Preparation method of Th1 cell subset and use of Th1 cell subset in preparation of anti-tumor cell preparation

A technology of cell subgroups and cell groups, which is applied in the direction of antineoplastic drugs, animal cells, vertebrate cells, etc., can solve the problems of metastatic drug resistance, stimulate the body's immune rejection, and the number of CTLs is small, and achieve strong immune activity and significant Effect of antitumor activity

Inactive Publication Date: 2014-07-09
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

Despite the continuous development of surgery, radiotherapy, chemotherapy and other treatment techniques in recent years, the recurrence, metastasis or drug resistance of malignant tumors have not been fundamentally resolved.
However, this culture protocol was used for CD4 + Immunomagnetic Beads for Cell Screening Cell sorting procedures are cumbersome, which increases the possibility of contamination during the culture process; although the immunomagnetic beads can be automatically decomposed, it is inevitable that some of the immunomagnetic beads will enter the human body, which may stimulate the body's immune rejection. possibility of reaction
In addition, using CD3 / CD28ClinEx Vivo Dynal Beads for cell culture, Th1 accounts for a high percentage of the total number of cells, but the number of CTLs is very small. The ability of Th1 cells to directly kill tumor cells is lower than that of CTL cells, and Th1 promotes CTLs to specifically kill tumors. The capacity of cells is also underutilized
Moreover, the high cost of immunomagnetic bead products will greatly hinder the clinical application of Th1 cells

Method used

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  • Preparation method of Th1 cell subset and use of Th1 cell subset in preparation of anti-tumor cell preparation
  • Preparation method of Th1 cell subset and use of Th1 cell subset in preparation of anti-tumor cell preparation
  • Preparation method of Th1 cell subset and use of Th1 cell subset in preparation of anti-tumor cell preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Preparation of CD4 monoclonal antibody-coated culture flasks with CD4 + T cell-dominated cell population

[0051] (1) Add the coating solution containing 1-10 μg / mL CD4 monoclonal antibody to the culture bottle made of polystyrene, polyvinyl chloride or polyethylene, and wrap the culture bottle with tinfoil Protected from light, lay it flat, and incubate overnight at 4°C; the type of CD4 monoclonal antibody used can be any one of IgG1, IgG2a, IgG2b, IgG3, IgM and IgA;

[0052] (2) On the first day, draw peripheral blood from healthy people or tumor patients under aseptic conditions, or take peripheral blood mononuclear cells from normal people or tumor patients obtained by preliminary separation of blood collection (that is, apheresis), or collect umbilical cord blood, or Collect bone marrow, transfer it to a 50mL centrifuge tube, add an equal volume of normal saline to dilute to obtain a diluted sample; Transfer the sample to the top of the lymphocyte sepa...

Embodiment 2

[0057] Example 2: Using the CD4 monoclonal antibody coating method described in Example 1 to obtain a higher proportion of CD4 in mononuclear cells + T cell

[0058] (1) The mononuclear cells prepared according to the method described in Example 1 step (2) were divided into the following two groups:

[0059] Group 1: CD4 monoclonal antibody coated culture flask method:

[0060] Mononuclear cells were divided into 2~5×10 6 The density per mL was planted in culture bottles made of polystyrene, polyvinyl chloride or polyethylene material coated with CD4 monoclonal antibody prepared according to the method described in Example 1 step (1); 5% CO 2 , After incubating at 37°C for 2-6 hours, discard the supernatant, wash the bottle wall with saline, D-Hanks solution or PBS, and obtain CD4 + T cell-dominated cell population;

[0061] Group 2: Ordinary mononuclear cell extraction method:

[0062] directly collect the mononuclear cells prepared according to the method described in E...

Embodiment 3

[0068](1) Coat the culture flask with a coating solution containing 1-10 μg / mL CD3 mAb and 1-10 μg / mL CD28 mAb, and incubate overnight at 4°C in the dark;

[0069] (2) The CD4 prepared according to the method in Example 1 + The T cell-based cell population was adjusted to 1~5×10 with GT-T551 / AIM V / X-VIVO medium containing 500~3000U / mL IFN-γ 6 / mL of the cell suspension was transferred to the culture flasks coated with CD3mAb and CD28mAb in step (1) overnight, 5% CO 2 , and incubated overnight at 37°C to obtain activated CD4 + T cell-dominated cell population;

[0070] (3) Collect the activated CD4 obtained in step (2) + For the T cell-based cell population, use GT-T551 / AIM V / X-VIVO medium containing any of the following cytokine combinations and 1% to 5% autologous plasma or AB plasma, adjusted to 1 to 3 ×10 6 / mL of cell suspension, seeded into culture flasks:

[0071] The first cytokine combination is as follows:

[0072] 10~100ng / mL IL-7, 10~100ng / mL IL-15, 10~100ng / ...

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Abstract

The invention discloses a preparation kit and preparation method of a cell group mainly comprising CD4<+>T cells and a Th1 cell subset, a use of the Th1 cell subset in preparation of an anti-tumor cell preparation, and belongs to the field of biotechnology. The preparation method utilizes a CD4 monoclonal antibody and a culture bottle made of polystyrene, polyvinyl chloride or polyethylene to realize preparation of the cell group mainly comprising CD4<+>T cells, and utilizes two cytokine combination to realize preparation of the Th1 cell subset. Through use of the kit and preparation method provided by the invention, high-ratio CD4<+>T cells in a single karyocyte are obtained. The obtained Th1 cell subset has strong immunocompetence, can secrete a large amount of cell granzyme B, perforin, IFN-gamma and TNF-alpha and has substantial anti-tumor activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing Th1 cell subgroups and its application in preparing anti-tumor cell preparations. Background technique [0002] In 2008, the number of deaths due to malignant tumors in the world reached 7.6 million (accounting for about 13% of all deaths), and it is estimated that the number of deaths due to malignant tumors in the world will exceed 13.1 million in 2030. Data show that 20% of the world's new malignant tumor patients are in China, and 24% of the malignant tumor death patients are in China. Despite the continuous development of surgery, radiotherapy, chemotherapy and other treatment techniques in recent years, the recurrence, metastasis or drug resistance of malignant tumors have not been fundamentally resolved. With the continuous progress of oncology and immunology, the research on the mechanism of tumor occurrence and development has been deepene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/14A61P35/00A61K35/15
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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