Skin substitutes are epidermal, dermal or complete bilayered constructs, composed by natural or synthetic scaffolds and by adherent cells such as fibroblasts, keratinocytes or mesenchymal stem cells. Silk fibroin is a promising polymer to realize scaffolds, since it is biocompatible, biodegradable, and exhibits excellent mechanical properties in terms of tensile strength. Moreover, fibroin can be added of others components in order to modify the biomaterial properties for the purpose. The aim of this work is to prepare silk fibroin films for adipose-derived stem cell (ADSCs) culture as a novel feeder layer for skin tissue engineering. Pectin has been added to promote the protein conformational transition and construct strength, while glycerol as plasticizer, providing biomaterial flexibility. Eighteen formulations were prepared by casting method using fibroin, pectin (range 1-10% w/w), and glycerol (range 0-20% w/w); films were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry assay, to select the optimal composition. A stable fibroin conformation was obtained using 6% w/w pectin, and the best mechanical properties were obtained using 12% w/w glycerol. Films were sterilized, and human ADSCs were seeded and cultured for 15 days. Cells adhere to the support assuming a fibroblastic-like shape and reaching confluence. The ultrastructural analysis evidences typical active-cell features and adhesion structures that promote cell anchorage to the film, thus developing a multilayered cell structure. This construct could be advantageously employed in cutaneous wound healing or where the use of ADSCs scaffold is indicated either in human or veterinary field.The development of skin substitutes dates back to 1975, when Rheinwald and Green (1) cultured keratinocytes on a layer of lethally irradiated 3T3 murine fibroblasts. Currently, this method remains the most reliable and widely used for in vitro keratinocyte culture, but some concerns are emerging about their safety (2-5). To overcome these limits, Sugiyama et al. (6) proposed the use of human irradiated adipose-derived stem cells (ADSCs) as a feeder cell layer: results showed that the morphology of irradiated stem cells was similar to 3T3, and both cell lines (stem and 3T3 cells) expressed genes promoting keratinocyte proliferation. Other researchers also showed that non-irradiated ADSCs can boost epithelialization, angiogenesis (7), capillary density and granulation thickness of transplanted cell-enriched collagen sponges (8). Altman et al. (9) observed that ADSCs, once seeded on acellular dermal matrix and applied on murine wound, spontaneously differentiated along vascular endothelial, fibroblastic and epidermal epithelial lineages and significantly improved wound healing; moreover, cells localize at implantation site.Current cutaneous substitutes are epidermal, dermal or
