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Method for serum-free culturing of 293T cells for lentivirus package

A technology of serum-free culture and lentivirus packaging, which is applied in the field of serum-free culture of 293T cells, and can solve the problem of mixing bovine serum

Inactive Publication Date: 2016-06-08
SHENZHEN PREGENE BIOPHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] For this reason, an object of the present invention is to propose a method for culturing 293T cells without serum for lentivirus packaging, which can solve the problem of bovine serum components mixed in the lentivirus packaging process to meet the requirements of GMP products

Method used

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  • Method for serum-free culturing of 293T cells for lentivirus package
  • Method for serum-free culturing of 293T cells for lentivirus package
  • Method for serum-free culturing of 293T cells for lentivirus package

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Embodiment 1

[0030] Step S1: Using DMEM medium containing 10% (volume ratio) fetal bovine serum, 293T cells were placed at 37°C, 5% CO 2 cultured in the incubator until the cells became confluent.

[0031] Step S2: use CDM medium containing 5% (volume ratio) fetal bovine serum (in step 2, replace the medium with Pro293a-CDM, referred to as CDM medium, which is a serum-free 293 cell medium, LONZA Company, product number: 12-764Q), place the 293T cells cultured in step S1 at 37°C, 5% CO 2 cultured in the incubator until the cells became confluent. At this time, the cell morphology and proliferation rate did not change significantly.

[0032] In an orderly manner, subculture is carried out after the cells are overgrown, and after 5 consecutive passages (about 10-12 days for 5 passages), some cells are sampled for cryopreservation.

[0033] Step S3, use CDM medium containing 2% (volume ratio) fetal bovine serum, and place the 293T cells cultured in step S2 at 37°C in 5% CO. 2 cultured in t...

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Abstract

The invention provides a method for serum-free culturing of 293T cells for lentivirus package. The method includes sequentially using DMEM culture medium containing 10% (volume ratio) of fetal bovine serum (FBS), CDM culture medium containing 5% (volume ratio) of FBS, CDM culture medium containing 2% (volume ratio) of FBS, CDM culture medium containing 1% (volume ratio) of FBS, CDM culture medium containing 0.5% (volume ratio) of FBS, CDM culture medium containing 0.1% (volume ratio) of FBS, and CDM culture medium containing 0.05% (volume ratio) of FBS in an incubator containing 5% CO2 at 37oC, and using CDM culture medium to culture 293T cells until the serum-free 293T cells for lentivirus package is obtained. With the method for serum-free culturing of 293T cells for lentivirus package, the problem that bovine serum is blended in during lentivirus packaging can be solved, and requirements for GMP (good manufacture practice) products are met.

Description

technical field [0001] The present invention relates to a method for culturing 293T cells without serum for lentivirus packaging. Background technique [0002] With the development of the immune cell therapy industry, more and more research institutions, pharmaceutical companies and start-up companies are devoted to the research and development, clinical trials and industrial preparation of CAR-T, the star of immune cell therapy technology. The full name of CAR-T is chimeric antigen receptor T cells. It is a kind of CAR gene designed and synthesized by genetic engineering, transferred to lentiviral vector, packaged into lentivirus using 293T, and then infected with T cells of tumor patients to obtain CAR. -T cells, infused back into patients, used for immune cell therapy of tumor patients. Because the CAR gene is designed with a single-chain antibody that recognizes tumor cells and an intracytoplasmic activation domain that activates T cells, CAR-T has an excellent effect o...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0686C12N5/0625
Inventor 何昱栗红建何嵘姜冬冬
Owner SHENZHEN PREGENE BIOPHARMA CO LTD
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