0.8 μm sterile filter

To obtain conditioned medium (CM) generated from hAMTCs, freshly isolated hAMTCs were cultured for 3 days in 24‐well plates (Corning, NY, USA) at a density of 3 × 10⁵ cells/well in 0.5 ml Roswell Park Memorial Institute (RPMI) complete medium, composed of RPMI 1640 medium (Sigma‐Aldrich, St Louis, MO, USA) supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma‐Aldrich), 2 mm l‐glutamine (Sigma‐Aldrich) and 100 U/ml penicillin and 100 mg/ml streptomycin (pen–strep, herein referred to as P/S; both from Sigma‐Aldrich).
To obtain CM without prostaglandins (CM – PG), hAMTCs were cultured as described above in the presence of 10 μm indomethacin (Sigma‐Aldrich), a cyclooxygenase inhibitor. PGE2 quantification in CM and CM – PG was obtained using a Prostaglandin E2 EIA Kit (Cayman Chemical Co., Ann Arbor, MI, USA), according to the manufacturer's instructions. Absorbance was measured at 405 nm using a microplate reader.
At the end of the culture period, CM and CM – PG were collected, centrifuged at 300 × g, filtered through a 0.8 μm sterile filter (Sartorius Stedim, Florence, Italy) and kept frozen at −80 °C until use.
To obtain results that were least influenced by single donor variability and more representative of soluble factors released by hAMTCs, 10 pools, each containing CM from three to six different cell preparations, were used.

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.