Efficient purification of neonatal porcine gonocytes with Nycodenz and differential plating

Gonocytes are the only type of germ cells present in the postnatal testis and give rise to spermatogonial stem cells. Purification of gonocytes has important implications for the study and manipulation of these cells and may provide insights for the ongoing investigation of the male germline stem cells. To obtain a pure population of gonocytes from piglet testis cells, a wide range of Nycodenz concentrations were investigated for density gradient centrifugation. We also examined differential plating of testis cells for various culture durations with different extracellular matrix (ECM) components (fibronectin, poly-d-lysine, poly-L-lysine, laminin and collagen Types I and IV). Gonocytes were highly enriched in pellets of testis cells after using 17% Nycodenz centrifugation to a purity of 81±9%. After culturing testis cells on plates precoated with different ECM components for 120 min, the proportion of gonocytes increased among non-adherent cells (suspended in the medium), with fibronectin or poly-D-lysine resulting in the greatest (up to 85%) and laminin in the lowest (54%) gonocyte proportion. Combining the most promising ECM coatings (fibronectin and poly-d-lysine) and further extension of their culture duration to 240 min did not improve final gonocyte purity. However, centrifugation with 17% Nycodenz followed by differential plating with fibronectin and poly-d-lysine coating further purified gonocytes among the collected cells to >90%. These results provide a simple, quick and efficient approach for obtaining highly enriched populations of piglet gonocytes for use in the study and manipulation of these germline stem cells.