Abstract
Cells cultured in a monolayer have been a central tool in molecular and cell biology, toxicology, biochemistry, and so on. Therefore, most methods for adherent cells in cell biology are tailored to this format of cell culturing. Limitations and disadvantages of monolayer cultures, however, have resulted in the ongoing development of advanced cell culturing techniques. One such technique is culturing cells as multicellular spheroids, that had been shown to mimic the physiological conditions found in vivo more accurately. This chapter presents a novel method for separation of the spheroid rim and core in mature spheroids (>21 days) for further analysis using advanced molecular biology techniques such as flow cytometry, viability estimations, comet assay, transcriptomics, proteomics and lipidomic. This fast and gentle disassembly of intact spheroids into rim and core fractions, and further into viable single-cell suspension provides an opportunity to bridge the gap from 3D cell culture to current state-of-the-art analysis methods.
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Acknowledgments
The authors acknowledge the financial support from the Sino Danish Research and Education Center for PhD project for HSF and JMVN, Slovenian Research Agency [research core funding J1-2465, and grant to young researchers MR-MStampar P1-0245], and COST Actions CA16119 (In vitro 3-D total cell guidance and fitness).
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Frandsen, H.S., Štampar, M., Vej-Nielsen, J.M., Žegura, B., Rogowska-Wrzesinska, A. (2021). Method to Disassemble Spheroids into Core and Rim for Downstream Applications Such as Flow Cytometry, Comet Assay, Transcriptomics, Proteomics, and Lipidomics. In: Brevini, T.A., Fazeli, A., Turksen, K. (eds) Next Generation Culture Platforms for Reliable In Vitro Models . Methods in Molecular Biology, vol 2273. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1246-0_12
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DOI: https://doi.org/10.1007/978-1-0716-1246-0_12
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