Today, more and more biological laboratories use 3D cell cultures and tissues grown in vitro as a 3D model of in vivo tumours and metastases. In the last decades, it has been extensively established that multicellular spheroids represent an efficient model to validate effects of drugs and treatments for human care applications. However, a lack of methods for quantitative analysis limits the usage of spheroids as models for routine experiments. Several methods have been proposed in literature to perform high throughput experiments employing spheroids by automatically computing different morphological parameters, such as diameter, volume and sphericity. Nevertheless, these systems are typically grounded on expensive automated technologies, that make the suggested solutions affordable only for a limited subset of laboratories, frequently performing high content screening analysis. In this work we propose AnaSP, an open source software suitable for automatically estimating several morphological parameters of spheroids, by simply analyzing brightfield images acquired with a standard widefield microscope, also not endowed with a motorized stage. The experiments performed proved sensitivity and precision of the segmentation method proposed, and excellent reliability of AnaSP to compute several morphological parameters of spheroids imaged in different conditions. AnaSP is distributed as an open source software tool. Its modular architecture and graphical user interface make it attractive also for researchers who do not work in areas of computer vision and suitable for both high content screenings and occasional spheroid-based experiments. 
AnaSP: A software suite for automatic image analysis of multicellular spheroids