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Preparation method of high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell

A proliferative and cytotoxic technology, which is applied in the field of CIK cell preparation, can solve the problems of low proliferative capacity, low cytotoxic activity, and low purity of CIK cells, and achieve the effect of increasing the number and enhancing the anti-tumor effect

Active Publication Date: 2012-12-19
郑骏年
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  • Claims
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AI Technical Summary

Problems solved by technology

Solve the shortcomings of low purity, low proliferation and low cytotoxic activity in CIK cells prepared by existing methods

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: the preparation of CIK cell

[0025] 1. Preparation of peripheral blood mononuclear cells (PBMC)

[0026] Use a blood cell separator to collect anticoagulant blood from the patient under sterile conditions, centrifuge at 1500rpm / min for 10 minutes to absorb the upper layer of plasma, inactivate at 56°C for 30 minutes and centrifuge for later use, dilute the blood cell pellet with normal saline, and the human lymphocytes with a specific gravity of 1.077 The separated liquid and the 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, washed twice with normal saline, and obtained PBMC after low-speed centrifugation.

[0027] 2. Mini MACS to remove CD4 + CD25 + Treg cells

[0028] Take the PBMC cell suspension to adjust to an appropriate cell concentration, add PE-labeled AntiHuman CD25, incubate at 4°C for 30 minutes, take it out, centrifuge and wash...

Embodiment 2

[0029] Example 2: Properties of CIK cells

[0030] 1. Morphology, cell viability and immunophenotype detection of CIK cells

[0031] After adding PHA and INF-γ, most of the cells were still in a suspended state; after 3 days, the volume of the cells increased, the cells gradually aggregated into clusters, the cells were translucent, and the cytoplasm was abundant; from the 7th day, the cells began to proliferate in large quantities, and the cell shapes were diverse. Cell clusters increased; take 100 μl of cells cultured for 10 days, add 100 μl of 0.4% placenta blue staining solution, live cells will not be stained, and dead cells will be stained blue. The viability of the cells prepared by the invention is greater than 90%. On the 14th day, a small amount of dead cells could be seen. Cell flow detection analysis showed that CIK cells belonged to a heterogeneous cell population. With the prolongation of culture time, CD4 + , CD8 + , CD3 + CD56 + The ratio of T cells was s...

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Abstract

The invention belongs to the technical field of cell culture in vitro, and particularly relates to a preparation method of high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell. The method comprises the following steps: collecting and separating peripheral blood mononuclear cell of a patient, eliminating CD4+CD25+Treg cell by means of Mini MACS (magnetic active cell sorting) method, and sorting to obtain CD3+, CD4+ and CD8+T cells; and putting the obtained cells into culture solution containing phytohemagglutinin (PHA), so that the PHA concentration in the suspension liquid is 100ng / ml, hatching for 24h under the culture condition of 5% CO2 at 37 DEG C, transferring the hatched suspension liquid into a cell culture bottle coated by CD3 monoclonal antibody (1mug / ml), adding IFN (interferon)-gamma (1000U / ml), adding IL (interleukin)-2(500U / ml) and IL (interleukin)-21(1000U / ml) after 48h, compensating sodium selenite-containing (0.005mg / L)cell culture after four days, and continuously culturing for 7-14 days to obtain the high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell. The quantity, the activity and the purity of the CIK cell which is prepared by the method and amplified in vitro are improved, so that the antineoplastic function of the CIK is enhanced.

Description

technical field [0001] The invention belongs to the technical field of in vitro cell culture, in particular to a method for preparing CIK cells with high purity, high proliferative power and high cytotoxic activity. In this method, Treg cells were removed by MiniMACS indirect immunomagnetic bead forward sorting method before in vitro culture of patient mononuclear cells, and the Treg cell inhibitor IL-21 was added during the culture process to inhibit the production of Treg cells. In addition to routine addition of IFN-γ, CD3 monoclonal antibody, and IL-2, mitogen (PHA) was also added to promote lymphocyte proliferation, and sodium selenite was added to improve the cytotoxicity of CIK cells. The obtained CIK cells include CD3 + CD56 + Double positive, CD4 + , CD8 + The amplification factor reaches 400 times, and the total ratio reaches 83%-95%; at the same time, CD4 is removed + CD25 + Treg cells. It has stronger anti-tumor and anti-viral effects. Background of the in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783A61K35/14A61P35/00A61P31/00A61P37/02A61K35/17
Inventor 郑骏年李连涛李慧中刘俊杰徐为陈菲菲程乾章龙珍裴冬生
Owner 郑骏年
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