Effects of medium and hypothermic temperatures on preservation of isolated porcine testis cells

Abstract

The effects of medium and hypothermic temperatures on testis cells were investigated to develop a strategy for their short-term preservation. Testes from 1-week-old piglets were enzymatically dissociated for cell isolation. In Experiment 1, testis cells were stored at either room (RT) or refrigeration (RG) temperature for 6 days in one of 13 different media. Live cell recovery was assayed daily using trypan blue exclusion. In Experiment 2, three media at RG were selected for immunocytochemical and in vitro culture studies. Live cell recovery was also assayed daily for 6 days using both trypan blue exclusion and a fluorochrome assay kit. For all media tested, significantly or numerically more live cells were maintained at RG than RT. On preservation Day 3 at RG (cell isolation day as Day 0), 20% FBS-Leibovitz resulted in the highest live cell recovery (89.5 ± 1.7%) and DPBS in the lowest (60.3 ± 1.9%). On Day 6 at RG, 20% FBS- Leibovitz also resulted in the best preservation efficiency with 80.9 ± 1.8% of Day 0 live cells recovered. There was no difference in live cell recovery detected by the two viability assays. After preservation, the proportion of gonocytes did not change, whereas that of Sertoli and peritubular cells increased and decreased, respectively. After 6 days of hypothermic preservation, testis cells showed similar culture potential to fresh cells. These results show that testis cells can be preserved for 6 days under hypothermic conditions with a live cell recovery of more than 80% and after-storage viability of 88%.