Assay and Drug Development Technologies - 21

3D APPROACHES
data. The creation of a more natural cellular environment may
influence intracellular interactions and downstream signalling
pathways and possibly challenge current hypotheses generated
by results obtained from 2D cell-based assays.74-76
3D CELL CULTURE TECHNOLOGY
FOR HTS AND HCS
There are many options in today's market for those wishing
to generate 3D models in vitro, however, there are still many
concerns regarding these technologies. There have been numerous
publications which highlight these issues,34,77-80 for
example, in 2010, HTStec conducted a survey which attempted
to understand the drive behind the transition from 2D
monolayers to 3D.81 They found that there was no complete
system, no one relatively simple method that could transform
single cells into robust 3D structures, which could be easily
manipulated, treated, imaged, analyzed, and upscaled. The
survey highlighted issues such as (i) poor reproducibility, (ii)
labor intensiveness, (iii) overcomplicated systems, (iv) inability
to upscale, (v) difficulty in recovering cultures, (vi)
incompatibility with automated systems, (vii) lack of flexibility,
(viii) poor imaging quality, and (ix) long-term stability
problems.77 Industrial drug development requires a universal
3D culture system. However, many emerging technologies
focus on specific applications and fail to provide standardized
3D models compatible with automation. Routine 3D cell culture
relies on technologies that have considered cost, ease of
use, application, and reproducibility. These factors are especially
crucial for the field of drug discovery.34,79,80,82
The methods chosen for discussion in the later sections of
this review are purely examples of
the type of technology in question
and are bynomeans an exhaustive
list and include anchorage dependent
and independent methods
(Fig. 3). We will also briefly describe
a novel technology, currently
under development, that we
have engineered specifically for
3D culture in HTS and HCS settings.
Anchorage-Dependant
Technologies
Anchorage-dependent
scaffolds
represent the space available
for a tissue to develop. The cross
talk between the scaffold and the
cells is controlled by the scaffold
characteristics and the properties of the material from which it
is made. Scaffolds offer physical support for cell growth
and accommodate cell adhesion, proliferation and ECM
production.83
Hydrogels. Hydrogels are cross-linked networks composed largely
ofwater,84 engineered to represent basement membranes.
They are primed with ECM proteins such as laminin, collagen
IV, and entactin.85 Many hydrogels also contain growth factors,
which promote cellular interactions and 3D model formation.
Further applications of hydrogels include co-culture, regenerative
medicine and drug screening. Their ability to mimic essential
features ofthe ECMallow them to be bioengineered on a
molecular level, offering scientists the possibility to adapt 3D
models to specific cell types and to investigate different aspects
of cell-matrix interactions.77 For the most part hydrogels are
available in a liquid form. Cells are suspended in the hydrogel
and the user is required to carefully coat tissue culture plates
with the cell/hydrogel mixture while ensuring gelation does not
occur before the plate is fully prepared. An alternative overlay
method can also be employedwhich involves precoating plastics
with a layer of the hydrogel and allowing gelation to occur
before adding the second hydrogel/cell layer.86 While both
methods are sufficient for 3D culture, the latter is generally favored.
Hydrogels are effective in generating 3Dmodels, however
certain issues exist which may make them unsuitable for large
scale data generation. Hydrogels often need to be dispensed at
specific temperatures to prevent premature gelation. This factor
may disqualify them from use with some liquid handling systems
associated with high throughput screens. This coating of
Fig. 3. Chart detailing the principle methods involved in 3D cell culture model production.
ª 2022 MARY ANN LIEBERT, INC. ASSAY and Drug Development Technologies 21

Assay and Drug Development Technologies

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