Development of a human 3D co-culture model of the airway blood barrier to investigate cell:cell cross talk

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Barrier integrity is crucial for maintaining tissue homeostasis. In the lung, epithelial cells form a barrier to the inhaled environment and endothelial cells to the circulation. Cellular crosstalk between these different cell types is highly coordinated and maintains tissue homeostasis. Damage of the epithelium by inhaled environmental agents results in an inflammatory response in order to contain intruding agents, prevent further tissue damage and initiate tissue repair. Tight regulation of inflammation is essential to prevent chronic inflammation and fibrosis that can affect lung function and respiratory health. There is little knowledge about the mechanisms of cellular crosstalk to maintain barrier homeostasis in the human airway. This is mainly due to a lack of suitable human in vitro models of the airway which recapitulate the complex in vivo situation accurately. There is an unmet need for in vitro 3D tissue co-culture models of the human airway in order to understand the mechanisms of cellular crosstalk in detail. To address this, interdisciplinary research at Southampton has developed a microfluidic platform that enables the long term culture of human airway cells to create an ex-vivo 3D tissue construct. The technology is based on a microfluidic platform which recapitulates the interstitial flow mimicking the in-vivo cellular environment. The epithelial cells are cultured on materials similar to the widely accepted Transwell system. This consists of a thin nonporous polyester scaffold with limited porosity that sits at the air-liquid surface to support the tissue construct and aid epithelial cell differentiation. However, this system does not allow the infiltration of immune cells into the tissue construct. This PhD project will refine this complex 3D in vitro model of the airway-blood barrier by(i)Developing a biocompatible membrane support material that delivers a more physiological representative barrier model which allows for a high porosity supporting a much higher extent of cell-cell communication than in commonly used scaffolds.(ii)Incorporating immune cells (neutrophils) into the complex 3D co-culture model using microfluids to investigate cellular cross talk that initiates inflammation.(i)For in vitro epithelial barrier formation, epithelial cells are currently grown on extracellular matrix coated nanoporous membranes which aids their polarisation. However, these membranes are stiff and the small pore size hinders their use for the assessment of immune cell migration. These synthetic membranes, as well as many natural and synthetic biomaterials, are limited as they cannot mimic the mechanical properties, including strength and stiffness of the airway mucosa. This project will investigate the suitability of a new range of biodegradable polymers which are mechanically tunable with high strength and toughness. Using these biodegradable polymers, epithelial cell polarisation and endothelial cell barrier formation will be determined and their response to environmental challenges will be investigated. (ii)After tissue injury neutrophils are the first immune cells infiltrating the tissue within hours. The loss of epithelial barrier integrity can initiate cellular crosstalk between the epithelial and endothelial barriers to regulate the influx of neutrophils into the tissue during the initiation phase of inflammation. The kinetics of neutrophil infiltration is regulated at many levels, in particular through the release of chemokines and lipid mediators. In our current 3D model, neutrophils cannot transmigrate into the tissue construct. The PhD project aims to incorporate biodegradable scaffold that allows the formation of epithelial and endothelial barriers and direct immune cell infiltration the 3D model. Following environmental challenge, the adhesion and influx of neutrophils will be monitored in real time, temporal mediator release and expression of endothelial adhesion molecules will be determined.