CHEMICALLY MODIFYING GLASS SURFACES TO STUDY SUBSTRATUM-GUIDED NEURITE OUTGROWTH IN CULTURE

We describe here a modification procedure for chemically fabricating n euron adhesive substrates to study the substratum-guided neurite outgr owth in culture. These substrates were fabricated by chemically attach ing a synthetic peptide derived from a neurite-outgrowth-promoting dom ain of the B2 chain of laminin. The attachment was carried out by coup ling the peptide to an amine-derived glass surface using a heterobifun ctional crosslinker. Hippocampal neurons were dissociated from embryon ic rats and placed on the substrate at low-density in a chemically def ined medium to examine the direct effect of the modified surface on th eir outgrowth. We observed that the neurons developed a morphology typ ical to that of hippocampal neurons having multiple short and single l ong processes within 24 h in culture. The chemical modification proced ure was then combined with a UV-photo-masking technique to fabricate p atterns of peptide surfaces on glass substrates. By culturing the hipp ocampal neurons on substrates having alternate stripes of peptide surf ace and non-adhesive surface, we demonstrated substratum-controlled ch anges in the neuronal morphology. The modification procedure presented here can be easily achieved in the standard culture facility and shou ld be useful in fabricating an in vitro tool for studying substratum-g uided neurite outgrowth.