Abstract
Mainstream adoption of physiologically relevant three-dimensional models has been slow in the last 50 years due to long, manual protocols with poor reproducibility, high price, and closed commercial platforms. This chapter describes high-throughput, low-cost, open methods for spheroid viability assessment which use readily available reagents and open-source software to analyze spheroid volume, metabolism, and enzymatic activity. We provide two ImageJ macros for automated spheroid size determination—for both single images and images in stacks. We also share an Excel template spreadsheet allowing users to rapidly process spheroid size data, analyze plate uniformity (such as edge effects and systematic seeding errors), detect outliers, and calculate dose-response. The methods would be useful to researchers in preclinical and translational research planning to move away from simplistic monolayer studies and explore 3D spheroid screens for drug safety and efficacy without substantial investment in money or time.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Ivanov DP, Parker TL, Walker DA et al (2014) Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres. PLoS One 9:e103817
Astashkina A, Mann B, Grainger DW (2012) A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity. Pharmacol Ther 134:82–106
Hickman JA, Graeser R, de Hoogt R et al (2014) Three-dimensional models of cancer for pharmacology and cancer cell biology: capturing tumor complexity in vitro/ex vivo. Biotechnol J 9:1115–1128
Moscona A, Moscona H (1952) The dissociation and aggregation of cells from organ rudiments of the early chick embryo. J Anat 86:287–301
Sutherland RM, McCredie JA, Inch WR (1971) Growth of multicell spheroids in tissue culture as a model of nodular carcinomas. J Natl Cancer Inst 46:113–120
Tung Y-C, Hsiao AY, Allen SG et al (2011) High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst 136:473–478
Kelm JM, Timmins NE, Brown CJ et al (2003) Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83:173–180
Vinci M, Gowan S, Boxall F et al (2012) Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol 10:1–21
Ivascu A, Kubbies M (2006) Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen 11:922–932
Wenzel C, Riefke B, Gründemann S et al (2014) 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions. Exp Cell Res 323:131–143
Falkenberg N, Höfig I, Rosemann M et al (2016) Three-dimensional microtissues essentially contribute to preclinical validations of therapeutic targets in breast cancer. Cancer Med 5:703–710
Anastasov N, Höfig I, Radulović V et al (2015) A 3D-microtissue-based phenotypic screening of radiation resistant tumor cells with synchronized chemotherapeutic treatment. BMC Cancer 15:466
da Motta LL, Ledaki I, Purshouse K et al (2016) The BET inhibitor JQ1 selectively impairs tumour response to hypoxia and downregulates CA9 and angiogenesis in triple negative breast cancer. Oncogene 36(1):122–132
McIntyre A, Hulikova A, Ledaki I et al (2016) Disrupting hypoxia-induced bicarbonate transport acidifies tumor cells and suppresses tumor growth. Cancer Res 76:3744–3755
Bell CC, Hendriks DFG, Moro SML et al (2016) Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease. Sci Rep 6:25187
Ivanov DP, Al-Rubai A, Grabowska AM et al (2016) Separating chemotherapy-related developmental neurotoxicity from cytotoxicity in monolayer and neurosphere cultures of human fetal brain cells. Toxicol In Vitro 37:88–96
Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
Schindelin J, Rueden CT, Hiner MC et al (2015) The ImageJ ecosystem: an open platform for biomedical image analysis. Mol Reprod Dev 82:518–529
Ivanov DP, Parker TL, Walker DA et al (2015) In vitro co-culture model of medulloblastoma and human neural stem cells for drug delivery assessment. J Biotechnol 205:3–13
Sutherland RM, Eddy HA, Bareham B et al (1979) Resistance to adriamycin in multicellular spheroids. Int J Radiat Oncol Biol Phys 5:1225–1230
Friedrich J, Seidel C, Ebner R et al (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4:309–324
O’Brien J, Wilson I, Orton T et al (2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267:5421–5426
Walzl A, Unger C, Kramer N et al (2014) The resazurin reduction assay can distinguish cytotoxic from cytostatic compounds in spheroid screening assays. J Biomol Screen 19:1047–1059
Chan GKY, Kleinheinz TL, Peterson D et al (2013) A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays. PLoS One 8:e63583
Friedrich J, Eder W, Castaneda J et al (2007) A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay. J Biomol Screen 12:925–937
Ivanov DP, Coyle B, Walker DA et al (2016) In vitro models of medulloblastoma: choosing the right tool for the job. J Biotechnol 236:10–25
Zhang J-H (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73
Hall MD, Telma KA, Chang K-E et al (2014) Say no to DMSO: dimethylsulfoxide inactivates cisplatin, carboplatin, and other platinum complexes. Cancer Res 74:3913–3922
Acknowledgments
Delyan Ivanov was supported by an EPSRC Doctoral Prize award hosted by the University of Nottingham (DP2014/DI). The authors would like to thank Pamela Collier and Alan McIntyre for their help with manuscript editing. Special thanks to Neli Garbuzanova, Janhavi Apte, Arundhati Dongre, Parminder Dhesi, and Amarnath Pal for testing the macros and providing user feedback.
Supplementary Files
The macro files and Excel spreadsheet are available through the Figshare database:
Macro 1 link: https://figshare.com/s/32f81784ee28e3fde015 (DOI: 10.6084/m9.figshare.3487919).
Macro 2 link: https://figshare.com/s/9952d072c3238a60e134 (DOI: 10.6084/m9.figshare.3487943).
Volume analysis template: https://figshare.com/s/6c57cede1d940f6fd952 (DOI: 10.6084/m9.figshare.3487940).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Ivanov, D.P., Grabowska, A.M., Garnett, M.C. (2017). High-Throughput Spheroid Screens Using Volume, Resazurin Reduction, and Acid Phosphatase Activity. In: Gilbert, D., Friedrich, O. (eds) Cell Viability Assays. Methods in Molecular Biology, vol 1601. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6960-9_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6960-9_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6959-3
Online ISBN: 978-1-4939-6960-9
eBook Packages: Springer Protocols