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Preparation method and application of NK (Natural Killer) cell for over-expressing CD16a

A NK cell and overexpression technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problems of low transfection efficiency, excessive cell death, etc. Survival rate, the effect of enhancing the killing effect

Pending Publication Date: 2022-07-29
GUANGDONG PROCAPZOOM BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Electroporation has the advantages of simple operation, low immunogenicity and genotoxicity, and low safety risk, but its existing problems are also obvious: it is easy to cause excessive cell death under transient high voltage, and the transfection efficiency is low. type and electroporation conditions

Method used

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  • Preparation method and application of NK (Natural Killer) cell for over-expressing CD16a
  • Preparation method and application of NK (Natural Killer) cell for over-expressing CD16a
  • Preparation method and application of NK (Natural Killer) cell for over-expressing CD16a

Examples

Experimental program
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Effect test

Embodiment 1

[0058] Example 1: Preparation of NK916 cells (NK92MI cells overexpressing CD16)

[0059] (1) Prepare a three-plasmid system. The three-plasmid system is a target plasmid for overexpressing CD16, a psPAX2 plasmid and a pMD2.G plasmid.

[0060] (2) Preparation of basic medium: DMEM complete medium containing 5-20 vol% fetal bovine serum and 1 vol% penicillin-streptomycin.

[0061] (3) about (1~10)×10 6 293T cells were inoculated into T75 culture flasks, basic medium was added, and cultured overnight. When the cell density reached 50-90%, the supernatant in the culture flask was aspirated, and 10 mL of DMEM containing only 0.1-2vol% fetal bovine serum was added. The culture medium starves the cells for 1-3 hours to obtain tool cells;

[0062] (4) Pipette the Opti-MEM medium in an amount of 1-10 mL per virus, and divide it into two equal parts. One part is added with lentiviral packaging plasmids psPAX2, pMD2.G and the target expression plasmid, and mixed evenly to obtain a pla...

Embodiment 2

[0077] The operation mode of embodiment 2 is basically the same as that of embodiment 1, and the main difference is that the first, second and third culture medium in embodiment 1 are replaced as follows; and the mass ratio of pMD2.G, psPAX2, and the target expression plasmid The ratio of polyethyleneimine to the total amount of the three plasmids was 1:1, and the final concentration of polybrene was 8 μg / mL.

[0078] The specific composition of the first cell culture medium is as follows: 0.1 mM inositol, 0.2 mM folic acid, 0.1 mM mercaptoethanol, 2 vol % fetal bovine serum, 2 vol % horse serum, and 2 vol % double antibody are added to the MEM basal medium.

[0079] The specific composition of the second cell culture medium is as follows: 1 mM inositol, 0.05 mM folic acid, 0.1 mM mercaptoethanol, 25 vol% fetal bovine serum, 25 ol% horse serum, and 1 vol% double antibody are added to the MEM basal medium.

[0080] The specific composition of the third cell culture medium is as...

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Abstract

The invention provides a method for preparing an over-expressed CD16a NK (Natural Killer) cell. The method comprises the following steps: (1) constructing a lentivirus transfection system of a target plasmid with CD16a; (2) culturing the HEK 293T cell; (3) uniformly mixing the transfection reagent and plasmids; (5) carrying out lentivirus packaging; (6) collecting, filtering and concentrating virus liquid; (7) transfecting the NK cells resuspended by the second cell culture medium, adding polybrene, and preheating; (8) centrifuging; (9) adding a second cell culture medium containing polybrene, uniformly mixing, and continuously culturing by adopting a third cell culture medium; and (11) taking the cells, carrying out flow-type detection on the positive rate, carrying out flow-type sorting, screening out positive cells, inoculating a corresponding system, continuing multiplication culture, and building a library. The NK cell overexpressing CD16a prepared by the method is high in positive rate and high in cell survival rate, the NK cell can still stably overexpress CD16a after multiple passage, and the NK cell has good killing performance on cancer cells.

Description

technical field [0001] The invention belongs to the field of cellular immunotherapy, and in particular, relates to a preparation method and application of NK cells overexpressing CD16a. Background technique [0002] Antibody-dependent cell mediated cytotoxicity (ADCC) refers to the ability of immune cells with killing activity to recognize target antigens (such as bacteria or tumors) bound by monoclonal antibodies through the binding of FC receptors on the cell surface and monoclonal antibodies. cells), which directly kill target cells. Studies have found that ADCC is one of the important mechanisms and means of monoclonal antibody therapy for tumors or other diseases. [0003] Immune cells and their receptors that mediate ADCC: The immune cells that play the role of ADCC mainly include monocytes (such as natural killer cells (NK cells), macrophages and γδT cells) and polymorphonuclear cells (neutrophils). cells, basophils, and eosinophils). Among them, NK cells are the m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/12C12N5/10C12N5/0783
CPCC12N15/86C07K14/70535C12N5/0646C12N2510/00C12N2800/107C12N2740/15043
Inventor 曾皓宇沈振波蒋碧愉卫佳琦刘康志
Owner GUANGDONG PROCAPZOOM BIOSCIENCES CO LTD
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