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Culture method for in-vitro-amplified-gene edited and activated T cells, kit and application

A gene editing and in vitro amplification technology, applied in the field of biotechnology and cell culture, can solve the problems of inability to meet scientific research needs, low efficiency, relying on natural recombination, etc., and achieve the effect of fast and efficient induction method, economical simplicity, and stable effect.

Active Publication Date: 2017-07-18
CHENGDU MEDGENCELL CO LTD
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Problems solved by technology

However, this method relies too much on natural recombination, which is extremely inefficient and cannot meet the growing scientific research needs of human beings.

Method used

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  • Culture method for in-vitro-amplified-gene edited and activated T cells, kit and application

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Effect test

Embodiment 1

[0024] Embodiment 1 (experimental group)

[0025] Isolation and efficient expansion of human PD-1 gene knockout T cells using CRISPR-Cas9 technology

[0026] Human peripheral blood is now used as the processing sample, and the human PD-1 gene is knocked out by using CRISPR-Cas9 technology to construct PD-1 knockout T cells, and the gene-edited activated T cells are efficiently expanded and cultured in vitro. Specific steps are as follows:

[0027] Step 1: Isolation of peripheral blood mononuclear cells from blood

[0028] Directly extract 80-100ml of venous blood from the donor, and add anticoagulant-heparin;

[0029] Centrifuge the collected blood sample at 2000rpm for 10min at room temperature, and carefully absorb the upper plasma layer for later use; restore the blood sample to its original volume and mix well; slowly add the diluted blood to Ficoll, and centrifuge at 1000rpm for 20min; absorb milky white mononuclear cells at the interface of the separation solution lay...

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Abstract

The invention provides a culture medium for in-vitro-amplified-gene edited and activated T cells. The culture medium is characterized by containing a T-cell basal culture medium and further containing: 200IU / ml to 1,000IU / ml of interleukin 7 (IL-7), 300IU / ml to 1,000IU / ml of interleukin 21 (IL-21), 80ng / ml to 120ng / ml of anti-CD40 monoclonal antibody, 0.2mmol / ml to 1.0mmol / ml of serine and 0.3mmol / ml to 1.0mmol / ml of S-2-hydroxyglutarate. The invention further provides a kit containing the culture medium, a method for carrying out in-vitro-amplified-gene editing and activating on T cells by using the culture medium and application in culture of the T cells. By using the culture medium provided by the invention, the gene edited and activated T cells can be extensively amplified under the condition of not adding foreign serum and other T cell activating agents. According to the method provided by the invention, the amplified T cells can be activated by 500 to 1,000 times, and the times of activation are apparently higher than that in the prior art.

Description

technical field [0001] The invention belongs to the field of biotechnology and cell culture technology, and in particular relates to a culture method, kit and application of in vitro amplified gene-edited activated T cells. Including, but not limited to, functionally activated T cell culture methods, kits and applications constructed using genome editing technologies such as ZFN, TALEN, and CRISPR-CAS9. Background technique [0002] With the deepening of immunology research, tumor immunotherapy has made great progress. Tumor immunotherapy stimulates or mobilizes the patient's own immune system and enhances the anti-tumor immunity of the tumor microenvironment, thereby controlling and killing tumor cells. Tumor immunotherapy is also considered to be the fourth major tumor treatment technology after surgery, radiotherapy, and chemotherapy. In the past decade, tumor immunotherapy methods represented by adoptive cell immunotherapy (ACT) and immune checkpoint therapy (immune che...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0783
CPCC12N5/0636C12N2500/30C12N2500/32C12N2501/2307C12N2501/2321C12N2501/52C12N2510/00
Inventor 邓涛喻堃徐国锋卢铀
Owner CHENGDU MEDGENCELL CO LTD
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