Date on Master's Thesis/Doctoral Dissertation

8-2016

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Mechanical Engineering

Degree Program

Mechanical Engineering, PhD

Committee Chair

Williams, Stuart

Committee Co-Chair (if applicable)

Kynton, Robert

Committee Member

Soucy, Patricia

Committee Member

Sharp, Michael

Committee Member

Berfield, Thomas

Author's Keywords

Impedance; Endothelial cells; Shear stress; Microelectrodes; Microfluididcs

Abstract

Endothelium dysfunction has been associated with many pathophysiological processes leading to cardiovascular diseases. Studying endothelium behavior is vital to understand the onset, prevention, and treatment of such diseases. Electrical impedance spectroscopy has been shown to provide a real-time in vitro evaluation of cell behavior including cell monolayer permeability. However, the majority of published work has been primarily with static cell culture models or macro-scale models that do not properly represent the physiological sizes, structures, and environmental conditions of human blood vessels. Within this dissertation, the design, fabrication, characterization, and application of a microfluidic impedance platform is presented for the in vitro characterization of HUVECs undergoing different hydrodynamic shear stress conditions (static, 2.5, 17.6 and 58.1 dyne/cm2). Electrodes diameters of 50, 100, and 200 µm were incorporated to monitor different subpopulations sizes of HUVECs. Initial characterization experiments with relevant biological solutions indicated that electrodes smaller than 50 µm in diameter suffered from significant interfacial impedance and were unsuitable for the sensing application. Impedance spectra (102-106 Hz) were collected for HUVECs at the different shear conditions for 14 hours. Equivalent circuit fits were implemented to derive the different electrical cell monolayer parameters including the trans-endothelial resistance, cell membrane capacitance, constant phase element, and the resistance of cell culture medium. Results confirmed that while the trans-endothelial resistance and cell membrane capacitance were suitable measurements for cell permeability and confluency respectively, the constant phase element did not identify any discernible cell behavior. Resistance of cell culture medium was strongly influenced by cell attachment and values should be extracted from control cell-free measurements. Initial trans-endothelial resistance measurements showed a shear magnitude dependent increase at the sudden onset of flow. This increase was greatest for the largest shear condition (58.1 dyne/cm2). After 14 hours of shear, trans-endothelial resistance measurements were largest for HUVECs sheared at 58.1 dyne/cm2 and lowest for the 17.6 dyne/cm2 shear condition and the difference showed to be statically significant (p <0.05). Monitored HUVECs were stained for nuclei, F-actin and VE-cadherin. Quantification of immunofluorescence of VE-cadherin showed a similar trend to the extracted trans-endothelial resistance values. Immunofluorescence images of F-actin showed significant cytoskeleton remodeling of sheared HUVECs. While cells sheared at 17.6 dyne/cm2 aligned parallel to the direction of flow, HUVECs sheared at 58.1 dyne/cm2 were angled in the direction of flow and sometimes even perpendicular to flow direction.

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