Sertoli cells cultured under high-temperature and hypoxic conditions

Sertoli cells (SCs) isolated from adult C57Bl/6 mice were characterized under four different cell-culture conditions: standard conditions (34°C, 21% O 2 -34_21), high-temperature conditions (37°C, 21% O 2 -37_21), hypoxic conditions (34°C, 5% O 2 -34_5), and a combination of these conditions (37°C, 5% O 2 -37_5). Hypoxia facilitated cell proliferation and improved their viability: 28.5 and 24.6% of SCs in 34_5 and 37_5 cultures, respectively, were BrdU-positive, and 92.7 and 92.7% of SCs were viable after 15 days versus 20.2 and 88.9%, respectively, in 34_21 culture. The proliferation of SCs grown under high-temperature conditions was slightly increased, whereas the viability decreased: 23.1% of SCs were BrdU-positive, and only 74.9% of SCs were viable at 37_21. At the same time, cultivation of SCs at 37°C promoted their dedifferentiation: after 15 days in culture, 98.8 and 98.6% of cells in 37_5 and 37_21 cultures, respectively, expressed cytokeratin-18, a marker of immature SCs, as compared to 26.5% in 34_5 and 6.6% in 34_21. Expression of Wt1, a transcription factor controlling cell-cell junction and germ-cell development, disappeared in most cells after 3 days under all culture conditions. However, SCs forming colonies restored Wt1 expression at day 15 in culture under high-temperature conditions: 59.1 and 29.5% of SCs were Wt1-positive in 37_21 and 37_5, respectively, versus 11.1 and 3.6% in 34_21 and 34_5, respectively. Cultured SCs expressed other SC markers (vimentin, clusterin, GATA-4) under all culture conditions. Our results show that cultured SCs may be useful for reproductive biology and regenerative medicine.