Preparation method of cytokine-induced killer cells

A technology for killing cells and cytokines, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of high cost, easy pollution, large cell damage, etc., to achieve increased yield, increased killing potential, The effect of reducing the ratio

Pending Publication Date: 2021-01-05
北广再生医学科技(广东)有限公司
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Problems solved by technology

However, immunomagnetic bead sorting is not only costly, but also cumbersome to operate, causes great damage to cells, and is easy to contaminate

Method used

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  • Preparation method of cytokine-induced killer cells
  • Preparation method of cytokine-induced killer cells
  • Preparation method of cytokine-induced killer cells

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Embodiment 1

[0027] 1. Peripheral blood samples and reagents

[0028] 1.1 Peripheral blood samples

[0029] The peripheral blood of the 9 healthy donors mentioned above was collected through the median cubital vein with a sodium citrate anticoagulant tube, according to "blood collection volume (mL) = 1X10 8 / Per mL peripheral blood lymphocyte count" calculation, the total number of lymphocytes contained in the collected peripheral blood shall not be less than 3X10 7 .

[0030] Peripheral blood samples from 9 healthy donors were included in this example, 6 males and 3 females; the average age (years) was 29.22±5.58 (21~42); the results of blood routine examination before blood collection showed that LYM# (*10 9 / L) was 1.93±0.44 (1.08~2.82); the average blood volume (mL) was 18.88±3.60 (14~27).

[0031] 1.2 Reagents

[0032] Sodium citrate anticoagulant tube (Nanger Biomedical Co., Ltd.);

[0033] Human peripheral blood lymphocyte separation medium (LTS1077, Tianjin Haoyang Biological...

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Abstract

The invention discloses a preparation method of cytokine-induced killer cells. The method comprises the following steps of: separating a collected human peripheral blood sample to obtain PBMNC; inoculating the PBMNC into a culture flask coated with a recombinant humanized anti-CD25mAb monoclonal antibody for culture; and transferring non-adherent cells obtained in the step S2 and culture supernatant into a culture flask coated with a recombinant humanized anti-CD3 epsilon monoclonal antibody, and supplementing INF-gamma for continuous culture. According to the preparation method of the cytokine-induced killer cells, CIK cells are induced and amplified by adopting a traditional culture process of CD25 monoclonal antibody improved CIK cells, so that the yield of CD3+ CD56+ cells is increased, the proportion of CD3+ CD4+ cells is reduced, the killing potential of the CIK cells on K562 and SK-MEL-5 cells is effectively improved, and the preparation method has important significance for formulating a preparation method and quality standard of a clinical-grade CIK cell preparation.

Description

technical field [0001] The invention relates to the technical field of immune cell preparation, in particular to a preparation method of cytokine-induced killer cells. Background technique [0002] In 1991, Schmidt Wolf et al. found that a group of CD3+CD56+ cells with high proliferation potential and high lethality could be obtained by step-by-step induction and expansion of PBMNC with a combination of INF-γ, anti-CD3Mab, IL-1a, and IL-2 factors. For active NK-like T lymphocytes, the culture system used in a large number of experimental studies or clinical applications at home and abroad is similar to that of Schmidt Wolf et al. Previous studies have shown that CIK cells induced and expanded by unsorted PBMNCs are highly heterogeneous, including a group of non-MHC-restricted CD3+CD56+ cells with high killing activity and low proliferative ability and another group with low proliferation ability. CD3+CD56- cells with killing activity and high proliferative capacity. The 48...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2501/2302
Inventor 苑春慧陈革
Owner 北广再生医学科技(广东)有限公司
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