Insulin like growth factor

Human umbilical vein endothelial cells (HUVECs) from an infant male Caucasian donor were obtained cryopreserved at passage 1 (Pro-moCell GmbH) and expanded to passage 7 in a 5% CO2/37°C atmosphere. This study abides by all criteria of the UK Human Tissue Act. HUVECs were expanded using MCBD 131 medium (Life Technologies) supplemented with 5% v/v fetal bovine serum (FBS; ThermoFisher Scientific); 1% v/v L-glutamine; 1% v/v penicillin/streptomycin (Life Technologies); 1 mg/L hydrocortisone; 50 mg/L ascorbic acid (Sigma); 2 μg/L fibroblast growth factor (PeproTech); 10 μg/L epidermal growth factor (PeproTech); 2 μg/L insulin-like growth factor (PeproTech); and 1 μg/L vascular endothelial growth factor (PeproTech). All cells were subsequently cultured with this complete media.

Human alveolar epithelial cell line (A549) was obtained from ATCC (CCL-185). The cells were cultured in DMEM with 10% fetal bovine serum (GIBCO Laboratories, Grand Island, NY), penicillin [100 U/ml] and streptomycin [100 mg/ml] at 37°C in a gas mixture of 5% CO₂ / 95% air in 25 cm² culture flasks (T-25; Corning, Costar). After reaching early confluence the cells were trypsinized and plated for experiments.
Lung epithelial cell lines from mice (MLE-12; CRL-2110) and rat (RLE-6TN; CRL-2300) were purchased from ATCC (Manassas, VA). MLE-12 cells were cultured in DMEM: F-12 medium with insulin (5 ng/ml), transferrin (0.01 mg/ml), sodium selenite (30 nM, hydrocortisone (10 nM), B-estradiol (10 nM), HEPES 10 nM, glutamine and 10% FBS (GIBCO Laboratories, Grand Island, NY). RLE-6TN cells were grown in Ham's F12 medium supplemented with bovine pituitary extract (0.01 mg/ml), insulin (5 ng/ml), insulin-like growth factor (2.5 ng/ml), transferrin (1.25 μg/ml), EGF (2.5 ng/ml), and 10% SFB (GIBCO Laboratories, Grand Island, NY).