Exploring axonal biology using a novel microfluidics-based neuronal culture platform.

摘要

This dissertation describes the development, characterization, and use of a novel micro fluidics-based neuronal culture platform to investigate axonal biology. The culture platform was fabricated using micro fabrication and soft lithography technologies and consists of a microgroove-embedded barrier which allows axons to penetrate the barrier, but prevents the passage of larger cell bodies. The use of passive microfluidics allows the isolation of distinct micro environments to axons or cell bodies. Advantages of this culture platform include, but are not limited to, biocompatibility, optical transparency, batch processing, simplified processing, and reproducibility. In this dissertation, the culture platform is used for investigation of axonal biology in the central nervous system (CNS). Long axonal tracts are characteristic of the human brain and axonal deficits have been linked to numerous neurodegenerative diseases, including Alzheimer's, Huntington's, Parkinson's, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis. The study of axonal biology is also relevant to research in traumatic brain injury (TBI) and stroke, where axonal regeneration is prevented due to secondary damage mechanisms. In Chapter 1, I describe the motivation for developing an in vitro method to study CNS axons. Chapter 2 focuses on the development and characterization of the platform and includes analytical and experimental results showing that microenvironments of small molecular weight compounds can be established for extended periods of time using passive microfluidics. Chapter 3 demonstrates several experimental paradigms using the platform. The platform is an improvement over current techniques for the study of axonal injury and regeneration, axonal transport, and for biochemical analysis of axons. The platform also permits the establishment of axonally restricted cocultures. In Chapter 4, I use the platform to compare gene expression levels in axons as they mature using real-time PCR. In Chapter 5 Affymetrix GeneChips® are used to analyze and categorize CNS axonal mRNAs. The identification of axonal mRNAs is an important step to investigate functions of local protein synthesis and has not been possible using previous methods. Chapter 6 concludes with a discussion of possible future directions of this work.