Fabio Bucchieri1,2,3,4, Alessandro Pitruzzella2,3, Alberto Fucarino2,3, Antonella Marino Gammazza2,3, Celeste Caruso Bavisotto2,3, Vito Marcianò2, Massimo Cajozzo5, Giorgio Lo Iacono5, Roberto Marchese6, Giovanni Zummo2, Stephen T Holgate1,7, Donna E Davies1,7. 1. a Academic Unit of Clinical and Experimental Sciences , University of Southampton Faculty of Medicine, University Hospital Southampton , Southampton , United Kingdom. 2. b Dipartimento BIONEC , University of Palermo , Palermo , Italy. 3. c Istituto Euro-Mediterraneo di Scienza e Tecnologia (IEMEST) , Palermo , Italy. 4. d Institute of Biomedicine and Molecular Immunology (IBIM), Italian National Research Council (CNR) , Palermo , Italy. 5. e Dipartimento di Discipline Chirurgiche, Oncologiche e Stomatologiche , University of Palermo , Palermo , Italy. 6. f Interventional Pulmonology Unit , La Maddalena Cancer Center , Palermo , Italy. 7. g Southampton NIHR Respiratory Biomedical Research Unit, Sir Henry Wellcome Laboratories , University of Southampton School of Medicine, University Hospital Southampton , Southampton , United Kingdom.
Abstract
BACKGROUND/AIM: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. MATERIALS AND METHODS: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). RESULTS: The outgrowths formed as a multicellular sheet with motile cilia becoming evident as the Matrigel was remodeled to provide an air interface; cultures were viable for more than one year. Immunofluorescence and electron microscopy (EM) identified an upper layer of mucociliary epithelium and a lower layer of highly organized extracellular matrix (ECM) interspersed with fibroblastic cells separated by a basement membrane. EM analysis of the mucosal construct after repetitive exposure to CSE revealed epithelial damage, loss of cilia, and ECM remodeling, as occurs in vivo. CONCLUSIONS: We have developed a robust bronchial mucosal model. The structural changes observed following CSE exposure suggest the model should have utility for drug discovery and preclinical testing, especially those targeting airway remodeling.
BACKGROUND/AIM: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. MATERIALS AND METHODS: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). RESULTS: The outgrowths formed as a multicellular sheet with motile cilia becoming evident as the Matrigel was remodeled to provide an air interface; cultures were viable for more than one year. Immunofluorescence and electron microscopy (EM) identified an upper layer of mucociliary epithelium and a lower layer of highly organized extracellular matrix (ECM) interspersed with fibroblastic cells separated by a basement membrane. EM analysis of the mucosal construct after repetitive exposure to CSE revealed epithelial damage, loss of cilia, and ECM remodeling, as occurs in vivo. CONCLUSIONS: We have developed a robust bronchial mucosal model. The structural changes observed following CSE exposure suggest the model should have utility for drug discovery and preclinical testing, especially those targeting airway remodeling.
Entities:
Keywords:
ECM; bronchial; cigarette smoke; electron microscopy; epithelium; fibroblast; in vitro model
Authors: Kolene E Bailey; Michael L Floren; Tyler J D'Ovidio; Steven R Lammers; Kurt R Stenmark; Chelsea M Magin Journal: Am J Physiol Lung Cell Mol Physiol Date: 2018-11-21 Impact factor: 5.464
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