Dissociation and separation method for shellfish spermary spermatogenic cells

Abstract

The invention provides a dissociation and separation method for shellfish spermary spermatogenic cells, and belongs to the technical field of the separation of ocean shellfish cells. The dissociationand separation method comprises the following steps of: the spermary of a shellfish during a proliferating phase is broken and is digested by a collagenase IV solution of which the mass concentrationis 0.1% and a trypsin solution of which the mass concentration is 0.25% in sequence, and spermary dissociation is carried out to obtain a single-cell suspension; and Percoll solutions of which the volume concentration is independently 10%, 22% and 35% is prepared, and the Percoll solutions are loaded in the same centrifuge tube in a descending order, the single-cell suspension of the shellfish spermatogenic cells is added into the top layer of the centrifuge tube, and centrifugation is carried out to independently collect three layers of cells. By use of a two-step enzymolysis method, spermatogenic cells in spermary tissues can be successfully dissociated into the single-cell suspension, a cell survival rate can be as high as 96.62%, and the cells can be normally subjected to in vitro normal culture. Meanwhile, high-purity spermatogonia and spermatocytes can be separated, an important material is provided for researching a shellfish gamete generating and breeding mechanism, and the dissociation and separation method has a high guidance meaning and a good application value.

Description

technical field

[0001] The invention belongs to the technical field of separation of marine shellfish cells, and in particular relates to a method for dissociation and separation of shellfish testicular germ cells. Background technique

[0002] Sperm cells are collectively referred to as male germ cells, which can form spermatozoa through graded differentiation, transmit genetic information to offspring, and maintain species stability. According to the process of development and differentiation, germ cells can be divided into spermatogonia, primary spermatocytes, secondary spermatocytes, sperm cells and spermatozoa. The study of germ cells has always been a hot spot in developmental biology and reproductive biology. The in vitro isolation and purification of germ cells is conducive to exploring the mechanism of spermatogenesis and realizing the in vitro culture and transplantation of germ cells. However, due to the small size and difficulty of identifying germ cells, it is ...

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