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Method for producing functional sperms by 3D in-vitro culture of bostrichthys sinensis spermatogonium

A technology of Channa sinensis and spermatogonia, applied in 3D culture, biochemical equipment and methods, tissue culture, etc., can solve the problem that spermatogonia cannot produce sperm, achieve the goal of promoting proliferation and differentiation, and improving production efficiency Effect

Active Publication Date: 2022-05-13
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the inventors successfully induced mature testicular cells to produce sperm in this method, there are still obvious shortcomings in this method: after subsequent experiments, it was found that when the method was applied to spermatogonia in the developing testis, the spermatogonia could not produce sperm

Method used

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  • Method for producing functional sperms by 3D in-vitro culture of bostrichthys sinensis spermatogonium
  • Method for producing functional sperms by 3D in-vitro culture of bostrichthys sinensis spermatogonium
  • Method for producing functional sperms by 3D in-vitro culture of bostrichthys sinensis spermatogonium

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Experimental program
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Effect test

Embodiment 1

[0056] (1) Isolation and identification of spermatogonial cells of Channa sinensis:

[0057] a. Preparation of testis single cell suspension:

[0058] Separation of the testes of Channa sinensis in the developing stage; figure 1 as shown, figure 1 A is the macroscopic structure of the gonad; figure 1 B and figure 1 C is the expression of vasa in the testis detected by immunofluorescence, wherein figure 1 C is figure 1 Enlarged view of the box in B, the Vasa signal is green (brighter color), and the cell nucleus is red; figure 1 In C, SG refers to spermatogonia, PSP refers to primary spermatocytes, SSP refers to secondary spermatocytes, SPD refers to sperm cells, and SPZ refers to spermatozoa.

[0059] Sterilize the testis of Channa sinensis in the developing stage in 70% ethanol for 30 seconds, wash it three times with phosphate buffered solution (PBS), cut the testis into pieces with medical scissors, add 1 ml of testis digestion solution, and incubate at 37°C Digested...

Embodiment 2

[0071] (1) Isolation and identification of spermatogonial cells of Channa sinensis:

[0072] a. Preparation of single-cell suspension of testis: separate the testis of Channa sinensis in the developing stage, disinfect it in 70% ethanol for 30 seconds, wash it with phosphate buffered solution (PBS) for 3 times, cut the testis with medical scissors, Add 1 ml testis digestion solution, digest at 37°C for 1 h, and filter the digested cells through a 70-micron filter to remove undigested cell clusters to prepare testis single-cell suspension.

[0073] b. Separation and identification of spermatogonia of Channa chinensis: preparation of percoll gradient solution 2: its composition is 1.5 ml of 22% percoll and 1.5 ml of 35% percoll solution. Add testis single cell suspension into percoll gradient solution 2 and centrifuge horizontally at 1500 rpm for 15 minutes. Testis cells are divided into upper, middle and lower layers again, and spermatogonia are obtained from the middle layer; ...

Embodiment 3

[0084] (1) Isolation and identification of spermatogonial cells of Channa sinensis:

[0085] a. Preparation of single-cell suspension of testis: separate the testis of Channa sinensis in the developing stage, disinfect it in 70% ethanol for 30 seconds, wash it with phosphate buffered solution (PBS) for 3 times, cut the testis with medical scissors, Add 1 ml testis digestion solution, digest at 37°C for 1 h, and filter the digested cells through a 70-micron filter to remove undigested cell clusters to prepare testis single-cell suspension.

[0086] b. Separation and identification of spermatogonial cells of Channa chinensis: preparation of percoll gradient solution 3: its composition is 1.5 ml of 30% percoll and 1.5 ml of 45% percoll solution. Add testis single cell suspension into percoll gradient solution 3 and centrifuge horizontally at 1500 rpm for 15 minutes. Testis cells are re-divided into upper, middle and lower layers, and spermatogonia are obtained from the middle lay...

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Abstract

The invention discloses a method for producing functional sperms by 3D in-vitro culture of bostrichthys sinensis spermatogonium, which comprises the following steps: (1) disinfecting, cleaning and cutting separated bostrichthys sinensis testis, adding a testis digestive juice for digestion, and filtering by a filter screen to remove undigested cell debris or cell clusters to prepare a testis single-cell suspension; adding the testis single-cell suspension into a percoll gradient solution for centrifugation, and separating to obtain spermatogonium; the percoll gradient solution is composed of a percoll solution with two concentrations of 22%-30% and 35%-45%; (2) transferring the spermatogonium into a 3D culture dish, and culturing the spermatogonium with a sperm induction culture medium; the sperm induction culture medium comprises a basic sperm culture medium and sex hormone, and further comprises melatonin. According to the method, the separation, culture and induction conditions of the spermatogonium are optimized and improved, and the efficiency of generating functional sperms by culturing the spermatogonium in vitro is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for producing functional sperm by 3D in vitro culture of spermatogonial cells of Channa sinensis. Background technique [0002] Fish farming makes a vital contribution to the world's sustainable supply of food, especially animal protein. In the past decade, the innovation of biotechnology has made important progress in the analysis and genetic improvement of some important economic traits of fish. Fish genetics and breeding have developed from traditional selective breeding and hybrid breeding to precise design breeding such as cell engineering breeding, sex control breeding, molecular marker-assisted selection breeding and genome-wide selection breeding. Advances in basic research and technology of fish genetics and breeding promote the formation and vigorous development of China's fish seed industry. With the expansion of aquaculture scale, fish farming is fa...

Claims

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Application Information

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IPC IPC(8): C12N5/076
CPCC12N5/061C12N2513/00C12N2501/30Y02A40/81
Inventor 刘威易梅生张洪贾坤同
Owner SUN YAT SEN UNIV
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