A crucial step of Tissue Engineering (TE) approach is the fabrication of 3-D biodegradable scaffolds. It has been achieved using various techniques, such as gas foaming, fiber bonding, solvent casting/particulate leaching, phase separation and 3D-printing. Each technique presents specific advantages and disadvantages; but, all of them share the difficulty to obtain simultaneously the macro, micro and nanostructure. In this work, a Supercritical Freeze Extraction Process (SFEP) is proposed for the formation of chitosan structures suitable for TE applications. We showed that it is possible to produce chitosan scaffolds characterized by a micrometric cellular structure, nanofibrous sub-structure and porous surfaces. The low process temperature allows to obtain 3-D solids, whose structure is preserved during supercritical drying. Preliminary results on cell cultivation confirmed that the generated chitosan scaffolds are characterized by a morphology that is potentially suitable for TE applications. A good cell adhesion was obtained and a large percentage of living cells was observed. This result can depend on the micrometric morphology of the scaffolds, that assures a good nutrient diffusion, and on the nanometric sub-structure that allows an adequate cells adhesion. (C) 2014 Elsevier B.V. All rights reserved.