A method for 3D cultured in vitro spermatogonia of Chinese Wutang snakehead to produce functional sperm

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

Active Publication Date: 2022-07-05
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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  • A method for 3D cultured in vitro spermatogonia of Chinese Wutang snakehead to produce functional sperm
  • A method for 3D cultured in vitro spermatogonia of Chinese Wutang snakehead to produce functional sperm
  • A method for 3D cultured in vitro spermatogonia of Chinese Wutang snakehead to produce functional sperm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] (1) Isolation and identification of spermatogonia of Wutang sinensis:

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

[0058] Separation of the developmental testis of Snakehead sinensis; such as figure 1 shown, figure 1 A is the macroscopic structure of the gonad; figure 1 B and figure 1 C is the expression of vasa detected by immunofluorescence in the testis, where figure 1 C is figure 1 A magnified view of the box in B, the Vasa signal is in green (brighter color) and the nucleus is in red; figure 1 In C, SG is spermatogonia, PSP is primary spermatocyte, SSP is secondary spermatocyte, SPD is spermatocyte, and SPZ is sperm.

[0059] After sterilizing the developing testis in 70% ethanol for 30 seconds, after washing with phosphate buffered saline (PBS) for 3 times, cut the testis into pieces with medical scissors, add 1 ml testis digestive solution, and heat at 37°C. After digestion for 1 h, the digested cells were filtered through a 70-micron filter...

Embodiment 2

[0071] (1) Isolation and identification of spermatogonia of Wutang sinensis:

[0072] a. Preparation of testis single-cell suspension: isolate the testis of Snakehead sinensis in the developmental stage, sterilize it in 70% ethanol for 30 seconds, wash it three times with phosphate buffered saline (PBS), and cut the testis into pieces with medical scissors. Add 1 ml of 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 from Snakehead sinensis: preparation of percoll gradient solution 2: its components are 1.5 ml of 22% percoll and 1.5 ml of 35% percoll solution. The testis single cell suspension was added to the percoll gradient solution 2, and after horizontal centrifugation at 1500 rpm for 15 minutes, the testis cells were re-divided into upper, middle and lower layers, and spermatogo...

Embodiment 3

[0084] (1) Isolation and identification of spermatogonia of Wutang sinensis:

[0085] a. Preparation of testis single-cell suspension: isolate the testis of Snakehead sinensis in the developmental stage, sterilize it in 70% ethanol for 30 seconds, wash it three times with phosphate buffered saline (PBS), and cut the testis into pieces with medical scissors. Add 1 ml of 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. Isolation and identification of spermatogonia from Snakehead sinensis: preparation of percoll gradient solution 3: its components are 1.5 ml of 30% percoll and 1.5 ml of 45% percoll solution. The testis single cell suspension was added to the percoll gradient solution 3, and after horizontal centrifugation at 1500 rpm for 15 minutes, the testis cells were re-divided into upper, middle and lower layers, and spermatogon...

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Abstract

The invention discloses a method for producing functional sperm by 3D in vitro culturing of spermatogonia of C. sinensis. , and then filtered through a filter to remove undigested cell debris or cell clusters to obtain a testis single cell suspension; the testis single cell suspension was added to the percoll gradient solution for centrifugation to separate spermatogonia; the percoll gradient solution was changed from 22 to 30 % and 35~45% two concentrations of percoll solution; (2) spermatogonia are transferred to 3D culture dishes and cultured with sperm induction medium; sperm induction medium includes basal sperm medium and sex hormones, and further includes spermatogonia Melanin. The method optimizes and improves the separation, culture and induction conditions of spermatogonia, and improves the efficiency of in vitro cultured spermatogonia to produce functional sperm.

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 culturing of spermatogonia of Snake chinensis sinensis. Background technique [0002] Fish farming makes a vital contribution to the continued supply of world 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-controlled breeding, molecular marker-assisted selective breeding and genome-wide selective breeding. Advances in basic research and technology of fish genetics and breeding have promoted the formation and vigorous development of China's fish seed industry. With the expansion of aquaculture scale, fish aqua...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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