Comparative evaluation of multicellular tumor spheroid fabrication platforms for reliable drug screening

Abstract Multicellular tumor spheroids (MTS) are widely used three-dimensional tumor models in various biomedical fields. Despite the increasing use of MTS, systematic comparisons providing practical information for selecting fabrication platforms according to experimental objectives remain limited. In this study, four widely used MTS fabrication platforms (U-bottom, UB; ultra-low attachment, ULA; hanging drop, HD; and droplet microfluidics, DM) were compared in terms of fabrication performance, structural characteristics, and their potential influence on drug screening. Among the platforms, ULA showed the highest fabrication rate, while HD exhibited relatively high size uniformity. In contrast, DM demonstrated balanced performance in both fabrication rate and size uniformity. Structural characteristics of MTS, including morphology and compactness, also varied depending on the fabrication platform. Although these structural differences did not lead to significant differences in drug responses under static states, clear differences in structural stability were observed under dynamic states. MTS with lower compactness exhibited pronounced morphological deformation and size changes. These findings demonstrate that the choice of MTS fabrication platform can influence not only fabrication efficiency but also the structural properties of MTS and endpoint drug response readouts. This study provides practical information for considering fabrication platform-dependent effects in MTS-based drug screening studies.