archives@tulane.edu / Acute respiratory distress syndrome (ARDS) is a life-threatening, non-carcinogenic inflammatory pulmonary conditions characterized by the collection of fluids in the air sacs of the lungs. When fluid-filled airways are ventilated, the stresses of repetitive recruitment-decruitment (Rec-Der) causes cellular damage to the epithelial surface, leading to ventilator induced lung injury (VILI). The objective of this study was to establish a foundation for use of electric cell-substance impedance sensing (ECIS) in real-time analysis of cell membrane morphology and RecDer-induced damage. NCI H441 papillary adenocarcinoma human pulmonary epithelial cells are cultured onto custom 1F8x10E PC Flow Array. 10mM cysteine and 1% gelatin surface treatments demonstrated strong results for improved cell-substrate adhesion strength. RecDer insults were introduced at a velocity of 0.5mm/s through FBS-enhanced RPMI164 growth media. Experimental trials for 0 (n=1), 1 (n=1), 5 (n=1), 10 (n=1), 20 (n=7), and 50 (n=1) RecDer insults were analyzed using Annexin-V/PI flow cytometry; results showed monolayer health of 97.76%, 93.152%, 91.801%, 72.495%, 66.88% and 60.812% respectively. Trials for 20 (n=1), 30 (n=1), and 40 (n=1) RecDer insults were analyzed using ECIS; Frequency-dependent impedance modeling of the acquired data suggested increased damage to both cell-cell junction health and cell membrane integrity with increased RecDer insults. Results established a strong foundation for ECIS analysis of RecDer-induced monolayer damage. / 1 / Joshua Erwa Yao

The interactions between neighboring cells and between cells and their attached substrate have long been studied in tissue culture. These in vitro studies may provide information regarding cell behavior in vivo including cell movement, cell proliferation, tissue development and wound healing. Transcellular resistance (or impedance) measurements, using various dc or ac techniques have been used to study the barrier function of epithelial and endothelial cell layers. With an appropriate equivalent circuit used for data analysis, junctional resistance between cells and other cellular properties, including cell membrane capacitance, can be determined. However, these techniques have seldom been applied to fibroblastic cell layers because the transcellular resistance is so small that it is difficult to measure it accurately. This research is based on detecting the invasive activities of metastatic cells in vitro using electric cell-impedance sensing (ECIS). The metastatic cells where added over the established endothelial cells and were observed to attach and invade the cell layer. Human umbilical vein endothelial cells (HUVECs) were first grown and then loaded on eight well gold electrodes. The impedance of these electrodes was followed after the suspension of different sublines of cancer cells (SKOV3, OVCA429). For highly metastatic sublines, within an hour after being challenged, the impedance of confluent HUVECs layer was substantially reduced. In addition the conditioned cancer media and heat-killed cancer cells was also suspended which had no substantial effect on the impedance. This result suggests that ECIS based assay might be used with primary human cultures to establish the metastatic abilities of cells.

Adhesion signaling is a field of cell biology studied mostly on adherent cell types. However, hematopoietic cells grow in suspension, and use adhesion to the extracellular matrix (ECM) only in their early development, or - in case of differentiated cells - to perform the tasks they are specialized for. Peripheral leukemic cells are derived from more or less immature hematopoietic precursors that have, among other alterations, defects in adhesion to the bone marrow microenvironment. On the other hand, leukemic stem cells (LSC) use adhesion to the bone marrow ECM as a mean to evade chemotherapy, and are a source of the minimal residual disease, and of the disease relapses. Kinases of the Src family (SFK) are known regulators of adhesion signaling in adherent cell types, and their overexpression and/or hyperactivation is often seen in malignant diseases. They are also involved in hematooncologic disease progression and resistance to therapy, particularly in several types of leukemias. In the present work, we used a variety of methods including microimpedance measurement, fluorimetric measurement of adhered cell fraction, immunoblotting, confocal microscopy, and interference reflection microscopy. Our results indicate that active Lyn kinase, a hematopoietic SFK, is present in adhesion structures of...

Cigarette smoke's influence on cancer has primarily been a subject of epidemilogic and tumorigenic studies. There have been no proper investigations with interests focused on how cigarette smoke affects the cellular mechanics of metastasis. Gathering an understanding of how smoke influences metastatic invasion could be vital in regulating or possibly eliminatings cancer's ability to initiate new tumor growth sites. This project focuses on cigarette smoke's influence on cellular mechanics of endothelial cells, and the invasive potential of cancer against a fully active endothelium. It is already known that cigarette smoke has a carcinogenic effect, but it is hypothesized that the cigarette smoke causes the endothelium to exhibit pro-invasive characteristics. Cancer cells are often ignorant to extra-cellular stimuli. It is suspected that there will be a less pronounced degradation of cellular mechanics of cancerous cells than endothelial cells when exposed to similar concentrations of cigarette smoke.

This research focuses on the detection and characterization of cells using impedance-based techniques to understand the behavior and response of cells to internal/environmental changes. In combination with impedimetric sensing techniques, the biosensors in this work allow rapid, label-free, quantitative measurements and are very sensitive to changes in environment and cell morphology. The biosensor design and measurement setup is optimized to detect and differentiate cancer cells and healthy (normal) cells. The outcome of this work will provide a foundation for enhanced 3-dimensional tumor analysis and characterization; thus creating an avenue for earlier cancer detection and reduced healthcare costs. The magnitude of cancer-related deaths is a result of late-diagnosis and the fact that cancer is challenging to treat, due to the non-uniform nature of the tumor. In order to characterize and treat individual cells based on their malignant potential, it is important to have a measurement technique with enhanced spatial resolution and increased sensitivity. This requires the study of individual or small groups of cells that make up the entire tissue mass. The overall objective of this research is to optimize a microelectrode biosensor and obtain statistically relevant data from a cell culture using an independent multi-electrode design. This would provide a means to explore the feasibility of electrically characterizing cells with greater accuracy and enhanced sensitivity.

No description available.

Bioimpedance presents a versatile, label-free means of monitoring biological cells and their responses to physical, chemical and biological stimuli. Breast cancer is the second most common type of cancer among women in the United States. Although significant progress has been made in diagnosis and treatment of this disease, there is a need for robust, easy-to-use technologies that can be used for the identification and discrimination of critical subtypes of breast cancer in biopsies obtained from patients. This dissertation makes contributions in three major areas towards addressing the goal. First, we developed miniaturized bioimpedance sensors using MEMS and microfluidics technology that have the requisite traits for clinical use including reliability, ease-of-use, low-cost and disposability. Here, we designed and fabricated two types of bioimpedance sensors. One was based on electric cell-substrate impedance sensing (ECIS) to monitor cell adhesion based events and the other was a microfluidic device with integrated microelectrodes to examine the biophysical properties of single cells. Second, we examined a panel of triple negative breast cancer (TNBC) cell lines and a hormone therapy resistant model of breast cancer in order to improve our understanding of the bioimpedance spectra of breast cancer subtypes. Third, we explored strategies to improve the sensitivity of the microelectrodes to bioimpedance measurements from breast cancer cells. We investigated nano-scale coatings on the surface of the electrode and geometrical variations in a branched electrode design to accomplish this. This work demonstrates the promise of bioimpedance technologies in monitoring diseased cells and their responses to pharmaceutical agents, and motivates further research in customization of this technique for use in personalized medicine. / Ph. D.