Pcs 100 041

Human umbilical vein endothelial cells (HUVEC) were obtained from American Type Culture Collection, ATCC (PCS-100-010; Manassas, VA, USA). Cells were grown in the presence of 10 mM glutamine as a supplement in the growth media to render them more resistant to radiation effects in order to better modulate ceramide related cell signalling. Under these conditions an 8 Gy dose of radiation results in 50-60% cell survival (an order of magnitude of protection compared to an 8 Gy dose in the absence of glutamine). Cells were cultured in flasks pre-coated with 0.1% gelatin and grown using endothelial cell growth kit-VEGF medium (PCS-100-041 from ATCC).
Cells were grown and maintained under humidity at 37°C, 5% CO2, and were used for experiments between passages three and six. Confluent cells were harvested using 0.05% Trypsin-EDTA from Invitrogen (25300-062, Carlsbad, CA) or Trypsin-EDTA for primary cells (PCS-999-003) and trypsin neutralizing solution (PCS-999-004), both obtained from ATCC (Manassas, VA, USA). Cells were then collected by centrifugation at 4°C for 7 min (180 × G/1000 rpm) and were counted using a hemocytometer. To ensure reproducibility, all experiments were carried out in triplicate.

MSCs were isolated from the decidua basalis (DBMSCs) of human term placenta, whereas HUVEC (human umbilical vein endothelial cells) were isolated from umbilical cord veins as previously described by us.¹⁰, ¹² DBMSCs were cultured in complete DBMSC culture medium [DMEM‐F12 medium containing 10% foetal bovine serum (MSC‐FBS, catalogue number 12‐662‐011, Life Technologies), and antibiotics (100 µg/mL streptomycin and 100 U/mL penicillin)], whereas HUVEC were cultured in complete endothelial cell growth medium (Catalogue number PCS‐100‐041^™, ATCC). Cells (DBMSCs and HUVEC) were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air (a cell culture incubator).
The viability of DBMSCs and HUVEC was determined using Trypan blue. DBMSCs (passage 3) and HUVEC (passages 3‐5) of twenty placentae and umbilical cords, respectively, were used in this study.

Human umbilical cord vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC). HUVECs were cultured in vascular cell basal medium (PCS100030; ATCC) supplemented with vesicle-free endothelial cell growth kit components (PCS100041; ATCC) with the addition of 1% penicillin-streptomycin (PCS999002; ATCC) and 2% exosome-free fetal bovine serum (FBS) (EXO-FBSHI-250A-1; Systems Biosciences). HUVECs at passage 3 were cultured in 6-well plates until they reached 80% confluence.