Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air–liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.

The airway epithelium is a key protective barrier whose integrity is preserved by the selfrenewal and differentiation of basal progenitor cells. Epithelial cells are central to the pathogenesis of multiple lung diseases. In chronic diseases, increasing age is a principle risk factor. In acute diseases, such as COVID-19, children suffer less severe symptoms than adults and have a lower rate of mortality. Few studies have explored differences between airway epithelial cells in children and adults to explain this age dependent variation in diseases. Here, we perform bulk RNA sequencing studies in laser-capture microdissected whole epithelium, FACS-sorted basal cells and cultured basal cells, as well as in vitro cell proliferation experiments, to address the intrinsic molecular differences between paediatric and adult airway basal cells. We find that, while the cellular composition of the paediatric and adult tracheobronchial epithelium is broadly similar, in cell culture, paediatric airway epithelial cells displayed higher colony forming ability, better in vitro growth and outcompeted adult cells in competitive proliferation assays. In RNA sequencing experiments, we observed potentially important differences in airway epithelial gene expression between samples from children and adults. However, genes known to be associated with SARS-CoV-2 infection were not differentially expressed between children and adults. Our results chart cell-intrinsic differences in transcriptional profile and regenerative capacity between proximal airway epithelial cells of children and adults.

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Advances in stem cell biology and the understanding of factors that determine lung stem cell self-renewal have enabled long-term in vitro culture of human lung cells derived from airway basal and alveolar type II cells. Improved capability to expand and study primary cells long-term, including in clonal cultures that are recently derived from a single cell, will allow experiments that address fundamental questions about lung homeostasis and repair, as well as translational questions in asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and lung cancer research. Here, we provide a brief history of post-natal lung epithelial cell culture and describe recent methodological advances, including some culture systems that now permit clonal cell culture. We further discuss the applications of primary cultures in defining 'normal' epithelium, modelling lung disease and in future cell therapies.