Objective    To isolate and culture spermatogonial stem cells (SSCs) from human testes with a two-step two-enzyme digestion method.     Methods    A suspension of human SSCs was obtained through enzymatic digestion of human testes with type Ⅰ collagenase and trypsin

and the SSCs in the suspension were isolated and cultured with Percoll density gradient centrifugation and different adherence method. Immunofluorescence assay was employed to identify SSCs and

in combination with flow cytometry

to screen Oct-4-positive cells and detect the purity of the SSCs. A co-culture system for SSCs and Sertoli cells was established.     Results    The viability of the obtained cells was 91.07% in the suspension. Immunofluorescence assay results demonstrated that the cells purified by Percoll density gradient centrifugation and different adherence method were Oct-4 positive. The flow cytometry results indicated the purity of the Oct-4-positive cells was 86.7%. The Oct-4-positive cells could stably grow for 30 days on the feeder layer of Sertoli cells.     Conclusion    The Oct-4-positive cells are SSCs. The two-step two-enzyme digestion method is economical

simple

and feasible for isolating and culturing human SSCs with reduced cell contamination and cell damage. The isolated human SSCs can form a stable cell colony on a supporting cell layer.