Preparation method of DCs, and application of DCs in preparing anti-tumor cell preparation

A cell and nuclear cell technology, applied in the application field of mature DC cells in the preparation of anti-tumor cell preparations, can solve the problems of failing to achieve clinical efficacy, inhibiting the secretion of IL-12 by DC cells, etc., achieving strong cell migration ability and increasing the number of cells , high maturity effect

Inactive Publication Date: 2014-02-26
山东迪博生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in a phase III melanoma DC vaccine clinical trial, although the so-called "gold standard" COCKTAIL was used to stimulate DC cell maturation, no significant clinical efficacy was achieved
The researchers believe that a possible reason is the use of PEG2, which can significantly inhibit the ability of DC cells to secrete IL-12, although it can increase the expression of CCR7 in DC cells and enhance its ability to metastasize to secondary lymph nodes

Method used

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  • Preparation method of DCs, and application of DCs in preparing anti-tumor cell preparation
  • Preparation method of DCs, and application of DCs in preparing anti-tumor cell preparation
  • Preparation method of DCs, and application of DCs in preparing anti-tumor cell preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of adherent mononuclear cells

[0047] (1) Add the coating solution containing 1-10 μg / mL CD14 monoclonal antibody to the culture bottle dish made of polystyrene, polyvinyl chloride, polyethylene, etc., and wrap the culture bottle dish with tin foil. Store in the dark, lay flat, and incubate overnight at 4°C; wherein, the type of CD14 monoclonal antibody used can be any one of IgG1, IgG2a, IgG2b, IgG3, IgM and IgA;

[0048] (2) On the first day, draw 50-100 mL of peripheral blood from normal people or tumor patients under sterile conditions, or take 80-100 mL of peripheral blood mononuclear cells from normal people or tumor patients preliminarily separated by blood collection (ie, apheresis). into a 50mL centrifuge tube, add an equal volume of normal saline to dilute to obtain a diluted sample; then transfer the diluted sample to a 2:1 or 1:1 volume ratio of the diluted sample to the lymphocyte separation solution. Above the lymphocyte separation...

Embodiment 2

[0053] Example 2: The CD14 monoclonal antibody coating method can increase the adherence ability of mononuclear cells and increase the number of adherent cells, thereby helping to reduce the influence of impurity cells on their transformation into immature DCs

[0054] (1) Prepare mononuclear cells according to the method of Example 1, grouping:

[0055] Group 1: Press the mononuclear cells into 2~4×10 6 / mL was planted in a culture bottle dish made of polystyrene, polyvinyl chloride or polyethylene and other materials coated with CD14 monoclonal antibody;

[0056] Group 2: Press the mononuclear cells into 2~4×10 6 / mL planted in petri dishes made of polystyrene, polyvinyl chloride or polyethylene;

[0057] After 2 to 4 hours, the supernatant was discarded, and the wall of the bottle was washed with normal saline, D-Hanks solution or PBS, and the adherent mononuclear cells were obtained;

[0058] (2) Comparison of CD14 obtained by the two methods + Number and purity of mon...

Embodiment 3

[0061] Example 3: Culture of Adherent Cells Using a Combination of GM-CSF, IL-4 and IFN-α Cytokines

[0062] (1) After adhering the mononuclear cells prepared by the method of Example 1 for 2 to 4 hours, aspirate and discard the supernatant, and gently rinse the wall of the bottle with normal saline, D-Hanks solution or PBS for 3 times, and aspirate and discard the supernatant. Add the original volume of medium GT-T551 / AIM V / X-VIVO, 500-1000U / mL GM-CSF, 500-1000U / mL IL-4, 500-1000U / mL IFN-α and 1-5% Autologous plasma or AB plasma, in 5% CO 2 , incubate at 37°C;

[0063] (2) On the third day, add double volume of medium GT-T551 / AIM V / X-VIVO, 500-1000U / mL GM-CSF, 500-1000U / mL IL-4, 500-1000U / mL IFN- Alpha and 1 to 5% autologous plasma or AB plasma in 5% CO 2 , incubate at 37°C;

[0064] (3) On the 5th day, the adherent cells were differentiated into immature DC cells; the immature DC cells grown in suspension were collected and washed by centrifugation at 600×g to obtain imm...

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Abstract

The invention discloses a preparation kit and a preparation method of adherent mononuclear cells, immature DCs (Dendritic Cells) and mature DCs, and an application of the mature DCs prepared by the method in preparing an anti-tumor cell preparation, and belongs to the biological technical field. According to the preparation kit, the preparation method and the application of the mature DCs, which are disclosed by the invention, the adherent mononuclear cells are prepared from CD14 monoclonal antibodies through a culture bottle dish made of a polystyrene, polyvinyl chloride or polyethylene material; the immature DCs are prepared from GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-4 (interleukin-4) and IFN (interferon)-alpha; the mature DCs are prepared from TNF (tumor necrosis factor)-alpha and OK-432. The method disclosed by the invention is capable of increasing the adherence ability of the adherent mononuclear cells and increasing the number of the adherent cells, and thus being advantageous for reducing the influence of impurity cells on the transformation of the adherent mononuclear cells into the immature DCs; besides, the method is capable of increasing the yield of the immature DCs; the method is capable of obtaining mature DCs which are higher in maturity, and better in cell migration ability and IL-12p70 secretion ability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular, relates to a preparation kit and preparation method for adherent mononuclear cells, immature DC cells and mature DC cells, and the use of the mature DC cells prepared by the above methods in the preparation of anti-tumor cell preparations Applications. Background technique [0002] Tumor immunotherapy is considered to be the fourth most important treatment after surgery, radiotherapy and chemotherapy. Tumor immunotherapy is to stimulate and enhance the immune function of the body to control and kill tumor cells. Dendritic cells are the most powerful antigen-presenting cells discovered and named by Ralph Steinman, the winner of the 2011 Nobel Prize in Physiology or Medicine in 1973. They can pass tumor antigen information through major tissues. Compatible complexes (MHC I and MHC II)-restricted delivery of polypeptide complexes as antigens to CTL and Th1 cells play a key role in i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12N5/0784A61K35/14A61K35/28A61P35/00A61K35/15A61K35/32A61K35/51
Inventor 罗昀曾钢张立媛王敏杰
Owner 山东迪博生物科技股份有限公司
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