Overview

When developing a HCS assay, a critical aspect is the choice of cellular model. Multicellular 3D models (e.g., spheroids, organoids) have the potential to better predict the effects of drug candidates in vivo. However, they tend to make all steps of the workflow more complex compared to 2D cultures.

Introduction

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

Methods

Essentials for 3D applications: Harmony software and water immersion objectives Uniform spheroid formation using CellCarrier Spheroid ULA 96 microplates Optical clearing improves imaging depth PreciScan acquisition saves time and data Analysis of incompletely imaged spheroids

Results

In 3D imaging, extracting meaningful data is more difficult compared to 2D imaging. Often, 3D objects are not completely imaged through, and biological readouts remain incomplete.

Conclusion

re not completely imaged through, and biological readouts remain incomplete. Here we have shown that: - High NA water immersion objectives substantially improve 3D image quality and nuclei segmentation when compared directly to air objectives - Harmony software a offers a wide range of 3D analysis and visualization tools - Harmony enables the analysis of large 3D objects including hollow spaces as well as single cells - The PreciScan tool increases acquisition speed and decreases data volume