Sertoli cells play critical roles in regulating spermatogenesis and testis development by providing structural and nutritional support. This study aimed to develop a standard protocol for canine Sertoli cell isolation and culture and to characterize its biological features, functionality, and applications in compound toxicity testing. Canine testicles were received from the neuter clinic, and a three-step enzymatic digestion process was applied to isolate Sertoli cells. We characterized the growth and purity of Sertoli cells by the expression of SOX9, GATA4, and Clusterin. In addition, cadmium was selected as a model toxicant to evaluate toxic responses in newly established Sertoli cells using High-Content Analysis (HCA). With our optimized protocol, the purity of isolated Sertoli cells was above 95%, as determined by Sertoli cell-specific protein markers SOX9 and GATA4.
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