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Configuration method for obtaining memorable T lymphocyte subset culture medium

A lymphocyte and configuration method technology, applied in the field of biomedicine, can solve the problem of limited expansion effect of memory T cells, and achieve the effects of strong practicability, simple proportioning, and improved efficiency

Inactive Publication Date: 2019-04-19
THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, the serum-free medium sold in the market, such as Corning’s KBM581 serum-free medium, Lonza’s X-Vivo15 serum-free medium or Gibco’s AIM-V serum-free medium can expand the number of cells, but Limited expansion of memory T cells

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  • Configuration method for obtaining memorable T lymphocyte subset culture medium
  • Configuration method for obtaining memorable T lymphocyte subset culture medium
  • Configuration method for obtaining memorable T lymphocyte subset culture medium

Examples

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example 1

[0029] Example 1 1000ml serum-free medium is configured as follows

[0030] Step 1: Weigh 20 mg of insulin powder (Baying Biotech, 11061-68-0) and dissolve it in 4 ml of phosphate buffer solution to prepare a stock solution with a final concentration of 4 mg / ml, filter and sterilize with a 0.22um filter membrane , stored at -20°C.

[0031] Step 2: Weigh 5 mg of transferrin powder (Sigma, T8158) and dissolve it in 5 ml of phosphate buffer solution to prepare a storage solution with a final concentration of 1 mg / ml, filter and sterilize with a 0.22um filter membrane, and store at -20°C save.

[0032] Step 3: Take 8ml of ethanolamine (Sigma, E0135) and place it in a 15ml centrifuge tube, filter and sterilize with a 0.22um filter membrane, and store at -20°C.

[0033] Step 4: Weigh 2 g of catalase powder and dissolve it in 10 ml of phosphate buffer solution to prepare a stock solution with a final concentration of 0.2 g / ml, filter and sterilize with a 0.22 μm filter, and store a...

example 2

[0037] Example 2 Evaluation of different media on CAR-T cell culture

[0038] The preparation and treatment process of traditional CAR-T cells is as follows: a. PBMC cell acquisition; b. T cell activation; b. CAR gene introduction into T cells; d. In vitro expansion of CAR-T cells; reinfusion of cells.

[0039] During the preparation of the CAR-T cells, KBM581, X-Vivo15 and AIM-V were used as references to evaluate the expansion of T cells and the ratio of memory T cells in the serum-free medium configured in the present invention. The preparation of CAR-T cells and the evaluation of the medium adopt the traditional CAR-T cell preparation process:

[0040] 1) PBMC cell acquisition: collect 60-80ml of peripheral blood from the patient, dilute the peripheral blood with PBS buffer at 1:1, mix well, and slowly add it to a centrifuge tube with 15ml of human lymphocyte separation medium (Ficoll) at room temperature. Centrifuge at 1200g for 20 minutes, slowly rise and fall slowly. ...

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Abstract

The invention discloses a configuration method for obtaining a memorable T lymphocyte subset culture medium. The serum-free medium is formed by adding 40 micrograms / ml of insulin, 10 micrograms / ml oftransferrin, 101 micrograms / ml of ethanol amine, 2 mg / ml of catalase and other cell factors into an RPM1 1640 basic culture medium and using microelements of a Trace element A and a Trace element B cooperatively. The serum-free medium can increase the amplification multiple of T cells and remarkably increase the proportion of memorable T cells to the total T cells, is suitable for in-vitro production of memorable T lymphocyte subset cells, easily improves the efficiency of clearing away tumors in the body of a patient through CAR-T cells, and has positive meanings for CAR-T cell immunotherapy.

Description

technical field [0001] The method relates to a method for configuring cell culture fluid in the biomedical field. More specifically, the present invention relates to a method for configuring a culture medium for cultivating memory T lymphocyte subsets for cellular immunotherapy. Background technique [0002] Compared with traditional tumor therapy drugs, chimeric antigen receptor T cells (CAR-T cells) are a living cell therapy drug. It modifies immune cells through chimeric antigen receptor genes, enabling them to specifically recognize and attack malignant tumor cells. It is the most promising tumor treatment method in the 21st century. [0003] The preparation of CAR-T cells mainly includes the following steps: firstly, the mononuclear cells (PBMC cells) of tumor patients are separated and obtained by apheresis, and then the chimeric antigen receptor gene (CAR gene) is introduced by genetic engineering technology, so that It becomes CAR-T cells that can specifically reco...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2500/24C12N2500/46C12N2501/33C12N2501/71
Inventor 孙洪伟陈必成李杰
Owner THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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