Institute for Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia.
Methods Mol Biol. 2021;2273:173-188. doi: 10.1007/978-1-0716-1246-0_12.
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.
在单层中培养的细胞一直是分子和细胞生物学、毒理学、生物化学等领域的重要工具。因此，细胞生物学中大多数用于贴壁细胞的方法都是针对这种细胞培养形式量身定制的。然而，单层培养的局限性和缺点导致了先进细胞培养技术的不断发展。其中一种技术是将细胞培养成多细胞球体，这已被证明可以更准确地模拟体内的生理条件。本章介绍了一种从成熟球体（>21 天）中分离球体边缘和核心的新方法，以便进一步使用先进的分子生物学技术进行分析，如流式细胞术、活力估计、彗星试验、转录组学、蛋白质组学和脂质组学。这种快速而温和的方法可以将完整的球体拆分成边缘和核心部分，并进一步分离成有活力的单细胞悬液，为从 3D 细胞培养到当前最先进的分析方法架起了桥梁。
