Abstract: BACKGROUND AND AIM: To obtain highly purified cultured Sertoli cells from rat testis. MATERIALS AND METHODS: Testes from 18－22 day old SD rats were removed and decapsulated，then chopped and sequentially digested with two enzymes at 37 ℃：first with 0.25％ trypsin for 20－40 minutes，then with 0.05％ collagenase V for 40－60 minutes．The speed of centrifugation was 800 r/min. The isolated cells were incubated at 35 ℃ in a humidified atmosphere of 5％ CO2．To increase the purity of Sertoli cells，cultured cells were subjected to hypotonic shock(treatment)with 20 mmol Tris_HCl after 48 h of incubation．After 0.25％ trypsin digestion and adherent culture, the cultured cells were identified by HE staining，oil red O staining and transmission electron microscope. The growth curve of Sertoli cells was evaluated as well. RESULTS: Over 95％ of the cultured cells were Sertoli cells．The exponential phase of growth was 4－9 days. CONCLUSION: Highly purified Sertoli cells could be obtained by two step enzymes digestion and hypotonic shock, and identification of Sertoli cells by oil red O was a convenient method.

Key words: rat, Sertoli cell, isolation, identification