Evaluating Fibroblast Viability on Hydrogel Coatings

Dermal fibroblasts were harvested from skin biopsies collected from the experimental animals, as previously described [20 (link)]. Cells were cultured under controlled atmosphere (37 °C, 5% CO₂, humidity) using Dulbecco’s modified Eagle medium (DMEM) enriched with 10% fetal bovine serum and 1% of antibiotic-antimycotic cocktail for cell culture (Life Technologies, Carlsbad, CA, USA). To assess the cytocompatibility of the hydrogels, fibroblasts were seeded in 6-well plates (2.5 × 10⁵ cells per well) with uncoated meshes or meshes coated with HApN or Rif-HApN (n = 3 each). Following a 24-h incubation, cells were harvested with 0.25% trypsin-EDTA (Life Technologies) and centrifuged at 200 g for 7 min. The resulting pellets were resuspended in 2 mL of flow cytometry buffer (R&D Systems, Minneapolis, MN, USA) and centrifuged again. Cells were resuspended in 400 μL of buffer mixed with 10 μL of propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) and the rate of cell viability was determined using a MACSQuant 10 flow cytometer equipped with a 488 nm laser (Miltenyi Biotec, Bergisch Gladbach, Germany). Data were analyzed using the MACSQuant software provided by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany).