Canine testes thin section culture

Abstract

Testes tissue culture systems would provide a tool to elucidate spermatogenesis mechanisms, with the aim of genetic preservation of mammals, especially endangered species. Our experiment aims to develop a culture system capable of producing viable mammalian sperm cells in vitro. Dogs were chosen as the model organism because testes are readily available. Canine testes were obtained from a local veterinary clinic. Thin sections were generated using a commercial electric slicer. They then were cleaned using Dulbecco’s Phosphate-Buffered Saline (DPBS) supplemented with antibiotics then cultured in a modified Tissue Culture Medium 199 (TCM-199). Sections were cultured in an environment aimed to best reflect realistic physiological conditions, that is 7%CO2 : 7%O2 : balanced N2 at 37 degrees Celsius. Finally, the sections were stained with live/dead cell stain and observed under a fluorescence microscope to determine viability. Numerous live stained nuclei were observed, proving their high viability (~100% viability) after 21 days of culturing. Sections reformed during culture to assume a tiny testes-like morphology. Fungal contamination was detected in all culture dishes at various time points during the experiment from unknown sources. The sections then were washed with DPBS supplemented with antimycotic before being again cultured in fresh medium. For ongoing experiments, the culture system will be revised to prevent fungal contamination. While spermatogenesis takes approximately 60 days in vivo, testes thin sections were maintained for 21 days, therefore culture duration will be extended in the future. Overall, our result demonstrated a cost-effective culture system to potentially obtain viable mammalian sperm cells