Can substrate stiffness nano-gradient drive glial cell migration?
Nanoscience Foundries&Fine Analysis (NFFA) ID321
Principal investigator: doc.dr.sc. Jelena Ban
Summary
Cell response to enviromental cues has been investigated for many years and was mainly focused on chemical factors. Recently the role of mechanical factors, such as substrate topography, rigidity, roughness and the role of forces applied to the cell has been increasingly considered. Indeed, the field of mechanobiology has grown considerably in the last decade and in our previous study we showed that the substrate stiffness is the key factor for the neuronal differentiation of embryonic stem cells(1). Therefore, the role of substrate stiffness is of particular importance for neurons, while the effect on glial cell growth and differentiation is less known. There are several reasons why glial cells deserve more attention. Astrocytes, CNS glial cells, are fundamental for maintaining the homeostasis of neuronal cells both in vivo and in vitro. During development, neurons migrate using radial glia as platform. The number, dimension and complexity of glial cells progressively increases with phylogeny. Following injury, formation of glial scar occurs, inhibiting neuroregeneration. Finally, most of the brain tumors develop from glial cells. For all these reasons, in this project, primary glial cultures (postnatal cortical astrocytes) will be used and role of the substrate stiffness on their adhesion, migration and differentiation will be investigated. In particular, focal adhesions (FAs) formation, structure and composition, stress fibers orientation and glial cell migration will be analysed, and that could be of fundamental importance to get more reliable in vitro cell model of the CNS.
Polydimethylsiloxane (PDMS) will be the material of choice because of its biocompatibility (previously tested and confirmed), but mostly for the possibility to modulate the stiffness with the substrate geometry. Array of pillars with different height, ranging from 10 to 100 µm will produce a gradient of different stiffness.
For fabrication of PDMS substrates with gradient of stiffness, Electron beam lithography (EBL, step 1), ultraviolet litography (UVL, step 2) nanoimprinting litorgraphy (NIL, step 3), inductively coupled plasma (ICP, step 4) and atomic force microscopy (AFM, step 5) will be used.
For control experiment, standard glass coverslips (hard substrate) will be compared with PDMS with different degree of stiffness.
Primary glial cells will be morphologically tested using markers for FAs (such as integrin, paxillin, vinculin) and for cytoskeleton (such as filamentous actin, vimentin, glial fibrillary acidic protein (GFAP)) by immunocytochemistry using fluorescent microscopy. We will analyze glial cell shape, size, process extension, adhesion, proliferation and survival along stiffness gradient. For glial cell migration, time lapse videoimaging experiments will be performed. We expect different migration rate depending on substrates stiffness.
The results obtained could be applied to different cell types and for different cell processes, such as neuronal stem cells proliferation and differentiation, important for development of new approaches in treatments of CNS lesions (cell-based regenerative therapy).
1: Migliorini et al. Biotechnol Bioeng. 2013 Aug;110(8):2301-10.