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Method for expanding and culturing peripheral blood T cells in vitro

A technology of in vitro expansion and culture method, applied in the field of in vitro expansion and culture of peripheral blood T cells, can solve the problems of increasing the burden and disadvantage of patients and CAR-T, saving the initial blood supply, reducing difficulty and cost low effect

Active Publication Date: 2020-02-04
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the immunomagnetic bead method to stimulate and culture T cells has certain limitations in clinical application, especially in the future industrialized large-scale production.
In 2017, the FDA approved the marketing of two CAR-T products, bringing new hope to patients with hematomas, but their prices were as high as US$473,000 (Kymriah) and US$373,000 (Yescarta), which undoubtedly increased the number of patients and CAR-T. - Burden of industry development

Method used

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  • Method for expanding and culturing peripheral blood T cells in vitro
  • Method for expanding and culturing peripheral blood T cells in vitro
  • Method for expanding and culturing peripheral blood T cells in vitro

Examples

Experimental program
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Effect test

Embodiment 1

[0040] (1) Pre-prepared peripheral blood T cell in vitro expansion medium: use serum-free immune cell culture medium X-VIVO15 (1L / bottle, Lonza, product number: 04-418Q), add 10% FBS (Gibco, product number: 10091- 148), and sequentially added interleukin-2 (IL-2, final concentration 75 μg / L, Sino, product number: 11848-HNAY1) with a concentration of 250 μg / mL, interleukin-15 (IL-15, final concentration 40 μg / L, Sino, Cat. No.: 10360-H07E), 1 mg / mL human CD3 monoclonal antibody (Biolegend, Cat. / L), mixed well as the T cell in vitro expansion medium.

[0041] (2) Collect blood (sodium heparin anticoagulant) using traditional methods and routine blood collection methods, and collect 20 mL of whole blood;

[0042] (3) Ficoll density gradient centrifugation was used to separate the peripheral blood lymphocytes in the above-mentioned whole blood, the method is as follows:

[0043] i) The 1640 culture is based on a 37°C water bath for warming;

[0044] ii) Take out the Ficoll se...

Embodiment 2

[0056] T cells that had been cryopreserved for 7 days were taken out from liquid nitrogen (5×10 6 ), using the peripheral blood T cell in vitro expansion medium in Example 1 to quickly revive T cells, and to culture at a density of 1 × 10 6 cells / mL to re-stimulate T cells (culture conditions 37°C, 5% CO 2 ), which promotes the reactivation and expansion of T cells. 5×10 after 2-3d 5 -5×10 6 The medium was inoculated at a culture density of 1 cell / mL, and the resuscitated T cells were infected with the lentiviral vector PCDH lentiviral vector to obtain chimeric antigen receptor T cells targeting EGFR (EGFR CAR- T), using the above-mentioned T cell in vitro expansion medium to carry out routine culture of the prepared CAR-T, every 2-3d with 5 × 10 5 -5×10 6 The culture density of cells / mL was inoculated and changed, and it was obtained by cell counting ( Figure 5 ) CAR-T cells can be expanded to 60-80 times the initial amount on the 22nd day in vitro, that is, from the i...

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Abstract

The invention discloses a method for expanding and culturing peripheral blood T cells in vitro. In the method, a culture medium consists of a basic culture medium, fetal bovine serum, interleukin-2, interleukin-15, a human CD3 monoclonal antibody and a human CD28 monoclonal antibody. According to the method, T cells isolated from peripheral blood are activated and cultured in one cycle, a sufficient amount of cells for in vitro functional experiments can be obtained; after 7 days of expansion, number of the T cells can reach 40-50 times the initial amount; after 14-22 days of expansion, the number of the cells can reach 80-200 times the initial amount; furthermore, the CAR-T cells difficult to be expanded can be expanded to 60-80 times the initial amount on the 22nd day after in vitro infection, and the number is much greater than the initial isolation amount. According to the method, activation / expansion of the T cells and CAR-T cells is realized through stimulation of monoclonal antibodies and cytokines in the culture medium, no antigen-presenting cells or specific antigens or magnetic beads are needed, operation is simple, and culture cost is greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for expanding and culturing peripheral blood T cells in vitro. Background technique [0002] Human cancer cells are, by their very nature, derived from normal cells that have undergone genetic or epigenetic changes to become cancerous cells. Cancer cells often express their unique proteins, commonly known as tumor-specific antigens or tumor-associated antigens. These abnormal tumor antigens can be specifically recognized by the body's innate immune system and further kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells such as T and B lymphocytes from recognizing cancer cells. Human T cell therapy relies on in vitro enrichment and modification of human T cells to target and kill cancer cells in a subject (eg, a patient). Various techniques have been developed to enrich naturally occurring T cells targeting tumor an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2501/2302C12N2501/2315C12N2501/51C12N2501/515
Inventor 刘文夏琳郑早早刘珺懿陈宇洁
Owner XIAMEN UNIV
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