Immunocompetent 3D Model of Human Upper Airway for Disease Modeling and In Vitro Drug Evaluation

Abstract

The development of more complex in vitro models for the assessment of novel drugs and chemicals is needed because of the limited biological relevance of animal models to humans as well as ethical considerations. Although some human-cell-based assays exist, they are usually 2D, consist of single cell type, and have limited cellular and functional representation of the native tissue. In this study, we have used biomimetic porous electrospun scaffolds to develop an immunocompetent 3D model of the human respiratory tract comprised of three key cell types present in upper airway epithelium. The three cell types, namely, epithelial cells (providing a physical barrier), fibroblasts (extracellular matrix production), and dendritic cells (immune sensing), were initially grown on individual scaffolds and then assembled into the 3D multicell tissue model. The epithelial layer was cultured at the air–liquid interface for up to four weeks, leading to formation of a functional barrier as evidenced by an increase in transepithelial electrical resistance (TEER) and tight junction formation. The response of epithelial cells to allergen exposure was monitored by quantifying changes in TEER readings and by assessment of cellular tight junctions using immunostaining. It was found that epithelial cells cocultured with fibroblasts formed a functional epithelial barrier at a quicker rate than single cultures of epithelial cells and that the recovery from allergen exposure was also more rapid. Also, our data show that dendritic cells within this model remain viable and responsive to external stimulation as evidenced by their migration within the 3D construct in response to allergen challenge. This model provides an easy to assemble and physiologically relevant 3D model of human airway epithelium that can be used for studies aiming at better understanding lung biology, the cross-talk between immune cells, and airborne allergens and pathogens as well as drug delivery.

The impact of surface chemistry modification on macrophage polarisation

“…Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats by Histopaque-1077 density gradient centrifugation. Monocytes were isolated from PBMCs using the MACS magnetic cell separation system (positive selection with CD14 MicroBeads and LS columns, Miltenyi Biotec) as described before (Garcia-Nieto et al, 2010;Harrington et al, 2014). Purified monocytes were suspended in RPMI-1640 medium supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin.…”

A 3D in vitro model of the dermoepidermal junction amenable to mechanical testing

“…S1). To gain an accurate measure of TEER of KC of the model DEJ, an alternative assay is needed, which could include, a custom‐designed platform or microfluidic device . Nevertheless, TEER confirmed the difference of forming KC integrity between WTKC and pKC.…”

A paper-based in vitro model for on-chip investigation of the human respiratory system

“…All results were compared with conventional static ALI transwell flasks. For conventional transwell ALI cultures, 12 well format transwells from Corning® with 0.4 µm pore size were used as described before 4 . Same number of CALU-3 cells (2 × 10 5 cells in 200 µl) were seeded in both systems.…”

“…However, exposure to insults such as hazardous substances, allergens, pathogens, and smoking can alter the integrity of the epithelial cells, resulting in severely impaired respiratory function, which may lead to many life threatening respiratory diseases (e.g., viral respiratory tract infections, asthma, chronic obstructive pulmonary disease, and pneumonia) 3 . Treatment for these conditions is challenging, primarily due to an incomplete understanding of the disease etiologies and the fundamental underpinnings of epithelial tissue physiology 4 . Such scarcity of therapies urges the need for developing more clinically relevant models of epithelial tissue that facilitate studying of the respiratory system and drug screening 5 .…”