Megalobrama amblycephala bone tissue culture method

A technique for tissue cells and bream, applied in the field of cells, can solve problems such as limited use of trypsin digestion, and achieve the effects of low cost, solving technical difficulties, and simple and easy methods.

Inactive Publication Date: 2017-07-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The object of the present invention is to provide a method for culturing the bone tissue cells of bream, which is easy to implement and easy to operate, and solves the problems of limited sample size and limited use of trypsin digestion.

Method used

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  • Megalobrama amblycephala bone tissue culture method
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  • Megalobrama amblycephala bone tissue culture method

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Embodiment 1

[0034] A method for culturing bream bone tissue cells, the steps are:

[0035] 1) Choose 3-month-old bream in good growth condition as the material (fresh or dead), and soak it in 0.01% potassium permanganate solution for 20 minutes before the experiment. Put it into the aseptic operating table, and disinfect the fish body with 75% (volume ratio) alcohol;

[0036] 2) Use a sterilized scalpel to cut off the caudal fin under aseptic conditions, cut off the entire tail from the direction behind the cloaca, carefully peel off the skin with tweezers, soak the tissue block in PBS, and use ophthalmic forceps and tweezers under a dissecting microscope. Peel off the muscles around the tailbone and ribs with a scalpel, and take the connective tissue close to the tailbone and ribs of bream (fresh or dead);

[0037] 3) After rinsing the tissue block with PBS 3 times in the petri dish, cut it to 2mm with ophthalmic scissors 3 ; Wet the tissue pieces one by one in fetal bovine serum, care...

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Abstract

The invention discloses a megalobrama amblycephala bone tissue culture method. The method includes steps: 1), selecting megalobrama amblycephala of 3 months old as an experimental material; 2), taking caudal vertebra, costa and its connective tissue around of megalobrama amblycephala; 3), flushing with PBS, wetting a tissue block in fetal calf serum, and using forceps to put the tissue block at the bottom of a culture bottle; 4), putting the culture bottle into a culture box for inverted culture; 5), daily observing cell growth and replacing a culture medium; 6), performing trypsin digestion passage after cell convergence degree reaches 80% to obtain megalobrama amblycephala primary bone tissue cells; 7), using trypsin for digestion passage of the cells when convergence degree of the megalobrama amblycephala primary bone tissue cells reaches 70-80%, centrifuging before re-suspending the cells, and inoculating the cells into two new culture bottles to continue culture to obtain passage cells. The method is easy to implement and simple and convenient to operate, the problems of limitation on sample individual size and limitation on using of a trypsin digestion method are solved, and a base material is provided for related study on fish skeleton growth and development and the like in the future.

Description

technical field [0001] The invention belongs to the field of cell technology, and more specifically relates to a method for culturing bone tissue cells of bream, which is suitable for future research on the differentiation process of bone tissue cells, bone development, bone tissue mineralization process, gene screening and functional analysis, etc. . [0002] technical background [0003] Compared with mammals, fish has many technical advantages, so more and more researchers choose to use fish as research models (see: Rafael M S, Marques C L, Parameswaran V, et al. Fishbone-derived cell lines:an alternative in vitro cell system to study bonebiology[J].Journal of Applied Ichthyology,2010,26(26):230-234), so far some fish have been successfully used as research models for bone development and related diseases Research. There have been reports abroad that the tissue block culture method has been used to culture fish bone tissue cells. For example, Vijayakumar et al. have succ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0654C12N2501/11C12N2501/115
Inventor 高泽霞关柠楠兰天陈宇龙
Owner HUAZHONG AGRI UNIV
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