Isolation of rat Leydig cells with combined low-concentration type II and IV collagenases

Objective To establish a simple and effective method to isolate and purify rat Leydig cells. Methods Leydig cells were digested with a mixture of 0.1% types II and IV collagenases, purified by differential adherence, and identified by 3β-HSD immunofluorescence staining. The testosterone-secreting capability of Leydig cells was determined by ELISA. Results The optimal enzyme digestion time for Leydig cells was about 1 hour. The best adherence time for the purification of Leydig cells was 1-2 h. After 24 hours of culture, the purity of Leydig cells was (98.1 ± 1.5)% as revealed by 3β-HSD immunofluorescence staining, and the survival rate of Leydig cells was (97.3 ± 1.6)%. After incubation for 12 hours, the Leydig cells reached the peak of secretion. Conclusion In this study, a simple, stable, and reliable method for isolation and purification of primary Leydig cells was established. This method allows to obtain high-purity Leydig cells with high activity. Key words: Leydig cell; Isolation; Purification; Identification; Rats