Chemically modifying glass surfaces to study substratum-guided neurite outgrowth in culture

We describe here a modification procedure for chemically fabricating neuron adhesive substrates to study the substratum-guided neurite outgrowth in culture. These substrates were fabricated by chemically attaching a synthetic peptide derived from a neurite-outgrowth-promoting domain of the B2 chain of laminin. The attachment was carried out by coupling the peptide to an amine-derived glass surface using a heterobifunctional crosslinker. Hippocampal neurons were dissociated from embryonic rats and placed on the substrate at low-density in a chemically defined medium to examine the direct effect of the modified surface on their outgrowth. We observed that the neurons developed a morphology typical to that of hippocampal neurons having multiple short and single long processes within 24 h in culture. The chemical modification procedure was then combined with a UV-photo-masking technique to fabricate patterns of peptide surfaces on glass substrates. By culturing the hippocampal neurons on substrates having alternate stripes of peptide surface and non-adhesive surface, we demonstrated substratum-controlled changes in the neuronal morphology. The modification procedure presented here can be easily achieved in the standard culture facility and should be useful in fabricating an in vitro tool for studying substratum-guided neurite outgrowth.