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A method for dissociation and separation of shellfish testis germ cells

A technology for spermatogenic cells and shellfish, which is applied in the field of separation of marine shellfish cells, can solve the problem of difficulty in separation of shellfish spermatogenic cells, and achieves the effects of great application value, guaranteed thoroughness and high content.

Active Publication Date: 2022-01-25
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of this, the purpose of the present invention is to provide a method for dissociation and separation of shellfish testicular germ cells, which can effectively solve the problem of difficult separation of shellfish spermatogenic cells and obtain higher purity

Method used

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  • A method for dissociation and separation of shellfish testis germ cells
  • A method for dissociation and separation of shellfish testis germ cells
  • A method for dissociation and separation of shellfish testis germ cells

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Take the testes of Chlamys farreri in the proliferating stage, and cut the testes into 1mm after cleaning and disinfection 3 The small pieces were then digested with 0.1% collagenase IV, and then digested with 0.25% trypsin. The time of the two enzymes for digestion is shown in Table 1. The testis was dissociated to obtain a single-cell suspension of Chlamys farreri germ cells.

[0048] The results are shown in Table 1. The results showed that at 25°C, when the concentrations of the two enzymes (0.1% collagenase IV and 0.25% trypsin) were fixed, the number of cells obtained by dissociation of 0.1 g of testis gradually increased with the prolongation of the treatment time of the two enzymes. increased, but the viability of cells gradually decreased after dissociation. Among them, experimental group 1 (digested with 0.1% collagenase IV for 60 min and digested with 0.25% trypsin for 5 min) had the highest survival rate of cells after dissociation, which was 98.23%, but th...

Embodiment 2

[0054] 1. Obtaining single cells from the testis of Chlamys farreri

[0055] ①Treatment of testicular tissue

[0056] Select the proliferating male clamshell scallop with good vitality and slightly white testes, wipe the surface of the double shell with 75% alcohol to disinfect, and then dissect out the testis tissue in an ultra-clean workbench, use calcium and magnesium ion-free phosphate buffer for cells (1 ×CPBS) rinsed 3 times, transferred to citrate-modified phosphate buffer solution (1×CPBS solution) containing double antibodies (350IU / ml penicillin and 350IU / ml streptomycin) and soaked for 1 hour for sterilization and disinfection, and then rinsed with 1×CPBS solution 3 times, use ophthalmic scissors to cut the testis tissue into a volume of 1mm 3 Little pieces left and right.

[0057] ② Preparation of single cell suspension by two-step enzymatic hydrolysis

[0058] Collect 0.1g of the shredded testis tissue into a 2ml EP tube, add 1ml of 0.1% collagenase IV, digest ...

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Abstract

The invention provides a method for dissociation and separation of shellfish testicular germ cells, and belongs to the technical field of separation of marine shellfish cells. The shellfish testis in the proliferating stage was broken, digested with 0.1% collagenase IV solution and 0.25% trypsin solution in sequence, and dissociated testis to obtain single cell suspension; the volume concentration of 10%, 22% and 35 % Percoll solution, put into the same centrifuge tube in descending order of concentration, add shellfish germ cell single-cell suspension to the top layer of the centrifuge tube, centrifuge, and collect three layers of cells respectively. The spermatogenic cells in the testis tissue can be successfully dissociated into a single cell suspension by two-step enzymatic hydrolysis, the cell survival rate can be as high as 96.62%, and normal culture can be carried out in vitro. At the same time, the spermatogonia and spermatocytes of higher purity are separated, which provides important materials for studying the mechanism of shellfish gametogenesis and reproduction, and has high guiding significance and 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07
CPCC12N5/0601C12N2509/00
Inventor 秦贞奎王庆张志峰刘丹雯
Owner OCEAN UNIV OF CHINA