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A method for isolation, purification and primary culture of grass carp intestinal macrophages

A technology of macrophages and grass carp, applied in the field of fish cell culture, can solve the problems of cell death, decreased cell viability, unclear conditions of grass carp intestinal macrophages, etc., and achieve high purity and activity, strong operability, and growth good effect

Active Publication Date: 2019-06-25
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, our previous research found that this method is not suitable for grass carp, a temperature-changing animal living in a water environment. DTT-EDTA can damage grass carp macrophages, and the cell viability is greatly reduced. After 12 hours of culture, the cells die
Secondly, little is known about the surface marker molecules of grass carp intestinal macrophages, and there is no specific monoclonal antibody for marker molecules, which brings difficulties to the purification of grass carp intestinal macrophages
Finally, the conditions suitable for the primary culture of grass carp intestinal macrophages are also unclear

Method used

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  • A method for isolation, purification and primary culture of grass carp intestinal macrophages
  • A method for isolation, purification and primary culture of grass carp intestinal macrophages
  • A method for isolation, purification and primary culture of grass carp intestinal macrophages

Examples

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Effect test

Embodiment 1

[0052] Example 1 Isolation of grass carp intestinal macrophages

[0053] 1.1 Removal of intestinal mucus and intestinal epithelial cell layer of grass carp

[0054] Select about 250g of healthy grass carp, rinse the body surface with tap water, beat the head to death, and quickly soak in 75% ethanol for 20s. Aseptically remove the middle and posterior intestinal tract on the ultra-clean workbench, put it in a petri dish containing pre-cooled sterile PBS buffer solution (0.01mol / L, pH 7.4), and remove extra-intestinal fat and mesentery with sterile elbow tweezers , dissect the intestine longitudinally and wash the feces. Afterwards, scrape the intestinal mucus and epithelial cell layer on the inner side of the intestinal wall repeatedly with sterile elbow tweezers until no mucus is scraped off (it takes 15 minutes), and then wash it with PBS buffer (0.01mol / L, pH 7.4) for 3 times with slight shaking. At this time, the intestinal mucus and epithelial cells can be seen to fall ...

example 2

[0059] Purification of example 2 grass carp intestinal macrophages

[0060] The isolated intestinal macrophage suspension was further purified by differential attachment method. That is, resuspend the isolated intestinal macrophages with RPMI1640 basal medium, and adjust the cell density to 3×10 6 cells / mL and spread in 96-well cell culture plate, 100 μL / well, 28°C, 5% CO 2 Cultivate in the incubator, change the medium for the first time after culturing 4h, discard the old nutrient solution when changing the medium, add fresh RPMI 1640 complete nutrient solution containing grass carp autologous serum (84% RPMI 1640 nutrient solution (Gibco product), 10% FBS, 5% grass carp autologous serum, 1% antimicrobial solution) continued to culture for 12 hours, changed the medium again, discarded the old culture medium, and added RPMI 1640 complete culture medium containing grass carp autologous serum to continue the culture. As a result, it was found that the macrophages began to adhe...

example 3

[0061] Primary culture of example 3 grass carp intestinal macrophages

[0062] The concentration of purified macrophages was adjusted to 3×10 with RPMI 1640 complete culture solution containing grass carp autologous serum (84% RPMI 1640 culture solution, 10% FBS, 5% grass carp serum, 1% antimicrobial solution). 6 cells / mL, inoculate 96-well cell culture plate, 100 μL / well, 28°C, 5% CO 2 Culture in an incubator. After the cells start to adhere to the wall, replace the medium with RPMI 1640 complete culture medium containing 20 μg / mL LPS, and then change the medium every 2 days. When changing the medium, suck out 500 μL of the old culture medium, and then add 500 μL of RPMI 1640 complete culture solution containing grass carp autologous serum was continued in the incubator. The results showed that after 1 day of culture, most macrophages began to stretch and grow, and a few cell clumps appeared ( figure 2 A); 2-3 days after culture, the cells continued to stretch and grow in ...

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Abstract

The invention relates to separation, purification and primary culture methods of intestinal macrophages in Ctenopharyngodon idella. The separation method comprises: taking out the rear middle segment of an intestine by aseptic operation, removing intestinal outer fat and mesentery, longitudinally cutting the intestine to clear excrement, and using aseptic bent-tip tweezers to scrape off the intestinal mucus and epithelial cell layer for 15 min; cutting the segment with the mucus and epithelial cell layer removed to obtain fragments, and digesting the fragments in collagenase IV digestive juice to obtain lamina propria single-cell suspension; using a separation kit of fish organ monocytes to separate the intestinal macrophages. The purification method comprises: using a differential wall attachment method to purify the intestinal macrophages. The primary culture method comprises: using RPMI (Roswell Park Memorial Institute medium) 1640 complete culture solution containing autoserum of Ctenopharyngodon idella to culture the intestinal macrophages, changing the solution once with lipopolysaccharide-containing RPMI 1640 complete culture solution after cells attach to the wall, and changing the solution once every other two days so that cells may survive for at least 20 days. The separation, purification and primary culture methods established herein have good operability and repeatability.

Description

technical field [0001] The invention belongs to the technical field of fish cell culture, and in particular relates to a method for the separation, purification and primary culture of grass carp intestinal macrophages. Background technique [0002] The intestine is not only the digestive and absorption organ of teleosts, but also its mucosal immune organ. Studies have shown that fish intestines do not have mammalian organized lymphoid organs (such as Peyer's Peyer's lymph nodes and follicular lymph nodes), but a large number of macrophages (Macrophages), lymphocytes and granulocytes are distributed in the middle and rear mucosa of the intestine. Intestinal macrophages, which are rich in mucosal lamina propria, not only have the functions of phagocytosis, killing and clearing intestinal pathogenic microorganisms, but also play the role of antigen-presenting cells, and play the role of initiating and regulating intestinal mucosal immune response. effect. [0003] Grass carp ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0786
CPCC12N5/0645C12N2500/34C12N2509/10
Inventor 李槿年肖宁陶会竹赵雨婷李琳刘雪兰
Owner ANHUI AGRICULTURAL UNIVERSITY
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