TSRC, Tob. Sci. Res. Conf., 2018, 72, abstr. 008

Use of cilia beat frequency in an in vitro airway model (EpiAirway-FT) as a tool for assessing viability

Covance, Harrogate, UK

Human respiratory tract cilia play an essential role in clearing mucus and debris from the airway (mucociliary clearance). This important defence mechanism process protects the lungs from aerosolised pathogens and pollutants and can be directly attributed to cilia beat frequency (CBF). The Sissons-Ammons Video Analysis (SAVA) system (Ammons Engineering, USA) is a combination of high-speed video, microscopy and image analysis which makes it possible to evaluate CBF in vitro. In a Proof-of-Concept study using EpiAirway™ tissues (Mattek Corporation, Ashland, USA) and a basolateral treatment regime, we optimised the CBF analysis parameters and recorded 2.6 second movies at 10x magnification. We then applied either chemicals with known ciliomotility effects (Adenosine triphosphate [ATP], Procaterol Hydrochloride [USAN], Hydrogen Peroxide [H2O2] or Benzalkonium Chloride [BC]) or adjusted the culture medium pH from 4 to 10 and analysed tissues after 30 minutes and 2 hours. Our results showed an untreated tissue average CBF of 17.62 Hz n=40. ATP (0.01mM) and USAN (0.01mM) increased CBF (as expected) to 19.68 Hz and 20.42 Hz respectively and BC (3mM) decreased CBF (as expected) to 2.5 Hz however H2O2 (0.1mM) caused an unexpected increase in CBF (19.55 Hz) even when tested at 10mM (19.37 Hz). From the pH testing, only pH4 adversely affected CBF yielding ciliostasis. To expand this work, EpiAirway™ AIR-200 tissues were exposed to 3R4F cigarettes (Health Canada Intense puffing regime) for 88 minutes using a Vitrocell® VC10® machine (VitroCell GmbH, Germany). CBF was slightly elevated at 2L/min compared to the air control and was undeterminable at higher concentrations. These data demonstrate CBF analysis could be useful for studying cytotoxic effects of chemicals, pH and aerosol exposure on airway tissue in vitro.