Developing a 3D model of the airways

Abstract

Chronic cigarette smoke exposure leads to chronic obstructive pulmonary disease (COPD) in susceptible individuals where goblet cell metaplasia, ciliated cell hypoplasia and extra cellular matrix (ECM) thickening are observed. Current in vitro COPD models such as air-liquid interface (ALI) cultures, cannot model the complex morphology and the cell-ECM interaction seen in vivo. No organoid models of COPD currently exist and, therefore, a lung organoid model termed bronchosphere of COPD was generated. Basal epithelial cells from normal healthy and COPD donors were cultured into bronchospheres over 20 days in 25% Matrigel. Bronchospheres were characterised using quantitative PCR, immunofluorescence and RNA sequencing. The effect of stromal cells on basal epithelial cell-derived bronchosphere structure and function were investigated through a triple culture of bronchial epithelial, lung fibroblast and airway smooth muscle cells. COPD bronchospheres developed more slowly displaying goblet cell hyperplasia and ciliated cell hypoplasia with reduced cilial beat frequencies compared to normal healthy controls. Normal healthy basal cells chronically treated with cigarette smoke condensate formed bronchospheres with lumens lacking a differentiated epithelium. RNA-seq analysis of bronchospheres showed up-regulation of the club cell markers mucin 5B (muc5b) and secretoglobin family 3A member 1 (scgb3a1) in healthy vs COPD bronchospheres. Pathway analysis revealed increased extracellular matrix function and decreased fibroblast growth factor signalling in COPD and cigarette smoke-treated healthy bronchospheres, which may be a possible driver of disease phenotype. Epithelial-stromal cross talk enabled formation of epithelial cell-driven branching tubules consisting of luminal epithelial cells surrounded by stromal cells. Addition of agarose to the Matrigel scaffold (Agrigel) altered the matrix viscoelasticity and stiffness and prevented tubule collapse. This thesis described the development of a novel bronchosphere model of COPD derived from primary human airway cells that recapitulates many functions of human COPD. Generating large numbers of these bronchospheres provides opportunities for future personalised drug testing.