High-Throughput Spheroid Screens Using Volume, Resazurin Reduction, and Acid Phosphatase Activity.

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.

[1]  M. Ingelman-Sundberg,et al.  Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease , 2016, Scientific Reports.

[2]  B. Coyle,et al.  In vitro models of medulloblastoma: Choosing the right tool for the job. , 2016, Journal of biotechnology.

[3]  Michael Rosemann,et al.  Three‐dimensional microtissues essentially contribute to preclinical validations of therapeutic targets in breast cancer , 2016, Cancer medicine.

[4]  Cameron Alexander,et al.  In vitro co-culture model of medulloblastoma and human neural stem cells for drug delivery assessment. , 2015, Journal of biotechnology.

[5]  A. Grabowska,et al.  Separating chemotherapy-related developmental neurotoxicity from cytotoxicity in monolayer and neurosphere cultures of human fetal brain cells. , 2016, Toxicology in vitro : an international journal published in association with BIBRA.

[6]  Delyan P. Ivanov,et al.  Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres , 2014, PloS one.

[7]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[8]  Shuichi Takayama,et al.  High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. , 2011, The Analyst.

[9]  Maria Vinci,et al.  Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation , 2012, BMC Biology.

[10]  Juergen Friedrich,et al.  Spheroid-based drug screen: considerations and practical approach , 2009, Nature Protocols.

[11]  Matthias Gutekunst,et al.  Three‐dimensional models of cancer for pharmacology and cancer cell biology: Capturing tumor complexity in vitro/ex vivo , 2014, Biotechnology journal.

[12]  Johannes E. Schindelin,et al.  The ImageJ ecosystem: An open platform for biomedical image analysis , 2015, Molecular reproduction and development.

[13]  C. Unger,et al.  The Resazurin Reduction Assay Can Distinguish Cytotoxic from Cytostatic Compounds in Spheroid Screening Assays , 2014, Journal of biomolecular screening.

[14]  A. Ivascu,et al.  Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis , 2006, Journal of biomolecular screening.

[15]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[16]  Grace Ka Yan Chan,et al.  A Simple High-Content Cell Cycle Assay Reveals Frequent Discrepancies between Cell Number and ATP and MTS Proliferation Assays , 2013, PloS one.

[17]  Martin Fussenegger,et al.  Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. , 2003, Biotechnology and bioengineering.

[18]  R. Sutherland,et al.  Resistance to adriamycin in multicellular spheroids. , 1979, International journal of radiation oncology, biology, physics.

[19]  Jan Lichtenberg,et al.  A 3D-microtissue-based phenotypic screening of radiation resistant tumor cells with synchronized chemotherapeutic treatment , 2015, BMC Cancer.

[20]  Karsten Parczyk,et al.  3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions. , 2016, Experimental cell research.

[21]  A. Harris,et al.  The BET inhibitor JQ1 selectively impairs tumour response to hypoxia and downregulates CA9 and angiogenesis in triple negative breast cancer , 2016, Oncogene.

[22]  R. Sutherland,et al.  Growth of multicell spheroids in tissue culture as a model of nodular carcinomas. , 1971, Journal of the National Cancer Institute.

[23]  I. Wilson,et al.  Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. , 2000, European journal of biochemistry.

[24]  L. Kunz-Schughart,et al.  A Reliable Tool to Determine Cell Viability in Complex 3-D Culture: The Acid Phosphatase Assay , 2007, Journal of biomolecular screening.

[25]  K. Gull,et al.  Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis , 2012, BMC Biology.

[26]  M. Gottesman,et al.  Say no to DMSO: dimethylsulfoxide inactivates cisplatin, carboplatin, and other platinum complexes. , 2014, Cancer research.

[27]  D. Grainger,et al.  A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity. , 2012, Pharmacology & therapeutics.

[28]  A MOSCONA,et al.  The dissociation and aggregation of cells from organ rudiments of the early chick embryo. , 1952, Journal of anatomy.

[29]  A. Harris,et al.  Disrupting Hypoxia-Induced Bicarbonate Transport Acidifies Tumor Cells and Suppresses Tumor Growth. , 2016, Cancer research.