Penicillin streptomycin antibiotic antifungal cocktail

Silver nanoparticles, obtained by chemical reduction method (patent RU 2 638 716 C2), were provided by LLC "M9". Polyvinyl alcohol 0.7% (PVA), sodium carboxymethylcellulose 0.01% (CMC), sodium dodecyl sulfate 0.15% (SDS), 0.15% sodium oleate (Ole Na) and 2% agarose (AgA) provided by LLC "M9" were used as stabilizers. Minimum Essential Medium (MEM, Sigma-Aldrich), fetal bovine serum (FBS, Hyclone), penicillin-streptomycin antibiotic antifungal cocktail (Sigma-Aldrich), 0.05% trypsin with EDTA (Life technologies). Ultrapure water (resistivity >18.2 MΩ·cm) was used for all experiments.

The hospital’s committee of ethics approved this study, and informed consent was obtained from all subjects. Primary normal human dermal fibroblasts (NHDF) were isolated from skin biopsy of a healthy young male donor (Municipal Clinical Hospital No. 7, Saratov, Russian)by sequential trypsin and collagenase digestion and were expanded in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA), containing 10% fetal bovine serum (FBS, Hyclone), 2 mM l-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and 1% penicillin–streptomycin antibiotic antifungal cocktail (Sigma-Aldrich, St. Louis, MO, USA). Cell stocks were frozen in complete DMEM containing 10% dimethylsulfoxide and kept in liquid nitrogen until use. Cells were revived by thawing at 37 °C and were further propagated in DMEM complete. NHDF were used in passages 2–6. The media were replaced every three days, and the cells were maintained in a humidified incubator at 37 °C with 5% CO₂. Cell cultures with 75–85% confluence were harvested using 0.25% trypsin (Life Technologies, Delhi, India) and counted with a hemocytometer.