Isolation and Culture of Human Skin Fibroblasts

Fibroblasts were isolated from human skin, obtained by donation with written informed consent. The skin was taken from healthy voluntary donors, using a cylindrical scalpel for 5 mm biopsies, in septic and antiseptic conditions; the skin thus obtained was immediately deposited in Hank solution with an antibiotic. In a laminar flow hood, the skin samples were cut into smaller fragments, and each fragment was grown in Dulbecco’s modified Eagle’s low-glucose medium (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin, and 100 mg/mL gentamicin), all from Gibco BRL (Rockville, MD, USA), and incubated at 37 °C and in 5% CO₂. The culture medium was replaced every two days; after two weeks of culture, the explants (skin fragments) were removed. The fibroblasts were cultured to approximately 80% confluence, and the cells were separated with 0.05%/0.02% trypsin/EDTA and reseeded to generate sufficient cells for the subsequent tests [17 (link)].