A method for isolating and culturing fish high-fat stored hepatocytes

Abstract

The invention discloses a method for separating and culturing fish high-fat storage hepatocytes. a. Select 45-55d juvenile gobies, obtain their liver tissue under aseptic conditions, disperse the liver tissue into pieces, and obtain tissue pieces; b. Place a cell sieve with a pore size of 20 μm in trypsin stop solution, and A cell sieve with a pore size of 50 μm is set above the cell sieve with a pore size of 20 μm; c. Place the tissue block in the cell sieve with a pore size of 50 μm, and continuously add trypsin solution to the cell sieve with a pore size of 50 μm to digest the tissue block, flow and continue to digest, and collect at The cells on the cell sieve with a pore size of 20 μm were washed with 10% FBS to obtain high-fat storage hepatocytes; d, the high-fat storage hepatocytes were inoculated into the medium for culture. The high-fat storage hepatocytes of the present invention can be stable for one week after adhering to the wall, and the adipocytes can survive for about one month, during which they can be used for experiments at the cell level, thereby solving the problem of difficulty in separating and culturing fish mature adipocytes.

Description

Technical field [0001] The present invention relates to the field of cell biology, more particularly to a fish high-fat storage hepatocyte isolation and culture method. Background [0002] In scientific research, in order to further verify the accuracy and authenticity of experimental results, it is generally necessary to carry out verification experiments at the cellular level, and the cell sources used at the cellular level are commercialized cells and primary cell culture. Primary cell culture is the first culture performed in vitro after cells are taken from donor biopsy, allowing them to grow and multiply under artificial conditions. At this time, the cells maintain the basic properties of the original cells, which is the basis of various experimental research and the first step in the establishment of various cell lines. Primary cell culture also includes differentiatable primary stem cell culture and primary mature cell culture; The key to culturing cells in vitro is how t...

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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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