Abstract
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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References
Pampaloni F, Reynaud EG, Stelzer EHK (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8:839–845
Wang W, Itaka K, Ohba S, Nishiyama N, Chung U-i, Yamasaki Y et al (2009) 3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells. Biomaterials 30(14):2705–2715
Fey SJ, Wrzesinski K (2012) Determination of drug toxicity using 3D spheroids constructed from an immortal human hepatocyte cell line. Toxicol Sci 127(2):403–411
Wrzesinski K, Fey SJ (2013) After trypsinisation, 3D spheroids of C3A hepatocytes need 18 days to re-establish similar levels of key physiological functions to those seen in the liver. Toxicol Res (Camb) 2(2):123–135
Mehta G, Hsiao AY, Ingram M, Luker GD, Takayama S (2012) Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J Control Release 164(2):192–204
Sirenko O, Mitlo T, Hesley J, Luke S, Owens W, Cromwell EF (2015) High-content assays for characterizing the viability and morphology of 3D cancer spheroid cultures. Assay Drug Dev Technol 13(7):402–414
Piccinini F, Tesei A, Arienti C, Bevilacqua A (2017) Cell counting and viability assessment of 2D and 3D cell cultures: expected reliability of the trypan blue assay. Biol Proced Online 19(1):8
Madoux F, Tanner A, Vessels M, Willetts L, Hou S, Scampavia L et al (2017) A 1536-well 3D viability assay to assess the cytotoxic effect of drugs on spheroids. SLAS Discov Adv Sci Drug Discov 22(5):516–524
De Witt Hamer PC, Jonker A, Leenstra S, Ruijter JM, Van CJF N (2005) Quantification of viability in organotypic multicellular spheroids of human malignant glioma using lactate dehydrogenase activity: a rapid and reliable automated assay. J Histochem Cytochem 53(1):23–34
Friedrich J, Eder W, Castaneda J, Doss M, Huber E, Ebner R et al (2007) A reliable tool to determine cell viability in complex 3-D culture: the acid phosphatase assay. J Biomol Screen 12(7):925–937
Perard M, Tricot-Doleux S, Pellen-Mussi P, Meary F, Pérez F (2011) Evaluation of the cytotoxicity of pulp floor perforation filling materials by using in parallel 2d and 3d culture models. Bull Group Int Rech Sci Stomatol Odontol 50(2):42–43
Kim YE, Jeon HJ, Kim D, Lee SY, Kim KY, Hong J et al (2018) Quantitative proteomic analysis of 2D and 3D cultured colorectal cancer cells: profiling of Tankyrase inhibitor XAV939-induced proteome. Sci Rep 8(1):1–12
Jones KH, Senft JA (1985) An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J Histochem Cytochem 33(1):77–79
Weber K, Fehse B (2010) Diva-fit: a step-by-step manual for generating high-resolution graphs and histogram overlays of flow cytometry data obtained with FACSDiva software. Cell Ther Transplant 1(4)
Acknowledgments
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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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. https://doi.org/10.1007/978-1-0716-1246-0_11
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DOI: https://doi.org/10.1007/978-1-0716-1246-0_11
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