21. If the plate reader has a temperature-controlled chamber let it warm up to 37 °C before the measurements. Alternatively, wrap the plates in aluminum foil and leave them to equilibrate to room temperature for 20 min before measuring fluorescence.
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.
58
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/6c57cede1d940 f6fd952 (DOI: 10.6084/m9.figshare.3487940).
References
1. 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 2. Astashkina A, Mann B, Grainger DW (2012) A
critical evaluation of in vitro cell culture models for high-throughput drug screening and toxic- ity. Pharmacol Ther 134:82–106
3. Hickman JA, Graeser R, de Hoogt R et al (2014) Three-dimensional models of cancer for pharmacology and cancer cell biology: cap- turing tumor complexity in vitro/ex vivo.
Biotechnol J 9:1115–1128
4. Moscona A, Moscona H (1952) The dissocia- tion and aggregation of cells from organ rudiments of the early chick embryo. J Anat 86:287–301
5. 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
6. 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
7. Kelm JM, Timmins NE, Brown CJ et al (2003) Method for generation of homogeneous multi- cellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83:
173–180
8. Vinci M, Gowan S, Boxall F et al (2012) Advances in establishment and analysis of three-dimensional tumor spheroid-based func- tional assays for target validation and drug evaluation. BMC Biol 10:1–21
9. 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
10. 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
11. Falkenberg N, Hửfig I, Rosemann M et al (2016) Three-dimensional microtissues essen- tially contribute to preclinical validations of therapeutic targets in breast cancer. Cancer Med 5:703–710
12. Anastasov N, Hửfig I, Radulović V et al (2015) A 3D-microtissue-based phenotypic screening of radiation resistant tumor cells with syn- chronized chemotherapeutic treatment. BMC Cancer 15:466
13. 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
14. McIntyre A, Hulikova A, Ledaki I et al (2016) Disrupting hypoxia-induced bicarbonate trans- port acidifies tumor cells and suppresses tumor growth. Cancer Res 76:3744–3755
15. 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
16. 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
17. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological- image analysis. Nat Methods 9:
676–682 Delyan P. Ivanov et al.
18. Schindelin J, Rueden CT, Hiner MC et al (2015) The ImageJ ecosystem: an open plat- form for biomedical image analysis. Mol Reprod Dev 82:518–529
19. 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
20. Sutherland RM, Eddy HA, Bareham B et al (1979) Resistance to adriamycin in multicellu- lar spheroids. Int J Radiat Oncol Biol Phys 5:1225–1230
21. Friedrich J, Seidel C, Ebner R et al (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4:309–324 22. O’Brien J, Wilson I, Orton T et al (2000)
Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mamma- lian cell cytotoxicity. Eur J Biochem 267:
5421–5426
23. Walzl A, Unger C, Kramer N et al (2014) The resazurin reduction assay can distinguish cyto- toxic from cytostatic compounds in spheroid
screening assays. J Biomol Screen 19:
1047–1059
24. 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
25. 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
26. 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
27. Zhang J-H (1999) A simple statistical parame- ter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73
28. Hall MD, Telma KA, Chang K-E et al (2014) Say no to DMSO: dimethylsulfoxide inacti- vates cisplatin, carboplatin, and other platinum complexes. Cancer Res 74:3913–3922
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Daniel F. Gilbert and Oliver Friedrich (eds.), Cell Viability Assays: Methods and Protocols, Methods in Molecular Biology, vol. 1601, DOI 10.1007/978-1-4939-6960-9_5, © Springer Science+Business Media LLC 2017
Chapter 5
A Protocol for In Vitro High-Throughput Chemical
Susceptibility Screening in Differentiating NT2 Stem Cells
Ann-Katrin Menzner and Daniel F. Gilbert
Abstract
The incidence of neurological diseases including learning and developmental disorders has increased in recent years. Concurrently, the number and volume of worldwide registered and traded chemicals have also increased. There is a broad consensus that the developing brain is particularly sensitive to damage by chemicals and that evaluation of chemicals for developmental toxicity or neurotoxicity is critical to human health. Human pluripotent embryonal carcinoma (NTERA-2 or NT2) cells are increasingly considered as a suitable model for in vitro developmental toxicity and neurotoxicity (DT/DNT) studies as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and allow toxicity assessment at different stages of maturation. Here we describe a protocol for cell fitness screening in differentiating NT2 cells based on the analysis of intracellular ATP levels allowing for the identification of chemicals which are potentially harmful to the developing brain. The described method is suitable to be adapted to low-, medium-, and high-throughput screening and allows multiplexing with other cell fitness indicators. While the presented protocol focuses on cell fitness screening in human pluripotent stem cells it may also be applied to other in vitro models.
