Development of an In Vitro Exposure System for Live Visualization of the Health Impacts of Oily Marine Aerosol on the Human Respiratory System

Abstract

Aerosolization of oily water droplets has recently been recognized as a potential respiratory health threat to oil spill cleanup workers, communities near spills, and marine mammals in oil-polluted waters. These sub-micron to millimeter scale droplets may be aerosolized by bursting bubbles, breaking waves, and splashing raindrops. Furthermore, dispersant applied to oil slicks also may become aerosolized as oil-water-dispersant emulsion droplets and subsequently inhaled, with unknown health consequences. With the goal of investigating the effects of inhaled oily marine aerosol on human lung health, we present the design of a novel in vitro bioreactor which mimics the conditions and exposures that human lungs might experience in the field. The bioreactor provides the ability to expose human lung cell cultures to laboratory-produced, well-characterized and chemically analyzed oily marine aerosols. A major advance over similar systems currently used to study the effects of smoking is the incorporation of optical access to allow visualization of the cells throughout exposure. In the bioreactor, differentiated, primary human bronchial epithelial cell cultures reside on membranes at the air-liquid interface between the flow-through test atmosphere and a temperature-controlled bath of culture media, thereby simulating the human lung. Oily marine aerosol is produced by a 1-Jet Collison Nebulizer (Mesa Labs Inc.) to match realistic concentrations produced and measured in a wave tank and is sampled via scanning mobility particle sizer (TSI Inc) to characterize its size distribution. The aerosol-laden air is humidified and injected at a controlled flow rate of ~1 ml/s into a module containing the cell culture, allowing particles to deposit on the cells. The module has sealed glass windows to allow optical access. An optical setup incorporating a 20× long-distance objective, 1× tube lens, and camera is used to visualize the cells over time. Preliminary testing involves determining the effectiveness of deposition of oily marine aerosol droplets at various concentrations onto the cell culture surface. Phase contrast microscopy is used to examine contact between cells as a determinant of monolayer integrity. Immunofluorescence of live cells is used with FITC- or mCherry-labelled cytoskeletal and cell-cell adhesion proteins, such as actin and E-cadherin, to determine underlying mechanisms disrupting the monolayer. A system such as this allowing for live visualization of cells during the exposure currently does not exist and will provide significant understanding of how changes within the epithelium may disrupt tissue integrity in response to inhalation of oily marine aerosol.