How to dissociate porcine ovaries: towards efficient retrieval of putative female germline stem cells.

The discovery of female germline stem cells in the mature murine ovary reignited discussion of postnatal oogenesis. Due to extremely low numbers, the use of an appropriate, highly reliable dissociation method is crucial in guaranteeing the recovery of these putative cells. This study aimed to determine which mechanical and enzymatic dissociation method would give an ovarian cell suspension with the highest number of viable cells in a prepubertal porcine model, followed by flow cytometric detection of a rare DDX4-positive cell population indicative of putative female germline stem cells. It was discovered that viability was increased using the GentleMACS homogeniser as compared to the Ultra-Turrax T18 homogeniser, and that viability increased further when using Liberase DH as compared to collagenase IV digestion. No further improvement in viability of recovered cells resulted from altering other experimental parameters such as enzyme incubation time (30 min or 90 s), blocking buffer (DPBS or HBSS), overnight incubation temperature (37 °C or 38.5 °C), cell detachment with or without trypsin, and centrifugation speed (1000 g, 300 g, or no centrifugation). The optimised retrieval protocol therefore used GentleMACS homogenisation with 30 min Liberase DH digestion, DPBS for blocking steps, incubation at 38.5 °C, trypsin recovery of cells, and centrifugation at 300 g. Flow cytometry identified a rare viable DDX4-positive cell population. This study provides an optimised approach for generating highly viable porcine ovarian single-cell suspensions, which may facilitate subsequent analysis of rare ovarian cell populations, including DDX4-positive cells consistent with putative female germline stem cells. Further studies, including additional marker analysis and functional assays, are required to determine the identity and functional properties of the DDX4-positive population.