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Method for cultivating Chinese rhesus monkey gammadelta T cells

A technology of cell culture and culture method, applied in the field of culture of Chinese rhesus monkey γδT cells, to achieve the effect of less steps, reduced interference and high purity

Active Publication Date: 2019-09-03
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention is made in view of the lack of in vitro culture of non-human primate peripheral blood-derived γδT cells. One purpose of the present invention is to provide a A reproducible method for isolating and culturing peripheral blood-derived γδT cells, using OpTmizer cell culture medium with low concentration of monkey autologous serum, IL-2 necessary for T cell culture, and has been reported to promote γδT cell expansion IL-18 can quickly and stably cultivate high-purity Chinese rhesus macaque γδT cells with morphological and immunological characteristics of γδT cells in vitro

Method used

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  • Method for cultivating Chinese rhesus monkey gammadelta T cells
  • Method for cultivating Chinese rhesus monkey gammadelta T cells
  • Method for cultivating Chinese rhesus monkey gammadelta T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Isolation and culture of Chinese rhesus monkey peripheral blood mononuclear macrophages

[0029] Intramuscularly anesthetize healthy adult (4-5 years old) Chinese rhesus monkeys with ketamine (10mg / kg), collect venous blood 5-10ml with EDTA anticoagulant vacuum blood collection tubes and non-anticoagulant vacuum blood collection tubes respectively Whole blood 5mL. Anticoagulated blood was used for the separation of PBMCs, and non-anticoagulated blood was used for the separation of monkey serum after self-coagulation. Anticoagulated whole blood (5-10mL) was double-diluted with phosphate buffered saline (PBS, pH 7.4), then slowly added to the upper layer of lymphocyte separation solution (Ficoll) diluted to 1 / 2 volume of the blood, and kept at 22°C Centrifuge at 1800g for 30 minutes, and suck out the PBMCs layer between the plasma dilution and Ficoll. The collected cells were resuspended and washed twice with an equal volume of PBS, and the washed cells were r...

Embodiment 2

[0032] Example 2 Effects of Different Concentrations of Zoledronic Acid on the Differentiation of Peripheral Blood-derived γδT Cells from Chinese Rhesus Monkeys

[0033] After culturing monkey PBMCs with 1, 5, and 10 μmol / L zoledronic acid special culture medium for 8 days, the cells were collected and washed twice with PBS, then resuspended in 50 μl PBS to make a single cell suspension, and CD3-PerCP- Cy5.5 (BD Pharm, USA) and TCR Vγ9-FITC antibody (Thermo, USA) each 2 μ l, negative control tube plus isotype (Isotype) antibody IgG1-Percp-Cy5.5 (BD Pham, USA) and IgG1-FITC ( eBioscience, USA) each 2 μl, incubate at 4°C for 15 minutes in the dark, wash twice with PBS, remove excess antibody, add fixative (2% paraformaldehyde) to resuspend cells, and use flow cytometry (BD FACS Verse, USA) The expression levels of CD3 and CR Vγ9 on the cell surface were detected. After testing, the double-positive expression rates of CD3 / TCR Vγ9 on the surface of Chinese rhesus monkey periphera...

Embodiment 3

[0034] Example 3 Identification of Immune Activation of Peripheral Blood-derived γδT Cells from Chinese Rhesus Monkeys by Flow Cytometry

[0035] After cultivating monkey PBMCs in a special medium containing 5 μmol / L zoledronic acid for 8 days, the cells were collected and washed twice with PBS, then resuspended in 50 μl PBS to make a single cell suspension, and TCR Vγ9-FITC antibody (Thermo, USA), CD56-PE antibody (BD Bio, USA), CD69-APC antibody (BD Bio, USA) and HLA-DR-PE-Cy7 antibody (BD Bio, USA) each 2μl, negative control tube plus isotype (Isotype) Antibody IgG1-FITC (eBioscience, USA), IgG1-PE (Biolegend, USA), IgG1-APC (Biolegend, USA) and IgG2a-PE-Cy7 (BD Pham, USA) 2 μl each, incubated at 4°C in the dark for 15 minutes Wash 2 times with PBS, remove excess antibody, add fixative (2% paraformaldehyde) to resuspend cells, and use flow cytometry (BD FACS Verse, USA) to detect the expression of cell surface markers CD56, CD69 and HLA-DR Level. After testing, the propor...

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Abstract

The invention discloses a method for cultivating Chinese rhesus monkey gammadelta T cells, which belongs to the technical field of cell biology. An OpTmizer cell culture medium used in the present invention is added with low-concentration monkey autologous serum, essential IL-2 for T cell culture, and an IL-18 substance which is reported to promote amplification of the gammadelta T cells, the high-purity gammadelta T cells can be cultured in vitro quickly and stably, and the cultured cells have typical morphological characteristics and immunological characteristics of the gammadelta T cells. In addition, the method uses the monkey autologous serum to culture cells, reduces the interference of exogenous serum on the experimental results, has fewer steps, and is suitable for the research work of Chinese rhesus gammadelta T cells.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a method for cultivating Chinese rhesus monkey γδT cells. Background technique [0002] γδT cells are T cells with both innate immunity and acquired immunity (D'Ombrain M.C., Hansen D.S., Simpson K.M., et al., gammadelta-T cells expressing NK receptorspredominate over NK cells and conventional T cells in the innate IFN- gammaresponse to Plasmodium falciparum malaria[J].Eur J Immunol,2007.37(7):p.1864-73), mainly distributed in the skin, small intestine, lung and other mucous membranes and subcutaneous tissues, and plays an important role in the body's anti-pathogenic microbial infection, anti-tumor and Both play an important role in the treatment of autoimmune diseases, with immune killing and immune regulation functions (BonnevilleM., O'Brien R.L., Born W.K., Gammadelta T cell effector functions: a blend ofinnate programming and acquired plasticity[J].Nat Rev I...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2509/00C12N2501/2302C12N2501/2318Y02A50/30
Inventor 张毅周立龚睿付慧敏
Owner HUBEI UNIV OF TECH
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