Reassembly of somatic cells and testicular organogenesis in vitro

Testicular organogenesis in vitro requires an environment allowing a reassembly of testicular cell types. Previous in vitro studies using male murine germ cells cultured in a defined three-dimensional environment demonstrated tubulogenesis and differentiation into spermatozoa. Combining scaffolds as artificial culture substrates with testicular cell culture, we analysed the colonization of collagen sponges by rat testicular cells focusing on cell survival and reassembly of tubule-like-structures in vitro. Isolated testicular cells obtained from juvenile Sprague Dawley and eGFP transgenic rats were cultured on collagen sponges (DMEM high glucose + Glutamax, 35 °C, 5% CO2 with or without gonadotropins). Live cell imaging revealed the colonization of cells across the entire scaffold for up to 35 days. After two days, histology showed cell clusters attached to the collagen fibres and displaying signs of tubulogenesis. Clusters consisted mainly of Sertoli and peritubular cells which surrounded some undifferentiated spermatogonia. Flow cytometry confirmed lack of differentiation as no haploid cells were detected. Leydig cell activity was detected by a rise of testosterone after gonadotropin stimulation. Our approach provides a novel method which is in particular suitable to follow the somatic testicular cells in vitro an issue of growing importance for the analysis of germ line independent failure of spermatogenesis.