Development and Characterization of Analytical Tools to Study Immunity Ex Vivo 251 views

Biological function is governed by well-defined chemical signals occurring with a high degree of spatial and temporal specificity. The field of bioanalytical chemistry is concerned with measuring these signals quantitatively. While simplifying the biological system to well-defined protein solutions, or cell cultures, allows for precise manipulation and detection, removing the signals from the tissue matrix also removes the biological context. In vivo measurements maintain the spatial distribution and other biological functions, such as fluid flow; however, they can be extremely complicated, often requiring genetic modifications. Ex vivo platforms maintain the spatial organization of in vivo while providing the increased experimental accessibility of in vitro studies. Ex vivo sections have been used to characterize functions of different tissues, including immune tissue. Immunity is a highly dynamic and systemic process that is constantly working to clear the body of harmful pathogens. The immune system is generally separated into two components, the innate and adaptive responses. The innate response is immediate but non-specific, while the adaptive is slower but much more targeted. The success of the adaptive immune response is determined in a secondary immune organ, the lymph node. Measuring chemical signals within the lymph node tissue requires the development of novel analytical tools. Chapter 2 describes the characterization of live lymph node tissue slices. These slices were viable and able to function as an acute culture platform to study immunological events. Lymph node slices processed whole-protein antigen and were able to respond to antigen challenge after vaccination. This work provided the platform that novel analytical tools were then developed for. Chapters 3 and 4 are focused on these tools. The work in chapter 3 focuses on the development of a novel cell labelling reagent. Bright-blue polymer nanoparticles were generated in collaboration with the Fraser lab at UVA, and used to label phagocytic immune cells. The labelling did not affect cellular viability or function. The bright-blue fluorescence allowed labelled cells to be easily distinguished from the tissue background for cell tracking. Chapter 4 characterizes magnetic beads as an embedded platform for immunoassays. Beads were functionalized with model antibodies and polyethylene glycol (PEG) and delivered to lymph node slices. The addition of the PEG was critical to an even delivery of the beads within the tissue slices. The work in this dissertation focuses on setting the foundation for analytical measurements in lymph node tissue. In the future, we anticipate that these platforms will provide additional insight into immunological functions.

PHD (Doctor of Philosophy)

Analytical Chemistry; Immunology; Lymph Nodes; Ex Vivo Tissue; Cell Labeling; In-Tissue Detection

English

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2020-05-11