Abstract:

This study introduces and implements a novel device for Real-Time Examination of Cell Exposure (RTECE), which enables direct in situ assessment of functional responses of cells in culture, such as migration, as well as other more specialized cellular responses such as ciliary beat frequency (CBF) both during and following an exposure to an environmental agent. Data consists of transepithelial resistance, cell permeability, and CBF of Normal Human Bronchial Epithelial (NHBE) cells exposed to cigarette smoke in Vitrocell and RTECE system using the same data collection and analysis procedures. It also includes Real-time measurements of CBF and cell velocities using RTECE.

Suggested Citation:

Lakshmana chandrala, Kristine Nishida, Nima Afshar-Mohajer, Yury Ronzhes, Ramana Sidhaye, Kirsten Koehler, Joseph Katz. 2019. Health impact of crude oil-dispersant aerosols. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-xpky-ss66

Publications:

Chandrala, L. D., Afshar-Mohajer, N., Nishida, K., Ronzhes, Y., Sidhaye, V. K., Koehler, K., & Katz, J. (2019). A Device for measuring the in-situ response of Human Bronchial Epithelial Cells to airborne environmental agents. Scientific Reports, 9(1). doi:10.1038/s41598-019-43784-5

Purpose:

To study the health impacts of aerosols on Normal Human Bronchial Epithelial (NHBE) cells.

Data Parameters and Units:

The dataset contains three excel files with multiple worksheets. In the excel files “Bilogical responses of NHBE cells in vitrocell and RTECE” and "Real-time measurements of Ciliary beating frequency using RTECE", worksheet "TEER" contains: case (1, 2, 3); Control exposure; Cigarette exposure in RTECE; and Cigarette exposure in Vitrocell. Here, NHBE = Normal Human Bronchial Epithelial; RTECE = Real-Time Examination of Cell Exposure, and TEER = Transepithelial electrical resistance. All these headers are measures of TEER and have the unit of ohms. Vitrocell is a system of introducing smoke that closely resembles real-life exposure. The worksheet "CBF" contains: Case (1, 2, 3); Vitrocell before exposure; Vitrocell after exposure; RTECE before exposure; and RTECE after exposure; which are measures of Ciliary Beat Frequency (CBF) with units of hertz (Hz). The worksheet “Permeability” contains: case (1, 2, 3); Control exposure; Cigarette exposure in RTECE, and Cigarette exposure in Vitrocell; which are measures of Epithelial monolayer permeability with the unit of cm/sec. The excel file “Real-time measurements of cell migration using RTECE contains the data on exposure to smoke and clean air, and includes: time (min); Mean velocity (case 1); RMS velocity (case 1); Mean velocity (case 2); and RMS velocity (case 2). The unit of velocity is micrometer per second. RMS means Root Mean Square. Using oblique illumination microscopy, the RTECE is used for in situ measurements of the time evolution of CBF (Hz) and cell velocities (micrometer per second ) during and after two cigarette exposures.

Methods:

Primary NHBE cells (MatTek corp.) have been grown on collagen-coated transwell inserts (Coring®) immersed in bronchial epithelial growth media (Lonza, Stemcell) at 37°C and 5% CO‌2. Once the cells form a confluent monolayer, they are transferred from a submerged condition to an air-liquid interface (ALI) condition by removing the apical media. The cells are maintained at the ALI condition for 4-6 weeks, at which time they are terminally differentiated and comprised of clara, basal, goblet, and ciliated cells forming a tight monolayer. The resulting culture is then inserted in the exposure chamber while maintaining an ALI condition. Paracellular permeability is assessed using a 4kD FITC dextran assay (Sigma-Aldrich. After each exposure test, 2-3mL of phosphate-buffered saline (PBS) solution is added to the basal compartment of the transwells, and 1-2mL of 4kD FITC dextran at a concentration of 0.5mg/mL is added to the apical side of the cells. The cells are then incubated at 37°C, 5% CO‌2, and 95% RH for 30-45 min. Following incubation, a fluorimeter is used for determining the fluorescence of the sample solution taken from the basolateral compartment. In all the experiments, cells are subjected to two exposures, separated by a rest period of 60 min, and the duration of each exposure is 16 minutes.

Instruments:

The oblique illumination microscopy is designed and developed in house to have a long-working distance between the sample and objective. A 20x infinity-corrected long-working distance objective (Mitutoyo) combined with a 200 mm focal length tube lens (Edmund Optics) is used for magnifying the images.

Individual Files: