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Separating and culturing method for feeder-layer-free rat spermatogonial stem cells

A spermatogonial stem cell, feeder-free technology, applied in the direction of germ cells, animal cells, vertebrate cells, etc., can solve the problems of increased cost of cultured cells, high cost of separation, high requirements for medium nutritional conditions and ion stability, etc. Achieve the effect of saving the cost of cell culture and the method of separation and culture is simple

Active Publication Date: 2019-04-02
JILIN UNIV
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Problems solved by technology

However, there are many disadvantages in the existing methods: First, rats do not have the same compatible and stable STO trophoblast cell line as mice, and most of the existing experiments use mouse STO as the compatibility of rat spermatogonial stem cells trophoblast; second, the use of serum-free medium has higher requirements on the nutritional conditions and ion stability of the medium; third, the separation cost is high, because the number of isolated spermatogonial stem cells is small and the purity is insufficient, if you want to get more The spermatogonial stem cells need to be cultured for a long time, so the cost of culturing cells increases

Method used

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  • Separating and culturing method for feeder-layer-free rat spermatogonial stem cells
  • Separating and culturing method for feeder-layer-free rat spermatogonial stem cells
  • Separating and culturing method for feeder-layer-free rat spermatogonial stem cells

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Embodiment Construction

[0018] Introduce real-time example and experimental determination of the present invention in detail below:

[0019] A method for isolating and culturing rat spermatogonial stem cells without a feeder layer, comprising the following steps:

[0020] Step 1. Separation of primary rat spermatogonial stem cells: Separation of spermatogonial stem cells from the testes of male rat suckling rats 7-9 days after birth, and obtaining rat spermatogonial stem cells through differential adherence and percoll separation, specifically:

[0021] Take 7-9 day old WISTAR male rats, dissect the testicular tunica, separate and expose the seminiferous tubules, add 4ml of HBSS dissolved in 10μg / ml type IV collagenase, place at 37°C for 15 minutes, shake gently every 5 minutes Mix well to speed up digestion. Then use 3ml volume of 300μg / ml DNAse to wash 2-4 times, centrifuge at 600g for 5 minutes, then add 4ml of 0.25% trypsin containing 1mM EDTA to digest gently at 37°C for 5 minutes. 10% serum t...

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Abstract

The present invention discloses a separating and culturing method for feeder-layer-free rat spermatogonial stem cells. The spermatogonial stem cells are separated from testicles of suckling male ratsin 7-9 days after birth, differential adherence and percoll separation are conducted to obtain the rat spermatogonial stem cells with a higher purity, the obtained rat spermatogonial stem cells are cultured in a trophoblast-free culture system, can be stably passaged for a long time and can differentiate into spermatogenesis. The separating and culturing method is low in cost and simple in operation, the number of the obtained spermatogonial stem cells can be doubled, and the separating and culturing method is of great significance for treatment of infertility and protection of endangered species resources.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isolating and culturing rat spermatogonial stem cells without a feeder layer. Background technique [0002] Spermatogonia stem cells are the source of maintaining spermatogenesis in the testes of male mammals. They are a group of reproductive stem cells that can maintain their own number while maintaining their own differentiation properties. They can proliferate and differentiate to produce a large number of sperm. They belong to adult stem cells and can pass through in vitro. Induced, reprogrammed into pluripotent stem cells. In 2003, the Kanatsu-Shinohara laboratory successfully established the in vitro culture system of mouse spermatogonial stem cells. After more than 10 years of development, the isolation and culture technology of mouse spermatogonial stem cells has become more and more mature, but there is no stable culture system for rat and human spermatogonial s...

Claims

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Application Information

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IPC IPC(8): C12N5/076
Inventor 郭丽尹飞郭柯君
Owner JILIN UNIV
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