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Primary culture method for liver cells of crucian carp

A technology of primary culture and culture method, applied in the field of cell biology and biotechnology, can solve the problems of low activity and low purity of liver cells, achieve high environmental requirements, eliminate blood cells, and reduce pollution.

Inactive Publication Date: 2012-01-04
SHANGHAI OCEAN UNIV +1
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0005] Although there are some methods for isolating and identifying hepatocytes, the purity of the isolated hepatocytes is relatively low and their activity is relatively low.

Method used

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  • Primary culture method for liver cells of crucian carp
  • Primary culture method for liver cells of crucian carp
  • Primary culture method for liver cells of crucian carp

Examples

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Embodiment 1

[0062] Example 1 Primary culture method of crucian carp hepatocytes

[0063] 1 Formula and source of main materials

[0064] 1) Serum-free medium: Add 100U / ml penicillin, 100U / ml streptomycin and 20ug / ml hepatocyte growth-promoting factor to DMEM / F12 medium.

[0065] 2) The medium formula is: add newborn bovine serum, 100U / ml penicillin, 100U / ml streptomycin and 20ug / ml hepatocyte growth factor to DMEM / F12 medium (Shanghai Xufei Biological Co., Ltd.).

[0066] 3) Digestive enzymes: 0.25% trypsin (Shanghai Xufei Biological Co., Ltd.), 0.02% EDTA and 0.1% type II collagenase (Shanghai Xufei Biological Co., Ltd.), 3mmol / LCaCl 2 .

[0067] 4) Red blood cell lysate was purchased from Shanghai Xufei Biological Co., Ltd.

[0068] 5) Percoll separation solution: Use Percoll stock solution (Shanghai Xufei Biological Co., Ltd.) and 10×D-hank’s at a ratio of 9:1 to prepare separation solution Ⅰ; :3 volume ratio to prepare the separation liquid II.

[0069] 6) D-hank's solution: mix ...

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Abstract

The invention provides a new primary culture method for liver cells of crucian carp, which mainly comprises the following steps: a, separating liver tissues from crucian carp; b, digesting cut liver tissues by using a stepwise digestion process and obtaining inner cell mass; and c, purifying and culturing obtained inner cell mass. In the method disclosed by the invention, the raw material is obtained from common crucian carp aquatic experimental animals, in-vitro tissue blocks are soaked in chlorhexidine gluconate (chlorhexidine) to be disinfected for reducing pollution. The tissues are digested by a stepwise digestion process, blood cell lysing solution is used to reduce the blood cell content in cells, and the liver cells are purified by percoll gradient centrifugation. The liver cells are purified by combined material preparation, the stepwise digestion process, blood cell lysing solution and percoll. The method can culture adequate liver cells, the survival rate of the liver cellsis over 90 percent, and the method meets requirements of primary culture.

Description

technical field [0001] The invention relates to a new primary culture method of crucian carp liver cells, which belongs to the field of cell biology and biotechnology. Background technique [0002] The liver is the main detoxification organ of the body, the target organ damaged by many poisons, and an important organ for the metabolism of chemicals in the body. The complex and changeable environment in the body brings certain difficulties to the study of liver damage caused by chemicals in vivo. The time of primary cultured cells in vitro is short, and the biological characteristics have not changed much. To a certain extent, they can reflect the state in vivo. Using primary cultured cells Cultivate the stability of physiological, biochemical and genetic characteristics of cells, and study the toxicity of chemicals and the response and detoxification mechanism of liver cells to poisons. [0003] Fish are vertebrates that live in water bodies, and are good biological indicat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 魏华郭敏吴婷婷孙杨
Owner SHANGHAI OCEAN UNIV
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