Epithelial Electrophysiology Using Intracellular Robotics and Extracellular Impedance Spectroscopy

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LEWALLEN-DISSERTATION-2021.pdf (50.89 MB)
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Lewallen, Colby Foster
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http://hdl.handle.net/1853/66173
Abstract
Epithelia are barrier-type cells that regulate the transport of materials into and out of the body. Dysfunction of these cells is implicated in numerous diseases such as cystic fibrosis, age-related macular degeneration, and diabetes. Since the discovery of induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka in 2006, scientists around the world have begun utilizing iPSC-based therapies to halt, and potentially reverse, the progression of these diseases. For epithelia-based therapies, validation of tissue polarity and function is an essential component of a thorough physiological exam, and are commonly performed electrochemically, but existing methods are some combination of (1) destructive to the cells, (2) incomplete, (3) extremely difficult, and (4) low throughput (e.g., 1-2 tissues/day). Therefore, in this work, novel tools and measurement techniques were developed to study epithelial cell function that addresses these issues. For example, an algorithm that automates the insertion of a pipette into the cytoplasm of a cell was developed (chapter 2). This algorithm outperforms a highly trained expert at a much lower operator skill level; enabling more labs to explore the physiology, drug toxicity, and disease processes of epithelia. In addition, a new mathematical model was developed that combines a technique called electrochemical impedance spectroscopy (EIS) with intracellular voltage data to extract membrane-specific properties of epithelia (chapter3). Furthermore, experimental data demonstrating an inverse relationship between tissue capacitance and the median cell cross-sectional area is presented (chapter 4). Finally, a mathematical link between membrane-specific properties of epithelia and the time constant ratio, a property that does not require intracellular voltage data, is derived and experimentally tested. This link can act as a bridge between the comprehensive, yet slow and invasive, intracellular measurements and the fast, yet simple, extracellular measurements of epithelial function (chapter 4).
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2021-12-14
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