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

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...