Human dermal microvascular endothelial cells

Human dermal microvascular endothelial cells (PromoCell, Heidelberg, Germany) were seeded in quadruplicates in a 96-well plate at 12,000 cells/cm² in Endothelial Cell Growth Medium MV2 (MV2; PromoCell). After reaching confluency, the monolayers were scratched using the Wounding Pin Tool (V&P Scientific, Radway Green, United Kingdom), and the MV2 media was added as negative control, α-MEM supplemented with 10% FCS and antibiotics was added as vehicle control, and the conditioned media from P2 ASCs (C_FSC) or sorted subpopulations (SP1–4) were added for testing the wound-healing properties. The conditioned media were based on α-MEM supplemented with 10% FCS and antibiotics and produced during a 3-day culture period prior to the end of P2. All FCS used in this study originated from the same batch and lot. The progress of damage reparation was monitored by time-lapse microscopy every 2 hours for a total of 12 hours using a Zeiss Axio Observer.Z1 microscope equipped with an AxioCam MRm camera and AxioVision software package (Carl Zeiss, Birkerød, Denmark). Relative wound size at each time point was analyzed using the TScratch software [38 (link)].

Human dermal microvascular endothelial cells (Promo Cell, Heidelberg, Germany) were incubated in endothelial cell growth medium (Promo Cell). N/TERT-1 keratinocytes derived from normal human epidermal keratinocytes and immortalized with the telomerase catalytic subunit were used for experiments. Cells were cultured in keratinocyte serum-free medium (Life Technologies) supplemented with 0.2 ng/ml of epidermal growth factor (EGF), 25 mg/ml of bovine pituitary extract, 0.4 mM CaCl2 and penicillin/streptomycin. Both cells then incubated for 24 h in fresh growth medium containing 2% BSA in the absence or presence of 500 μmol/l palmitic acid, stearic acid, oleic acid and linoleic acid (Nacalai Tesque). These doses are well within the physiological range for free fatty acid concentrations reported for rodents and humans^{35 (link)}. Each fatty acid was dissolved in ethanol and diluted 1:100 in endothelial cell growth medium containing 2% (wt/vol) fatty acid–free BSA (Calbiochem, San Diego, Calif) before being added to the cells. Control cells received vehicle.