Isolation Of Leydig Cells From Juvenile Sd Rat Testis By Combined Digestion Method With Low Concentration Type Ii And Iv Collagenase
Keywords
Leydig Cells,Isolation,Purification,Identification,Rat
Abstract
Abstract Objective To explore a simple and effective method for the isolation and purification of rat testicular Leydig cells. Methods: Leydig cells were isolated from testicular tissue by digestion of tissue blocks with 0.1% type II and IV collagenase mixture. Leydig cells were purified by differential adhesion method. Leydig cells were identified by 3¦Â-HSD immunofluorescence staining and assayed by enzyme-linked method. Activity of Leydig cells secreting testosterone. Results (1) The optimal enzymatic digestion time for isolating Leydig cells using 0.1% type II and IV collagenase combined digestion method is about 1 hour. (2) The optimal attachment time for purifying Leydig cells using differential attachment is 1 to 2 hours. (3) After 24 hours of culture, 3¦Â-HSD immunofluorescence staining determined that the purity of Leydig cells reached (98.1¡À1.5)%, and pan blue staining determined that the survival rate of Leydig cells reached (97.3¡À1.6)%. (4) After 12 hours of culture after attachment, the Leydig cells of the testis will reach the peak of secretion. Conclusion This experiment established a simple, stable and reliable method for the isolation and purification of primary testicular Leydig cells. Through this method, sufficient high-activity and high-purity testicular Leydig cells can be obtained, and the proliferation ability of testicular Leydig cells is relatively high. powerful
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Original research was done by Liu Chun, Li Yang, Wang Yu, Cheng Kang
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