Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Chemistry

Language

English (en)

Date of Award

7-30-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Joshua A Maurer

Abstract

The following work discusses the development of several techniques and new methods for the production of patterned surfaces for protein and cell confinement. These well-defined substrates allow us to study the mechanism of axonal differentiation in neurons confined to a two-dimensional starburst pattern. We utilize self-assembled monolayer: SAM) chemistry in conjunction with microcontact printing to create stable patterned substrates for cell culture. Photolithography is employed in the fabrication of patterned masters, which are used to create elastomeric stamps for microcontact printing.

Initially, trichlorosilanes were employed in our patterned SAMs because they react rapidly with glass. These patterned surfaces confined protein and cells to a defined pattern; however, trichlorosilane monomers were difficult to work with because of their extreme reactivity with moisture in the air. An alternative to this highly sensitive system was required to develop stable SAMs. Alkanethiols on gold have traditionally been stable for just 5−7 days in cell culture, but modifications to the linkage between the alkane chain and glycol termination led to the formation of a stable self-assembled monolayer for over five weeks. This is a tremendous advance in the field of SAM chemistry and allows for the study of cellular processes that occur over the course of several weeks.

While long-term stability is necessary for the study of developmental events, there are many researchers who do not have the resources to fabricate their own patterned substrates. This led to the development of recyclable, reusable patterned SAMs for cell culture. By utilizing two different methods, either a trypsin analog or detergent, these substrates can be reused up to 11 times over the course of two weeks. This allows investigators to perform several studies on the same patterned substrate, which leads to rapid, reproducible results.

The interesting biological question we set out to answer was whether axonal differentiation was an innate process or one that was environmentally determined. We cultured E18 mouse hippocampal neurons on starburst patterned substrates. The starburst consisted of twelve paths of equal width; eleven were short, 20 μm paths and one was longer, ranging from 40 μm to 160 μm. We observed which path the axon grew along by immunostaining for the microtubule-associated tau protein, bound to microtubules in the axon. Our data showed that the axon grows along the long path ~58% of the time for the smallest starburst pattern and the distance a neurite is allowed to extend down a path is linearly correlated to the likelihood of finding the axon on the long path. This points toward axonal differentiation being an environmentally determined process.

This combination of photolithography, microcontact printing, and self-assembled monolayer chemistry has led to important advances in the production of stable, patterned substrates for cell culture. We have successfully used this technology to study axonal differentiation and have found that this process is environmentally determined.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7B27S97

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