Micrometer resolution silane-based patterning of hippocampal neurons: critical variables in photoresist and laser ablation processes for substrate fabrication.

Toward the goal of creating patterns of primary hippocampal neurons in low density culture, we investigated techniques to fabricate microminiature grids of organofunctional silanes on glassy surfaces. A new photoresist (PR) process, Selective Silane Removal (SSR), was developed and compared to two previously developed techniques which use PR and laser patterning. The grid patterns consisted of 27 combinations of path width, length, and intersection (node diameter). The background consisted of squares bounded by the paths. The best neuron patterning was observed on substrates produced by the SSR process where cytophilic aminosilane is uniformly deposited and selectively removed from the background. Controlling water during aminosilane deposition was critical to good neuronal growth and patterning. Oxygen plasma etching of background regions prior to cytophobic phenylsilane binding significantly reduced off-pattern cell growth. Up to 90% of somata grown on these substrates complied to the pattern, and an average of 77% of background regions were free of neurites or cells connected to the pattern. The highest laser energy density, 120 mJ/cm2, produced the best compliance on lased substrates, with an average of 35% of background regions free of connected cells and neurites, but considerable variation across the surface. On substrates with excellent patterning, compliance to nodes was found to be dependent on pattern dimensions, with 20-micron node diameters and 80-micron internodal path lengths increasing compliance.