Erythroculter ilishaeformis spermatogonia stem cell separation and culture method

Abstract

The invention relates to an erythroculter ilishaeformis spermatogonia stem cell separation and culture method. The method comprises the following steps: (1) collecting spermatic tissues: collecting spermary of erythroculter ilishaeformis with an age of seven months in a sterile environment, then rinsing spermary in PBS containing double-antibody, and finally grinding the spermary to disperse the spermary tissues; (2) digesting and separating spermatogonia stem cell: adding IV type collagenase (0.1%) into the dispersed spermary tissues, subjecting the digestive fluid to centrifugal separation, collecting the precipitate, digesting the precipitate by trypsin (0.25%), stopping the digestion by a culture medium containing FBS (10%), and collecting the cells; (3) carrying out primary culture of spermatogonia stem cell: using a DMEM / F12 complete medium containing cell factors to re-suspend the cells obtained in the step (2), adjusting the cell concentration, then paving the suspension liquid on a 24-hole cell culture plate coated by gelatin, and culturing the cells at a temperature of 26 DEG C. Trough the provided method, the in-vitro growth of primary cells of erythroculter ilishaeformis spermatogonia stem cell becomes easier, and an effective cell platform is provided for the research on reproduction and growth of erythroculter ilishaeformis.

Description

technical field [0001] The invention belongs to the fish cell culture technology in the aquatic biology technology, and is a method for separating and culturing spermatogonial stem cells under artificial culture conditions in vitro and using the testis tissue of the red catfish as a material. Background technique [0002] As a cultured fish species, red bream is an important cultured species in the middle and lower reaches of the Yangtze River. Its artificial breeding and breeding technology are very mature, but there are few studies on its reproductive development. At present, the output of red chrysalis in natural waters is very low, and the wild resources are decreasing day by day. Therefore, researches on the biological characteristics, artificial reproduction, and breeding techniques of red chrysalis are carried out to protect the natural red chrysalis for the development and utilization of species. The wild resources of carp are of great significance. Understanding th...

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Problems solved by technology

In addition, some researchers have successfully isolated SSCs from eel (Miura, Development, 2002, 129 (11)) and zebrafish (Leal, Belo Horizonte, MG, Brazil. 2006, 3: 181.) testis by tissue block culture method. However, the number of SSCs isolated by this method is less and there are more miscellaneous cells, which is not conducive to the later purification

However, no invention patent application for the method of isolating and culturing spermatogonial stem cells from the red-headed catfish was found

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