Controlled Outgrowth of Dissociated Neurons on Patterned Substrates

The cytoarchitecture of neurons and their support cells observed in vivo is lost during the procedures used to form primary cell cultures. As a step toward establishing an ordered arrangement of these cells in vitro, we developed a set of procedures for patterning the outgrowth of cells cultured on two-dimensional substrates. These procedures used a combination of surface chemistry and photolithographic techniques. The adhesive properties of either silicon or quartz surfaces were controlled by covalently binding organic molecules to the surface with silane coupling agents. The attachment and growth of either embryonic mouse spinal cells or perinatal rat cerebellar cells were found to be promoted by binding certain amine derivatives to the surface. The attachment of cells to a substrate was inhibited by binding alkane chains to the surface and plating the cells in media containing serum. Patterns of selected adhesivity were formed using photochemical resist materials and lithographic masking techniques compatible with the silane chemistry. Cultures of either cell type could be confined to square regions on the scale of 50microns. Cerebellar cells could be confined to grow on lines with widths less than 10microns. This width is comparable to the diameter of granule cell somata. The patterned growth of cerebellar cells was maintained at this resolution up to 2 weeks in vitro. Over this time period the granule cells developed electrical excitability and immunoreactivity for neuron specific enolase. Immunochemical reactivity of the patterned cultures for glia fibrillary acid protein showed that glia are patterned along with the associated granule cells.