Trypsin versene

Human placentas were provided at the Department of Obstetrics and Gynecology from healthy mothers under written consent approved by the Ethics Committee from Hospital Universitario 12 de Octubre. DMSCs were isolated and cultured from extraembryonic membranes as described previously [30] . Briefly, placental tissue was digested with trypsin-versene (Lonza, Spain), cells were seeded at 1.16×10⁵ cells/cm² and cultured at 37°C, 5% CO₂ and 95% humidity in Dulbecco's modified Eagle Medium (Lonza) supplemented with 2 mM L-glutamine, 0.1 mM sodium pyruvate, 55 µM of B-mercaptoethanol, 1% non-essential amino acids, 1% penicillin/streptomycin, 10% fetal bovine serum and 10 ng/ml of Epidermal Growth Factor (Sigma-Aldrich Química, Spain). Non-adherent cells were eliminated by washing off and adherent cells were grown to confluence and passaged at a density of 4–5×10⁴ cells/cm².

HUVECs were purchased from BD Biosciences (San Jose, CA, USA), and cultured on gelatin-coated T-75 tissue culture flasks with supplemented M199 medium (Life Technologies, Carlsbad, CA, USA). M199 was supplemented with 1 % (v/v) 0.2 M L-glutamine, 1.5 % (v/v) 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid from Lonza Walkersville, Walkersville, MD, USA), 1.8 % PSG (penicillin-streptomycin-glutamine from Lonza Walkersville, Walkersville, MD, USA), 15 % (v/v) fetal calf serum (FBS), sodium bicarbonate (Lonza Walkersville, Walkersville, MD, USA), and heparin salt (Fisher Bioreagents, Fair Lawn, NJ, USA). Endothelial cell growth supplement (Alfa Aesar, Ward Hill, MA, USA) was added to the supplemented M199 to achieve a final concentration of 40 μg/ml. Full media was stored at 4 °C for use up to 4 weeks. HUVECs were grown to 80 % or greater confluency and were passaged using a 50:50 mixture of trypsin-versene and HBSS (Hank’s Balanced Salt Solution from Lonza Walkersville, Walkersville, MD, USA) before being used in experiments. Only cells between passages 3 and 6 were used.

To stain cells using fluorescence-activated cell sorting antibodies for analysis or sorting, cells were first dissociated into single cells. To obtain single cells from organoids, cell pellets were incubated in 2 ml of Trypsin-Versene (Lonza) for 2 min at 37 °C, mechanically dissociated with rapid pipetting, then incubated with 1 μg/ml DNase (New England BioLabs, Ipswich, MA, USA) for 5 min at 37 °C. Cells were washed with complete media and spun at 1500 rpm for 5 min, then strained through a 0.45-μm cell strainer to obtain single cells. Cells were washed with 1× PBS and resuspended in 400 μl of sorting buffer (2.5% FBS in PBS). To stain cells, 3 μl of each directly labelled antibody was added per 10 million cells and incubated on ice for 1 h, washed with sorting buffer, resuspended in sorting buffer, and sorted using a BD FACSAria cell sorter (BD Biosciences). Antibodies used for sorting experiments were as follows: epithelial cell adhesion molecule (EpCAM)-allophycocyanin (APC) (175791; eBioscience, San Diego, CA, USA), CD24-APC (170242; eBioscience), β1-integrin-eFluor 450 (48-0291; eBioscience), and α6-integrin-eFluor 450 ( 48-0495; eBioscience).

Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA), heat inactivated fetal bovine serum (FBS) from Invitrogen (Carlsbad, CA, USA), and RPMI-1640, Dulbecco’s phosphate buffered saline (DPBS), l-glutamine, trypsin-versene and Hank’s buffered salt solution (HBSS) from Lonza, (Braine-l’Alleud, Belgium). Human recombinant IL-1α ELISA kit was purchased from R&D systems, Minneapolis, MN, USA, CytoTox 96®. The non-radioactive cytotoxicity assay, CellTiter-Glo luminescent cell viability, Cell proliferation ELISA and BrDU chemiluninescent were supplied by Roche (Mannheim, Germany), and Caspase-3/7 assay by Promega (Madison, WI, USA). BDH Chemical Ltd. (Poole, England) supplied Triton-x100 and ICN Biomedicals Inc (Santa Ana, CA, USA) supplied Tween®-20.