Isolation and Culture of Human Fibroblasts

Redundant skin tissue samples from abdominoplasty were obtained from three consenting healthy patients (n = 3) and were processed as described elsewhere [36 (link)]. The samples were digested with 0.6% type I collagenase (Worthington, NJ, USA) for 4–6 h, followed by 8–10 min cell dissociation using 0.05% trypsin-EDTA (Gibco, Carlsbad, CA, USA). The cells were then re-suspended in co-culture medium (equivalent mixture of EpiLife (Gibco) and F12:DMEM (1:1; FD; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Gibco)) and seeded in 6-well culture plates (Greiner Bio-One, Monroe, NC, USA) at 37 °C in 5% CO₂. The medium was replaced every 2–3 days. The fibroblasts were removed when the cells were 70–80% confluent and were sub-cultured in a T75 flask (Nunc, Rochester, NY, USA) using FD+10% FBS until passage 3 (P3).