Impact of mouse tracheal basal cell expansion medium on formation and metabolic characteristics of tracheospheres
Year: 2025
Type: article
Abstract: BACKGROUND: Airway basal cells have been shown to play important roles in lung disease, prompting their use in research involving 3-dimensional (3D) organoid culture. Mouse tracheal basal cells (MTBC) represent a key model in understanding the differentiation of airway basal cells in normal development and diseased states. The isolation, expansion, and culture media of basal cells can affect their capacity to maintain stemness and properly differentiate, thereby introducing unknown experimental variables. As MTBC use in 3D organoid culture proceeds, there is an increasing need for comparative studies to identify unknown experimental variables. In this study, we examined the effects of MTBC expansion media on the differentiation and tracheosphere-forming ability in 3D culture. METHODS: MTBC were isolated from mouse tracheas, and three commonly used airway basal cell media were compared during MTBC expansion in cell culture (hereafter referred to as Media 1, 2, and 3). Following expansion, MTBC were cultured identically in 3D conditions to analyze tracheosphere-forming capability. Image analyses were performed to characterize tracheosphere count, size, and morphology, while metabolic and transcriptomic signatures were analyzed to assess MTBC differentiation in 3D culture. RESULTS: Expansion of MTBC in the three distinct media resulted in subsequent 3D tracheospheres that displayed unique metabolite and gene expression profiles, accompanied by overall changes in tracheosphere count and size. MTBC expanded in Medium 1 displayed lower levels of TCA cycle intermediates, along with lowered expression of differentiation markers Foxj1 and Scgb1a1, compared to MTBC expanded in Media 2 or 3. Pathway analysis showed an upregulation of idiopathic pulmonary fibrosis signaling pathways in tracheospheres cultured from MTBC expanded in Media 2 or 3. CONCLUSION: This study identified several key differences in tracheosphere-forming ability of MTBC resulting from MTBC expansion media and highlights the importance of transparency in cell culture protocols, particularly those involving stem cells or 3D culture.
Source: BMC Molecular and Cell Biology
Language: English
Subfield: Pulmonary and Respiratory Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,390
Funders: BrightFocus Foundation, Leona M. and Harry B. Helmsley Charitable Trust, University of Cincinnati, Japan Agency for Medical Research and Development, National Sanitarium Association, UK Research and Innovation, Ministerio de Ciencia, Innovación y Universidades, Joint Fire Science Program, National Institute of Forest Science, Shanghai Municipal Health Commission, Ontario Centre of Innovation, National Institute for Health and Care Research, Diabetes UK, Imperial College London, European Commission, Council of Scientific and Industrial Research, India, Ulster University, Deutsche Forschungsgemeinschaft, Japan Science and Technology Corporation, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, National Natural Science Foundation of China, ZonMw, King Saud University, Chung-Ang University, Carl Tryggers Stiftelse för Vetenskaplig Forskning, China Postdoctoral Science Foundation, Daiichi-Sankyo, Ministero della Salute, National Research Foundation of Korea, Knut och Alice Wallenbergs Stiftelse, Max-Planck-Gesellschaft, Shanxi Datong University, Vetenskapsrådet, China Scholarship Council, Ministerio de Ciencia e Innovación, Österreichische Forschungsförderungsgesellschaft, Natural Science Foundation of Liaoning Province, Norges Forskningsråd, Kungliga Fysiografiska Sällskapet i Lund, Yangzhou University, Natural Science Foundation of Shandong Province, Conseil National de la Recherche Scientifique, King Khalid University, Beijing Municipal Science and Technology Commission, Taichung Veterans General Hospital, Pontificia Universidad Católica del Perú, Ministry of Science and Higher Education of the Russian Federation, Foundation for Innovative Research Groups of the National Natural Science Foundation of China, Shanghai Rising-Star Program, Shanxi Agricultural University, Ipsen, Natural Science Foundation of Sichuan Province, South-Central University for Nationalities, Renji Hospital, National Institutes of Health, Health Resources and Services Administration, California Department of Fish and Wildlife, Wu Jieping Medical Foundation, National Outstanding Youth Science Fund Project of National Natural Science Foundation of China, Canadian Institutes of Health Research, Biotechnology and Biological Sciences Research Council, Economic and Social Research Council, Australian Research Council, Japan Society for the Promotion of Science, Japan Science and Technology Agency, European Regional Development Fund, Natural Science Foundation of Tianjin Municipal Science and Technology Commission, National Key Research and Development Program of China, Fundamental Research Funds for the Central Universities, Spanish National Plan for Scientific and Technical Research and Innovation, Science and Technology Program of Gansu Province, College of Petroleum Engineering and Geosciences, King Fahd University of Petroleum and Minerals, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, National Institute on Deafness and Other Communication Disorders, National Institute of Diabetes and Digestive and Kidney Diseases, Basic Energy Sciences, Southern Research Station, Strategic International Collaborative Research Program, Air Force Office of Scientific Research, H2020 European Research Council, Alagappa University
Award: G9000045247
OpenAlex ID: W4415353519