Ebm 2 plus singlequots of growth supplements

The process of ECFC isolation from PBMCs is illustrated in Figure S1. PBMCs were plated on collagen I‐coated (Corning, Corning, NY) 25‐cm² tissue culture–treated Falcon flasks (Thermo Fisher Scientific, Waltham, MA) at a density of 5.0×10⁶ cells/flask following standard protocols.6 (link), 31 (link) Cultured cells were maintained in a standard cell culture condition (humidified chamber, maintained at 37 °C with 21% O₂ and 5% CO₂), using complete endothelial cell growth basal medium‐2 (EBM‐2 plus SingleQuots of Growth Supplements; Lonza, Basel, Switzerland) supplemented with 1% penicillin/streptomycin (Gibco by Life Technologies) and 10% FBS (Invitrogen). Media were changed every 2 or 3 days, and cells were maintained for up to 30 days in culture. PBMC cultures were observed daily from days 6 to 30 to determine the first day of cobblestone patterned ECFC colony formation (Figure S1). Once ECFC colonies were identified, they were expanded for no longer than 10 days to control the time of colony formation. These cells were then passaged and plated in similar conditions for all individuals (ie, same surface area in collagen‐coated flasks and equal cell density). ECFC colonies were then passaged and further expanded under similar conditions. ECFC function was assessed using cells from the second passage.

Human umbilical vein endothelial cells (HUVEC) (Lonza Walkersville, Inc.), human small airway epithelial cells (SAEC) (Lonza Walkersville, Inc., Walkersville, MD), and skeletal muscle cells (SkMC) (Lonza Walkersville, Inc.) were harvested from cryopreserved batches from our laboratory, plated and grown confluent with the following mediums: EGM-2 Bulletkit, EBM-2 plus SingleQuots® of growth supplements (CC-3162) (Lonza Walkersville, Inc.), SAGM Bulletkit, SABM plus SingleQuots® of growth supplements (CC-3118) (Lonza Walkersville, Inc.), and skeletal muscle cell supplemented growth medium (Cat No. 151–500) (Cell Applications, Inc.).