Neuronal cell growth on polymeric scaffolds studied by CARS microscopy

For studies of neuronal cell integration and neurite outgrowth in polymeric scaffold materials as a future alternative for the treatment of damages in the neuronal system, we have developed a protocol employing CARS microscopy for imaging of neuronal networks. The benefits of CARS microscopy come here to their best use; (i) the overall three-dimensional (3D) arrangement of multiple cells and their neurites can be visualized without the need for chemical preparations or physical sectioning, potentially affecting the architecture of the soft, fragile scaffolds and (ii) details on the interaction between single cells and scaffold fibrils can be investigated by close-up images at sub-micron resolution. The establishment of biologically more relevant 3D neuronal networks in a soft hydrogel composed of native Extra Cellular Matrix (ECM) components was compared with conventional two-dimensional networks grown on a stiff substrate. Images of cells in the hydrogel scaffold reveal significantly different networking characteristics compared to the 2D networks, raising the question whether the functionality of neurons grown as layers in conventional cultivation dishes represents that of neurons in the central and peripheral nervous systems.