Controlling the Physico-Chemical Microenvironment for Low-Density Neuronal Analysis in Cultures and Microfluidics

129 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.From the inception of neuron cultures, in vitro, technological advancements have emerged to improve access to developing cells and tissues. The fundamental technological shifts in the ability to culture neurons over the past century are addressed, beginning with the first cultures in the hanging drop by Ross G. Harrison to the modern methods of microislands, Campenot chambers, patterned substrates and microfluidics.Through direct experimentation, we develop conditions for controlling neurons and the extracellular environment in vitro, to include, culturing neurons in nanoliter-scale microfluidic devices, defining neuronal interactions and conditions for mass spectrometry, and manipulating substrates and fluids with spatiotemporal control.Here, we demonstrate that postnatal primary hippocampal neurons from rat can be cultured at low densities within nanoliter-volume microdevices fabricated using polydimethylsiloxane (PDMS). We found that this extraction procedure improves the biocompatibility of PDMS microfluidic devices significantly. Comparisons made to autoclaved and native PDMS reveal the advantages of solvent-extracted PDMS.By developing a novel neuron-to-neuron, serum-free, co-culture approach, the higher-level cellular peptidome of individual primary mammalian neurons can be determined. In this approach, early postnatal rat magnocellular neurons (MCN) from the supraoptic nucleus are isolated and cultured in low-density co-cultures with hippocampal neurons. This approach permits local access to individual neurons within the culture for mass spectrometry. Through direct mass spectrometry, 27 peptides are repeatedly detected from profiles that correspond to MCN or hippocampal neurons.We also demonstrate a simple microfluidic device that has enabled us to produce planar substrate-bound gradients that guide neuronal development. This same device enables adlayers of patterned lines produced in conjunction with surface gradients to provide an additional level of growth control. Furthermore, spatiotemporal chemical manipulation and co-cultures can be achieved with the same device.Lastly, these results are briefly reviewed and in context with other research approaches and discussed in relation to future directions.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD