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Preparation method of CIK in three-dimensional environment

An environmental and three-dimensional technology, applied in the biological field, can solve the problems of poor cell proliferation and insufficient cell activity, and achieve the effect of increasing the expansion multiple, strong cell activity, and promoting growth

Inactive Publication Date: 2016-02-10
SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 provides a preparation method of CIK with significantly increased cell amplification multiple and stronger cell activity

Method used

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  • Preparation method of CIK in three-dimensional environment
  • Preparation method of CIK in three-dimensional environment
  • Preparation method of CIK in three-dimensional environment

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preparation example Construction

[0029] Specifically, the preparation method under the CIK three-dimensional environment provided by the present invention includes the following steps:

[0030] 1. Take aprotinin to dissolve freeze-dried fibrinogen to obtain solution A;

[0031] 2. Add thrombin into the soluble calcium salt solution to obtain solution B;

[0032] 3. Obtain mononuclear cells, and mix the mononuclear cells with solution A and solution B and place them at 10-40°C until solidified to obtain a pre-induction mixture;

[0033] 4. Inducing and culturing the mononuclear cells in the pre-induction mixture to produce CIK.

[0034] Among them, thrombin can enzymatically cleave fibrinogen, release fibrin A peptide and B peptide, and form fibrin monomers, which can be polymerized into unstable soluble fibrin fibers by hydrogen bonds and electrostatic attraction Thrombin can also activate factors at the same time, and participate in the cross-linking of fibrin polypeptides in the presence of calcium ions, ...

Embodiment 1

[0055] A preparation method under a CIK three-dimensional environment provided by the invention comprises the following steps:

[0056] (1) Prepare 2000KIU / ml aprotinin, take 1ml of prepared aprotinin to dissolve polypeptide-coupled freeze-dried fibrinogen, and prepare solution A with a final concentration of fibrinogen of 2.0g / L;

[0057] (2) Add thrombin to 40mmol / LCaCl 2 In the solution, a solution B with a thrombin concentration of 250-400 U / ml was prepared;

[0058] (3) separating mononuclear cells from blood;

[0059] (4) Set the concentration to 1*10 4 After the mononuclear cell solution per ml is fully mixed with solution A and solution B, place it in a 37°C incubator until the mixture is completely solidified to obtain a pre-induction mixture;

[0060] (5) Place the pre-induction mixture with mononuclear cells in the pre-coated culture flask of 50ng / ml CD3McAb and 10g / ml RetroNectin, and add the IFN containing 1% volume fraction of PPP and 1000IU / ml concentration ...

Embodiment 2

[0088] The preparation method under another CIK three-dimensional environment provided by the present invention comprises the following steps:

[0089] (1) Prepare 1000KIU / ml aprotinin, take 1ml of prepared aprotinin to dissolve polypeptide-coupled freeze-dried fibrinogen, and prepare solution A with a final concentration of fibrinogen of 1.5g / L;

[0090] (2) Add thrombin to 40mmol / LCaCl 2 In the solution, a solution B with a thrombin concentration of 250-400 U / ml was prepared;

[0091] (3) separating mononuclear cells from blood;

[0092] (4) Set the concentration to 1*10 2 After the mononuclear cell solution per ml is fully mixed with solution A and solution B, place it in a 37°C incubator until the mixture is completely solidified to obtain a pre-induction mixture;

[0093] (5) Place the pre-induction mixture with mononuclear cells in the pre-coated culture flask of 10ng / ml CD3McAb and 50g / ml RetroNectin, and add the IFN containing 0.5% volume fraction of PPP and 1500IU / ...

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Abstract

The invention discloses a preparation method of CIK in three-dimensional environment. The preparation method includes following steps: taking aprotinin to dissolve fibrinogen to obtain a solution A; preparing a solution B containing thrombin; obtaining a mononuclear cell, and mixing the mononuclear cell with the solution A and the solution B to be solidified to obtain a pre-induction mixture; performing induced culture on the pre-induction mixture containing the mononuclear cell to obtain CIK. The preparation method has the advantages that the mononuclear cell is mixed with the solution A and the solution B to enable the mononuclear cell to be positioned in fibrin gel environment, and fibrin gel can simulate three-dimensional growing microenvironment of in-vivo cells, so that sufficient growing space and spatial structure supportive of intercellular connection are provided for the cells, number of amplification times of the cells is increased remarkably, and activity of the cells obtained is higher.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a preparation method of CIK in a three-dimensional environment. 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 ...

Claims

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Application Information

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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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