Authors

W Schaefer¹; N Hedemann²; A Willms¹; B Werdelmann¹; M Stoehr¹; S Sebens³; R Geisen¹; M Pirsch¹; 
¹ SYNENTEC GmbH, Germany;  ² Department of Gynaecology and Obstetrics, CAU + UKSH, Kiel, Germany, Germany;  ³ Institute for Experimental Cancer Research Kiel, Germany

Discussion

Two-dimensional (2D) cell culture models have been widely used in cancer research and drug discovery, but in the last years, three-dimensional (3D) cell culture models like spheroids have become an innovative tool as they better mimic the physiology of tissues and tumors. However, generation, application and evaluation of assays using 3D models are usually more difficult. Therefore, simple endpoint assays using plate readers are required as a reliable read-out for high-throughput screening or confocal or time-lapse microscopes are used for high content analysis. Here, we show a convenient method to analyze death inducing effects of the drug staurosporine in 2D and 3D models of colorectal cancer cells (HCT116) over time using SYNENTEC’s automation system. Therefore, cells were grown in multiwell plates, treated with different staurosporine concentrations, and stained with the CellTox™ Green Cytotoxicity Assay for 3D models and with a live cell DNA probe for 2D models. Cells were automatically imaged and analyzed every 2 h/6 h using SYBOT-1000®, CELLAVISTA® and YT-SOFTWARE®. From these data, growth rates were calculated and plotted as dose response curves. The resulting IC50 values were lower in the 2D model (nuclei count: 19.5 nM, confluence: 23.6 nM) compared to the 3D model (CellTox: 502 nM). Additional dose response curves for selected time points were calculated of CellTox signaling. Comparing these IC50 values, we saw a large variation (661 nM after 24 h vs. 193 nM after 72 h). This demonstrates the difficulty of determining the optimal endpoint and the benefit of evaluating kinetic measurements. Finally, we used an endpoint Live/Dead assay after 72 hours of treatment with Calcein-AM and propidium iodide again resulting in lower IC50 values in the 2D model compared to the 3D model. Altogether, we show a straightforward methodology of imaging and quantifying cytotoxicity in 2D and 3D cell cultures suitable for automation and high content imaging.