Assay and Drug Development Technologies - 19

3D APPROACHES
Fig. 1. The combination of HTS and HCS strategies will enhance drug development. HCS, high-content screening; HTS, high-throughput screening.
The geometry of and pathophysiological gradients exhibited
by multicellular 3D models are comparable to in vivo
tumors. Beyond a critical size (‡100 mM), most 3D models
from cell lines develop a necrotic core surrounded by a viable
rim of cells (>100 mM). The periphery of the 3D model is
composed of proliferating cells, which resemble actively cycling
tumor cells adjacent to capillaries in vivo. In contrast, the
innermost cells become quiescent and eventually die by apoptosis
or necrosis, forming the necrotic core (Fig. 2). This cell
death is a consequence of limited inward and outward diffusion
ofpositive and negative regulators, resulting in decreased
oxygen (hypoxia) and/or nutrients, accumulation of waste
products, and low pH.48,49 Such heterogeneity in cellular
physiology and tumor milieu are linked to alterations in
sensitivity to many antitumor therapies.20,21,23,48,50-52
One ofthe biggest challenges in visualizing 3D structures is
overcoming the inherent practical limitations imposed by
their geometry. With regard to HCS and imaging approaches
to 3D cellular assays, many have implemented the use of
confocal analyzing modalities to obtain detailed information
regarding cellular behavior in 3D. However, even with these
advanced automated imaging systems, physical limitations
are imposed such as light scattering and absorption.32 We
have found that these limitations are often manifested as poor
light penetration of the sample, which in many cases can lead
to incomplete imaging of the multicellular structures, and
long acquisition times. These issues can be further exacerbated
if cells are grown on scaffold materials such as gels or
biocompatible polymers, which may further reduce optical
clarity. There has, however, been some exciting recent developments
that may improve the capabilities of light microscopy
platforms, in particular light sheet microscopy.53
Although this approach offers improved image quality and
resolution, it has not yet been routinely adopted by laboratories
focused on HTS. In addition, much emphasis has been
placed on methods for improving the optical characteristics of
the specimen, for example, assay systems where highly
Fig. 2. Cellular gradients in 3D cell culture models. Cells originate
on the outer proliferative layer (1) and ultimately undergo apoptosis/necrosis
in the core (3). This migration is initiated by cell-tocell
and cell-to-extracellular matrix interactions, which regulate the
movement of cells from the proliferative (1) to the quiescent (2)
zones. From here, cells generally enter the necrotic core either as a
direct result of necrotic cell death or through accumulative apoptosis
followed by secondary necrosis. Hypoxia is one of several
factors that contributes to the development and maintenance of
the necrotic core. 3D, three dimensional.39
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