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

Joel Mario Vej-Nielsen1, Adelina Rogowska-Wrzesinska2

  • 1Institute for Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

Methods in Molecular Biology (Clifton, N.J.)
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PubMed
Summary

The 3D-ViaFlow method quantifies cell viability in multicellular spheroids using dual staining and flow cytometry. This technique enhances 3D cell culture analysis for improved biological research.

Keywords:
3D cell culture3D-ViaFlowFACSFlow cytometryFluorescein diacetateHigh throughputMorphologyPropidium iodideSpheroidViability

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • Three-dimensional (3D) cell culture models offer enhanced functionality and physiological relevance compared to traditional 2D in vitro methods.
  • Multicellular spheroids, a form of 3D culture, promote tissue-like growth by enabling cell-generated extracellular matrices and intercellular connections.
  • Assessing cell viability within these complex 3D structures is crucial for accurate biological interpretation.

Purpose of the Study:

  • To present the 3D-ViaFlow method for quantitative cell viability assessment in multicellular spheroids.
  • To optimize a dual live-dead cell staining protocol combined with flow cytometry for 3D cell populations.
  • To adapt the method for analyzing both monolayer cultures and spheroids derived from human hepatocytes and endothelial cells.

Main Methods:

  • Development of the 3D-ViaFlow method integrating optimized dual live-dead cell staining (fluorescein diacetate and propidium iodide) with flow cytometry.
  • Protocol includes spheroid disassembly, cell staining, and specific flow cytometry gating strategies for heterogeneous spheroid cell populations.
  • Method validation on HepG2/C3A human hepatocytes and co-cultures with HMEC-1 endothelial cells.

Main Results:

  • Successful quantitative estimation of cell viability in multicellular spheroids using the 3D-ViaFlow method.
  • Optimized protocols for spheroid dissociation and staining suitable for flow cytometric analysis.
  • Demonstrated applicability to both single cell type spheroids and co-culture spheroids.

Conclusions:

  • The 3D-ViaFlow method provides a robust and quantitative approach to assess cell viability in 3D spheroid cultures.
  • This technique improves the analysis of complex cellular environments, advancing 3D cell culture research.
  • The optimized protocol facilitates more accurate interpretation of cell behavior and drug responses in physiologically relevant models.