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Live-Cell Analysis Handbook — Third Edition

Kinetic Assays for Utilizing Complex Models

Live-cell microplate assays for studying growth and health of three-dimensional cultures

Introduction

It is well established that conventional monolayer cultures of tumor is desirable for drug testing. Multi-spheroid models capture the
cells grown on plastic do not adequately reflect the in vivo situation. inherent heterogeneity of tumor cells and are considered by many
1,2
Yet, in vitro culture models of cancer cells are still an integral to be more physiologically relevant.
component of clinical drug development and for the advancement
of our understanding of cancer cell biology. 3D cell culture models, Employing these complex models is challenging due to inherent
although still relatively new compared to traditional 2D monolayer limitations for studying cell growth, kinetics, and other
cultures, are increasingly adopted as advanced tools to accelerate characteristics in this 3D setting. These include time-consuming,
drug discovery efforts. This shift is most notable in cancer biology, expensive or laborious workflows, labeling requirements that can
3
immuno-oncology and hepatotoxicity, where organoids and 3D affect cell biology, and single time-point or indirect read-outs that
microtissues can represent more relevant biological features such do not report the full experimental time-course and potentially
4,5
as hypoxic regions or tumor-immune cell interactions. There miss valuable information about size or growth, or shifts in the
are a multitude of approaches to generating these models and characteristics of cell populations.
they can generally be described as scaffold-free (media-based) or
scaffold-based. The IncuCyte® S3 Live-cell Analysis System, for both scaffold-free
and scaffold-based 3D spheroid models, avoids such limitations by
Scaffold-free models are easily achieved using round-bottom providing simple, cost-conscious protocols that result in 3D cultures
ultra-low attachment (ULA) microplates to promote spheroid self- amenable to imaging, employing either label-free techniques or
assembly. These models generally yield a single tumor spheroid use non-perturbing reagents, and enabling real-time monitoring
per well and exhibit key features of solid tumors; larger spheroids of morphological and cell health measurements without removing
consisting of proliferating, quiescent, and necrotic zones resulting precious samples from the incubator. The approach works both with
from a radial gradient of nutrients, metabolites and oxygen. single spheroids grown in round-bottomed ULA wells in 96- and
384-well formats, and multi-spheroid cultures grown on plates
Current scaffold-based models rely primarily on an extracellular coated with Matrigel® to reproduce the ECM scaffold.
matrix (ECM) such as Matrigel® or collagen and attempt to
recapitulate both physical and biochemical characteristics of the The IncuCyte® approach has many advantages over traditional cell
tumor microenvironment. Generally, these models employ the use analysis methods (summarized in Table 1). In the following chapter,
of flat bottom plates and result in multiple tumor spheroids per well. the assays are described in detail, demonstrating their potential as
a valuable tool for insightful scientific discovery and drug screening
Choosing a model depends on the scientific question or objective and development.
at hand. Single spheroid models may be more representative of
large solid tumors and can be made with high reproducibility which

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