Fish high-fat storage hepatocyte separation and culture method

Abstract

The invention discloses a fish high-fat storage hepatocyte separation and culture method. The method comprises the following steps: a, selecting 45-55d young gobius giurinus, obtaining liver tissues of the young gobius giurinus under a sterile condition, and dispersing the liver tissues into blocks to obtain tissue blocks; b, putting the cell sieve with the pore diameter of 20 microns into a trypsin stop solution, and arranging the cell sieve with the pore diameter of 50 microns above the cell sieve with the pore diameter of 20 microns; c, putting the tissue blocks into a cell sieve with the pore diameter of 50 microns, continuously adding trypsin liquid into the cell sieve with the pore diameter of 50 microns to digest the tissue blocks, continuously digesting by flowing, collecting cellson the cell sieve with the pore diameter of 20 microns, and cleaning with 10% FBS to obtain high-fat storage liver cells; and d, inoculating the high-fat storage hepatocytes into a culture medium forculturing. The high-fat storage hepatocytes can be stabilized for one week after adherence, adipose cells can survive for about one month, and the high-fat storage hepatocytes can be used for cell level experiments in the period so that the problem that mature fish adipose cells are difficult to separate and culture is solved.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a method for separating and culturing fish high-fat storage hepatocytes. Background technique [0002] In scientific research, in order to further verify the accuracy and authenticity of experimental results, verification experiments at the cell level are generally required, and the sources of cells used at the cell level are commercialized cells and primary cell culture. Primary cell culture is the first culture in vitro of cells obtained from donor living tissue, allowing them to grow and multiply continuously under artificial conditions. The cells at this time maintain the basic properties of the original cells, which is the basis of various experimental studies and the first step in establishing various cell lines. Primary cell culture includes differentiated primary stem cell culture and primary mature cell culture; the key to culturing cells in vitro is how to separate cells wi...

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Problems solved by technology

So far, the methods for cell culture mainly contain the following: tissue block adherent culture method (Wu Ning, Wu Chenglong. (2007)), mixed culture method (Bhatia, S.N., Balis, U.J., (1999). The FASEBjournal ), single-layer collagen culture method, co-culture system, etc., the above-mentioned commonly used primary mature cell culture can only maintain the shape and function of the cells within 1-2 weeks, and then gradually degenerate and die, so that the primary mature cell culture App is restricted

There are few reports on the isolation and culture methods of liver mature adipocytes. In general, primary stem cells are selected for culture, and then differentiated to induce the formation of hepatic adipocytes, and then experimentally verified. This conventional method can basically It satisfies the experimental requirements, but there are also problems such as difficulty in the separation and culture of primary cells and long modeling time, especially for the primary culture of fish hepatocytes. In view of this, the present invention is proposed

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