Adhesive micro-lines of various sub-cellular geometries were created using a non-traditional micro stamping technique. This technique employed the use of commercially available diffraction gratings as the molds for the micro stamps, a method which is quick and inexpensive, and which could easily be adopted as a patterning tool in a variety of research efforts. The atypical saw-tooth profile of the micro stamps enabled a unique degree of control and flexibility over patterned line and gap widths. Cortical neurons cultured on patterned poly-lysine micro-lines on PDMS exhibit a startling transition in axonal guidance: From the expected parallel guidance to an unexpected perpendicular guidance that becomes dominant as patterned lines and gaps become sufficiently narrow. This transition is most obvious when the lines are narrow relative to gaps, while the periodicity of the pattern is reduced. Axons growing perpendicular to micro-lines exhibited ‘vinculated’ growth, a unique morphological phenotype consisting of periodic orthogonal extensions along the axon.
Studying how individual neuronal cells grow and interact with each other is of fundamental importance for understanding the functions of the nervous system. However, the mechanism of axonal navigation to their target region and their specific interactions with guidance factors such as membrane-bound proteins, chemical and temperature gradients, mechanical guidance cues, etc. are not well understood. Here we describe a new approach for controlling the adhesion, growth and interconnectivity of cortical neurons on Au surfaces. Specifically, we use Atomic Force Microscopy (AFM) nanolithography to immobilize growth-factor proteins at well-defined locations on Au surfaces. These surface-immobilized proteins act as a) adhesion proteins for neuronal cells (i.e. well-defined locations where the cells “stick” to the surface), and b) promoters/inhibitors for the growth of neurites. Our results show that protein patterns can be used to confine neuronal cells and to control their growth and interconnectivity on Au surfaces. We also show that AFM nanolithography presents unique advantages for this type of work, such as high degree of control over location and shape of the protein patterns, and application of proteins in aqueous solutions (protein buffers), such that the proteins are very likely to retain their folding conformation/bioactivity.
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