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Separating and culturing methods and induction medium of chicken functional macrophage

A technology for inducing culture medium and macrophages, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of difficult cultivation, high cost, unfavorable large-scale cultivation, etc., and achieve strong phagocytic function, shape stabilization effect

Inactive Publication Date: 2016-02-24
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing chicken macrophages (such as the chicken macrophage cell line HD-11) are difficult to cultivate, cost high, are extremely prone to morphological changes, are not conducive to large-scale cultivation, and many experiments have proved that their phagocytic ability is very low

Method used

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  • Separating and culturing methods and induction medium of chicken functional macrophage
  • Separating and culturing methods and induction medium of chicken functional macrophage
  • Separating and culturing methods and induction medium of chicken functional macrophage

Examples

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Embodiment 1

[0027] This embodiment provides a method for isolating and culturing chicken functional macrophages derived from blood, comprising the following steps:

[0028] (1) Preparation of medium containing macrophage stimulating factors:

[0029] Chicken macrophage cell line HD-11 (purchased from Shanghai Cell Bank) (LinigerM1, SummerfieldA, ZimmerGetal.ChickencellssenseinfluenzaAvirusinfectionthroughMDA5andCARDIFsignalinginvolvingLGP2.JVirol.2012Jan; 86(2):705-17.doi:10.1128 / NVI.00742.901.E ), placed at 10cm 2 Petri dish, 37°C, CO 2 In an incubator with a volume concentration of 5%, culture with RPMI-1640 complete medium for 5-7 days, collect the cell supernatant when the cell supernatant turns yellow, and obtain the HD-11 cell supernatant, and the HD-11 cell supernatant After filtering through a 0.22um filter, add it to RPMI-1640 complete medium to obtain a medium containing macrophage-stimulating factors; wherein, the RPMI-1640 complete medium contains 10% volume ratio of fetal b...

Embodiment 2

[0035] This embodiment provides a method for isolating and culturing chicken functional macrophages derived from bone marrow, comprising the following steps:

[0036](1) Preparation of medium containing macrophage stimulating factor: same as Example 1

[0037] (2) Isolation of bone marrow-derived macrophage precursor cells:

[0038] Place the 12-22-day-old chicks killed by blood drawing in 75% absolute ethanol for 5 minutes, place them on the fixed plate of the ultra-clean workbench, fix the limbs, and separate the tibia and femur with sterile surgical instruments, Remove residual tissue and cartilage as much as possible to obtain bone marrow; wash the outside of the bone marrow with sterilized PBS buffer, and then use a disposable 10ml syringe to draw 10ml of PBS buffer each time to slowly rinse the bone marrow cavity until the bone marrow cavity becomes colorless, and collect the bone marrow cavity Rinsing fluid placed at 25cm 2 In a culture bottle; use a 1ml pipette gun t...

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Abstract

The invention discloses separating and culturing methods and an induction medium of chicken functional macrophage. The separating and culturing methods comprise the following steps: (1) preparing the medium containing macrophage stimulating factors; (2) directly obtaining chicken marrow resource macrophage precursor cells from shank or tibia of young chickens of 12-21 days, or separating chicken blood cells from chicken lymphocyte separating liquid, thus obtaining the chicken blood resource lymphocyte precursor cells; and (3) carrying out induced differentiation by adopting the medium containing the macrophage stimulating factors to obtain the chicken macrophage. The induction medium is RPMI-1640 complete medium containing the macrophage stimulating factors. According to the invention, the separating and culturing processes of the chicken functional macrophage are established for the first time, thus the mature macrophage with the phagocytic function is obtained, and a foundation is laid for the further research on the chicken macrophage on the aspects of immunity, metabolism and regulation.

Description

technical field [0001] The invention relates to a cell separation and culture method, in particular to a chicken functional macrophage separation and culture method and its induction medium. Background technique [0002] Macrophages are important immune cells in vertebrates. Functional macrophages are divided into classically activated M1 type and alternatively activated M2 type. In inflammation, Th1 stimulated and activated by the M1 type releases intermediates such as reactive nitrogen and reactive oxygen through respiratory bursts, killing pathogens, and plays an important role in bacterial, viral, and parasitic diseases; while the M2 type is stimulated by Th2 Stimulated activation of cytokines plays an important role in parasite immunity, enhanced tissue repair and immune regulation. The balance between the two types of macrophages also affects the body's immune response and neurological diseases such as bipolar disorder, muscular dystrophy and Rett's syndrome. In add...

Claims

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Application Information

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IPC IPC(8): C12N5/0786
Inventor 陈芳芳金星余为一
Owner ANHUI AGRICULTURAL UNIVERSITY
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