A Device for measuring the in-situ response of Human Bronchial Epithelial Cells to airborne environmental agents

Abstract

Measuring the time evolution of response of Normal Human Bronchial Epithelial (NHBE) cells to aerosols is essential for understanding the pathogenesis of airway disease. This study introduces a novel Real-Time Examination of Cell Exposure (RTECE) system, which enables direct in situ assessment of functional responses of the cell culture during and following exposure to environmental agents. Included are cell morphology, migration, and specialised responses, such as ciliary beat frequency (CBF). Utilising annular nozzles for aerosol injection and installing windows above and below the culture, the cells can be illuminated and examined during exposure. The performance of RTECE is compared to that of the commercial Vitrocell by exposing NHBE cells to cigarette smoke. Both systems show the same mass deposition and similar trends in smoke-induced changes to monolayer permeability, CBF and transepithelial resistance. In situ measurements performed during and after two exposures to smoke show that the CBF decreases gradually during both exposures, recovering after the first, but decreasing sharply after the second. Using Particle image velocimetry, the cell motions are monitored for twelve hours. Exposure to smoke increases the spatially-averaged cell velocity by an order of magnitude. The relative motion between cells peaks shortly after each exposure, but remains elevated and even increases further several hours later.

Effect of sub-chronic exposure to cigarette smoke, electronic cigarette and waterpipe on human lung epithelial barrier function

“…We have previously demonstrated a 5 to 10 times increase in cellular velocity of epithelial cells exposed to CS compared to control [ 20 ], and here we showed evidence of 2.5 and 2.6 times increase after exposure to CS and EC (1.2%), respectively. The underlying molecular mechanisms that control cell velocity after CS or EC (1.2%) exposures are beyond the scope of this report, and it will be examined in the future, but this observation correlates with the loss of cell-cell adhesion proteins and altered actin-myosin contractility [ 11 ].…”

“…We have recognized several instances of similar responses to CS in vitro [ 42 , 55 ], ex-vivo [ 56 , 57 ], and in vivo [ 58 ]. Moreover, we have previously shown that continuous monitoring of CBF in lung epithelial cells exposed to CS revealed temporal fluctuations, with the appearance of CBF curve inflections in cells, minutes and hours into the recovery time [ 20 ]. Thus, while it could be attributed to distinct components present in the TW smoke [ 59 ], it is also entirely plausible that chronic exposures to TW would cause decreases in CBF, similar to that seen with EC and CS.…”

“…Briefly, on cells exposed to EC or CS, time-lapse videos are taken at phase contrast using the 32X dry objective with one frame taken every 5 min for 2 h. We did not measure cell velocity in cells exposed to TW. Cell migration was quantified by performing Particle Image Velocimetry (PIVlab) on Matlab using multi-pass cross-correlation analysis with decreasing interrogation window size on image pairs to obtain the spatial velocity field as described previously [ 20 ].…”

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“…The 2.5 µm droplets transported like passive tracers, and a large fraction of the 40 µm and 60 µm droplets remained suspended for at least 600 m. The model enables estimations of the order of magnitude of oil droplet concentration near unsteady waves, which are challenging for existing models. Chandrala et al (2019) introduced a novel exposure chamber for use in the Real-Time Examination of Cell Exposure (RTECE) system that enables in situ imaging of lung cell cultures during and after exposure to airborne particles. The unique features of the enhanced system include three independently sealed chambers that are adaptable for housing different cell culture optimized, dis-posable nozzles that provide uniform aerosol deposition and prevent crosscontamination between experiments.…”

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“…Cellular velocity. Cellular velocity was quantified by performing Particle Image Velocimetry (PIVlab) on Matlab (R2020a), using a multi-pass cross correlation analysis with decreasing interrogation window size on image pairs to obtain the spatial velocity as described previously by us (40). Using a phase contrast microscopy at 3i Marianis/Yokogawa Spinning Disk Confocal Microscope at 32X, and 37°C and 5% CO 2 incubation, we captured time-lapse videos of the pseudostratified monolayer epithelium for every 5 minutes for 2 hours and average velocity was computed for the area.…”