Assay and Drug Development Technologies - 18

RYAN ET AL.
Table 1. The Advantages and Disadvantages of 2D
and 3D Cell Culture
Advantages
2D Cost effective
Easy to use
Convenient
Easy downstream processing
3D More accurate representation
of the in vivo scenario
Increased relevant cell-to-cell and
cell-to-ECM signalling
More applicable drug resistance
models
Added expense
More complex culture system
Further complex downstream
processing
Endpoint assay dependant on
culture method utilized
2D, two dimensional; 3D, three dimensional; ECM, extracellular matrix.
Disadvantages
Less biologically relevant models
Reduced cell-to-cell interactions
iaturize, and speed up assays.30 Active compound hits, identified
by HTS and HCS screens, act as templates for further
drug development.31 The features of both HTS and HCS, and
the potential impact of combining these technologies, are
summarized below (Fig. 1).
HTS and HCS technologies are, for the most part, conducted
and optimized with cells cultured in 2D monolayers. However,
creating the means to facilitate screening of 3D models using
the same technologies is essential. Unfortunately, not many
3D cell culture technologies are compliant with both HTS and
HCS. We believe that this is perhaps a consequence of technological
development being promoted above design principles.33
Very few commercially available products are readily
available technologies designed to improve the accuracy of
in vitro 3D cell culture analyses in a routine and cost-effective
manner.34 In this study, we discuss the merits of 3D cell culture
models and the technologies that are currently attempting
to mainstream their utilization in HTS. The need to identify the
optimal method to facilitate their generation and use in
translational research in the most effective and efficient way
possible, is essential.
limited. This unpredictability is attributable to the fact that
such systems do not accurately mimic the response of cells in
the 3D microenvironment present in vivo.23
Billions are spent every year on developing targets identified
from in vitro systems through to Phase III trials in patients.
The vast majority of these compounds fail due to either
unacceptable toxicities or limited efficacy in humans.13 This
in itselfdemonstrates that the more traditional 2D cell systems
are ineffective in predicating clinical responses. Indeed, 3D
models tend to have better drug predicative value compared
with 2D.13,24,25 Furthermore, issues are associated with the
ECM component in 2D culture, which appears to be overcome
in a number of 3D systems.25-29
Incorporating 3D cell culture with in vitro screening processes
such as high-throughput screening (HTS) and highcontent
screening (HCS) is necessary to identify clinically
relevant compounds. Drug discovery is heavily reliant on
HTS; the process of identifying hits by testing a large number
of diverse chemical structures against disease targets and is
characterized by its simplicity, efficacy, low cost per assay,
and high efficiency.30 In addition, HCS-facilitated phenotypic
screens yield more complex biologically relevant information
and increased data generation relative to conventional in vitro
assays, such as protein enzyme assays, binding assays, and
endpoint assays.31,32 Highly sensitive fluorescence-based HCS
assays are important for and complement HTS, therefore
contributing to the industry-wide initiative to simplify, min18
ASSAY and Drug Development Technologies
3D CELL CULTURE OF IN VITRO MODELS
To create an environment that mimics more closely
that found in tumors in vivo, 3D systems must simulate a
pathophysiological cellular microenvironment in a tumor,
reconstruct a tissue-like cytoarchitecture with cell-to-cell
and cell-to-ECM interactions, and exhibit growth, differentiation,
and therapy responses similar to those observed
in vivo. Multicellular structures appear to be the best described
3D tumor model system, which meets all of these
criteria.23,35-38
As mentioned previously, 2D models are not a reflection of
the in vivo state as only a portion of the cell membrane engages
with neighboring cells, while the remainder of the cell
is exposed to the bulk culture medium.39 It is therefore a
crucial aspect of cancer research that tumor and normal cells
are cultured in a manner that most closely resembles the
in vivo environment. An important factor in the 2D culture
system is the absence of a true ECM. ECM is a critical cellular
factor important in normal and deregulated cellular homeostasis26,40-43
as it affects cellular properties such as structure,
adhesive potential, mechanotransduction, and outcomes of
exposure to soluble effectors.44 Indeed, it has been noted that
cells in 2D lose their ability to differentiate, while this property
can be recovered in 3D.44,45 Consequently, ECM-related
cellular morphology results in modifications in cellular functions
in 2D versus 3D, with 3D demonstrating physiological
relevance.44-47
ª 2022 MARY ANN LIEBERT, INC.
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