Adhesion and Proliferation of Normal Human Dermal Fibroblasts

Adhesion and proliferation assay was carried out using normal human dermal fibroblasts (NHDF) from juvenile foreskin (PromoCell, VWR, Milan, Italy) [9 (link),10 (link)]. Fibroblasts were grown in the presence of 150 µL Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich, Milan, Italy) supplemented with 10% v/v fetal bovine serum (Euroclone, Milan, Italy), and with penicillin/streptomycin solution (pen/strep, 100 UI/100 μg/mL, Sigma-Aldrich, Merck, Milan, Italy), at 37 °C in a 5% CO₂ atmosphere with 95% relative humidity. The 0.36 cm² circular portion scaffolds were placed at the bottom of the wells in a 96-well plate (flat bottom, Cellstar©, Greiner bio-one, Frickenhausen, Germany). Fibroblasts were seeded onto the scaffolds at a seeding density of 35,000 cells/well and grown for 3 or 6 days. The cell growth without scaffolds (35,000 cells/well) was considered the standard growth (growth medium (GM)). After 3 or 6 days, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays were performed. The fibroblasts that adhered and grew onto the scaffolds (growth for 6 days) were fixed for 2 h at 4 °C, using 3% w/v of glutaraldehyde in Dulbecco’s phosphate buffered saline (PBS, Sigma-Aldrich, Milan, Italy) and analyzed by SEM and confocal laser scanning microscopy (CLSM), as described in the following paragraphs.