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3D-ViaFlow: A Quantitative Viability Assay for Multicellular Spheroids

Part of the Methods in Molecular Biology book series (MIMB,volume 2273)


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.

Key words

  • 3D-ViaFlow
  • Viability
  • Spheroid
  • 3D cell culture
  • Morphology
  • FACS
  • Flow cytometry
  • Fluorescein diacetate
  • Propidium iodide
  • High throughput

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The authors acknowledge the financial support from the COST Action CA16119 (In vitro 3-D total cell guidance and fitness) and the Sino Danish Center for Education and Research for PhD grant to JMVN.

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Correspondence to Adelina Rogowska-Wrzesinska .

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Vej-Nielsen, J.M., Rogowska-Wrzesinska, A. (2021). 3D-ViaFlow: A Quantitative Viability Assay for Multicellular Spheroids. 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.

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1245-3

  • Online ISBN: 978-1-0716-1246-0

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