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Method used separating and purifying microglia cells

A technology for separation and purification of microglia, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of low cell yield, no separation and purification of microglia, and large damage. , to achieve the effect of high purity, maintaining biological characteristics and easy operation

Inactive Publication Date: 2014-04-09
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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Problems solved by technology

[0004] Based on the important role of microglia in the pathophysiology of the central nervous system, several recent studies have focused on the isolation of human microglia in order to explore their role in various central nervous system diseases; however, most of the current Separation methods all spend a considerable amount of time stimulating cultured cells with growth factors (such as M-CSF, GM-CSF); among them, Gibbons et al. believe that in order to obtain ideal purity and quality, it is necessary to analyze small cells from the perspective of molecular biology. Glial phenotypes (Int J Biochem Cell Biol, 2010, 42:844–856); methods for isolating microglia from fresh tissue were described by Dick and Hussain in 1997 and 2006, respectively, but did not address Isolation purity of microglia (AIDS, 1997, 11:1699–1708; Neuro Oncol, 2006a, 8:261–279); in 2010, Adam Wu used Percoll density gradient centrifugation to separate microglia from human glioma tissue Microglia (Neuro Oncol, 2010, 12(11): 1113-1125)
Recently, Dutch scientist Marta et al. provided a way to sort microglia from cadaveric brain tissue, and also adopted the method of percoll density gradient centrifugation combined with magnetic bead sorting (GLIA, 2012, 60: 96-111), but The mechanical shearing tissue used to prepare the cell suspension still has the disadvantages of low cell yield and large damage. Although Cardona et al. established a method for extracting microglia from mouse brain tissue, and achieved a nearly 100% yield Purity (Nat Protoc, 2006, 1:1947–1951), but so far, how to efficiently obtain high-purity microglia from fresh human glioma specimens still has great difficulties
[0005] So far, there is no method to effectively isolate and purify microglial cells from fresh glioma specimens by combining Percoll density gradient centrifugation and magnetic bead sorting, labeling CD11b with immunomagnetic beads, and culture and expand them in vitro

Method used

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  • Method used separating and purifying microglia cells
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  • Method used separating and purifying microglia cells

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1 Isolation and purification of microglial cells from glioma specimens

[0045] (1) Treatment of glioma specimens: Take about 2-3g of fresh glioma specimens (within 20 hours of surgical resection), put them in a 1640 culture medium culture dish filled with 5ml, cut them into pieces with scissors, and put them in 15ml centrifuge tube, centrifuge at 1500rpm for 5 minutes, discard the supernatant; add papain (1mg / ml) 4-5ml, digest at 37°C for 30 minutes, add serum to stop digestion, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and use 10ml of 1640 medium was resuspended, filtered through a 40 μm filter into a 15ml centrifuge tube to obtain a single cell suspension, centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded;

[0046] (2) Percoll density gradient centrifugation enrichment and separation of cells: Add 4ml of 70% Percoll solution, 8ml of 30% Percoll solution to the above 15ml centrifuge tube, and finally add 2ml of HBSS, 5...

Embodiment 2

[0048] Example 2 Optimizing Percoll Density Gradient Centrifugation to Enrich Microglia

[0049] Treatment of glioma specimens: take fresh glioma specimens (within 20 hours of surgical resection), put them in a petri dish containing 5ml of 1640 culture medium, cut them into pieces with scissors, add papain, and digest at 37°C for 30 minutes , add serum to stop the digestion, after mixing, centrifuge at 1500rpm for 5 minutes, discard the supernatant, resuspend with 20ml-30ml1640 culture medium, pass through a 40μm filter to obtain a single cell suspension, centrifuge, and discard the supernatant.

[0050] Microglial cells were enriched by Percoll density gradient centrifugation in the prior art: resuspended cells in 4ml of 70% Percoll solution, added to a 15ml centrifuge tube, then added 4ml of 40% and 30% Percoll solutions in sequence, and finally added 2ml of HBSS , centrifuged at 500g for 30 minutes; after centrifugation, the interface of HBSS / 30%Percoll was shown to be myel...

Embodiment 3

[0057] Primary culture and immunofluorescence identification of harvested microglial cells in Example 3

[0058](1) Primary culture: Inoculate the microglial cells obtained according to the method of the present invention on a culture dish, use RPMI1640 culture medium (including GM-CSF) containing 10% fetal bovine serum, place at 37°C, volume The fraction was cultivated in a 5% CO2 incubator, and then the medium was changed every 3 days;

[0059] (2) Cell immunofluorescence: after climbing CD11b+ microglial cells in a 12-well plate, wash 3 times with PBS, 5 minutes each time; fix with 4% paraformaldehyde for 30 minutes, wash 3 times with PBS, 5 minutes each time ; treated with 1% Triton for 30 minutes; washed 3 times with PBS, 5 minutes each time; blocked with goat serum for 20 minutes at 37°C; added Anti Iba1 primary antibody (0.5 μg / mL) (Wako Catalog No.019-19741), 4 overnight at ℃, washed 3 times with PBS, 5 minutes each time; add secondary antibody Alexa Fluor 594donkey...

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Abstract

The invention belongs to the field of tumour basic research, and relates to a method used separating and purifying microglia cells from glioma specimens. The method comprises following steps: a cell suspension is prepared from tumour tissues via mechanical method combined with enzymatic digestion method; the cell suspension is subjected to density gradient centrifugation so as to obtain the microglia cells of CD11B+ with a relatively high purity via enrichment and selection; and the microglia cells with a relatively high purity are obtained via further separation using CD11B+ magnetic beads. Wherein, Percoll separating medium is composed of 70% of Percoll, 30% of Percoll, and HBSS. The purity of the microglia cells obtained via percoll density gradient centrifugation enrichment can be more than 95%, and it is confirmed by immunofluorescence assay and phagocytosis assay that functions of the microglia cells are excellent. The method is simple and convenient for operation, is low in cost, is stable in quality, and is capable of providing basis for further research on immunologic functions microglia cells in brain glioma and effects of microglia cells in immune escape of tumour.

Description

technical field [0001] The invention belongs to the field of basic tumor research, and relates to a method for isolating and purifying microglial cells, in particular to a method for isolating and purifying microglial cells from isolated fresh glioma specimens; the method combines Percoll density gradient centrifugation and Magnetic bead sorting method, microglial cells were isolated and purified from fresh glioma specimens by immunomagnetic bead-labeled CD11b, and cultured and expanded in vitro, which provided a basis for the study of the relationship between microglial cells and tumor immune escape. Background technique [0002] The prior art discloses that glioma is currently the primary intracranial tumor with the highest incidence rate, and it has the characteristics of short survival period, high recurrence rate and mortality rate. Statistics show that brain tumors are the leading cause of death in children with solid tumors, gliomas account for about 1 / 5 of childhood ...

Claims

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Application Information

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IPC IPC(8): C12N5/078
Inventor 毛颖叶红星姚瑜岳琪莫链杰张新周良辅
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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