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Preparation method of enhanced CIK (Cytokine Induced Killer) cell, and cell preparation

A cell preparation and enhanced technology, which is applied in the direction of animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problem of CIK cell proliferation multiples and cytotoxic activity that are not ideal, and immune cells cannot achieve the effect of killing tumors, etc. problems, to achieve the effect of increasing the killing effect and increasing the toxicity

Active Publication Date: 2014-07-23
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

Therefore, the effect of killing tumors cannot be achieved by relying on one's own immune cells, and the absolute number of such cells can only be increased by culturing in vitro
[0004] CIK cells can proliferate in large quantities through in vitro induction. The general CIK preparation method is to treat isolated peripheral blood mononuclear cells (PBMC) with IFN-γ for 24 hours, and then add CD3mAb, IL-1α and IL-2 factors to stimulate and induce , and finally obtain a certain number of CIK cells, but the multiplier and cytotoxic activity of the finally obtained CIK cells are not ideal

Method used

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  • Preparation method of enhanced CIK (Cytokine Induced Killer) cell, and cell preparation
  • Preparation method of enhanced CIK (Cytokine Induced Killer) cell, and cell preparation
  • Preparation method of enhanced CIK (Cytokine Induced Killer) cell, and cell preparation

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Embodiment 1

[0040] A method for preparing enhanced CIK cells, comprising the following steps:

[0041] 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);

[0042] 2) Peripheral blood mononuclear cells were isolated from peripheral blood collected from patients; cultured in blood-free medium to obtain 1×10 6 nucleated cells / ml, add IFN-γ, phytohemagglutinin and PGE2, a...

Embodiment 2

[0062] A method for preparing enhanced CIK cells, comprising the following steps:

[0063] 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);

[0064] 2) Peripheral blood mononuclear cells were isolated from peripheral blood collected from patients; cultured in blood-free medium to obtain 1×10 6 nucleated cells / ml, add IFN-γ, phytohemagglutinin and PGE2, a...

Embodiment 3

[0069] A method for preparing enhanced CIK cells, comprising the following steps:

[0070] 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);

[0071] 2) Peripheral blood mononuclear cells were isolated from peripheral blood collected from patients; cultured in blood-free medium to obtain 1×10 6 nucleated cells / ml, add IFN-γ, phytohemagglutinin and PGE2, and...

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Abstract

The invention discloses a preparation method of an enhanced CIK cell, and a cell preparation. The method comprises the following steps: separating to obtain a peripheral blood mononuclear cell from peripheral blood collected from a sufferer; putting the peripheral blood mononuclear cells into a blood-free medium to culture, so as to obtain karyocyte of which the concentration is (1-3)*10<6> / ml; adding IFN (interferon)-gamma, phytohemagglutinin and PGE2 (Phenyl Glycidyl Ether), transferring to a culture bottle or a culture bag to culture for 24 hours, and then adding a CD3 monoclonal antibody, IL-2 (interleukin-2), IL-1alpha, IL-4 and GM-CSF (granulocyte-macrophage colony-stimulating factor); continuing to culture for 3-5 hours, and then controlling the cell density at (1-6)*10<6> / ml; and centrifugally colleting to obtain the enhanced CIK cell after continuously culturing for the 14th to 21st days. The enhanced CIK cell preparation comprises the enhanced CIK cell, human serum albumin, IL-2, amino acids, vitamins and inorganic salts. Compared with a preparation in the patient, the CIK preparation has the advantages that the preservation time of the CIK preparation is obviously prolonged, and is prolonged to 36 hours from 6 hours at room temperature. The CIK preparation is higher in reliability in clinical application, and the safety is effectively ensured.

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 CIK 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, CIK cells have tumoricidal activity High, broad tumor killing spectrum, equally sensitive to a variety of drug-resistant cells, and less toxic to normal bone marrow hematopoietic premise cells, etc., are widely used in clinical practice. [...

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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