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Method for separating and primary culture of zebrafish intestinal mucosal epithelial cells

A primary culture, intestinal mucosa technology, applied in cell dissociation methods, cell culture active agents, gastrointestinal cells, etc.

Active Publication Date: 2020-09-22
WUHAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on the isolation and primary culture of zebrafish intestinal mucosal epithelial cells

Method used

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  • Method for separating and primary culture of zebrafish intestinal mucosal epithelial cells

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Embodiment 1

[0052] Select healthy zebrafish and place them in water containing 80,000 U / L penicillin and 80,000 U / L gentamicin for fasting culture for 12 hours. Take the fasted zebrafish and wash them with ultrapure water Tap the zebrafish brain twice to kill it, and then quickly soak it in 70% ethanol for 20-30s. Open the zebrafish abdomen on an ultra-clean bench, take out the zebrafish foregut, and remove the surface attachments.

[0053] Cut the removed zebrafish foregut into pieces of 1-2 mm 3 Tissue blocks were washed 2-3 times with D-Hank's solution containing 80,000 U / L penicillin and 80,000 U / L gentamycin, and then added with 0.2 g / L collagenase Ⅰ and 0.2 g / L collagenase Ⅳ D-Hank's solution was shaken and digested in a water bath at 28°C for 15 minutes, then stop solution (5% FBS+95% DMEM medium) was added to stop the digestion, centrifuged at 400r / min for 5 minutes, the supernatant was discarded, and DMEM medium was added to make a cell suspension.

[0054] Centrifuge the above ...

Embodiment 2

[0056] Select healthy zebrafish and place them in water containing 80,000 U / L penicillin and 80,000 U / L gentamicin for fasting culture for 24 hours. Take the fasted zebrafish and wash them with ultrapure water. Tap the zebrafish brain twice to kill it, and then quickly soak it in 70% ethanol for 20-30s. Open the zebrafish abdomen on an ultra-clean bench, take out the zebrafish foregut, and remove the surface attachments.

[0057] Cut the removed zebrafish foregut into pieces of 1-2 mm 3 The tissue block was washed 2 to 3 times with D-Hank's solution containing 80,000 U / L penicillin and 80,000 U / L gentamicin, and then added with 0.25 g / L collagenase Ⅰ and 0.25 g / L collagenase Ⅳ D-Hank's solution was shaken and digested in a water bath at 27°C for 18 minutes, then stop solution (5% FBS+95% DMEM medium) was added to stop the digestion, centrifuged at 350r / min for 8 minutes, the supernatant was discarded, and DMEM medium was added to make a cell suspension.

[0058] The above-men...

Embodiment 3

[0060] Select healthy zebrafish and place them in water containing 80,000 U / L penicillin and 80,000 U / L gentamicin for fasting culture for 18 hours. Take the fasted zebrafish and wash them with ultrapure water Tap the zebrafish brain three times to kill it, and then quickly soak it in 70% ethanol for 20-30 seconds. Open the zebrafish abdomen on an ultra-clean bench, take out the zebrafish foregut, and remove surface attachments.

[0061] Cut the removed zebrafish foregut into pieces of 1-2 mm 3 Tissue blocks were washed 2-3 times with D-Hank's solution containing 80,000 U / L penicillin and 80,000 U / L gentamicin, and then added with 0.15 g / L collagenase Ⅰ and 0.15 g / L collagenase Ⅳ D-Hank's solution was shaken and digested in a water bath at 29°C for 20 minutes, then stop solution (5% FBS+95% DMEM medium) was added to stop the digestion, centrifuged at 500r / min for 4 minutes, the supernatant was discarded, and DMEM medium was added to make a cell suspension.

[0062]The above c...

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Abstract

The invention discloses a method for separating and primary culture of zebrafish intestinal mucosal epithelial cells. According to the method, firstly, the zebrafish is placed in a water body containing antibiotics for fasting culture to solve a problem of microbial self-pollution of zebrafish intestinal tract; then through selection of zebrafish intestinal parts, an intestinal sample that best reflects intestinal function of zebrafish is selected; and finally, through a separation technology of zebrafish intestinal mucosal epithelial cells and setting of culture conditions (primary cell culture medium, CO2 concentration, culture temperature) of cell primary culture, the zebra fish intestinal mucosa epithelial cells with high purity and good growth are obtained. The method provides a theoretical basis for subsequent establishment of the zebrafish intestinal mucosal epithelial cell line, and provides powerful research tools and methods for its extensive use as a fish cell line in genetics, neurobiology, developmental biology, immunology, human disease models, drug screening, toxicology and environmental testing.

Description

technical field [0001] The invention relates to the technical field of in vitro culture of fish intestinal mucosa cells, in particular to a method for isolating and primary culture of zebrafish intestinal mucosa epithelial cells. Background technique [0002] The intestine is an important digestive and absorption organ and the largest mucosal immune organ in humans and animals. Among them, the intestinal mucosal epithelial cells are the main functional cells of the intestinal tract, which play a major role in the digestion, absorption, and immune barrier of intestinal food. Therefore, intestinal epithelial cells are an important cell model for studying intestinal physiological functions, drug metabolism, and pathological changes. It is particularly necessary to use intestinal mucosal epithelial cell lines in various fields for experimental research. [0003] At present, the isolation and culture of intestinal mucosal epithelial cells has been successful in humans and mice,...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0679C12N5/0625C12N2509/00C12N2509/10C12N2501/115C12N2501/11C12N2500/44
Inventor 刘亚军刘文斌王旋方成
Owner WUHAN POLYTECHNIC UNIVERSITY
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