To establish a system for separation, enrichment, culture, and primary identification for the spermatogonial stem cells of Macaca fascicularis (MSSCs), and to explore its in vitro growth characteristics.

One testis was surgically removed from a two years and 14 days old male Macaca fascicularis; SSCs were obtained through the three-step enzyme digestion, then enriched by culturing the suspension of cells with differential adherence method. The enriched SSCs were then co-cultured over 20 days with mitomycin C-treated STO cells in serum-free medium with adding three important growth factors of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and GDNF family receptor α1 (GFRα1). The generation of SSCs were tentatively identified with CDH1 immunofluorescence staining and reverse transcription-polymerase chain reaction (RT-PCR).

The enriched SSCs acquired through differential adherence method began to divide and propagate one day later after seeding into mitomycin C-treated STO cells. Small clones emerged after culturing in medium containing growth, and these clones enlarged to remarkable size five days later. After 20-day culture, clusters of grape like cells appeared which were consistent with morphology features of SSCs; immunofluorescence staining and RT-PCR demonstrated that these cells express SSCs marker of CDH1.

A system of separating, purifying, culturing, and identifying SSCs for Macaca fascicularis has been successfully established. Serum-free culture system basing on feeder of STO cells and media with adding three growth factors of GDNF, bFGF, and GFRa1 is suitable for culturing MSSCs. CDH1 can serve as a primitive marker for identifying MSSCs.