Authors
K Harper 1;
1 REVVITY, UK
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