| Background and purpose: The research on Spermatogonial Stem Cells (SSC) is evoking more and more interests of people because of the development of other stem cells and the appearance of many new research tools and experimental techniques in recent years. The spermatogonia of some kinds of animals such as rat, mouse, pig and cattle have been isolation, culture and transplantation. Some kinds of different culture systems for the culture of SSC in vitro have been built such as the co-culture system with Sertoli cells, the germ cell culture system with STO (mouse embryonic fibroblast lines) as feeder layer and so on. Meanwhile, different culture media such as DMEM, DMEM/F12, serum-free medium and KSOM culture medium including ion of potassium have been used. The biochemical and molecular characteristics and detailed biological mechanism of differentiation and proliferation of SSC, which is the foundation and the premise of the complicated spermatogenesis, have not been deep studied. Long-term culture of human SSC in vitro has not been reported at present. Following the constantly development of researching methods to various kinds of stem cell, The study of the isolation, purification, culture and inducing differentiation of human spermatogonia becomes more and more important. According to the present situation for the research of SSC, this study planed to gain the higher purification of SSC from normal human fetal testicular tissue by several steps of isolation, and then to culture the SSC for a long time using the different culture media in different culture systems for seeking the long-time optimal culture system for human SSC in vitro. This study established a foundation for the further research on spermatogonial transplantation and functional assessment of human SSC. This study was divided into three parts: 1. Preparation for the feeder layer: Different feeder layers of mouse embryonic fibroblast (MEF) and mouse embryonic fibroblast lines (STO) for the culture of human spermatogonia were prepared. 2. Isolation and the purification of human testicular SSC: Sequential two-step enzymes digestion, Percoll density gradient centrifugation and isolation according to the different adhesiveness were used to purify the SSC. 3. Optimization of the culture system and long-time observation of the cultured spermatogonia in vitro: Variousculture media such as DMEM, DMEM/F12 and serum-free medium in different co-culture systems were used to culture and observe the SSC in vitro. Then the optimized culture system in which human SSC can survive stably was selected for a long period of culture and observation of the SSC in vitro.Methods: The SSC was taken from normal human testicular tissue (the testes were taken from the legal aborted fetus older than six months). Sequential two-step enzymes digestion, Percoll uncontinuous density gradient centrifugation and isolation according to the different adhesiveness were used to purify the SSC, then the purified SSC was planted into three kinds of culture media of DMEM, DMEM/F12 or DMEM-SF in which the STO or MEF were used as the feeder layer, or the single cell suspension originate from the two-step enzymes digestion was used as the Sertoli cell-germ cell co-culture in three kinds of culture media. The spermatogonia were identified by the characterized shape and size with microscope, and the proportion of the survival spermatogonia in the seeded cells in all groups were observed and assessed within a short-term culture. Meanwhile, The spermatogonia co-cultured with the Sertoli cell in the DMEM/F12 were observed for a long period of time, and the SSEA-1 antigen expressed by the spermatogonia was initially detected by immunohistochemistry.Results: 1. MEF which was taken from 12.5dpc embryo of mouse can proliferate fast in culture and suit for a feeder layer.2. The average percentages of live cell and dead cell of testicular cell suspension gained from the two-step enzymes digestion are 89.71% and 10.29%, respectively. 3. The methods such as Percoll uncontinuous den...
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