Method for efficiently separating mouse spermatogonial stem cells

Abstract

The invention discloses a method for efficiently separating mouse spermatogonial stem cells. The method includes the following steps that firstly, mouse testis are collected, and a testicular cell suspension is prepared through a two-step enzyme digestion method; secondly, the testicular cell suspension is inoculated in a porous plate according to a certain density, and a spermatogonial stem cell culture solution is added for in-vitro culture; thirdly, the culture solution is removed, testis cells of different times are cultured through tryptic digestion, and spermatogonial stem cells are sorted from the testis cells through the magnetic activated cell sorting; fourthly, the sorted spermatogonial stem cells are inoculated to an STO trophoblast, and a spermatogonial stem cell culture solution is added for primary culture and subculture of the spermatogonial stem cells. A mouse spermatogonial stem cell system is built through the method including the steps of separation, culture, re-separation and re-culture. The method can achieve the aim of efficiently enriching the spermatogonial stem cells in vitro, and is economical and easy to implement, so that a direction is provided for protection of rare animal varieties and treatment of male infertility.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for efficiently isolating mouse spermatogonial stem cells. Background technique [0002] Spermatogonia stem cells are the source of spermatogenesis in the testis of male mammals. Since 1998, the Brinster laboratory successfully cultured the spermatogonia stem cells in mice for the first time, and proved that the spermatogonia stem cells were separated from the seminiferous tubules. Since it can be cultured in vitro for a long time, many laboratories have also begun to study mammalian spermatogonial stem cells, but the number of spermatogonial stem cells in the mouse testis is small, and the purity of the isolated spermatogonial stem cells is also low, even though it is currently considered that the separation purity is relatively high. The purity of spermatogonial stem cells directly isolated from high THY-1 antibodies is only about 60%, which will affect their culture and e...

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Problems solved by technology

[0002] Spermatogonia stem cells are the source of spermatogenesis in the testis of male mammals. Since 1998, the Brinster laboratory successfully cultured the spermatogonia stem cells in mice for the first time, and proved that the spermatogonia stem cells were separated from the seminiferous tubules. Since it can be cultured in vitro for a long time, many laboratories have also begun to study mammalian spermatogonial stem cells, but the number of spermatogonial stem cells in the mouse testis is small, and the purity of the isolated spermatogonial stem cells is also low, even though it is currently considered that the separation purity is relatively high. The purity of spermatogonial stem cells directly isolated from high THY-1 antibodies is only about 60%, which will affect their culture and expansion. By optimizing the isolation and culture conditions, we can obtain high purity and doubling time from mice of different ages. shorter spermatogonial stem cells

[0003] The method of isolating spermatogonial stem cells in the prior art is that most of the commonly used methods for isolating spermatogonial stem cells in mice are based on male mice 5-7 days after birth, directly using the immunomagnetic bead method or by means of flow cytometry The cytometer sorts spermatogonial stem cells according to the antigen-antibody reaction. However, there are three disadvantages in the existing method: first, the experimental subjects are relatively limited, and only mice that have not entered the meiosis phase can be selected, because once they enter meiosis, During the sorting process, many somatic cells and spermatocytes at all levels will be brought in, reducing the purity of spermatogonial stem cells; second, the number of isolated spermatogonial stem cells is small, and a mouse that is 5-7 days after birth is usually isolated Only 1×10 can be obtained 4 -1.5×10 4 cells / ml, 2×10 can be obtained from an adult mouse 5 -3×10 5 third, the cost is high, because the number of spermatogonial stem cells obtained by separation is small and the proliferation rate is slow, in order to obtain more spermatogonial stem cells, it needs to be cultivated for a long time, so the various aspects of culturing cells Increased costs

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