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Goose primary hepatocyte isolation culture method

A primary hepatocyte, isolation and culture technology, applied to animal cells, vertebrate cells, artificial cell constructs, etc. The effect of uniform flow and less impurities

Inactive Publication Date: 2013-06-05
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention provides a method for isolating and culturing goose primary hepatocytes, which solves the problem that there is no perfect method for isolating and culturing goose primary hepatocytes

Method used

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  • Goose primary hepatocyte isolation culture method
  • Goose primary hepatocyte isolation culture method

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Embodiment 1

[0038] The isolation and culture method of goose primary hepatocytes, its operating steps are:

[0039] (1) Inject pentobarbital sodium intraperitoneally into geese fasting for 12 hours. The injection volume is 30 mg / kg body weight and 100 IU / kg body weight of heparin sodium. After it is anesthetized, the supine position is fixed and the abdomen is sterilized;

[0040] (2) Cut the abdominal cavity along the midline of the abdomen of the goose, quickly take out the complete liver, and wash the surface of the liver with 40°C normal saline;

[0041] (3) The washed liver was perfused with preperfusate at 40°C until the liver turned pale yellow;

[0042] (4) Replace with 40°C cleaning solution to wash out the preperfusate in the liver;

[0043] (5) Repeated perfusion with 0.05% enzyme perfusate at 40°C until the subhepatic capsule showed a turtle-like fissure;

[0044] (6) Put the liver into a glass petri dish, tear off the capsule, and cut the liver into pieces;

[0045] (7) Po...

Embodiment 2

[0059] 1. Experimental method

[0060] Isolation and Culture of Goose Primary Hepatocytes

[0061] (1) Disinfection anesthesia: intraperitoneally inject pentobarbital sodium (30 mg / kg body weight) and heparin sodium (100 IU / kg body weight) into the geese fasting for 12 hours, wait for anesthesia (15-30 min), fix the supine position, and the abdomen is covered with skin disinfection;

[0062] (2) Liver extraction: cut the abdominal cavity along the midline of the abdomen of the goose, quickly remove the complete liver, and wash the surface of the liver with 40°C normal saline;

[0063] (3) Lavage: The washed liver is rapidly perfused with 40°C preperfusate at a rate of about 30ml / min until the liver turns light yellow; then replaced with 40°C wash solution to wash out the preperfusate in the liver;

[0064] (4) Digestion: transfer the liver to another sterile plate, replace with enzyme perfusate (preheated at 40°C) and repeatedly perfuse and digest for 10-30min at a perfusion...

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Abstract

The invention discloses a goose primary hepatocyte isolation culture method, and relates to the field of biotechnology. The method comprises the following operation steps: cleaning a goose liver; performing liver perfusion; repeatedly perfusing the liver by enzyme perfusion liquid till the liver subcapsular tissue has tortoise back-like cracks; cutting the liver; continuing to digest the cut hepatic tissue with collagenase liquid in a water bath; adding a DMEM medium containing fetal calf serum to stop digestion; performing filtration to obtain a hepatocyte suspension; centrifuging the hepatocyte suspension, adding PBS into the precipitate to prepare a hepatocyte suspension again; adding the DMEM medium containing fetal calf serum into the DMEM medium containing fetal calf serum, blowing the hepatocytes uniformly; counting the hepatocytes, diluting with the DMEM medium, inoculating into a cell culture dish, culturing to allow the hepatocytes to adhere to the wall; after hepatocyte culture, washing with PBS to obtain live primary hepatocytes; adding the DMEM medium containing fetal calf serum to culture. The invention solves the problem that no improved goose primary hepatocyte isolation culture method is available currently.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating and culturing goose primary hepatocytes. Background technique [0002] Hepatocytes are the only parenchymal cells of the liver and are the most numerous and densest cell group in the liver. The primary cultured hepatocytes are not affected by the complex neuroendocrine system in the body, and can maintain many specific functions of the liver, and maintain the responsiveness to some hormones, which can better reflect the metabolism in the body. Therefore, the primary cultured liver cells Cells are ideal models for studying animal substance metabolism and its regulatory mechanisms. Goose has the excellent performance of producing fatty liver, and the research on the mechanism of producing fatty liver of goose is a hotspot at present. The foie gras cells were isolated, cultured in primary culture, and the cell morphology and activity were observed, which laid a...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 韩春春王继文潘志雄刘贺贺李亮许峰许恒勇康波魏守海何桦
Owner SICHUAN AGRI UNIV
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