Optimisation of an Equine Endometrial Cell Culture

Uterine inflammation is the most common cause of subfertility in the mare. A protocol was optimised for culturing isolated endometrial stromal and epithelial cells that may be used to measure in vitro responses to challenge. Equine uteri were collected after slaughter. Endometrium was dissected, cells were harvested by tissue digestion, re-suspended in HBSS, and the epithelial and stromal cells filtered and washed with centrifugation at 700 g three times for 7 min. A standard protocol was thereafter optimised (experiment 1) and once established was used to test the efficacy of different media (experiment 2). Indicators of optimal cell growth were: adherence and proliferation of cells; <5% cross-contamination of each cell type; cells achieving confluence <10 days after plating. In Experiment 1, cells were re-suspended in supplemented RPMI medium and 1·105 cells/ml/well were plated in 24-well plates. Once stromal cells had adhered, suspended epithelial cells were removed at 18, 24 or 36 h (n = 2) and re-plated allowing adherence of pure epithelial cells. Culture medium for each of the epithelial and stromal cell cultures was changed at 24, 48 or 72 h (n = 4) until the cells reached confluence. Optimal time for separating stromal and epithelial cells was 36 h and for medium change was 72 h. Experiment 2 used this optimised protocol from Experiment 1 to test supplemented RPMI, DMEM or Williams media (n = 3). Experiment 2 demonstrated that stromal and epithelial cell growth was optimal in DMEM medium. Endometrial cells cultured using this protocol may provide an in vitro model for investigating inflammation.