{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T01:24:34Z","timestamp":1792805074927,"version":"4.5.0"},"reference-count":38,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,4,11]],"date-time":"2023-04-11T00:00:00Z","timestamp":1681171200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Med."],"abstract":"<jats:p>The field of airway biology research relies primarily on <jats:italic>in vitro<\/jats:italic> and <jats:italic>in vivo<\/jats:italic> models of disease and injury. The use of <jats:italic>ex vivo<\/jats:italic> models to study airway injury and cell-based therapies remains largely unexplored although such models have the potential to overcome certain limitations of working with live animals and may more closely replicate <jats:italic>in vivo<\/jats:italic> processes than <jats:italic>in vitro<\/jats:italic> models can. Here, we characterized a ferret <jats:italic>ex vivo<\/jats:italic> tracheal injury and cell engraftment model. We describe a protocol for whole-mount staining of cleared tracheal explants, and showed that it provides a more comprehensive structural overview of the surface airway epithelium (SAE) and submucosal glands (SMGs) than 2D sections, revealing previously underappreciated structural anatomy of tracheal innervation and vascularization. Using an <jats:italic>ex vivo<\/jats:italic> model of tracheal injury, we evaluated the injury responses in the SAE and SMGs that turned out to be consistent with published <jats:italic>in vivo<\/jats:italic> work. We used this model to assess factors that influence engraftment of transgenic cells, providing a system for optimizing cell-based therapies. Finally, we developed a novel 3D-printed reusable culture chamber that enables live imaging of tracheal explants and differentiation of engrafted cells at an air-liquid interface. These approaches promise to be useful for modeling pulmonary diseases and testing therapies.<\/jats:p><jats:p><jats:fig><jats:label>Graphical abstract<\/jats:label><jats:caption><jats:p><jats:bold>1<\/jats:bold>,<jats:bold>2<\/jats:bold>. We describe here a method for differential mechanical injury of ferret tracheal explants that can be used to evaluate airway injury responses ex vivo. <jats:bold>3<\/jats:bold>. Injured explants can be cultured at ALI (using the novel tissue-transwell device on the right) and submerged long-term to evaluate tissue-autonomous regeneration responses. <jats:bold>4<\/jats:bold>. Tracheal explants can also be used for low throughput screens of compounds to improve cell engraftment efficiency or can be seeded with particular cells to model a disease phenotype. <jats:bold>5<\/jats:bold>. Lastly, we demonstrate that ex vivo-cultured tracheal explants can be evaluated by various molecular assays and by immunofluorescent imaging that can be performed live using our custom-designed tissue-transwell.<\/jats:p><\/jats:caption><\/jats:fig><\/jats:p>","DOI":"10.3389\/fmed.2023.1144754","type":"journal-article","created":{"date-parts":[[2023,4,12]],"date-time":"2023-04-12T11:50:31Z","timestamp":1681300231000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":4,"title":["Development and characterization of ferret ex vivo tracheal injury and cell engraftment model"],"prefix":"10.3389","volume":"10","author":[{"given":"Vitaly","family":"Ievlev","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Albert C.","family":"Pai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"Jr","given":"Drew S.","family":"Dillon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Spencer","family":"Kuhl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas J.","family":"Lynch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kyle W.","family":"Freischlag","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caitlyn B.","family":"Gries","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John F.","family":"Engelhardt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kalpaj R.","family":"Parekh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,4,11]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"766","DOI":"10.1038\/s41588-019-0388-9","article-title":"Lung regeneration by multipotent stem cells residing at the bronchioalveolar-duct junction","volume":"51","author":"Liu","year":"2019","journal-title":"Nat Genet"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"1302","DOI":"10.1039\/C4LC01252F","article-title":"A lung-on-a-chip array with an integrated bio-inspired respiration mechanism","volume":"15","author":"Stucki","year":"2015","journal-title":"Lab Chip"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"1065","DOI":"10.1096\/fasebj.24.1_supplement.1065.22","article-title":"A human breathing lung-on-a-chip","volume":"24","author":"Huh","year":"2010","journal-title":"FASEB 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long-term cultivation of ex vivo rat trachea","volume":"22","author":"Mir","year":"2022","journal-title":"Lab Chip"},{"key":"ref11","doi-asserted-by":"publisher","first-page":"95","DOI":"10.1007\/978-3-030-24108-7_6","article-title":"Heterogeneity of pulmonary stem cells","volume":"1169","author":"Lynch","year":"2019","journal-title":"Adv Exp Med Biol"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"1974","DOI":"10.1097\/TP.0000000000004148","article-title":"Ferret lung transplantation models differential lymphoid aggregate morphology between restrictive and obstructive forms of chronic lung allograft dysfunction","volume":"106","author":"Lynch","year":"2022","journal-title":"Transplantation"},{"key":"ref13","doi-asserted-by":"publisher","first-page":"524","DOI":"10.1089\/hum.2020.013","article-title":"Viral vectors, animal models, and cellular targets for gene therapy of cystic fibrosis lung disease","volume":"31","author":"Tang","year":"2020","journal-title":"Hum Gene 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