Page 92 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Kinetic Single Spheroid Assays

Reproducible quantitative analysis of single spheroid growth and health over time

Introduction

Single spheroids exhibit several physiological traits including available 3D techniques for generating and quantifying spheroids
relevant morphology, increased cell survival, and a hypoxic core are time consuming, laborious, costly and can lack reproducibility.
that make them ideally suited to the study of larger solid tumors.
In this chapter we describe the validation and application of
These scaffold-free (media-based) models are easily achieved using miniaturized IncuCyte live-cell single tumor spheroids formed
round-bottom ultra-low attachment (ULA) microplates to promote in ULA plates and monitored up to two weeks. These assays are
spheroid self-assembly. They generally yield a single tumor spheroid flexible, simple to run, and provide automated and direct measures
per well. By virtue of their size, they exhibit the key features of of tumor size and health in real time.
solid tumors: they are comprised of proliferating and quiescent
cells, and contain necrotic zones resulting from a gradient of IncuCyte S3 Single Spheroid assays at a glance
®
nutrients, metabolites and oxygen that recapitulates the authentic
heterogeneity within a tumor.
Cells of interest are harvested, counted, and plated onto ULA round
bottom 96- or 384-well plates and then centrifuged. Spheroid
Single spheroids formed in ultra-low attachment (ULA) plates also formation is monitored every six hours using DF Brightfield and
avoid the use of complex and poorly characterized biomatrices, HD phase contrast image acquisition until they reach the desired
and are ideal for studies that require high levels of well-to-well size. Test compounds are then added, and the spheroid growth
consistency.
and shrinkage assay is initiated. Automated monitoring continues
every six hours for up to two weeks, with tumor spheroid size
A growing body of evidence suggests that more relevant and measurements made available in real time. The simple protocol is
translational observations can be made in 3D multi-cell tumor shown below:
models compared to 2D monolayer models. Most currently

1 Cell seeding 2 Spheroid formation 3 Add treatments
(Day 0) (Day 0—3) (Day 3)

Seed cells into 96W or 384W Place inside the IncuCyte® S3 Add treatments to plate.
Ultra Low Attachment plate. and scan every six hours. Monitor spheroid growth
Centrifuge. and shrinkage.
Figure 1. Overview of workflow for generation and analysis of single spheroid cultures in an IncuCyte® S3 Live-Cell Analysis System.

Key advantages:
• Quantify growth and investigate morphology of single spheroids • Lab-tested protocols, high quality images, and unbiased analysis
grown in ultra-low attachment (ULA) round bottom plates delivers robust data suitable for pharmacological analysis.
without the need of labels, and leaving cells undisturbed inside a
standard tissue culture incubator. • Automated acquisition and analysis tools enable rapid creation
of concentration response curves from thousands of images
• Investigate mechanisms of action or immune modulation generated from 96- or 384-well plates.
with kinetic viability and toxicity measurements using non-
perturbing reagents.

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