Department of Urology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Reprod Biol Endocrinol. 2011 Oct 24;9:141. doi: 10.1186/1477-7827-9-141.
To isolate and characterization of human spermatogonial stem cells from stem spermatogonium.
The disassociation of spermatogonial stem cells (SSCs) were performed using enzymatic digestion of type I collagenase and trypsin. The SSCs were isolated by using Percoll density gradient centrifugation, followed by differential surface-attachment method. Octamer-4(OCT4)-positive SSC cells were further identified using immunofluorescence staining and flow cytometry technques. The purity of the human SSCs was also determined, and a co-culture system for SSCs and Sertoli cells was established.
The cell viability was 91.07% for the suspension of human spermatogonial stem cells dissociated using a two-step enzymatic digestion process. The cells isolated from Percoll density gradient coupled with differential surface-attachement purification were OCT4 positive, indicating the cells were human spermatogonial stem cells. The purity of isolated human spermatogonial stem cells was 86.7% as assessed by flow cytometry. The isolated SSCs were shown to form stable human spermatogonial stem cell colonies on the feeder layer of the Sertoli cells.
The two-step enzyme digestion (by type I collagenase and trypsin) process is an economical, simple and reproducible technique for isolating human spermatogonial stem cells. With little contamination and less cell damage, this method facilitates isolated human spermatogonial stem cells to form a stable cell colony on the supporting cell layer.
从精原干细胞中分离和鉴定人类精原干细胞。
采用Ⅰ型胶原酶和胰蛋白酶两步酶消化法分离精原干细胞（SSCs）。通过 Percoll 密度梯度离心和差速贴壁法分离 SSCs。采用免疫荧光染色和流式细胞术技术进一步鉴定 Octamer-4（OCT4）阳性 SSC 细胞。还确定了人 SSCs 的纯度，并建立了 SSCs 和支持细胞共培养系统。
两步酶消化法（Ⅰ型胶原酶和胰蛋白酶）分离的人精原干细胞悬液的细胞活力为 91.07%。从 Percoll 密度梯度与差速贴壁纯化分离的细胞为 OCT4 阳性，表明这些细胞是人精原干细胞。流式细胞术评估分离的人精原干细胞的纯度为 86.7%。分离的 SSCs 在支持细胞层上形成稳定的人精原干细胞集落。
两步酶消化（Ⅰ型胶原酶和胰蛋白酶）过程是一种经济、简单、可重复的分离人精原干细胞的方法。该方法污染少、细胞损伤小，有利于分离的人精原干细胞在支持细胞层上形成稳定的细胞集落。
