02121nas a2200289   4500000000100000000000100001008004100002260001500043653001300058653001500071653002500086653002900111653002300140100002100163700001700184700002000201700002000221700001700241700001700258700001700275700001700292245013300309856009200442490000600534520127700540022001401817       2025                            d  c2025-10-0110aFBS-free10aHeLa cells10aanimal free research10achemically defined media10afetal bovine serum1 aAlexandra Nessar1 aViola Röhrs1 aMathias Ziersch1 aAhmed S. M. Ali1 aJulia Moradi1 aAnke Kurreck1 aJohanna Berg1 aJens Kurreck00aPromoting ethical and reproducible cell culture: implementing animal-free alternatives to teaching in molecular and cell biology  uhttps://www.frontiersin.org/journals/toxicology/articles/10.3389/ftox.2025.1670513/full0 v73 aThe widespread use of fetal bovine serum (FBS) and other animal-derived reagents in cell culture raises ethical concerns and scientific limitations, including batch variability and undefined composition. To address these challenges and promote the adoption of xeno-free, human-relevant methods, we developed a graduate-level laboratory course based on animal-free workflows. The curriculum covers key molecular and cell biology techniques: HeLa cell culture and passaging, transfection, RNA interference (RNAi), quantitative RT-PCR (qRT-PCR), dual-luciferase assays, and Western blotting, using reagents selected to exclude animal-derived components. A chemically defined medium (CDM) was optimized for robust HeLa cell growth in the absence of FBS, and recombinant TrypLE was implemented as a substitute for porcine trypsin. Validated non-animal-derived antibodies are also introduced. The course has been successfully piloted and provides a scalable, ethical framework for modern bioscience education. A detailed, open-access protocol enables replication and dissemination. This initiative equips students with practical skills and educational foundation in animal-free methodologies, supporting a shift toward reproducible and ethically responsible biomedical research.  a2673-3080