Evaluation of tight junction disruption induced particles on airway epithelial cells using an automatized imaging method

There is strong evidence indicating that some inhaled particles can induce systemic effects. Translocation of particles from the airways or the lung is one of the possible ways for particles to induce systemic effects. Some nanomaterials have been proved to induce changes in the tight junctions of airway epithelial cells. In the present study we are using an automatized imager to evaluate changesintightjunctionsandcomparingwiththeresultsobtained by measuring the transepithelial electric resistance (TEER). We are usinghumanairwayepithelialcellline16HBEasamodelofepithelial barrier. To standardize the conditions of our cell cultures we seeded different cellular densities of 16HBE (8,000 to 33,000 cells) inside inserts with a surface of 0.3cm2. Oncethecultureslookconf luent,wemeasuredtheTEEReveryday.After5to7daysinculture, the higher density of cells did present a large increase in the TEER values,andthesevaluesremainhighduring3–5days.Whenexposing to NaHClO at concentrations from 0.0003 to 0.3% we observed drops in the TEER values. At the highest concentrations (0.3 and 0.03%) there was no recovery on the TEER values even after 24h of exposure. With the lowest concentrations, after the TEER values get reducedinthefirst2–5h,therewasarecoveryafter24h.Some particulate matter was used to evaluate the effect on the TEER values.TiO2,PM10 andPM2.5 weretestedwithtransientdropsonTEER values at concentrations above 10mg/cm2.Usingthesamecellular densities as on inserts, we seeded 96 well plates (with a surface of 0.3cm2/well). When we register an increase in the TEER values on the inserts, we treated the 96 well plates in the same way. To evaluate alterations in the tight junctions we did immunofluorescence staining for ZO-1 (tight junction protein) and evaluating changes in the tight junction patter using ImageXpress Micro equipment. With this method we are looking to automatize the evaluation of changes in tight junction patterns. Due to the variability on TEER values measurement, more efficient ways of evaluating changes in tight junctions is needed to evaluate and screen large amount of chemicals and particulate matter. Our preliminary results indicate that this approach is promising