Egf 1

The cutaneous squamous cell carcinoma A431(CRL–1555) and grade IV lung squamous cell carcinoma SW900 (HTB–59) cell lines, were purchased from American Type Culture Collection (ATCC) and grown according to the manufacturer’s suggested conditions in complete medium (1× DMEM (ATCC, 30–2002) for A431cells or 1× ATCC-formulated Leibovitz’s L–15 Medium, Catalog No. 30–2008 medium (ATCC, 30–2001) for SW900 cells) supplemented with 10% FBS and 1× penicillin-streptomycin grown at 37°C in 5% CO2. The cutaneous squamous cell carcinoma SCC13 cell line [81 (link), 82 (link)], were obtained from Harvard Skin Disease Research Center (HSDRC) and grown using the human keratinocyte culture methods provided by the HSDRC. The cutaneous squamous cell carcinoma COLO16 cell line [83 (link)] were grown in DMEM/Ham’s F12 50/50 media supplemented with cholera toxin (Sigma, C0852), insulin (Sigma, I5500-50MG), epidermal growth factor (Serotec/Bio-Rad, EGF-1), hydrocortisone (Sigma, H4881-1G), liothyronine (Sigma, T6397-100MG) and apo-transferrin (Sigma, T2252-100MG). All cell lines were verified human origin and free from pathogens and mycoplasma. Mycoplasma infection monitoring was performed using MycoAlert Detection Kit (Lonza) and only mycoplasma-free cultures were used.

Human placentas from healthy mothers were supplied by the Department of Obstetrics and Gynecology under written consent previously approved by the Ethics Committee at the Hospital Universitario 12 de Octubre. DMSC isolation and culture was performed as previously described [42 (link)]. Briefly, placental membranes were digested with trypsin-versene (Lonza, Spain), and the cells were seeded at 1.2 × 10⁵ cells/cm² and cultured at 37 °C, 5 % CO₂ and 95 % humidity in Dulbecco’s modified Eagle medium (DMEM; Lonza) supplemented with 2 mM L-glutamine, 0.1 mM sodium pyruvate, 55 μM B-mercaptoethanol, 1 % nonessential amino acids, 1 % penicillin/streptomycin, 10 % fetal bovine serum and 10 ng/ml epidermal growth factor 1 (EGF-1; Sigma-Aldrich Química, Spain). The morphology, phenotype and MSC characteristics of DMSCs have been previously reported [42 (link)]. Cells were cryopreserved and, before use, were thawed and passaged at a density of around 5 × 10⁴ cells/cm² until passage 6–8.

Human HT-1080 fibrosarcoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, 2 mM glutamine and penicillin/streptomycin (Corning, Somerville, MA, USA), supplemented with 10% fetal bovine serum (FBS; Capricorn Scientific GmbH, Ebsdorfergrund, Germany). The transformed microvascular endothelial cell line HMEC-1 was kindly supplied by Dr. Arjan W. Griffioen (Maastricht University, The Netherlands). HMEC-1 was cultivated in MCDB-131 (Corning, Somerville, MA, USA) medium supplemented with 1 µg/mL hydrocortisone, 10 ng/mL of EGF-1 (Sigma/Merck, Darmstadt, Germany), 1% penicillin/streptomycin solution, 2 mM L-glutamine and 10% FBS. Cells were kept in a humid incubator at 37 °C under conditions of 5% carbon dioxide. Subconfluent (at 75–80% of confluency) cells were used for subculturing and for treatments and experiments.