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However, there have been ongoing discussions about the limited use of these inhalation toxicity tests due to differences in the anatomical structure of the respiratory tract, difficulty in exposure, laborious processes, and ethical reasons. Alternative animal testing methods that mimic in vivo testing are required. Therefore, in this study, we established a co-culture system composed of differentiated epithelial cells under an air-liquid interface (ALI) system in the apical part and fibroblasts in the basal part. This system was designed to mimic the wound-healing mechanism in the respiratory system. In addition, we developed a multi-analysis system that simultaneously performs toxicological and functional evaluations. Several individual assays were used sequentially in a multi-analysis model for pulmonary toxicity. Briefly, cytokine analysis, histology, and cilia motility were measured in the apical part, and cell migration and gel contraction assay were performed by exposing MRC-5 cells to the basal culture. First, human airway epithelial cells from bronchial (hAECB) were cultured under air-liquid interface (ALI) system conditions and validated pseudostratified epithelium by detecting differentiation-related epithelial markers using Transepithelial Electrical Resistance (TEER) measurement, Hematoxylin and Eosin (H&amp;E) staining, and immunocytochemistry (ICC) staining. Afterward, the co-culture cells exposed to Transforming growth factor-beta 1 (TGF-\u03b21), a key mediator of pulmonary fibrosis, induced significant toxicological responses such as cytotoxicity, cell migration, and gel contraction, which are wound-healing markers. In addition, cilia motility in epithelial cells was significantly decreased compared to control. Therefore, the multi-analysis model with a 3D epithelial-fibroblast co-culture system is expected to be useful in predicting pulmonary toxicity as a simple and efficient high-throughput screening method and as an alternative to animal testing.<\/jats:p>","DOI":"10.5620\/eaht.2024024","type":"journal-article","created":{"date-parts":[[2024,9,27]],"date-time":"2024-09-27T05:04:11Z","timestamp":1727413451000},"page":"e2024024","update-policy":"https:\/\/doi.org\/10.5620\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development of a multi-analysis model using an epithelial-fibroblast co-culture system as an alternative to animal 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