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Culture method and application thereof for rhesus peripheral blood mononuclear macrophages

A technology of macrophages and peripheral blood, applied in the field of cell biology, to achieve the effect of less steps, less interference and less pollution

Inactive Publication Date: 2014-12-24
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention is made in view of the existing problems in culturing peripheral blood mononuclear macrophages of non-human primates in vitro. An object of the present invention is to provide a method for isolating and culturing peripheral blood mononuclear macrophages. Reproducible method, adding low concentration (2%-4%) autologous serum to the RPMI1640 medium used, without adding cell growth factors, can quickly and stably culture high-purity macrophages in vitro with typical characteristics Morphological and immunological characteristics of rhesus monocyte-macrophages

Method used

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  • Culture method and application thereof for rhesus peripheral blood mononuclear macrophages
  • Culture method and application thereof for rhesus peripheral blood mononuclear macrophages
  • Culture method and application thereof for rhesus peripheral blood mononuclear macrophages

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] [Example 1] Isolation and culture of rhesus monkey peripheral blood mononuclear macrophages

[0034] Intramuscularly anesthetize healthy adult (4-5 years old) Chinese rhesus monkeys with ketamine (10mg / kg), collect 5-10ml of venous blood with heparin sodium anticoagulant vacuum blood collection tubes and non-anticoagulant vacuum blood collection tubes respectively 5ml of whole blood was collected. Anticoagulated blood was used for the separation of PBMCs, and non-anticoagulated blood was used for the separation of monkey serum after self-coagulation. Dilute anticoagulated whole blood (5-10ml) with phosphate buffered saline (PBS), then slowly add to the upper layer of lymphocyte separation medium (Ficoll) that diluted 1 / 2 volume of the blood, and centrifuge at 1800rpm / min at 22°C for 30 minutes , Aspirate the layer of PBMCs between the plasma dilution and Ficoll. The collected cells were resuspended and washed twice with an equal volume of PBS, and the washed cells wer...

Embodiment 2

[0035] [Example 2] Identification of cultured rhesus monkey peripheral blood mononuclear macrophages by flow cytometry

[0036] Rhesus mononuclear macrophages on the 4th and 7th day of differentiation culture were first washed 3 times with PBS, digested with 0.25% trypsin for 5 minutes until most of the cells shrank and became round, and then added with 2% autologous monkey serum The complete medium terminates the digestion, the cells are suspended by pipetting, and the cells are collected. Continue to wash 2 times with PBS, then add 50 μl PBS to resuspend to make a single cell suspension, and add 2 μl of CD14-Percp-Cy5.5 (BD, USA) antibody, and add 2 μl of isotype (Isotype) antibody IgG2-Percp to the negative control tube -Cy5.5 (BD, USA), incubate at 4°C in the dark for 15 minutes, wash twice with PBS, remove excess antibody, add fixative (2% paraformaldehyde) to resuspend cells, and use flow cytometry (BD FACS Verse, USA) to detect the expression level of CD 14 on the cell...

Embodiment 3

[0037] [Example 3] Effects of different concentrations of monkey autologous serum or fetal bovine serum on the differentiation of rhesus monkey mononuclear macrophages

[0038] Place 96 wells (0.8×10 6 cells / well) or 48 wells (3×10 6 cells / well) monkey PBMCs in the culture plate, the non-adherent cells were washed away after 24 hours, and the pictures were taken after continuing to culture for 7 days. Such as image 3 As shown (the scales of all the figures in the figure are the same), the monkey autologous serum is better than the fetal calf serum, and the low-proportion autologous serum is better than the high-proportion serum for the differentiation of rhesus monkey mononuclear macrophages. As the serum ratio decreased, the adherent cells increased, and the morphology of differentiated macrophages was highly consistent. Under RPMI 1640 culture conditions containing 2% monkey autologous serum, most (>85%) monocytes could adhere to the wall and differentiate into macrophag...

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Abstract

The invention relates to a culture method for rhesus peripheral blood mononuclear macrophages. The method specifically comprises the following steps: separating peripheral blood mononuclear cells, resuspending and washing the collected peripheral blood mononuclear cells by a PBS (Phosphate Buffer Solution) twice, recovering the washed cells through centrifuging, centrifuging at a speed of 1200rpm for 8min each time, finally resuspending by an RPMI1640 culture solution containing 2-4% of rhesus autologous serum, 1% of mycillin and 0.1% of HEPES, controlling the density of the resuspended cells at 3*106 cells / ml, immediately adding into a 96-pore culture plate or a 48-pore culture plate of a CELLBIND Surface, culturing at a density of 0.8*106 cells per pore or 3*106 cells per pore, washing away suspended cells by the RPMI1640 culture solution after 24h, then continuously culturing adherent cells by a full culture medium containing 2-4% of rhesus autologous serum, changing the culture solution every two to three days, observing the cell morphology after seven days, and judging the cell differentiation result. The rhesus mononuclear macrophages obtained through differentiation culture are high in purity and have the typical macrophage morphology and immunology properties. The method is simple, economical and effective.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a method for culturing and identifying peripheral blood mononuclear macrophages of rhesus monkeys. Background technique [0002] Macrophages are distributed in various tissues of the body, and their functions are not limited to removing garbage in the body, but also perform various physiological functions such as monitoring invading pathogenic microorganisms and inducing natural immune responses in different tissues and organs of the body. There are a large number of macrophages in the immune frontier organs where the body is in close contact with pathogenic microorganisms, such as the lymphatic system, lungs, and lamina propria of the intestine. Macrophages "reside" in various tissues and organs, and exert their physiological balance and immune function by removing apoptotic and necrotic cells and foreign invading microorganisms (Gordon S, Taylor PR: Monocyte an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0786C12Q1/02
Inventor 桑明霍文哲刘金彪代明周立高建峰杨四军刘航
Owner WUHAN UNIV
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