Elastomeric Stamping: Design Considerations for Long -Term Maintenance of Neuronal Networks

This work describes the development of a technique for high-resolution patterning of proteins or biomolecules on glass substrates for the localization of neurons and their axons and dendrites. The new patterning process uses a microfabricated polydimethylsiloxane (PDMS) stamp with micrometer length features to transfer multiple types of biomolecules to surfaces prepared with (3-aminopropyl)silane (3-APS) or (3-mercaptopropyl)silane (3-MPS) using glutaraldehyde or N-gamma-maleimidobutyryloxysuccinimide ester (sulfo-GMBS) reagents as cross-linkers. PDMS microstamps were used to covalently attach poly-D-lysine (PDL) on glass and silicon dioxide substrates, where polyethylene glycol (PEG) or serine was applied to the background regions. The thickness of each film was measured using ellipsometry and atomic force microscopy (AFM), from which the surface concentrations and grafting density were calculated. Fourier transform infrared spectroscopy was used to examine the structure and organization of the organosilane surfaces. Long-term film stability was evaluated using ellipsometry and static water contact angles. As a biological assay, B104 neuroblastoma cells were plated on microstamped glass coverslips. Results indicated that the stamping and photoresist patterning procedures are equivalent, having an average pattern compliance of 52.6 +/- 4.4% compared to 54.6 +/- 8.1% for physisorbed substrates. For hippocampal pyramidal neurons to attach and remain in precise micropatterns for weeks in culture, background molecules that remain nonpermissive for extended culture durations need to be identified. Nonpermissive background molecules of either PEG or the amino acid serine (C3H7NO3) were evaluated. The foreground regions were microstamped with PDL. After 29 days in culture the foreground compliance in the PDL regions averaged 86% for serine and 90% for PEG, with only a small decline. The background compliance, or the fraction of square areas in the pattern background which were free of neurite extension, declined from highs of 40% and 55% (midculture) to 5.5% and 12% (29 days) for serine and PEG, respectively. Images of the cultures suggest that PEG is significantly more effective as a nonpermissive substrate. Ellipsometry confirmed long-term maintenance of background chemistry and grafted PEG, with decline in thickness occurring at day 25. In conclusion, these materials, especially PEG, are adequate for the maintenance of long-term patterned cultures of neurons.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics and Computational Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Branch, Darren Waltz

Contributors dc:contributor

• Wheeler, Bruce C.

• Biology, Cell

Language dc:language

eng

Identifier

(MiAaPQ)AAI9955591

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/85495