T cells occupy a central role in cell-mediated immunity and as such, deciphering the signaling events that govern T cell activity is critical in fully understanding the adaptive immune response. Current immunologic methodologies utilize either conventional cell culture techniques to analyze millions of cells over time, thereby averaging out rare signaling events, or technology that interrogates single cells at a single time point each, resulting in a loss of information regarding temporal signaling dynamics. To overcome these limitations, we have developed the multi-trap nanophysiometer, a novel, self-contained microfluidic platform fabricated of optically transparent, bio-inert PDMS designed to study signaling dynamics of multiple single T cells in parallel. This body of work describes the major accomplishments attained towards the development and validation of this platform. Cell viability analysis revealed that at flow rates of 100 nl/min, more than 70% of CD4+ T cells, held in place using only hydrodynamic forces, remained viable following 24 hours within the microfluidic environment. We then observed cytosolic calcium transients to demonstrate the ability to activate T cells within the multi-trap nanophysiometer using chemical, antibody, and cellular forms of stimulation. Applying this platform to study intercellular signaling events we were able to observe calcium transients in T cells in response to both contact- and non-contact-based interactions with dendritic cells. Further investigation revealed that in the absence of antigen, LPS-matured dendritic cells secrete chemical signals that induce calcium transients in naïve CD4+ T cells, but in such small concentrations that effects of these signals are not easily observed in normal cell culture conditions. Finally, utilizing the multi-trap nanophysiometer to study the immunological synapse between dendritic cells and T cells, we revealed the occurrence of bi-directional cytosolic dye transfer. This suggests that communication between dendritic cells and T cells during the immunological synapse may not be limited to cell surface interactions. Taken together, these results establish the multi-trap nanophysiometer as a powerful tool in the analysis of cell signaling dynamics.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07272007-082646 |
| Date | 02 August 2007 |
| Creators | Faley, Shannon Leigh |
| Contributors | John P. Wikswo, Jr., E. Duco Jansen, Franz Baudenbacher, David Wright, Derya Unutmaz |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-07272007-082646/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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