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Several aspects of the inflammatory response to ozone can be modeled in vitro using primary human bronchial epithelial cells (HBECs) cultured at an air\u2013liquid interface. We tested two commonly used HBEC culture media systems, one proprietary and one non-proprietary, to identify which system yielded the most in vivo-like pro-inflammatory response to acute ozone exposure as reflected by gene expression. Cells from 6 donors were grown in each culture system in parallel, followed by examination of epithelial morphology and cell type proportions prior to ozone exposure. Cultures grown in the proprietary system were notably thicker and contained more ciliated and secretory cells, as well as internal cyst-like structures. The transcriptomic response to acute ozone exposure (0.5 parts per million ozone \u00d7 2 h) was strongly affected by media type. HBECs grown in the proprietary system exhibited minimal changes after ozone, with only 7 differentially expressed genes (DEGs). In contrast, HBECs grown in the non-proprietary system exhibited a more dynamic response with 128 DEGs, including hallmark response genes indicative of inflammation (CXCL8) and oxidative stress (HMOX1). Gene set enrichment analysis using the 128 DEGs further corroborated upregulation of oxidative stress and inflammation pathways. In total, our results indicate that the choice of HBEC culture media should be carefully considered to best model the in vivo response to ozone.<\/jats:p>","DOI":"10.1093\/toxsci\/kfaf089","type":"journal-article","created":{"date-parts":[[2025,7,22]],"date-time":"2025-07-22T11:29:26Z","timestamp":1753183766000},"page":"31-36","source":"Crossref","is-referenced-by-count":4,"title":["Culture media influences primary human bronchial epithelial cell morphology, differentiation status, and transcriptional response to ozone exposure"],"prefix":"10.1093","volume":"207","author":[{"given":"Sarah A","family":"Lester","sequence":"first","affiliation":[{"name":"Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599-7249,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8745-570X","authenticated-orcid":false,"given":"Sabri H","family":"Abdelwahab","sequence":"additional","affiliation":[{"name":"Department of Genetics, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599-7248,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5351-2841","authenticated-orcid":false,"given":"Scott H","family":"Randell","sequence":"additional","affiliation":[{"name":"Marsico Lung Institute, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599-7248,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2676-9232","authenticated-orcid":false,"given":"Samir N P","family":"Kelada","sequence":"additional","affiliation":[{"name":"Department of Genetics, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599-7248,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2025,7,23]]},"reference":[{"key":"2025091914021752100_kfaf089-B1","doi-asserted-by":"publisher","first-page":"107004","DOI":"10.1289\/EHP3766","article-title":"Estimates of the global burden of ambient PM2.5, ozone, and NO2 on asthma incidence and emergency room visits","volume":"126","author":"Anenberg","year":"2018","journal-title":"Environ Health Perspect"},{"key":"2025091914021752100_kfaf089-B2","doi-asserted-by":"publisher","first-page":"1363","DOI":"10.1164\/ajrccm\/148.5.1363","article-title":"Ozone-induced airway inflammation in human subjects as determined by airway lavage and biopsy","volume":"148","author":"Aris","year":"1993","journal-title":"Am Rev Respir Dis"},{"key":"2025091914021752100_kfaf089-B3","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1093\/aje\/kwu115","article-title":"Who is more affected by ozone pollution? 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