Airway Epithelium-on-a-Chip as a Platform for Particulate Matter Study

Abstract

The purpose of this research is to develop a microfluidic Airway Epithelium-on-a-Chip, a novel in-vitro model for assessing effects of particulate matters (PM) on airway epithelium, and to evaluate its potential as a platform for PM study. The characteristic of human airway, which is the consecutive connection of different epitheliums, is established on upper channel of the chip, using a novel microfluidic cell-culture method. As airway epithelium is formed, on-chip cultured cells were exposed to diesel exhaust particles (DEP), and as a consequence, EMT (Epithelial-to-Mesenchymal Transition) and inflammatory reaction were observed on chip. In order to examine functionality and reliability of the chip, DEP experiments on cell culture plate and animal model are conducted. For animal model, asthma-induced mouse is used. Airway Epithelium-on-a-Chip is made of PDMS (Polydimethylsiloxane) and consisted of the upper channel, which enables in-series culture of different epithelial cells, ECM (Extra-cellular matrix)-coated membrane, and the lower channel, in which continuous perfusion is made. To mimic airway epithelium, human nasal epithelial cell line and human alveolar epithelial cell line are used and cells are co-cultured in-series. Moreover, fibronectin is coated on porous PET membrane in order to support cell attachments and proliferation. In order for long-term survival of on-chip cultured cells, the lower channel is connected to a syringe pump, which makes continuous flow of media. The confluency of two epithelial cells cultured inside the chip is observed using daily microscopic images and the epithelialization is verified by the formation of tight junctions between cells. Also, as the epithelial cell layer is exposed to sonicated DEP, the EMT ability and the release of pro-inflammatory cytokines are investigated using immunofluorescence staining and PCR (Polymerase Chain Reaction). To verify the on-chip results, in-vitro experiment and in-vivo study using asthma mouse are conducted simultaneously. During in-vitro experiment, human alveolar epithelial cells (A549) are cultured on cell culture plate and exposed to DEP and TGF-beta, and the formation of tight junctions and EMT ability are compared between control and experimental groups. During in-vivo study, an asthma-induced mouse is treated with DEP for 3 days using intra-nasal challenges and sacrificed. Its lung cells are then gone through PCR for investigating the secretion of inflammatory cytokines. Cells cultured inside the chip formed tight junctions in 2 days and achieve the full confluency in 4 days. As effects of DEP on epithelial cells, the breakage of tight junction, increased EMT ability and the secretion of IL-6 were observed. Significant similarity was found between on-chip results and those from in-vitro and in-vivo experiments. In vitro experiments resulted in the breakage of tight junctions and increased EMT ability of epithelial cell layers, while in vivo experiments resulted in the elevated level of IL-6 mRNA from DEP-treated asthma mouse model. In this study, a microfluidic airway epithelial chip was developed and tested for its reaction to Diesel Exhaust Particles. The formation of tight junction and gradual increase of cell layer in microscopic images prove the formation of epithelium on the chip, which also verifies the functionality of Airway Epithelium-on-a-Chip. Moreover, the elevation of IL-6, known inflammatory cytokine, was observed on the chip, which was supported by the in-vivo mouse model. By showing the agreement between on-chip results and in-vivo results, the reliability of the microfluidic chip has been assured.

Organ-on-a-Chip is an emerging 3D in-vitro model, which can provide tissue-level functions by reconstructing minimal functional units of organs. Applying this concept, Airway Epithelium-on-a-Chip is developed as a novel in-vitro model that mimics the consecutive lining of airway epithelium. The novel microfluidic in-series co-culture technique, introduced in this research, can be further applied to study interactions of different epithelium. For instance, the consecutive lining of intestinal epithelium can be mimicked using this microfluidic channel design. Further plans should be made to address limitations from this research. In order to improve the functional and environmental similarity to real tissue, the use of primary cells and their differentiation inside the chip are inevitable. Moreover, other air pollutants than DEP should be tested for enhancing the functionality of the chip. Despite these needs for further studies, however, this research clearly implies the potential of Airway Epithelium-on-a-Chip to be a platform for investigating underlying cellular mechanisms of PM.