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A kind of preparation method of trophoblast of limited generation and culture method of snk cell

A technology of trophoblasts and culture methods, which is applied in the fields of cell biology and molecular biology, immunology, and medicine, and can solve problems such as low NK activity, low killing activity of solid tumors, and low ratio of CD56+CD16+

Active Publication Date: 2021-07-06
BEIJING DCTY BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Common NK cell culture methods are as follows: 1. Isolate NK cells from autologous PBMC and culture them. The disadvantages are small number of expansion, low CD56+CD16+ ratio, and low NK activity; 2. Allogeneic stem cell source , Induction and culture of NK cells, disadvantages, risks of allogeneic sources, low ratio of CD56+CD16+, NK activity is not high; 3. The trophoblast cells used are K562, because K562 is a tumor cell line, there are certain risks in application, and must It can be used after irradiation, which increases the difficulty of use, and the ratio of CD56+CD16+ is not high, NK only has certain killing activity on B cells or lymphoma, and the killing activity on solid tumors is not high

Method used

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  • A kind of preparation method of trophoblast of limited generation and culture method of snk cell
  • A kind of preparation method of trophoblast of limited generation and culture method of snk cell
  • A kind of preparation method of trophoblast of limited generation and culture method of snk cell

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preparation example Construction

[0029] The invention provides a method for preparing trophoblasts with limited algebra, comprising the following steps:

[0030] 1) connecting the TAX2 gene to a lentiviral expression vector, and transferring it into a competent cell to obtain a lentivirus containing the TAX2 gene;

[0031] 2) culturing peripheral blood mononuclear cells with the lentivirus containing TAX2 gene obtained in step 1), and collecting CD3- cells;

[0032] 3) connecting the 41BBL-MICA fusion gene to the lentiviral expression vector, and transferring it into competent cells to obtain a lentivirus containing the 41BBL-MICA fusion gene;

[0033] 4) mixing the CD3-cells obtained in step 2) with the lentivirus containing the 41BBL-MICA fusion gene obtained in step 3) and then culturing to obtain trophoblast cells of limited generations;

[0034] There is no chronological order between said steps 1) and 3).

[0035] In the invention, the TAX2 gene is connected to the lentivirus expression carrier, and t...

Embodiment 1

[0054] Culture of limited expansion trophoblast cells:

[0055] 1. Limited expansion algebraic cells

[0056] Gene synthesis: TAX2 (SEQ ID No.1) and 41BBL-MICA fusion gene (SEQ ID No.2) were synthesized by Jinweizhi Company, connected to the lentiviral expression vector pCDH, harvested the virus supernatant through lentiviral packaging, concentrated After transfecting PBMC, the specific operation process is as follows:

[0057] Transform into E. coli competent cells, screen positive clones, and prepare for use after the sequencing is correct;

[0058] a) Inoculate 3 bottles of 293T cells with a cell density of about 3×10 6 cell / 10ml / T75, shake well and place at 37°C, CO 2 Overnight culture in the incubator: the next day, when the cell density grows to 80%, carry out lentivirus packaging;

[0059] b) Take two 15ml centrifuge tubes AB, add 1.5ml opti-MEM respectively, add 60μg plasmid to tube A, mix well, add 300ng liposome 3000 to tube B, mix well, and let stand at room tem...

Embodiment 2

[0081] 1. Culture of SNK cells:

[0082] a) separate human peripheral blood mononuclear cells, centrifuge and count, and resuspend with 1640+10% FBS+200IU / mLIL-2;

[0083] b) According to the ratio of 200:1, add the trophoblast cells with limited expansion generations obtained in Example 1; 37°C, CO 2 incubator cultivation;

[0084] c) According to the actual volume, add 50IU / mL~500IU / mL of IL-2 every day, and change the half volume of the medium when the medium turns yellow according to the growth of the cells;

[0085] d) SNK cells are obtained when cultured to 14-28 days;

[0086] 2. Flow cytometric detection of SNK phenotype

[0087] After the cells were cultured for 14 days, the expressions of CD3, CD4, CD8, CD56, and CD16 were detected by flow cytometry, and the phenotype of SNK was analyzed. Specific steps are as follows:

[0088] a) Take the SNK cells cultured for 14 days, centrifuge at 1000rpm for 5min, discard the supernatant, add 10ml PBS to wash once, and disc...

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Abstract

The invention provides a method for preparing trophoblast cells with limited algebra and a method for cultivating SNK cells, belonging to the technical fields of medicine, immunology, cell biology and molecular biology, comprising: linking TAX2 gene to a lentiviral expression vector, Transform into competent cells, infect peripheral blood mononuclear cells with lentivirus containing TAX2 gene and culture, collect CD3-cells; connect 41BBL-MICA fusion gene to lentiviral expression vector, transfer into competent cells, and CD3- The cells were mixed with the lentivirus containing the 41BBL-MICA fusion gene and cultured to obtain trophoblasts with a limited number of passages. The trophoblasts cultured by the culture method provided by the present invention have a limited number of passages, can be used for allogeneic use, and have no risk of tumorigenesis, and can efficiently stimulate the expansion of CD56+CD16+NK cells while ensuring safety.

Description

technical field [0001] The invention belongs to the technical fields of medicine, immunology, cell biology and molecular biology, and in particular relates to a method for preparing trophoblast cells with limited algebra and a method for cultivating SNK cells. Background technique [0002] Common NK cell culture methods are as follows: 1. Isolate NK cells from autologous PBMC and culture them. The disadvantages are small number of expansion, low CD56+CD16+ ratio, and low NK activity; 2. Allogeneic stem cell source , Induction and culture of NK cells, disadvantages, risks of allogeneic sources, low ratio of CD56+CD16+, NK activity is not high; 3. The trophoblast cells used are K562, because K562 is a tumor cell line, there are certain risks in application, and must It can be used after irradiation, which increases the difficulty of use. Moreover, the ratio of CD56+CD16+ is not high, and NK only has certain killing activity against B cells or lymphoma, and the killing activity...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10C12N5/0786
CPCC12N15/86C12N5/0646C12N2740/15043C12N2506/115A61K39/4644A61K2239/38A61K39/4613A61P35/00C12N2740/16043C07K14/70575C07K14/70539C12N2510/00C12N2501/2302C12N2501/515C12N2502/11C12N5/0605A61K2239/47A61K2239/50A61K2239/55A61K2239/54A61K2239/53A61K2239/49
Inventor 焦顺昌张嵘周子珊
Owner BEIJING DCTY BIOTECH CO LTD
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