When developing a HCS assay a critical aspect is the choice of cellular model. Multicellular “oids” (e.g. spheroids, organoids) have the potential to better predict the effects of drug candidates in vivo. However, 3D models tend to make all steps of the workflow more complex compared to 2D cultures. Generating large numbers of uniform spheroids at high quality for screening is one of the challenges. Using spheroids and cysts as examples, we show how to grow these models effectively using ULA coated U-bottom plates or low concentration gels. Careful selection of dyes and clearing strategies can improve image quality while targeted imaging of spheroids helps to shorten imaging time and minimize data volume. However, effective analysis of spheroids is still a major bottleneck.

Dedicated high-content software tools can help users to explore 3D data sets and quantify in 3D changes such as volume, shape and number of nuclei per spheroid to better understand drug effects.