Institute for Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Methods Mol Biol. 2021;2273:159-171. doi: 10.1007/978-1-0716-1246-0_11.
Three-dimensional cell culture became an essential method in molecular and cell biology research. Accumulating results show that cells grown in 3D, display increased functionality and are capable of recapitulating physiological functions that are not observed in classical in vitro models. Spheroid-based cell culture allows the cells to establish their own extracellular matrix and intricate intercellular connections promoting a tissue-like growth environment.In this paper we present the 3D-ViaFlow method that combines an optimised dual live-dead cell staining with flow cytometry to deliver a quantitative estimation of viability of cells in multicellular spheroids. The method is optimised for monolayer cultures and multicellular spheroids created from HepG2/C3A human hepatocytes or coculture of HepG2/C3A and endothelial cell line HMEC-1. It includes protocol for spheroids disassembling, labeling of cells with fluorescein diacetate and propidium iodide and instructions for flow cytometry gating optimized for analysis of heterogeneous cell populations form spheroids.
三维细胞培养成为分子和细胞生物学研究的重要方法。越来越多的结果表明，在 3D 中生长的细胞表现出增强的功能，并且能够再现经典体外模型中观察不到的生理功能。基于球体的细胞培养允许细胞建立自己的细胞外基质和复杂的细胞间连接，促进类似组织的生长环境。在本文中，我们提出了 3D-ViaFlow 方法，该方法将优化的双重活/死细胞染色与流式细胞术相结合，对多细胞球体中的细胞活力进行定量估计。该方法针对单层培养物和由 HepG2/C3A 人肝细胞或 HepG2/C3A 和内皮细胞系 HMEC-1 的共培养物形成的多细胞球体进行了优化。它包括用于球体分解、用荧光素二乙酸酯和碘化丙啶标记细胞的方案，以及针对从球体中分析异质细胞群进行优化的流式细胞术门控说明。
