Immune cell culture method capable of simultaneously activating CD4<+> and CD8<+>T cells

A culture method and technology of immune cells, which are applied in the field of induction and preparation of natural cytokine mixtures, can solve the problems of weakened therapeutic effect of immune cell therapy, weakened function of secreting cytokines, etc., and achieve strong direct killing function, enhanced killing activity and proliferation ability. improved effect

Inactive Publication Date: 2014-07-23
湖北华赛生物医药技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CIK cells can secrete a variety of high-level cytokines in the process of in vitro culture. After reinfusion into the human body, the cells are affected by the regulation of the human immune system and the immune suppression of the patient, and the function of secreting cytokines is weakened, resulting in Therapeutic effect of immune cell therapy weakens

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of immune cell EAIC and natural cytokine mixture NCM of the present invention

[0049] (1) Collection and separation of peripheral blood mononuclear cells (PBMC)

[0050] Under sterile conditions, use a 50mL syringe to draw heparin sodium anticoagulant, and use a scalp needle to collect 50mL of peripheral blood from tumor patients.

[0051] Dilute peripheral blood from tumor patients 1:1 with saline in a biosafety cabinet.

[0052] Add 5mL of lymphocyte separation medium with a specific gravity of 1.077 (product of Tianjin Haoyang Biological Products Technology Co., Ltd., medical grade) into an empty 15mL centrifuge tube, then add 10mL of diluted blood, and centrifuge at 1500rpm / 20min.

[0053] Take buffy coat cells, wash twice with PBS solution at 1000rpm / 20min, suspend cells with serum-free medium GT-T551 H3 and adjust the density to 1.0×10 6 cells / mL.

[0054]

[0055] (2) Induction and expansion method of EAIC immune cells 1

[005...

Embodiment 2

[0094] Example 2 Detection of EAIC cells and NCM prepared in Example 1 of the present invention

[0095] (1) Sterility test and heat source detection

[0096] The EAIC cells and NCM prepared in Example 1 were checked according to the sterility test method and pyrogen test method in "Chinese Pharmacopoeia 2010 Part III", and the results were all negative.

[0097] (2) Phenotype analysis of cellular immunity

[0098] The immunophenotype of the EAIC cells prepared in Example 1 was detected by flow cytometry. The proportion of each subpopulation in cells has a certain range of variation due to individual differences, generally T lymphocytes (CD3 + ) was 94-98%, natural killer cells (CD3 - CD56 + ) is 2-5%, CD3 + CD4 + 50-60% of cells, CD3 + CD8 + 35-50% of cells, CD3 + CD56 + Cells are 20-30%.

[0099] (3) Cell survival rate

[0100]The inspection method is a conventional method: aspirate 0.1 mL of cells from the cell preparation and stain with trypan blue dye. Normal...

Embodiment 3

[0117] Example 3 In vivo tumor inhibition detection of EAIC cells prepared in Example 1 of the present invention and NCM in animals

[0118] Thirty T and NK cell immunodeficiency-Beige nude mice with similar weight and age were randomly divided into 5 groups.

[0119] Will 1×10 6 K562 cells were intraperitoneally injected into the above-mentioned nude mice, and the growth of tumor mass was observed after inoculation.

[0120] On the 4th day, 6th day, 8th day, 10th day, and 12th day after inoculation of K562 cells, normal saline, traditional CIK cells, EAIC cells, NCM, EAIC+NCM were injected into the above tumor-bearing mice five times In vivo, the number of cells is 5 × 10 6 Each, the amount of NCM is 0.2mL.

[0121] On the 20th day, each mouse was dissected to take tumor mass and weighed. The results are shown in Table 4.

[0122]

[0123] Table 4 EAIC, NCM and traditional CIK animal tumor inhibition experiment results in vivo

[0124] therapy Tumor...

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PUM

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Abstract

The invention belongs to the field of biological treatment of tumors and relates to an immune cell culture method capable of simultaneously activating CD4 <+>and CD8<+>T cells. The immune cell culture method comprises the steps of placing single nuclear cells which are well separated in a culture solution containing rhIL-12 and IL-4 ligands and an IL-10 ligand, culturing for 8-12h, then adding levamisole, continuously culturing for 8-12h, adding Anti-CD3McAb and rhIL-2, continuously culturing for 2-3 days, and then alternately adding into the culture medium containing the rhIL-2 and the culture medium containing the Anti-CD3McAb and the rhIL-2, wherein the intermediate culture time is 2-3 days. The scheme for preparing a cell factor compound is as follows: collecting cell supernatant liquid within 3-14 days after the beginning of culture, performing low-temperature ultracentrifugation, performing ultrafiltration by an ultrafiltration centrifugal tube, and performing microfiltration by a microprous filtration membrane, wherein filtrate is a natural cell factor mixture. By adopting the culture method provided by the invention, sustained proliferation of cells can be kept for 30 days. NCM contains a variety of cell factors and can stimulate immune cells of a human body to play autoimmune anti-tumor activity.

Description

[0001] technical field [0002] The invention belongs to the field of in vitro cell culture, and also belongs to the field of tumor biotherapy, in particular to a method for simultaneously activating CD4 + & CD8 + The immune cell culture method of T cells and the induction preparation method of natural cytokine mixture. Background technique [0003] Tumor biotherapy is a kind of treatment method that uses cell biology and molecular biology methods to regulate the body's immune system or tumor growth, thereby inhibiting tumor growth or destroying it. It is an emerging tumor rehabilitation medicine with significant The curative treatment method has the advantages of strong targeting and no obvious side effects, and has been regarded as the fourth treatment method after surgery, radiotherapy and chemotherapy. Tumor biological therapy includes cytokine therapy, immune cell therapy, stem cell therapy, gene therapy, molecular targeted therapy, etc. [0004] Immune cell th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 汤威吴皖王雄伟邢文彦罗琳
Owner 湖北华赛生物医药技术有限公司
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