Development of a Primary Human Co-Culture Model of Inflamed Airway Mucosa

Lael M Yonker,Tim N Ford,Alexander D Eaton,Huimin Leung,J R Falck,Bryan P Hurley,T Bernard Kinane,Kengyeh K Chu,Dongyao Cui,Rhianna M Hibbler,Jayaraj Rajagopal,Michael A Pazos,Jinhyeob Ryu,Guillermo J Tearney,Hongmei Mou

doi:10.1038/s41598-017-08567-w

Abstract

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.

Highlights

A co-culture model has been developed to study neutrophil migration across the airway epithelial barrier[11]
Human airway basal cells cultured at an inverted air-liquid interface are able to maintain polarity and functional airway-specific micro-anatomy
In order to appropriately integrate Air-liquid interface (ALI) culturing with directionally relevant neutrophil transepithelial migration, we modified the conventional ALI by seeding the human airway basal cells on the underside of the transwell

Summary

Introduction

A co-culture model has been developed to study neutrophil migration across the airway epithelial barrier[11]. Current understandings of neutrophil transepithelial migration signaling pathways are based almost exclusively on immortalized human lung epithelial cell lines These cells polarize and form functional barriers, they do not feature many physiologically relevant characteristics, such as mucus or beating cilia. The novel features of this model system include the use of expandable human basal cell-derived epithelium to study neutrophil transmigration, generating a primary co-culture model that has not previously been described, combined with advanced cellular imaging, which offers a physiologically relevant platform to explore micro-anatomical processes at the mucosal surface. Combined, this advanced system represents a significant improvement over traditional human in vitro models

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Results

Conclusion