Ultraculture complete medium

Placentas were processed immediately after collection and cells were isolated and directly used. Specifically, human amniotic mesenchymal stromal cells (hAMSC) were obtained from the mesenchymal region of the amniotic membrane as previously described (44 (link)). Conditioned medium was generated by culturing hAMSC (CM-hAMSC) for 5 days in 24-well plates (Corning, NY, USA) at a density of 5 × 10⁵ cells/well in 0.5 ml of Ultraculture complete medium, composed of Ultraculture medium (Sigma-Aldrich, St Louis, MO, USA), supplemented with 2 mM L-glutamine (Sigma-Aldrich), and 100 U/ml penicillin and 100 mg/ml streptomycin (both from Sigma-Aldrich) as described (43 (link)). To obtain CM devoid of prostanoids (CM-hAMSC/PG), hAMSC were cultured in Ultraculture complete medium supplemented with 10 μM indomethacin (Sigma-Aldrich), a cyclooxygenase inhibitor (10 (link)). At the end of culture, CM-hAMSC and CM-hAMSC/PG were collected, centrifuged at 300 x g, filtered through a 0.8 μm sterile filter (Sartorius Stedim, Florence, Italy), and kept frozen at −80°C until use. Each experiment was performed by pooling together conditioned medium obtained from 3 to 4 different hAMSC donors.