Multi-protein Microcontact Printing of Surfaces for Neuronal Regeneration Studies

Central nervous system (CNS) injuries can be dramatically life altering and only 0.9% of the estimated 250 to 400 thousand individuals currently living with CNS impairment are expected to make a full recovery. The direction of neuronal outgrowth by the spatial distribution of axonal guidance molecules on aligned astrocytes suggests a novel way to achieve neuronal regeneration. Two proteins expressed by astrocytes that are of particular interest are laminin (permissive) and a chondroitin sulfate proteoglycan, aggrecan (inhibitory). This report addresses how to recreate their spatial distribution from the astrocyte’s membrane on artificial substrates for in vitro neuronal regeneration studies. The essential problem was to design novel surface patterning protocol that allows for patterning multiple proteins on surfaces in registry. We show that patterns of multiple proteins can be created by sequential microcontact printing (µCP) of laminin and aggrecan with µm precision. The biomimetic stamps were based on high resolution images of fluorescently tagged astrocytes and are fabricated out of PDMS using lithography techniques. The stamps were then mounted on a microscope stage while the surface to be stamped was placed on a microscope objective and kept at its focal distance. This geometry allowed for visualization of protein patterns during the multiple stamping events and resulted in multiple protein surface patterns in registry with µm precision.