Fibroblast Cell Culture Protocols

Fibroblast cell strains of three genetic backgrounds and two lineages were used in this study: normal human dermal fibroblasts (NHDF; Heidelburg, Germany), human diploid foetal lung fibroblasts (MRC-5; Coriell Institute for Medical Research) and neonatal foreskin fibroblasts (HF043; Dundee Cell Products, UK). Standard culture conditions were a seeding density of 6 × 10⁴ cells/cm² in media (C-23020, Promocell, Heidelburg, Germany) containing 1% penicillin and streptomycin, and a fibroblast-specific supplement mix consisting of foetal calf serum (3% v/v), recombinant fibroblast growth factor (1 ng/ml) and recombinant human insulin (5 μg/ml) (Promocell, Heidelburg, Germany). For the assays requiring senescent cultures, cells were counted and equal numbers of cells seeded at each passage until the growth of the culture slowed to less than 0.5 PD/week as previously described [2 (link)] (this occurred at PD = 64 (NHDF), 65 (MRC-5) and 64 (HF043). Viable cell numbers were determined at each passage by trypan blue staining. For cultures grown under serum starvation conditions, cells were maintained in DMEM (Sigma Aldrich, Dorset, UK) supplemented with 0.1% of serum and 1% penicillin and streptomycin in the absence of fibroblast-specific supplement, for 24 h prior to treatment.