, place it in a 37°C incubator until the fibrin gel is completely solidified, and then place the solidified fibrin gel with mononuclear cells in 10ng / ml of CD3McAb and 50g / ml of RetroNectin. Coated culture flask, and add the GTT551 medium containing the PPP of 0.5% volume fraction and the IFN-γ of 1500IU / ml concentration;

[0088] (3) On the second day, IL-1α and IL-2 were added, and their final concentrations in the GTT551 medium in step 2) were 50IU / ml and 100IU / ml respectively;

[0089] (4) On the third day, supplement the GTT551 medium in step 2) to a volume of 100ml, add IL-2 at the same time, and maintain its concentration in the medium at 100IU / ml;

[0090] (5) Rep...

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Abstract

The invention discloses a preparation method of CIK (cytokine-induced killer) cells. The preparation method includes the steps of 1, separating single nuclear cells from blood; 2, mixing single nuclear cells suspension with fibrin gel, disposing a mixture in a CD3McAb and RetroNectin pre-coated culture bottle, and adding a medium containing PPP and IFN-Gamma; 3, on a second day, adding IL-1Alpha and IL-2 for continual culturing, stopping culturing until the single nuclear cells are converted into CIK cells. The preparation method has the advantages that the fibrin gel is a polymer network system capable of absorbing a great amount of water and extremely similar to extracellular matrix in structure, an in-vivo cell growth microenvironment can be simulated so that sufficient growth space for cells and a spatial structure supporting inter-cell connection are provided, amplification times of the cells are significantly increased, and the obtained cells are more active.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of CIK. Background technique [0002] CIK (cytokine-induced killer, Chinese name: [autologous cell immunotherapy] multiple cytokine-induced killer cells) is a group of heterogeneous cell populations obtained from mononuclear cells cultured under the action of anti-CD3 monoclonal antibody and various cytokines , in which CD3+, CD56+ lymphocytes are the main effector cells. CIK can directly kill tumor cells and virus-infected cells; induce tumor cell apoptosis; inhibit or kill tumor cells by releasing a large number of inflammatory cytokines. CIK has the characteristics of high anti-tumor activity, broad anti-tumor spectrum, low toxicity to normal tissues, and highly expandable in vitro. It is a commonly used cell in clinical cell immunotherapy. [0003] However, the existing CIK preparation method is to use multiple cytokines to induce culture in a 2...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 曾宪卓鲁菲
Owner SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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