A passage and storage system for isolated bovine endometrial epithelial and stromal cells

To establish a storage system for isolated endometrial cells, we investigated the basal, oxytocin (OT)- and tumor necrosis factor (TNF) α-stimulated production of prostaglandin (PG) F_2α in bovine-passaged and frozen-thawed endometrial cells. Stromal and epithelial cells obtained from cows in the early stage of the estrous cycle (Days 2-5) were frozen at -80 C or further cultured and/or passaged until passage 4 in DMEM/Ham's F-12 supplemented with 10% calf serum. A fresh-unfrozen primary culture and one-time passaged fresh-unfrozen cells were used as the control. When both unfrozen and frozen cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA and the cells were stimulated with OT (100 ng/ml) or TNFα (1 ng/ml) for 4 h. The passage and freezing of the endometrial cells did not affect their morphology. In primary culture of frozen and unfrozen endometrial cells, OT strongly stimulated PGF_2α production in epithelial cells, and TNFα strongly stimulated PGF _2α production in stromal cells (P<0.05). The basal output of PGF_2α in frozen stromal cells was similar to that in unfrozen stromal cells. However, the basal output of PGF_2α in frozen epithelial cells was significantly lower than that unfrozen cells (P<0.05). On the other hand, in passaged cells, the basal level of PGF_2α production was retained until passage 1 in epithelial cells, whereas it was retained until passage 4 in stromal cells. Although epithelial cells responded to OT in PGF_2α production until passage 2 (P<0.05), the stromal cells showed a significant response to TNFα until passage 4 (P<0.05). These results suggest that stored cells could be used for studying the physiology of bovine endometrium in vitro until passage 1 in endometrial epithelial cells, and until passage 4 in stromal cells.