title: Research on the isolation of mouse Leydig cells using differential digestion with a low concentration of collagenase.
abstract: The aim of this study was to establish a novel method for isolating and purifying Leydig cells from mice testes. Testes of postpuberal mice were harvested and digested in a low concentration of collagenase NB4 for 15 min 2 times. Cells obtained were cultured in low glucose DMEM with 10% FBS. Immunofluorescence was used to detect the expression of Leydig cell biomarkers including 3β-hydroxysteroid dehydrogenase, cholesterol side-chain cleaving enzyme (CYP11A1) and 17α-hydroxylase/17,20-lyase (CYP17A1). It was found that the purity of the isolated Leydig cells was 69.6 ± 4.16%. After 7 days in primary culture, it increased to 90%. The testosterone synthase spectrum could be detected at the primary culture. In conclusion, the application of a low concentration of collagenase for differential digestion allows isolation of large quantities of viable Leydig cells.
authors.affiliation: Department of Urology, Shanghai Children's Medical Center, Shanghai, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.forename: Liang
authors.initials: L
authors.lastname: Zhong
authors.forename: Yingjian
authors.initials: Y
authors.lastname: Zhu
authors.forename: Guohua
authors.initials: G
authors.lastname: Liu
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism*; D007985:Leydig Cells / Q000166:cytology*; D008297:Male; D051379:Mice; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism; D013739:Testosterone / Q000096:biosynthesis
chemical_list: D013739:Testosterone; D015096:3-Hydroxysteroid Dehydrogenases; D013254:Steroid 17-alpha-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D017364:Collagenases