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Multicellular organisms are inherently three-dimensional. This leads to complex intercellular interactions that cannot be reproduced in two-dimensional cell culture. Instead, three-dimensional spheroids, ball-shaped cell aggregates, arise as model systems. Spheroids provide an accurate in vitro representation of the three-dimensional organization of cells in tissues, and compared to a real tissue, they excel with well-defined experimental conditions, easy handling, and suitability for high-quality imaging. Therefore, spheroids are an experimental system that can be readily combined with mathematical modeling. This chapter shows how image-based systems biology is implemented for multicellular spheroids to study three-dimensional cell–cell interactions. The chapter is intended for experimentalists and theoreticians who plan to extend their research by linkage with other disciplines. The relevant concepts for experimental approaches and quantitative imaging are introduced and linked to mathematical models of spheroids. This results in a list of potential systems biology workflows for typical spheroid research areas in cell biology, cancer biology, and bioprinting. In all three areas, there is a large gap between the details of the mathematical models and the available imaging data. The aim of this chapter is to encourage more interactions of experimentalists and theoreticians to fill this gap in spheroid research.
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