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Method for extracting and cultivating primary cyprinidae hepatocytes

A technology of primary hepatocytes and culture methods, applied in the directions of cell dissociation methods, biochemical equipment and methods, culture processes, etc., can solve the problems of unsatisfactory purity and activity of carp primary hepatocytes, and achieve simple and feasible operation, The effect of short time and high number of cells

Active Publication Date: 2016-07-13
SHANGHAI UNIV
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Problems solved by technology

Therefore, the extraction of primary carp hepatocytes is of great significance for environmental toxicology experiments, but the purity and activity of primary carp hepatocytes extracted by current methods are not ideal

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  • Method for extracting and cultivating primary cyprinidae hepatocytes
  • Method for extracting and cultivating primary cyprinidae hepatocytes
  • Method for extracting and cultivating primary cyprinidae hepatocytes

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Embodiment Construction

[0023] Preferred embodiments of the present invention are described in detail as follows:

[0024] In this example, see Figure 1~4 , a method for extracting and culturing carp primary hepatocytes, selecting carp with a uniform size and a strong body with an average body weight of 400-600g to raise in advance for 2 weeks, and the feeding conditions of carp are: water temperature 25 ± 1°C, and the photoperiod of light: Darkness=14h:10h, and keep feeding twice a day, then, carry out the following process under sterile conditions:

[0025] a. Aseptically take carp liver and carp liver pretreatment: anesthetize the fish with fish anesthesia for 5 minutes, draw blood from the tail vein with a sterile syringe until the gills are slightly white, and use scissors to cut forward from the anus along the midline of the abdomen to the mandible , then cut along the rear edge of the operculum to the lower jaw, open the muscles of the left body wall, find the location of the carp liver, us...

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Abstract

The invention discloses a method for extracting and cultivating primary cyprinidae hepatocytes, and is applicable to the field of bioengineering.The method includes carrying out dissection on selected cyprinidae, fetching and shearing livers under sterile conditions, digesting the livers by the aid of trypsin, then filtering the livers by the aid of hepatocytes strainers to obtain hepatocytes suspension, purifying the hepatocytes suspension by the aid of Perco111 and adding cultivation liquid with cyprinidae serum into the hepatocytes suspension to obtain new hepatocytes suspension; counting small blood spheres by the aid of small blood sphere counting plates, cultivating and proliferating the primary cyprinidae hepatocytes by the aid of plate laying processes under the most suitable cultivation conditions and carrying out microscopy identification on the obtained primary cyprinidae hepatocytes by the aid of diversified processes.The method for purifying and cultivating the primary cyprinidae hepatocytes has the advantages that the livers of the cyprinidae are directly fetched under the sterile conditions, hepatocytes are purified by the aid of the Perco111, and the large quantities of high-purity in-vitro primary cyprinidae hepatocytes can be truly obtained via identification; matched bovine serum in the traditional cultivation media is replaced by the cyprinidae serum, accordingly, the viability of the in-vitro primary cyprinidae hepatocytes can be improved, and foundation can be laid for further carrying out toxicology tests and other experiments on the primary cyprinidae hepatocytes.

Description

technical field [0001] The invention relates to a method for extracting and cultivating biological materials, in particular to a method for extracting and cultivating local tissue cells of fish, which is applied in the technical field of bioengineering. Background technique [0002] As the largest detoxification and metabolic organ in fish, the liver plays an important role in the digestion and absorption of nutrients and in ensuring the health of fish. Due to its complexity and importance, the research on fish liver has always been the key content of fish biology, and the results involve fish anatomy, histology, immunology, physiology, pathology, toxicology, nutrition and many other fields . Due to the large amount of animal samples required for in vivo liver toxicity experiments, high cost, and poor sensitivity and repeatability of experimental results, in vitro cell experiments are the direction to solve this problem. The biggest advantage of cell culture is that it can...

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/067C12N2500/84C12N2509/00
Inventor 杨明李意捷雷鹏辉蒋丽辉吴明红
Owner SHANGHAI UNIV
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