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

Joel Mario Vej-Nielsen, Adelina Rogowska-Wrzesinska*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review


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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Publication date2021
Publication statusPublished - 2021
SeriesMethods in Molecular Biology


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

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