Engineering the morphology and electrophysiological parameters of cultured neurons by microfluidic surface patterning.

The ability to control the orientation, morphology, and electrophysiological characteristics of neurons in culture allows the construction of neural circuits with defined physiological properties. Using microfluidic protein deposition onto chemically modified glass, we achieve the controlled growth of Aplysia neurons on geometrical patterns of poly-L-lysine and collagen IV, surrounded by nonadhesive regions of bovine albumin. We investigate the parameters essential for forming functional neuronal networks, the morphology, biochemistry, and electrophysiology under engineered cell culture conditions. We demonstrate that not only the orientation of neurite extension but also the number of primary neurites originating from the cell soma, their length, and branching pattern depend on the spatial constraints presented by the size and shape of the adhesion region on the patterned substrate. In addition, the physicochemical properties of the support layer influence the electrical activity of the cultured neurons. Substrate-dependent changes in the amplitude and in the dynamic parameters of the action potential cause decreased spike broadening in patterned neurons, which reflects changes in the number or functioning of active membrane ion channels. In contrast to morphology and electrophysiology, the neuropeptide content, as determined by mass spectrometry of individual patterned neurons, is not affected by the growth on patterned surfaces. Our results suggest that the morphological and electrophysiological parameters of neurons can be predictably altered/engineered by modulation of the chemical, physical, and topographical features of culture substrates. We also demonstrate that a full suite of techniques is required for functional characterization of neurons on engineered substrates.