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Microdrop Culture of Rat Spermatogonial Stem Cells
Huo Long1, Luo Fenhua2, Zhang Yan1,3, Quan Qiwei1, Wu Yingji1,2*
1College of Life Sciences, Inner Mongolia University, Hohhot 010021, China; 2Key Laboratory of China Education Ministry for the Research of Mammal Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010021, Chin
Abstract: Spermatogonial stem cells (SSCs) not only possess the capacity for self-renewal and differentiation， but also can pass on genetic information to offspring. It has great significance to verify proliferation and differentiation of SSCs for further application. Research on microdrop culture of mouse SSCs showed that it had unique advantages compared to conventional culture methods. However， feasibility of the microdrop culture in other species remains to be confirmed. The purpose of this study was to establish rat SSCs culture system in vitro by the microdrop culture method. We transfered 5， 8， 10， 20， 40 rat SSCs into microdrops containing 20 μL medium，respectively， in which STO cells treated with mitomycin were used as the feeder layer. The proliferation status of the rat SSCs were observed and recorded using microscopy. After cultured for one month， immunofluorescence doublelabel staining and induction of differentiation in vitro were performed for analysis of the cultured SSCs. The results showed that inoculating at least five SSCs in a microdrop was able to achieve the SSC proliferation. After cultured in microdrop for one month， the rat SSCs still expressed the marker molecules such as CDH1， OCT4， PLZF， Thy1 and Gfra1. And they also possessed the ability to differentiate into spermatocytes in vitro. The microdrop culture technique for proliferation of rat SSCs had been established. It offers reference for cultivation of SSCs in other species， and provides a technology platform for related research field in regenerative medicine and life science.
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