Isolation, Purification And Culture Of Human Spermatogonial Stem Cells In Vitro

ObjectiveTwo-step method and two enzymes isolates spermatogonial stem cells in human testis. In order to establish stable spermatogonial stem cell lines. we explore the method of culturing spermatogonial stem cells in vitro.MethodThe isolation and culture of spermatogonial stem cells were performed using enzymatic digestion of type I collagenase and trypsin, Percoll density gradient centrifugation, and differentiated wall-adherence method. Cytoimmunity fluorescence technology was employed for identification of stem spermatogonium and flow cytoimmunity fluorescence technology was employed for screening of Oct-4 labeled positive cells. The purity of the human spermatogonial stem cells was determined and a co-culture system for spermatogonial stem cells and Sertoli cells was established.ResultThe viability of the cells was 91.07% in the suspension of human spermatogonial stem cells digested in a two-step enzymatic process. The cells obtained after Percoll density gradient centrifugation coupled with differentiated adherence centrifugal purification, were confirmed to be human spermatogonial stem cells as assayed by immunocytochemistry against Oct-4. The purity of Oct-4 positive cells was 86.7% as detected by flow cytometry. Oct-4 positive cells can stable growth 30 days on the feeder layer of the sertoli cells.ConclusionThe Oct-4 positive cells is spermatogonial stem cells. The two-step enzyme digestion (by type I collagenase and trypsin) method is economical, simple, and feasible for isolating and culturing of 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.