Key words High-throughput screening, Cell-based assays, Cell viability, Developmental toxicity and neurotoxicity, DNT, Chemical susceptibility, Differentiation, Neurotoxicity, Cell fitness, Drug screen- ing, Drug discovery, Target validation, In vitro toxicity screening, CellTiter-Glo Luminescent Cell Viability Assay
1 Introduction
The incidence of learning and developmental disorders including attention-deficit or hyperactivity disorder, autism, mental retarda- tion, or other neurological diseases has increased in recent years [1–4]. Concurrently, the number and volume of worldwide regis- tered and traded chemicals have also risen. There is a broad con- sensus that the developing central nervous system is particularly sensitive to damage by chemical substances [5] and that evaluation of chemicals for developmental toxicity (DT) or neurotoxicity (DNT) is critical to human health [6, 7]. Nevertheless, only a minority of chemicals have yet been tested for DNT [8, 9],
presumably because first there is no legal obligation for DNT testing and second the present guidelines for DNT testing involve animal experimentation [10] (US EPA 712-C-98-239; US EPA, 1998) that is of poor reproducibility and predictive quality, low in throughput, and cost intensive as well as highly limited regarding mechanistic insights into the toxicant’s mode of action [11]. For the aforementioned reasons, in vitro-based alternatives to existing animal-dependent methods allowing for systematic DNT studies in high-throughput and large-scale format are urgently needed [12].
Human pluripotent teratocarcinoma (NTERA-2 or NT2) cells are increasingly considered as a suitable model for in vitro DT and DNT studies [13–18]. Upon treatment with retinoic acid, NT2 cells mimic the process of neuronal differentiation in the maturing brain and thus may allow testing for toxic effects at various devel- opmental stages ranging from non-differentiated stem cells and committed neural progenitors to differentiated neuronal cells [19–24].
NT2 and other stem cells have been reported to show specific changes in chemical susceptibility during differentiation potentially leading to consequences with significant impact on the cell’s fate, including cellular viability (e.g., metabolic activity), cell death (e.g., necrosis or apoptosis), or ability of the cells to undergo neu- ronal differentiation [25]. Thus, studying chemical susceptibility in differentiating stem cells in general and in human pluripotent NT2 cells in particular can be of great value in the context of devel- opmental toxicity or neurotoxicity testing and might contribute to a test battery of experimental strategies for stage-specific DNT prediction.
Here, we describe a protocol for elucidating chemical suscep- tibility in differentiating NT2 cells based on the analysis of cellular viability. The assay quantifies the viability of chemical-exposed dif- ferentiating NT2 cells using intracellular ATP as readout.
Intracellular ATP is an energy carrier driving many cell functions. Cell death is typically indicated by low ATP levels and persistent ATP depletion causes a cell to die. Due to its simple applicability, e.g., by an ATP-dependent luciferase-luciferin reac- tion (e.g., CellTiter-Glo Luminescent Cell Viability Assay Kit from Promega), the intracellular ATP level has been a long-serving, con- venient, and robust indicator of cellular viability. Although the approach is based on analysis of metabolic activity, assessed by a specific luminescence- based reporter system, it could also be replaced by as well as combined with any other luminescence- or fluorescence- based and biochemical cell viability indicator.
Examples are cellular integrity (membrane integrity: propidium iodide), cell proliferation (nucleus stain: Hoechst 33342), or meta- bolic activity (reductase- enzyme product: CellTiter 96 Aqueous One Solution Cell Proliferation Assay; ATP content: CellTiter-Glo Luminescent Cell Viability Assay Kit; esterase activity: Calcein-AM).
63 It is important to note that the fitness indicator employed in the present assay does not clearly and exclusively indicate cellular viability comprehensively, as it yields information on a very specific physiological condition that only indirectly and partially reflects cellular fitness. Although this limitation can be overcome by mul- tiplexing, i.e., the combination of multiple individual cell viability assaying methods in a single experiment, adding a level of effi- ciency and confidence for hit selection [26, 27], the presented methodology on its own is not suitable to reliably predict DT and/
or DNT.
2 Materials
Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 M Ω -cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials.