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Isolation and culture method of primary hepatocytes of bream

A technology for the isolation and cultivation of primary hepatocytes, which is applied in the field of separation and cultivation of primary hepatocytes of bream. Convenience and high cell viability

Active Publication Date: 2021-12-03
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Isolation and culture method of primary hepatocytes of bream
  • Isolation and culture method of primary hepatocytes of bream
  • Isolation and culture method of primary hepatocytes of bream

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Embodiment 1

[0030] Embodiment 1 Separation and culture method of primary hepatocytes of bream:

[0031] 1Main materials, sources and formulations:

[0032] 1) Fetal bovine serum was purchased from Sigma, USA.

[0033] 2) DMEM / F12, glutamine and double antibodies (penicillin and streptomycin) were purchased from gibco.

[0034] 3) DPBS was purchased from HyClone Company.

[0035] 4) Red blood cell lysate and type IV collagenase were purchased from biosharp.

[0036] 5) Trypan blue was purchased from Nanjing Kaiji Biological Company.

[0037] 6) Complete medium formula: DMEM / F12, 15% fetal bovine serum, 5% group head bream serum, double antibody (100IU / ml penicillin and 100μg / ml streptomycin), hepatocyte growth factor (20ng / ml), Bovine insulin (10 μg / ml) and glutamine (2 mM).

[0038] 7) Hepatocyte growth factor was purchased from R&D Systems.

[0039] 8) Fibronectin was purchased from Sciencell.

[0040] 9) Bovine insulin was purchased from Solarbio.

[0041] 2Operation steps:

[...

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Abstract

The invention discloses a method for isolating and culturing primary hepatocytes of bream. Healthy juvenile bream with a body weight of 30‑50 g were selected, and the whole body was disinfected with 1% potassium permanganate. After blood was collected from the tail vein, the liver was dissected aseptically. After digestion with collagenase, the cell suspension was filtered through a 200-mesh cell sieve, and excess red blood cells in liver cells were removed by red blood cell lysate, and cell debris was removed by gradient centrifugation. Add an appropriate amount of complete medium to the obtained cell pellet to suspend the cells, then use a cell counter to calculate and adjust the cell concentration, after plating, put it in 28°C, 5% CO 2 cultured in a cell incubator, and observed the adherence after 48 hours. The invention innovatively combines the characteristics of the researched species to isolate and cultivate hepatocytes, and the obtained cell viability is as high as 90%, which meets the requirements of primary culture, and provides theoretical basis and technical support for further experiments related to primary hepatocytes of bream .

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 Patents(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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