小鼠生精细胞的体外双室无血清培养

Establishment of culture system of mouse spermatogenic cells in vitro without serum

目的探讨无血清条件下应用插入式细胞培养皿(Transwell小室)对小鼠睾丸间质细胞-支持细胞-生精细胞的双室培养技术。方法取60日龄C57BL/6雄性小鼠睾丸间质细胞和15日龄雄性小鼠睾丸支持细胞与生精细胞混合细胞团双室共培养,不添加血清。每日在倒置显微镜下观察生精细胞的形态和生长情况,苏木精染色观察生精细胞形态,染色体倍性分析检测细胞分化情况。结果在培养1周后,可见圆形精子细胞出现,2周后可见长形精子细胞,3周后可见较短鞭毛,生精细胞可存活8周;培养1周时,流式细胞术可检测出单倍体细胞,单倍体细胞百分比随培养时间延长而增加。结论应用双室无血清培养体系体外培养小鼠生精细胞可获得精子且生精细胞存活时间较长。

Objective To establish a mouse Leydig cell-Sertoli cell-germ cell double-chamber coculture system in vitro without serum.Methods The Leydig cells were isolated from the testes of postnatal day 60 male C57BL/6 mice.Total germ cells and Sertoli cells were isolated from the testes of postnatal day 15 male C57BL/6mice.The cells in bicameral chambers culture system were grown in media without serum.The morphology and growth of culture cells were monitored daily under contrast phase microscope,and were identified by Hematoxylin staining.Ploidy analysis of cells was observed by flow cytometry.Results In bicameral culture system,sporadic round spermatids were detected after one week,and elongating spermatids or spermatids with flagella were observed after three weeks.The spermatogenic cells survived as long as eight weeks.The haploid peak was detected after one week;the percentage of monoploid increased with culture time.Conclusion The spermatogenic cells in bicameral culture system matured into morphologically normal spermatozoa and survived longer.