Successful long term culture of immature porcine sertoli cells in the reconstructed testicular cell cord

The ultimate goal of this study was to establish an in vitro system to produce sperms. To pursue this goal, immature porcine testicular cells were cultured in stereostructural form and cultured testicular cord was investigated morphologically. At 4 weeks of age, the seminiferous tubules of the porcine testes consisted of undifferentiated germ cells (gonocytes and undifferentiated spermatogonia) and immature Sertoli cells. The interstitial tissue was largely occupied by Leydig cells. The testes were enzymatically digested, and the dispersed cells were encapsulated with alginate either immediately or after freeze-thawing. The resulting testicular cell cords were cultured for up to 10 weeks. After 2 weeks of culture, Sertoli cells, which were identified by their inhibin-positive reaction in immunohistochemistry, and Leydig cells, which were identified by their morphological characteristics, were observed in the cords. Neither undifferentiated nor differentiated types of germ cells were detected. The number of cells in the cords progressively decreased during the culture period. In order to discover the fate of the Sertoli cells, the level of inhibin in the spent media was determined. Inhibin in the media was at a detectable level after 2 days of culture. The levels increased and peaked at 2 weeks. When frozen-thawed testicular cells were applied to the culture, the peak level was maintained for over 8 weeks, in contrast to the gradual decrease of inhibin level when fresh cells were cultured. These results indicate that the culture conditions can sustain the survival of Sertoli cells. Further improvement is required for proliferation and differentiation of germ cells.