Cell cultures

To evaluate the biological properties of HA hybrid complexes, human dermal keratinocytes (HaCat) and fibroblasts (HDF) cultures, were used as cellular models of human skin. HaCaT, a spontaneously transformed non-tumorigenic human keratinocytes cell line was provided by Istituto Zooprofilattico, Brescia, Italy and the cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% (v/v) heat inactivated Fetal Bovine Serum (FBS), penicillin 100 U/ml and streptomycin 100 μg/ml. DMEM, FBS, Pen-Strep PBS and Trypsin were provided by Gibco Invitrogen (Milan, Italy). A human dermal fibroblasts cell line immortalized with hTERT (HDF cells, BJ-5ta, ATCC CRL-4001), was cultured in a 4:1 mixture of DMEM and Medium199 supplemented with 0.01mg/ml hygromycin B and 10% (v/v) FBS. All materials for HDF culture were purchased from ATCC (USA). The cells were grown on tissue culture plates (BD Falcon, Italy), using an incubator with a humidified atmosphere (95% air/5%CO₂ v/v) at 37°C. For the gene expression analyses, human keratinocytes and fibroblasts were grown in different cell cultures. More in detail, 3.75x10⁴cells/cm² in a standard 24-well culture plate were seeded. For the immunofluorescence staining, HaCat/HDF co-cultures were used. In this model, human keratinocytes and fibroblasts were seeded, cultured together and then analyzed on a glass microscope slide. μ-dish (35 mm, high) culture-insert (Ibidi, Integrated BioDiagnostics, Munich, Germany) were used to separate the two cell populations and to allow cell interaction. Cells were seeded directly on chamber affixed to a specially glass microscope slide (BD Falcon™ BD Biosciences) at a ratio of 1:1.25 HaCat:HDF (1x10³ /cm² and 1.25x10³ /cm² respectively). In both models, cells were treated with H-HA 1400kDa (0.16% w/w), L-HA 100kDa (0.16% w/w) and hybrid cooperative complexes (H-HA/L-HA complexes 0.16% w/w). Treatments lasted 4 and 24h for the gene expression analyses and 24h for the fluorescence staining.