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Here, we examined how prolonged static culture affects lipid metabolism, mitochondrial bioenergetics, and viability in A549 cells. A549 cultures were maintained without passaging for up to 25 days in DMEM or Ham\u2019s F-12 and analyzed using lipid secretion assays, targeted lipidomics, [14C]-acetate incorporation, Seahorse bioenergetic profiling, and transcriptional analysis of stress-associated markers. Several surfactant-associated readouts were highest during early culture, peaking on day 7, as evidenced by elevated expression of ABCA3 and SP-A and maximal secretion of surfactant-associated phospholipids. With prolonged cultivation and increasing culture density, cellular phosphatidylglycerol levels declined progressively and became nearly undetectable by day 25, accompanied by reduced anabolic lipid metabolism, lower oxygen consumption, and impaired glycolytic activity. These changes coincided with increased reactive oxygen species, elevated intracellular Ca2+ levels, and increased expression of stress-associated transcripts, including CASP1, IL1B, and C3. Later stages were also associated with reduced mitochondrial respiration and decreased viability. Collectively, our findings show that prolonged static culture is associated with metabolic remodeling and reduced bioenergetic capacity in A549 cells.<\/jats:p>","DOI":"10.3390\/ijms27083417","type":"journal-article","created":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T17:05:51Z","timestamp":1775840751000},"page":"3417","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Long-Term Static Cultivation Alters Lipid Metabolism and Bioenergetic Capacity in A549 Cells"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4170-0615","authenticated-orcid":false,"given":"Ivana","family":"\u010euri\u0161ov\u00e1","sequence":"first","affiliation":[{"name":"Department of Membrane Biochemistry, Institute of Animal Biochemistry and Genetics, Centre of Biosciences, Slovak Academy of Sciences, 84104 Bratislava, Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8567-3794","authenticated-orcid":false,"given":"Lucia","family":"\u0160ofrankov\u00e1","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak University of Technology, 81237 Bratislava, Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5973-316X","authenticated-orcid":false,"given":"Ale\u0161","family":"Kvasni\u010dka","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry, Oslo University Hospital, 4950 Oslo, Norway"},{"name":"Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, Faculty of Medicine and Dentistry, Palack\u00fd University Olomouc, 77900 Olomouc, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miroslav","family":"Bal\u00e1\u017e","sequence":"additional","affiliation":[{"name":"Biomedical Research Center, Slovak Academy of Sciences, 84104 Bratislava, Slovakia"},{"name":"Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University, 84215 Bratislava, Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivana","family":"F\u00e1bryov\u00e1","sequence":"additional","affiliation":[{"name":"Department of Membrane Biochemistry, Institute of Animal Biochemistry and Genetics, Centre of Biosciences, Slovak Academy of Sciences, 84104 Bratislava, Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3448-9073","authenticated-orcid":false,"given":"David","family":"Friedeck\u00fd","sequence":"additional","affiliation":[{"name":"Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, Faculty of Medicine and Dentistry, Palack\u00fd University Olomouc, 77900 Olomouc, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8113-6855","authenticated-orcid":false,"given":"M\u00e1ria","family":"Bal\u00e1\u017eov\u00e1","sequence":"additional","affiliation":[{"name":"Department of Membrane Biochemistry, Institute of Animal Biochemistry and Genetics, Centre of Biosciences, Slovak Academy of Sciences, 84104 Bratislava, Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/S0925-4439(98)00060-X","article-title":"Pulmonary Surfactant: Functions and Molecular Composition","volume":"1408","author":"Goerke","year":"1998","journal-title":"Biochim. 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