A kind of in vitro expansion and culture method of peripheral blood T cells

A culture method and peripheral blood technology, applied in the field of biomedicine, can solve problems such as disadvantage, increase the burden on patients and CAR-T, and achieve the effect of reducing difficulty, saving initial blood supply, and alleviating serious shortages.

Active Publication Date: 2021-07-09
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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  • A kind of in vitro expansion and culture method of peripheral blood T cells
  • A kind of in vitro expansion and culture method of peripheral blood T cells
  • A kind of in vitro expansion and culture method of peripheral blood T cells

Examples

Experimental program
Comparison scheme
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 an in vitro expansion and culture method of peripheral blood T cells. The culture medium is composed of basic culture medium, fetal bovine serum, interleukin-2, interleukin-15, human CD3 monoclonal antibody and human CD28 monoclonal antibody. The T cells isolated from peripheral blood in the present invention can obtain sufficient cell volume for in vitro function experiments after a cycle of activation and expansion culture, and after 7 days of expansion, the T cell volume reaches 40-40% of the initial volume. 50 times, after 14-22 days of expansion, the number of cells can reach 80-200 times the initial amount, and for difficult-to-expand CAR-T cells, it can be expanded to 22 days after in vitro infection 60‑80 times the initial amount, the amount is far more than the initial separation amount. The activation / expansion of T cells and CAR-T cells in the present invention is achieved through the stimulation of monoclonal antibodies and cytokines in the culture medium, without the need for antigen-presenting cells or specific antigens or magnetic beads, and the operation is simple and the cost of culture is low. dramatically drop.

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 Patents(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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