Characterization of the Neuron/Substrate Interface of Long Term 2d Neural Networks Obtained by Nano Drop Printing

Abstract

Neuronal networks produced in cell culture are a unique tool to provide information about synapses formation, development and functionality. Several geometrical, physical and chemical cues play a role in neuron adhesion and neurite outgrowth. We have used a nano-drop method to pattern agarose coated surfaces with an adhesion protein (PDL) and we have subsequently monitored the growth of neural networks. Depending on its thickness, the intermixed agarose/PDL layer was proved to be a successful substrate to confine the neural network and to guide its growth. In particular, for thickness of the polymer cushion below 100 nm, neurons formed connections and remained in morphologically healthy conditions up to 21 DIV. The correlation between surface properties (morphology, stiffness, roughness, charge) and neuron adhesion and survival was investigated by AFM. Force-Volume mapping of the surface showed a dominating effect of surface stiffness vs topography. In order to allow connectivity, a critical distance of 80 μm between spots of adhesion protein was found. Finally, the functionality at 21 DIV of 2D networks grown on agarose/PDL substrates patterned by micro contact printing was proved by patch clamp and by the measurements of the basal activity on micro electrode arrays.