Nowadays, the culture surfaces used for in vitro testing must be capable of possessing an improved interface for cell interactions and adhesion. For this reason, the materials used need to have an appropriate chemistry and architecture of its surface, resembling to the extracellular matrix. Within this context, in this work we combined the advantages of natural biopolymer characteristics (chitosan) with the flexibility in surface texturing by ultrafast laser for creating functional microstructured surfaces for cell-substrate in vitro studies. A Ti:Sapphire femtosecond laser irradiation (lambda = 775 nm and 387 nm) was used for tailoring surface morphological characteristics of chitosan based films (i.e. polymer "bubbles", "fingertips" and "sponge-like" structures). These structures were investigated by scanning electron microscopy and atomic force microscopy. The morphology of the structures obtained was correlated with the response of oligodendrocytes cells line. In vitro tests on the patterned surface showed that early cell growth was conditioned by the microtopography and indicate possible uses of the structures in biomedical applications. (C) 2013 Elsevier B.V. All rights reserved.