Optimization of protein patterns for neuronal cell culture applications

Abstract

In the present study, we fabricated two-component extracellular matrix protein patterned substrates with fibronectin (FN) and laminin (LN) because of our interest in the mechanism of axonal regeneration and injury in the central and peripheral nervous systems. The authors investigated how the patterning order and method of attachment affected the spatial distribution and biological activity of the immobilized proteins. Micro-contact printing (μCP) techniques in concert with reactive surface chemistry were used to modify glass substrates with one- and two-component films of FN and LN, including micrometer-scale patterns of FN and LN. The composition and spatial distributions of both proteins on the patterned surfaces were characterized by x ray photoelectron spectroscopy, epi-fluorescence microscopy, atomic force microscopy, and time-of-flight secondary-ion mass spectrometry. The authors also characterized the biological activity of the top-most protein layer in a two-layer protein system as well as the ability of the top-most protein layer to mask the biological activity of an underlying protein layer using a fluorescence-based enzyme-linked immunosorbent assay. The order of protein deposition significantly affected the relative biological activity of the upper-most and underlying immobilized proteins. As a result of these optimization studies, maximum biological activity per surface protein was achieved by first immobilizing FN from solution, followed by μCP of LN on the FN. Addition of μCP LN films was able to mask ∼84% of the underlying FN activity, whereas μCP FN films were only able to mask ∼27% of the underlying LN activity.