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Separated culture method for primary hepatocyte of megalobrama amblycephala

A primary hepatocyte, separation and culture technology, which is applied in the field of separation and culture of primary hepatocytes of the bream bream, can solve the problems of inapplicability to the culture of bream bream primary hepatocytes, many cell impurities, and low cell viability.

Active Publication Date: 2019-01-08
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0004] At present, the culture of hepatocytes from some fishes has been studied, but the obtained cells have more impurities and lower cell viability. These methods are not suitable for the culture of primary hepatocytes of bream

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  • Separated culture method for primary hepatocyte of megalobrama amblycephala

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Embodiment 1

[0029] The present invention will be further described below in conjunction with embodiment. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the principles of the present invention, and these should also be considered within the protection scope of the present invention. Embodiment 1 Separation and culture method of primary hepatocytes of bream:

[0030] 1Main materials, sources and formulations:

[0031] 1) Fetal bovine serum was purchased from Sigma, USA.

[0032] 2) DMEM / F12, glutamine and double antibodies (penicillin and streptomycin) were purchased from gibco.

[0033] 3) DPBS was purchased from HyClone Company.

[0034] 4) Red blood cell lysate and type IV collagenase were purchased from biosharp.

[0035] 5) Trypan blue was purchased from Nanjing Kaiji Biological Company.

[0036] 6) Complete medium formula: DMEM / F12, 15% fetal bovine serum, 5% bream serum, double antibody (100IU / ml peni...

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Abstract

The invention discloses a separated culture method for primary hepatocyte of megalobrama amblycephala. The method comprises the following steps: selecting healthy megalobrama amblycephala, the weightof which is 30-50 g; fully disinfecting the megalobrama amblycephala with 1% potassium hypermanganate; sampling blood from a caudal vein; carrying out sterile dissection to take liver; digesting the liver with collagenase and filtering the liver with a 200-mesh cell screen to obtain a cell suspension; removing residual erythrocytes in liver cells by an erythrocuyte lysate; then carrying out gradient centrifugation to remove cell fragments; adding a proper amount of full culture medium suspension cells into the obtained cell precipitate; then calculating and adjusting the cell concentration with a cell counter; after paving a board, putting the board in a 5% CO2 cell incubator to be cultured at 28 DEG C; observing the adherence condition within 48 hours. The method provided by the inventionseparates and cultures liver cells by combining the characteristics of a researched species creatively, and the survival rate of the obtained cells reaches 90% or higher which meets the primary culture demand, thereby providing a theoretical foundation and a technical support for further carrying out related experiments of primary hepatocyte of megalobrama amblycephala.

Description

technical field [0001] The invention belongs to the field of cell biology and biotechnology, and relates to a method for separating and culturing local tissue cells of fish, in particular to a method for separating and culturing primary liver cells of bream. Background technique [0002] With the development of aquaculture industry, the breeding density continues to increase, unreasonable feed formula and environmental pollution have caused serious damage to the liver, and the liver is the most important physiological, biochemical and metabolic organ in fish, so it is realistic to cultivate fish liver cells significance. In addition, culturing primary hepatocytes in vitro has many advantages that cannot be replaced by in vivo experiments. Primary hepatocytes can be obtained in large quantities in a short period of time, which shortens the experimental time and is highly targeted. In vitro hepatocytes exclude the influence of interaction with other tissues in the body while ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2500/32C12N2501/18C12N2501/33C12N2509/00C12N2533/52
Inventor 刘文斌曹秀飞蒋广震戴永军袁向阳王聪聪黄洋洋
Owner NANJING AGRICULTURAL UNIVERSITY
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