Welsh M J, Wiebe J P
Endocrinology. 1975 Mar;96(3):618-24. doi: 10.1210/endo-96-3-618.
A method is described for obtaining populations of viable Sertoli cells from rat testes. Minced whole testes from rats of 15 to 29 days of age are sequentially treated with collagenase and pancreatin. The resulting suspension of cells is sedimented through a sucrose density gradient. Preparations are produced consisting of from 60% to 82% Sertoli cells, an enrichment of 2 to 5 times the proportion of Sertoli cells in whole testes of these ages. The preparations are free of interstitial cells, are essentially free of peritubular cells and contain reduced numbers of germinal cells; the main contaminating cell types are spermatogonia and spermatocytes. The Sertoli cells are considered to be 95% viable by their ability to exclude trypan blue and by subsequent culturing in vitro. The entire procedure requires 3 h. Maintenance of the Sertoli-enriched fraction in modified Eagle's minimal essential medium temporarily at 41 C allows preparations of Sertoli cell monolayer cultures consisting of 95%-98% Sertoli cells within 3 days.
本文描述了一种从大鼠睾丸中获取有活力支持细胞群体的方法。取15至29日龄大鼠的整个睾丸切碎，依次用胶原酶和胰蛋白酶处理。将所得细胞悬液通过蔗糖密度梯度离心沉淀。制备出的细胞制剂中支持细胞占60%至82%，这些细胞制剂中支持细胞的比例比这些年龄段大鼠整个睾丸中的比例富集了2至5倍。这些制剂不含间质细胞，基本不含睾丸周细胞，生精细胞数量减少；主要的污染细胞类型是精原细胞和精母细胞。通过台盼蓝排斥试验以及随后的体外培养，认为支持细胞的活力为95%。整个过程需要3小时。将富含支持细胞的组分在改良的伊格尔最低必需培养基中于41℃短暂保存，可在3天内制备出由95% - 98%支持细胞组成的支持细胞单层培养物。
