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Construction method of human stem memory T cell bank

A construction method and cell technology, which are applied in the construction of human stem memory T cell bank and the construction of human peripheral blood immune cell bank, can solve the problems of low efficiency, high preparation cost, large demand for peripheral blood, etc. Low, easy-to-use effects

Inactive Publication Date: 2016-08-03
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Since the proportion of TSCM in human circulating peripheral blood is low, accounting for about 2-4% of T cells, and even with flow cytometry, about 1 CD4 can be isolated from every 250 PBMC cells + TSCM cells, while 1 CD8 can be isolated from every 500-1000 PBMC cells + TSCM cells, for large-scale cell bank construction, this method is obviously inefficient, requires a large amount of peripheral blood, and is expensive to prepare

Method used

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  • Construction method of human stem memory T cell bank
  • Construction method of human stem memory T cell bank
  • Construction method of human stem memory T cell bank

Examples

Experimental program
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Effect test

Embodiment 1

[0082] A method for constructing a human stem memory T cell bank, comprising the following steps:

[0083] (1) Collection of human peripheral blood

[0084] The cell storage participants signed the informed consent form, and the physical examination results showed that their blood routine, hepatitis B, hepatitis C, syphilis, AIDS, and alanine aminotransferase were in line with the relevant blood collection regulations in the national "Basic Standards for Blood Stations"; 60 mL of peripheral blood was manually collected Store in a blood collection bag containing anticoagulant at 4°C and send it to the cell preparation room as soon as possible.

[0085] (2) Separation and freezing of plasma

[0086] Transfer the blood in the blood collection bag to the centrifuge tube on the clean bench, cut the plastic tube with sterilized scissors, carefully pour the peripheral blood in the bag into the centrifuge tube, and centrifuge at 1800rpm for 12 minutes at room temperature. Centrifuge...

Embodiment 2

[0100] Compared with Example 1, the difference is only in step (3), the specific operations are as follows, and the others are the same as in Example 1.

[0101] (3) Divide the collected peripheral blood into centrifuge tubes, centrifuge at room temperature at 1600rpm for 12min, take the supernatant to obtain autologous plasma, and store it at -20°C for later use; after separating autologous plasma, use normal saline at a ratio of 1:1 Dilute the lower layer of blood cells, and slowly and uniformly add the diluted blood cell suspension to the Ficoll-Paque TM Above the lymphocyte separation medium, centrifuge at room temperature at 1800rpm for 20min. After centrifugation, layers appear in the centrifuge tube. Carefully transfer the buffy coat layer to another centrifuge tube, and collect the obtained components to obtain PBMC.

Embodiment 3

[0103] Compared with Example 1, the difference is only in step (3), the specific operations are as follows, and the others are the same as in Example 1.

[0104] (3) Divide the collected peripheral blood into centrifuge tubes, centrifuge at room temperature at 1800rpm for 14min, take the supernatant to obtain autologous plasma, and store it at -20°C for later use; after separating autologous plasma, use normal saline at a ratio of 1:1 Dilute the lower layer of blood cells, and slowly and uniformly add the diluted blood cell suspension to the Ficoll-Paque TM Above the lymphocyte separation medium, centrifuge at room temperature at 1900rpm for 22min. After centrifugation, layers appear in the centrifuge tube. Carefully transfer the buffy coat layer to another centrifuge tube, and collect the obtained components to obtain PBMC.

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Abstract

The invention relates to a construction method of a human stem memory T cell bank. The method comprises the following steps of human peripheral blood collection, peripheral blood mononuclear cell separation, initial T cell sorting, stem memory T cell preparation, cell cryopreservation and coding and storage. According to the construction method of the human stem memory T cell bank, a method of conducting directionally inducing in vitro is adopted, enough stem memory T cells (TSCM) can be obtained in a short time and subjected to bank construction, the volume of needed peripheral blood is small, and the construction method of the human stem memory T cell bank has the advantages of being low in cost, low in requirement on lab condition, easy to operate and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a human peripheral blood immune cell bank, in particular to a method for constructing a human stem memory T cell bank. Background technique [0002] Human peripheral blood contains a variety of immune cells, among which lymphocytes are the main type of immune cells in the human body, including T cells, B cells and natural killer cells. Currently, the immune cell therapy in clinical research and application is mainly based on T cells. Such as TCR or CAR-T technology. Since autologous T cells have no immunogenicity, they can be directly used for reinfusion for the treatment of tumors and other diseases, and their memory characteristics can also inhibit the recurrence of the disease to a certain extent. T cells can be isolated from human peripheral blood mononuclear cells (PBMC), but because peripheral blood is usually collected from patients with tumors or vi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N5/0783A01N1/02
CPCC40B50/06A01N1/0221C12N5/0636C12N2500/32C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/51C12N2501/515C12N2506/11
Inventor 孙长斌张曦张家文周欣
Owner SHENZHEN HUADA GENE INST
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