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Culture medium and kit for massively amplifying PD-1 genic recombinant activated T cells and applications thereof

A technology of gene recombination and basal medium, which is applied in the field of PD-1 gene recombination and activated T cell medium, can solve the problems of difficulty in large-scale culture, and achieve the effect of economy, simplicity and stable effect.

Inactive Publication Date: 2017-01-11
安徽柯顿生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in order to realize the effective clinical application of this method, the effective large-scale culture of the PD-1 gene recombined activated T cells has become a major problem.

Method used

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  • Culture medium and kit for massively amplifying PD-1 genic recombinant activated T cells and applications thereof
  • Culture medium and kit for massively amplifying PD-1 genic recombinant activated T cells and applications thereof
  • Culture medium and kit for massively amplifying PD-1 genic recombinant activated T cells and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Pre-cultivation of human PD-1 gene recombination activated T cells and DC cells

[0031] Materials and reagents: lymphocyte separation medium (Ficoll) (Tianjin Haoyang Biotechnology Co., Ltd.); 1640 medium (Hyclone Company), fetal bovine serum (FBS, Hangzhou Sijiqing); cell counting kit (CCK-8) ( Japan Dojindo Company); apoptosis kit (Japan Dojindo Company); IL-2, INF-γ, CD3 monoclonal antibody, IL-1, IL-4 (Peprotech Company products); phytohemagglutinin (PHA) (US Sigma company); CD3-FITC / CD56-PE, PD1-PE mouse anti-human monoclonal antibody and the same subtype control antibody rabbit anti-mouse IgG (eBioscience, USA); FC500 flow cytometer and corresponding software (Beckman Coulter, USA) ; GM-CSF (Peprotech company product);

[0032] The culture medium for a large number of PD-1 gene recombined activated T cells of the present invention (referred to as the culture medium of the present invention), which contains the basal medium 1640 medium, based on the 16...

Embodiment 2

[0037] Example 2. Co-cultivation of human PD-1 gene recombination activated T cells and DC cells.

[0038] The T cells and DC cells activated by PD-1 gene recombination obtained in Example 1 were placed in the medium of the present invention and inoculated at 225 cm 2 In the culture flask, DC cells and T cells activated by PD-1 gene recombination were mixed and cultured at a cell number ratio of 1:100, and the seeding density was (3-5)×10 6 PD-1 gene recombination activated T cells / well, cultured with the medium of the present invention, cultured at 37°C, 5% CO2 in a cell culture incubator, half of the medium was changed for 3 days thereafter, typical PD-1 was seen after 14 days of culture Gene recombination activates T cell formation. During the culture process, at different time points, the growth morphology of PD-1 gene recombination-activated T cells was observed using an inverted phase-contrast microscope, and the expression of PD-1 gene recombination-activated T cell ma...

Embodiment 3

[0043] Example 3. Pre-cultivation of human PD-1 gene recombination activated T cells

[0044] The culture medium for massively expanding PD-1 gene recombination activated T cells of the present invention (referred to as the culture medium of the present invention) is the same as that in Example 1.

[0045] Method steps:

[0046] 1. Mononuclear cells were isolated from human autologous T cells (50 ml of blood was collected from a volunteer with liver cancer) by Ficoll two-step separation method, and the cell concentration was adjusted to 3 × 10 with serum-free medium. 6 .ml -1 , cultured in a 37°C, 5% CO2 incubator, and added a final concentration of 1000U.ml on the first day of culture -1 IFN-γ; on the second day, the final concentration was 100ng.ml -1 CD3 monoclonal antibody, 1000U.ml -1 IL-2, 100ug.ml -1 the PHA of the present invention; supplement the culture medium of the present invention every 1~2d afterwards, adjust the cell concentration to be 3×10 6 .ml -1 , the...

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Abstract

The invention discloses a culture medium and a kit for massively amplifying PD-1 genic recombinant activated T cells, and applications thereof. The culture medium contains a basal culture medium, and further contains OKT3, IL-2, PHA, IL-4 and GM-CSF, wherein the basal culture medium is applicable to generation of the PD-1genic recombinant activated T cells. Through application of the culture medium provided by the invention, the human PD-1genic recombinant activated T cells can be massively amplified under the condition that allogeneic serum is not required to be added, and amplification is fast, efficient, economical, simple, convenient, and stable in effect. The invention provides the culture medium and the kit capable of economically, simply, conveniently and efficiently transforming autologous or umbilical cord blood T cells into the PD-1genic recombinant activated T cells and massively amplifying the PD-1 genic recombinant activated T cells, and applications thereof. According to the invention, the transfection efficiency of the PD-1genic recombinant activated T cells is improved, the amplification speed of transfected T cells is increased, the amplification time is shortened, and thus the therapeutic effect of the activated T cells on tumors or other related diseases is improved.

Description

technical field [0001] The invention relates to the technical engineering field of biotechnology and cell therapy, in particular to a PD-1 gene recombination activated T cell culture medium, a kit and applications thereof. The details include: culture medium, kits and methods for massively expanding PD-1 gene recombined activated T cells. Background technique [0002] At present, in tumor cell immunotherapy, T cells and DC cells are the core roles of anti-tumor cell immunity. However, these immune cells reported so far generally suffer from high culture costs and limited number of expansions (the number of expansions can generally be in ( 1-3)×10 9 ), long amplification time, and unstable transfection efficiency directly affect its anti-tumor effect. [0003] Transfection of autologous or umbilical cord blood-derived T cells into PD-1 gene recombined activated T cells, and a large number of expansions, so as to be applied to clinical research is one of the important conten...

Claims

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Application Information

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IPC IPC(8): C12N5/10
CPCC12N5/0636C12N2501/22C12N2501/2302C12N2501/2304C12N2501/515C12N2501/59C12N2510/00
Inventor 孙昌秀
Owner 安徽柯顿生物科技有限公司
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