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Method for separation, purification, culture and passage of gill epithelial cells of hybridized prussian carp

A technology of epithelial cells and heterogeneous gibel carp, which is applied in the field of cultured aquatic biological tissue and cell culture engineering, can solve the problems of lack of separation, purification and culture technology in the primary culture technology of heterogeneous gibel carp gill epithelial cells, and achieve shortened culture the effect of time

Inactive Publication Date: 2013-05-29
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the prior art still lacks detailed isolation, purification and culture techniques for the primary culture of heterogeneous gibel carp gill epithelial cells

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] A method for isolating, purifying, cultivating and passing on gill epithelial cells of heterogeneous gibel crucian carp, comprising the following steps.

[0017] (1) Sampling of gill epithelial tissue: select 3 healthy heterogeneous gibel crucian carp (64 ± 5 ​​g), anesthetized (MS-222, Sigma; 1:2500) after fasting for 24 hours, and sterilized in 70% ethanol for 1 min. Take out the gills in a sterile room, rinse with phosphate buffer several times, remove the gill mucus, and cut the gills to a volume of 0.9-1.1 mm 3 organization block, spare.

[0018] (2) Preparation of culture medium: The culture medium was obtained according to the following feed ratio, and 0.1 μg of epithelial cell growth factor, 0.1 IU of insulin, 100 IU of penicillin, 100 μg of streptomycin and 0.1 ml of fetal bovine serum were added to each ml of DMEM medium.

[0019] (3) Tissue block culture: add 0.25% trypsin to the allogeneic gibel carp gill tissue block obtained in step (1), shake and digest ...

Embodiment 2

[0023] A method for isolating, purifying, cultivating and passing on gill epithelial cells of heterogeneous gibel crucian carp, comprising the following steps.

[0024] (1) Sampling of gill epithelial tissue: Select 3 healthy heterogeneous gibel crucian carp of 75 ± 4 g, anesthetized (MS-222, Sigma; 1:2500) after fasting for 24 hours, and sterilized in 70% ethanol for 1 min. Take out the gills in a sterile room, rinse with phosphate buffer several times, remove the gill mucus, and cut the gills to a volume of 0.9-1.1 mm 3 organization block, spare.

[0025] (2) Preparation of culture medium: Add 0.1 μg epithelial cell growth factor, 0.1 IU insulin, 100 IU penicillin, 100 μg streptomycin and 0.1 ml fetal bovine serum to each ml of DMEM (Dulbecco’s Modified Eagle’s Medium) medium.

[0026] (3) Tissue block culture: add 0.25% trypsin to the allogeneic gibel carp gill tissue block obtained in step (1), shake and digest in a water bath at 28°C for 20 minutes, pipette repeatedly un...

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Abstract

The invention discloses a method for separation, purification, culture and passage of gill epithelial cells of hybridized prussian carp. The method comprises the following steps of: sampling the gill epithelial cells, preparing a culture fluid, culturing tissue masses, purifying the gill epithelial cells, and subculturing the gill epithelial cells. According to the invention, the culture processes of separating, culturing and purifying the gill epithelial cells of the hybridized prussian carp, and components of the culture fluid are improved. According to a primary culture of the gill epithelial cells of the hybridized prussian carp, the normal connection time of culturing among cells can be shortened to 48 hours, thus the culture time is greatly shortened, and a established gill epithelial cell model of the hybridized prussian carp can be used for related researches of cytotoxicity of exogenous substances, ion exchange laws and the like, and provides convenience for the researches.

Description

Technical field [0001] The present invention involves the field of breeding aquatic biological tissue and cell culture engineering, especially the separation, purification, cultivation, and transmission of heterogeneous silver gill epithelial cells. Background technique [0002] Gills are important respiratory organs of animals in animals. They have a variety of physiological, biochemical and immune functions, which can ensure that the body has sufficient oxygen.The performance of these functions depends on the division, differentiation, and growth of gill epithelial cells. In fact, due to the interaction of many variable factors such as the type of aquatic animals, the body system and the local micro -environment,There are great difficulties in research. [0003] Cell culture is to separate the cells from the organism and simulate the physiological environment of the body in vitro, so that it survives and grows in the artificial environment, so that the cell structure, function,...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 王彦波傅玲琳
Owner ZHEJIANG GONGSHANG UNIVERSITY
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