A recent decline in the discovery of novel medications challenges the widespread use of 2D monolayer cell assays in the drug discovery process. As a result, the need for more appropriate cellular models of human physiology and disease has renewed the interest in spheroid 3D culture as a pertinent model for drug screening. However, despite technological progress that has significantly simplified spheroid production and analysis, the seeming complexity of the 3D approach has delayed its adoption in many laboratories. The present report demonstrates that the use of a spheroid model may be straightforward and can provide information that is not directly available with a standard 2D approach. We describe a cost-efficient method that allows for the production of an array of uniform spheroids, their staining with vital dyes, real-time monitoring of drug effects, and an ATP-endpoint assay, all in the same 96-well U-bottom plate. To demonstrate the method performance, we analyzed the effect of the preclinical anticancer drug MLN4924 on spheroids formed by VCaP and LNCaP prostate cancer cells. The drug has different outcomes in these cell lines, varying from cell cycle arrest and protective dormancy to senescence and apoptosis. We demonstrate that by using high-content analysis of spheroid arrays, the effect of the drug can be described as a series of EC50 values that clearly dissect the cytostatic and cytotoxic drug actions. The method was further evaluated using four standard cancer chemotherapeutics with different mechanisms of action, and the effect of each drug is described as a unique multi-EC50 diagram. Once fully validated in a wider range of conditions, this method could be particularly valuable for phenotype-based drug discovery.

Cell arrays are of foremost importance for many applications in pharmaceutical research or fundamental biology. Although arraying techniques have been widely investigated for adherent cells, organization of cells in suspension has been rarely considered. The arraying of non-adherent cells using the diamagnetic repulsive force is presented. A planar arrangement of Jurkat cells is achieved at the microscale above high quality microfabricated permanent magnets with remanent magnetization of J(r)≈ 1 T, in the presence of a paramagnetic contrast agent. The cytotoxicity of three Gd based contrast agents, Gd-DOTA, Gd-BOPTA and Gd-HP-DO3A, is studied. Among them, Gd-HP-DO3A appears to be the most biocompatible toward Jurkat cells. In close agreement with analytical simulations, diamagnetically 'suspended' cells have been successfully arrayed above square and honeycomb-like micromagnet arrays, which act as a "diamagnetophobic" surface. Living cell trapping is achieved in a simple manner using concentrations of Gd-HP-DO3A as low as 1.5 mM.