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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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in order to realize the effective clinical application of this method, the effective

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
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Example Embodiment

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

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

[0032] The medium of the present invention for a large number of expansion of PD-1 gene recombination activated T cells (referred to as the medium of the present invention), which contains the basic medium 1640 medium, based on the 1640 medium, and also contains 100ng·ml -1 OKT...

Example Embodiment

[0037] Example 2. Human PD-1 gene recombination activates the co-culture of T cells and DC cells.

[0038] The PD-1 gene recombination activated T cells and DC cells obtained by preculture in Example 1 were placed in the culture medium of the present invention and inoculated at 225 cm 2 In the culture flask, the DC cells and the T cells activated by the recombination of the PD-1 gene are mixed and cultured at a cell number ratio of 1:100, and the seeding density is (3~5)×10 6 PD-1 gene recombination activated T cells / well, cultured with the culture medium of the present invention, cultured in a cell incubator at 37°C, 5% CO2, and then half of the medium was changed for 3 days, and 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 with an inverted phase contrast microscope, and the expression of PD-1 gene r...

Example Embodiment

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

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

[0045] Method steps:

[0046] 1. The Ficoll two-step separation method separates mononuclear cells from human autologous T cells (50ml blood taken from liver cancer volunteers), and adjusts the cell concentration to 3×10 with serum-free medium 6 .ml -1 , Cultivate in a 37℃, 5% CO2 incubator, add the final concentration of 1000U.ml on the first day of culture -1 IFN-γ; add the final concentration of 100ng.ml on the second day -1 CD3 monoclonal antibody, 1000U.ml -1 IL-2, 100ug.ml -1 PHA; then supplement the culture medium of the present invention every 1 to 2 days and adjust the cell concentration to 3×10 6 .ml -1 , The final concentration of IL-2 is 1000U.ml -1 , Continue to cultivate ...

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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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