Page 85 - Live-cellanalysis handbook

Kinetic Multi-Spheroid Assays

Integrated turnkey solution for reproducible analysis of multi-spheroid cultures

Introduction

The use of multi-cell tumor spheroids as a model for oncology This chapter illustrates how IncuCyte’s multi-spheroid assays can
research has expanded rapidly in recent years. As 3D spheroid provide a rapid method for the pharmacological investigation of
protocols become more accessible to researchers, the experimental potential drug candidates. The approach allows the measurement
models have become more complex with correspondingly greater of real-time viability and toxicity measurements in a meaningful,
translational potential. multicellular in vitro model that better reflects the heterogeneous
nature of tumors and allows deeper study of their potential
Multi-spheroid models have a number of advantages over physiological interactions with the tumor microenvironment.
conventional cell culture. In 2D cell culture, tumor cells are
®
grown in monolayers under conditions that are quite different IncuCyte S3 Multi-Spheroid assays at a glance
from the physiological conditions of a tumor. They are grown on
a rigid, non-biological surfaces, have an abundance of oxygen, The IncuCyte® S3 Multi-Spheroid assays combine IncuCyte’s
and an excess of nutrients produces hyper-nourished cells with proprietary DF Brightfield image acquisition mode with a 3D multi-
unrestricted and non-physiological proliferation characteristics. spheroid model grown on a layer of extracellular matrix in a 96-
Using such cells for drug screening introduces unwanted bias, with well format. Multiple protocols have been developed to suit various
a tendency to identify molecules that work only against a uniform experimental objectives and cost considerations.
population of proliferative cells, and overestimating efficacy.
After coating either flat-bottom or round-bottom culture plates
1
A more realistic approach involves recreating more physiologic with Matrigel®, cells are added to the wells with or without
heterogeneity inherent to a 3D tumor structure and providing a IncuCyte® Cell Health Reagents. An optional layer of Matrigel
microenvironment more closely recapitulating in vivo conditions, can be added to “sandwich” the cultures and surround them with
which includes key interactions between the tumor and the ECM. The cells are then automatically scanned in the IncuCyte® S3
extracellular matrix (ECM). Live-Cell Analysis System every six hours to monitor multi-spheroid
formation.
Scaffold-based 3D spheroid models, in which tumor cell aggregates
are grown in ECM scaffolds such as Matrigel®, more closely After treatments are added, the spheroid growth and shrinkage
recapitulate physiological growth conditions of tumors, enabling the assay is initiated and monitored by repeat scanning every six
study of interactions between tumor cells and the microenvironment. hours in the IncuCyte® S3 system for up to two weeks. Spheroid
Tumor cells grown in 3D scaffold-based culture can form cell-cell size is reported based on DF Brightfield image analysis and can be
and cell-matrix interactions. The heterogeneous nature of the tumor accessed in real time or on demand as needed. The simple protocol
microenvironment can also be studied through the use of co-culture is shown below:
models, such as tumor cells with fibroblasts or immune populations.

1 Coat plate (Day 0) 2 Add cells (Day 0) 3 Add reagent 4 Monitor formation 5 Add treatments
(Day 0, optional) (Day 0—3) (Day 3)

Coat plate (50% Matrigel, Add cells in media (100 Add cell health reagent Place inside the IncuCyte Add treatments and
40 μl/well). Polymerize at or 150 μl/well) with (50 μl/well) at 3x final assay and scan every six hours continue to monitor
37°C for 30 minutes. or without cell health concentration. to monitor multi-spheroid growth in IncuCyte.
reagent, respectively. formation.
Figure 1. Overview of workflow for generation and analysis of multi-spheroid cultures in an IncuCyte® S3 Live-Cell Analysis System.

1. Protocols have been developed using either 96-well flat bottom or round bottom plates to address cost concerns when utilizing Matrigel. Round bottom plate
protocols use four times less Matrigel than flat bottom plate protocols.

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