Controlled outgrowth of dissociated neurons on patterned substrates

Abstract

The cytoarchitecture of nervous tissue is lost during the dissociation procedures used to form primary cell cultures. As a first step toward reestablishing an ordered arrangement of these cells in vitro, we developed a set of procedures for patterning the outgrowth of cells cultured on P-dimensional substrates. These procedures used a combination of sur-face chemistry and photolithographic techniques. The ad-hesive properties of either silicon or silicon dioxide (quartz) surfaces were controlled by covalently binding small 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 pro-moted by binding certain amine derivatives to the surface. In particular, cells grown on surfaces bound with diamines and triamines, but not with monoamines, formed cultures whose morphology was similar to that of cells cultured on conventional substrates, i.e., glass coated with poly(~-ly-sine). The attachment of cells to a substrate was inhibited by binding alkane chains (e.g., n-tetradecane) to the surface and plating the cells in media containing 5-10 % (vol/vol) serum. Patterns of selected adhesivity were formed using pho-tochemical resist materials and lithographic masking tech-niques compatible with the silane chemistry. Cultures of either spinal cord cells or cerebellar cells could be confined to square regions on the scale of 50 Am. Cerebellar cells could be confined to grow on lines with widths less than 10 Am. This width is comparable to the diameter of granule cell somata. The patterned growth of cerebellar cells was main-tained up to 12 d in vitro. Over this time period the granule cells were observed to develop electrical excitability and immunoreactivity for neuron-specific enolase. Purkinje neu-rons also developed electrical excitability when grown on the chemically modified surfaces. lmmunochemical reactiv-ity of the patterned cultures for glial fibrillary acid protei