The investigations conducted within this dissertation centers around the use of electric cell-substrate impedance sensing (ECIS). This system is able to characterize in real-time analysis, the adhesion of cells to their substrate and neighboring cells. With this, valuable information can be gathered with in-vitro experiments regarding a tissue culture's response to physiological stimulation. This dissertation has taken advantage of ECIS' ability to analyze toxicology, barrier function, and cancer invasion on a tissue culture. With proper analysis modifications, trans-epethelial resistance (TER) can be used as a cytotoxicity assay with higher sensitivity than previously thought. In vitro assessment of cytotoxicity based on TER needs more quantitative methods to analyze the alteration of cell morphology and motility.
Here, we applied ECIS to evaluate dose-dependent responses of human umbilical vein endothelial cells (HUVEC) and mouse embryonic fibroblasts (NIH 3T3) exposed to cytochalasin B and protein kinase inhibitor H7. To detect subtle changes in cell morphology, the frequency-dependent impedance data of the cell monolayer were measured and analyzed with a theoretical cell-electrode model. To detect the alternation of cell micromotion in response to cytochalasin B and H7 challenge, time-series impedance fluctuations of cell-covered electrodes were monitored and the values of power spectrum, variance, and variance of the increment were calculated to verify the difference. While a dose-dependent relationship was generally observed from the overall resistance of the cell monolayer, the analysis of frequency-dependent impedance and impedance fluctuations distinguished cytochalasin B levels as low as 0.1µM and H7 levels as low as 10 µM for HUVEC and 3T3 layers.
Even though overall resistance values are relatively small for 3T3 layers, and frequency scan measurements are negligible, impedance fluctuation analysis reveals significant micromotion for cytotoxic detection. Our results show that cytochalasin B and H7 causes a decrease of junctional resistance between cells and an increase of membrane capacitance. Cigarette smoke is cytotoxic and tumorigenic. Initial studies were conducted to evaluate the cytotoxicity of cigarette smoke condensate (CSC) on HUVEC layers. The focus was then turned to investigations involving in vitro cancer invasion assays with CSC on HUVEC layers. ECIS is an excellent investigative device that can be utilized to observe cancer invasion on normal tissue cultures due to the significantly higher impedance signature of cancer cells.
The investigation in this dissertation focused on cigarette smoke's influence on cellular mechanics of endothelial cells and the invasive potential of two ovarian cancer cell lines (ALST and OVCA429) against a fully active endothelium. The HUVEC cultures responded to CSC with an increase in junctional binding, where as ALST and OVCA429 relieved adhesion thereby providing an improved motility when evaluated in wound healing assays. Transmigration of the HUVEC layer by ALST cells exhibit a pre-CSC exposure time-dependence affecting the effectiveness of ALST transmigration. The HUVEC layer's decreased tight junction binding that resulted from CSC exposure, allowed for a more aggressive ALST layer formation that occurred during simulated intravasation. Increased HUVEC layer tight junction binding that occurred in the first five hours in response to CSC during extravasation contributes to impeding ALST transmigration at high concentrations of CSC.
Overall, CSC has an impeding effect on ALST transmigration during extravasation while causing aggressive transmigration during intravasation.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-3124 |
| Date | 01 June 2009 |
| Creators | Opp, Daniel |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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