4.3. Ex Vivo Skin Culture

Skin from plastic surgery was used for the development of the ex vivo skin culture model. After subcutaneous fat was removed, we obtained 4 mm biopsies which were rapidly placed into a 0.4 μm Transwell chamber (Corning, Somerville, MA, USA) and maintained under semi-liquid culture conditions in skin long-term culture medium (Biopredic, Saint-Grégoire, France) at 37 °C in a 5% CO₂ atmosphere. The biopsies were treated with two different concentrations of Ceramide AD™ cream (0.08% and 0.1%) and with a control cream (cream without Ceramide AD™) in the presence or absence of HDM 100 µg/mL (Stallergenes Greer, Lenoir, NC, USA) and with or without MMP-9 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"Ab142180","term_id":"62162998"}}Ab142180 (Abcam). HDM was administered on top of the skin for 30 min before the cream was brushed directly onto the skin. Twenty-four hours later, the biopsy was frozen in an OCT medium (VWR, Rosny-sous-Bois, France) for subsequent immunofluorescence staining on 7 μm tissue sections and supernatants collected for measurement of MMP-9 activity by ELISA (R&D Systems).