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Efficient multiplication CTL preparation method killing tumors in targeted mode

A high-efficiency proliferation and targeting technology, applied in the direction of blood/immune system cells, vertebrate cells, animal cells, etc., can solve the problems of reducing cell killing activity and high proportion of Treg cells, achieving high-efficiency proliferation, inhibiting transformation, and improving efficiency Effect

Inactive Publication Date: 2014-07-16
四川全组生命科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, when the peripheral blood mononuclear cells (PBMC) of tumor patients are collected in vitro, the proportion of Treg cells is high, and the IL-2 used in the induction and expansion of effector cells can also induce the proliferation of Treg cells and secrete a large amount of IL-10, thus Inhibit the proliferation of effector cells and reduce their cell killing activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation of CTL cells

[0045] 1. Preparation of PBMCs

[0046] Collect 50ml of peripheral blood from tumor patients, centrifuge at 2500rpm (rev / min) for 10min, and suck out the upper layer of plasma for later use. Add 0.9% physiological saline for injection to the blood cells, mix well, and dilute the blood cells. Slowly add the diluted peripheral blood onto the human lymphocyte separation liquid layer with a specific gravity of 1.077, the volume ratio of the two is 1:1. Centrifuge at 600g for 30 min, gently suck out the mononuclear cell layer in the form of white mist, and transfer it to another in a centrifuge tube. Diluted with 0.9% normal saline for injection, 400g, centrifuged for 5min, washed 3 times and centrifuged at low speed to obtain PBMC.

[0047] 2. Mini MACS immunomagnetic bead negative sorting method to remove CD4 + CD25 + Treg cells

[0048] The cells were resuspended in MACS (Magnetic-activated cell sorting, magnetic bead separation...

Embodiment 2

[0068] 1. Preparation of PBMCs

[0069] Collect 50ml of peripheral blood from tumor patients, centrifuge at 2500rpm for 10min, and suck out the upper layer of plasma for later use. Add 0.9% physiological saline for injection to the blood cells, mix well, and dilute the blood cells. Slowly add the diluted peripheral blood onto the human lymphocyte separation liquid layer with a specific gravity of 1.077, the volume ratio of the two is 1:1. Centrifuge at 600g for 30 min, gently suck out the mononuclear cell layer in the form of white mist, and transfer it to another in a centrifuge tube. Diluted with 0.9% normal saline for injection, 400g, centrifuged for 5min, washed 3 times and centrifuged at low speed to obtain PBMC.

[0070] 2. Mini MACS immunomagnetic bead negative sorting method to remove CD4 + CD25 + Treg cells

[0071] Resuspend the cells with MACS buffer, pass through a 100 μm filter to obtain suspended single cells, and adjust the cell density to 5×10 8 / ml. Add...

Embodiment 3

[0088] 1. Preparation of PBMC and 2. Mini MACS immunomagnetic bead negative sorting method to remove CD4 + CD25 + Treg cells adopt the same method as in Example 1;

[0089] 3. Separation of DC cells and T lymphocytes:

[0090] will result in CD3 + , CD4 + , CD8 + , CD14 + Mix the cells, aliquot into 30ml of serum-free AIM-Ⅴ medium, at 37°C, 5% CO by volume 2 After static incubation in the incubator for 2 hours, the suspension cells (ie, T lymphocytes) and adherent cells (ie, DCs) were obtained, and the suspension cells were transferred to a new culture flask.

[0091] 4. Preparation of mature DC cells:

[0092] Add 20 ml of serum-free AIM-Ⅴ medium containing 50 ng / ml GM-CSF and 25 ng / ml IL-4 to the adherent DC cells, place at 37 ° C, and the volume percentage is 5% CO 2 cultured in an incubator for 5 days. On the 6th day, 50 μg / ml of the whole tumor cell antigen of the tumor patient was added. On the 7th day, DC cells loaded with the whole tumor cell antigen were obta...

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Abstract

The invention discloses an efficient multiplication CTL preparation method killing tumors in a targeted mode. The CTL preparation method comprises the following steps: (a) removing CD4+CD25+Treg cells through immunomagnetic bead negative sorting; (b) arranging mixed cells in a serum-free medium for cultivation, and obtaining suspension cells and adherent cells; (c) adding GM-SCF and IL-4 in the adherent cells, culturing the cells for five days; in the sixth day, adding a tumour cell holoantigen, and in the seventh day, adding TNF-alpha and IL-27; (d) transferring the suspension cells to a culture flask wrapped by a CD3 monoclonal antibody and recombinant human fibronectin, adding IFN-gamma, in the second day, adding IL-2, IL-12 and the IL-27, and culturing the mixture till the eighth day to obtain CIK cells; (e) mixing the CIK cells and mature DC cells, and adding the IL-12, IL-7 and an anti-CD 28 monoclonal antibody for cultivation; in the third day, adding an anti-CTLA-4 monoclonal antibody, and then culturing the mixture for four days. According to the efficient multiplication CTL preparation method killing tumors in the targeted mode, efficiency of in-vitro CTL cell proliferation is improved, activity of killing the tumor cells in the targeted mode is improved, transformation of peripheral blood mononuclear cells to the CD4+CD25+Treg cells is inhibited.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to a method for preparing CTLs for efficient proliferation and targeted killing of tumors. Background technique [0002] At present, tumor has become the primary enemy that threatens human life and health, and its incidence rate is also increasing year by year. The "World Cancer Report" shows that global cancer cases have increased from 14 million in 2012 to 19 million in 2025, to 19 million in 2025. It will reach 24 million in 2035; the number of people who die from cancer every year will continue to increase, from 8.2 million to 13 million, and new cancer cases will increase by 50% by 2030, reaching 21.6 million per year. Cancer is one of the main causes of death among Chinese residents. According to the annual report on the registration of tumor incidence in my country, there are about 3.5 million new cancer cases every year, 6 people are diagnosed with cancer every minute...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 魏薇赵西娟魏丽婉孟祥玲
Owner 四川全组生命科技有限公司
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