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Spheroid-based drug screen: considerations and practical approach

Abstract

Although used in academic research for several decades, 3D culture models have long been regarded expensive, cumbersome and unnecessary in drug development processes. Technical advances, coupled with recent observations showing that gene expression in 3D is much closer to clinical expression profiles than those seen in 2D, have renewed attention and generated hope in the feasibility of maturing organotypic 3D systems to therapy test platforms with greater power to predict clinical efficacies. Here we describe a standardized setup for reproducible, easy-handling culture, treatment and routine analysis of multicellular spheroids, the classical 3D culture system resembling many aspects of the pathophysiological situation in human tumor tissue. We discuss essential conceptual and practical considerations for an adequate establishment and use of spheroid-based drug screening platforms and also provide a list of human carcinoma cell lines, partly on the basis of the NCI-DTP 60-cell line screen, that produce treatable spheroids under identical culture conditions. In contrast to many other settings with which to achieve similar results, the protocol is particularly useful to be integrated into standardized large-scale drug test routines as it requires a minimum number of defined spheroids and a limited amount of drug. The estimated time to run the complete screening protocol described herein—including spheroid initiation, drug treatment and determination of the analytical end points (spheroid integrity, and cell survival through the acid phosphatase assay)—is about 170 h. Monitoring of spheroid growth kinetics to determine growth delay and regrowth, respectively, after drug treatment requires long-term culturing (≥14 d).

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Figure 1: Tools for routine preparation of agarose-coated 96-well plates.
Figure 2: Routine spheroid analysis includes phase-contrast imaging at days 4 and 7 (before and after drug treatment) and every 48 h thereafter.
Figure 3: Routine monitoring of spheroid formation capacity and establishment of the spheroid initiation protocol for one representative cell line.
Figure 4
Figure 5: Routine monitoring of drug effects on cell survival in HT29 colorectal cancer spheroids following drug treatment as determined through the acid phosphatase (APH) assay.
Figure 6: Routine monitoring of spheroid growth/regrowth of drug-treated HT29 spheroids.

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Acknowledgements

We gratefully acknowledge the technical assistance of Marit Wondrak, Juana M Castaneda and Tammy Lawrence as well as Frank van Rey and Rupert Feldmeier. The experimental protocols and work documented herein were supported by the Society for Biomolecular Sciences (SBS) and by the German Federal Ministry of Education and Research (BMBF) through grants 0313143 and 01ZZ0502 to L.A.K.-S.

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Correspondence to Leoni A Kunz-Schughart.

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R. Ebuer is a minor stockholder C=<1% of Aoalon Pharmaceuticals Inc.

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Friedrich, J., Seidel, C., Ebner, R. et al. Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4, 309–324 (2009). https://doi.org/10.1038/nprot.2008.226

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