A Versatile Airway-On-Chip Model for High-Throughputs Scalable Pulmonary Toxicity Studies

Introduction:: Respiratory disease caused by smoking, infections, and genetic factors together account for the leading causes of death worldwide. To develop effective therapies for respiratory disease, it is therefore important to gain an in-depth understanding of the overall lung disease etiology, so as to improve the translational rate from benchtop successes to the human clinical trials in the drug discovery pipeline. Current in-vitro model systems have provided valuable data on toxicant-induced pulmonary injury. However, there is a need for new model systems with improved physiologic relevance, higher throughput, and automation to enhance the screening capability for existing and emerging toxicants. Recently, Airway-on-Chip (AoC) models have emerged as in vitro platforms that integrate additional tissue complexity with micro-engineering principles to mimic an in vivo-like lung microenvironment. However, the utility of AoC models can be limited because of their complex design, incompatibility with rapid prototyping, and the user’s limited capacity to retrieve biological material from microcompartments. These drawbacks highlight the need for innovative bioengineering to create a more user-friendly and user-modifiable high throughput platform for in-vitro pulmonary research. Therefore , a fundamental redesign of AoC platform to represent the intricacies of lung microenvironment would thus requires the system to be customizable, adaptable and elegantly manufacturable in order to overcomes the existing complexities of AoC engineering.


Materials and Methods:: We have developed an easy-to-fabricate, polymethyl methacrylate (PMMA) and adhesive thin film-based, HT-AoC (High Throughput – Airway-on-Chip) design that supports organotypic differentiation of airway epithelial cells in-vitro (figure.1). This design is based on 384-well spacing which allows for layout modifications that align with standard multifunction plate reader systems. PMMA and adhesive film layers were fabricated using a standard bench-top laser cutter and HT-AoCs were assembled with polyester porous membranes (PET; 0.4um pore size) to provide the cell culture growth surface. Chips were constructed under aseptic conditions, and UV light sterilized prior to use. To test effectiveness, primary human bronchial epithelial cells (hBECs) were seeded apically into a 40-well chip (n=40 replicate wells) and differentiated at an air-liquid interface (ALI) for 28 days. Basolateral medium (60ul) was replenished daily via microfluidic channels/pump. Cilia beat frequency (CBF), histology, and immunofluorescent labeling were used to characterize cell cultures. Further, the use of adhesive films also enables the on-demand detachment of the ensembles for conducting any post-processing assays such as histology or immunostaining as required.


Results, Conclusions, and Discussions:: After 28 days, all 40 wells of the HT-AoC plate produced differentiated hBECs with apical mucus and motile cilia with coordinated beating. Sisson Ammons Video Analysis (SAVA) quantified CBF at 8.40Hz ± 0.98Hz with an average of 23% ± 10.60% active area per field of view. Immunofluorescent labeling and subsequent morphometric characterization identified 39.33% ± 2.51% basal cells (P63 positive), 18.98% ± 1.67% club cells (CC10 positive), and 74.85% ± 5.34% ciliated cells (α-tubulin positive) in differentiated hBECs. All 40 wells of the HT-AoC functioned as expected with no failures. Few wells developed small air bubbles within the fluidics compartments. However, air bubbles were removed by bubble trap and did not have detrimental effects on the cultures. We also used HT-AoC system in two formats namely HT-AoC model for Aerosol Exposure and HT-AoC model for gas/nanoparticle exposure as potential drug screening/testing platform for boarder range of pulmonary toxicity studies.

Here, we describe a novel HT-AoC system, and demonstrate is use for the successful differentiation of primary hBEC cultures. At ALI day 28, hBECs cultures displayed normal epithelial layer morphology with ciliated, secretory, and basal cells. The fabrication techniques used in this work are ideal for rapid in-lab prototyping without the need of expensive cleanrooms. Furthermore, the integration of microfluidics channels enables high-throughput automation with liquid handlers and pumps. Based on the data presented here and the flexibility of this platform, we believe the HT-AoC model may provide an effective, affordable, and customizable way for researchers to enhance throughput and physiologic relevance in pulmonary toxicity studies. This multi-functional device has a strong potential for adaptation into applications that involve increased analysis throughput for conducting complex biomimetic assays of different organs.


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