223 Wharton’s jelly mesenchymal stromal/stem cell–derived conditioned medium effect on equine endometrial cell viability

Endometritis is the major cause of reduced fertility in mares, with a consequent measurable economic loss. Mesenchymal stromal/stem cells (MSCs) and their derivates gained extremely high attention in recent years for their promising effects in the treatment of inflammation (Carrade et al. 2012 Cell Med. 4, 1–12). Their application could help in the modulation of the immune inflammatory response, providing regeneration and remodeling of injured tissue. The aim of this preliminary study was to evaluate in vitro the effect of Wharton’s jelly (WJ) MSC-derived conditioned medium (CM) on equine endometrial cells with lipopolysaccharide (LPS)-induced inflammation. Equine WJMSCs were isolated from 3 samples, pooled and then frozen at passage (P) 3. The WJMSCs were thawed and maintained in culture medium (Dulbecco’s Modified Eagle Medium (DMEM) + 10% fetal bovine serum) until 80%–90% confluence, and then the culture medium was replaced with serum-free Ringer lactate solution. The CM was collected after 24 h of starvation and concentrated 10 times by centrifugation at 4000g for 30 minutes at 13°C using 3-kDa molecular weight cutoff filter units and then stored at −80°C until use. Endometrial cells were obtained from 3 diestrus mare uteri. Cells at P1 were plated in 24-well plates at a density of 20 000 cells/well. Four different conditions were tested: DMEM standard complete medium (CTRL), DMEM with the addition of (1) 10% of CM (CM), (2) 10 ng mL⁻¹ LPS (LPS) and (3) LPS combined with CM (LPS + CM). Cell viability, apoptosis and necrosis were assessed after 6, 12 and 24 h of incubation, using acridine orange and propidium iodide fluorescent staining. Data were analysed by analysis of variance and Tukey’s test. Results (mean ± s.d.) of 3 replicates are reported in Table 1. At each incubation time, CM gave similar results to the CTRL for all parameters. After 6 h of incubation, LPS increased the percentage of necrotic cells, while co-treatment with CM prevented the detrimental effect. After 12 h of incubation, LPS decreased cell viability and increased apoptosis and necrosis compared with the CTRL and CM groups, while the co-treatment LPS + CM gave intermediate results. In conclusion, these preliminary results suggest that equine WJMSCs with cytokines, chemokines, growth factors and microvesicles may in part mitigate the LPS-induced inflammation on endometrial cells, promoting further investigations on WJMSC-CM characterisation and its mechanisms of action.