Assay and Drug Development Technologies - 29

3D CULTURE AND HTS
patterning and differentiation. Naturally derived hydrogels
for 3D culture comprised proteins and ECM components, such
as collagen, laminin, and fibrin. Matrigel (Corning Matrigel
Matrix), notably, has been widely used since it contains many
of the common ECM components found in basement membranes.
3D culture of stem cells in ECM gels allows formation
of tissue organoids (organotypic culture) that recapitulate
key features of organ function. Accordingly, tissue organoids
representative of intestinal, retinal, pancreatic, mammary,
colonic, and cerebral tissues have now been generated. Historically,
these approaches were widely used to study tissuespecific
developmental processes. However, they also offer
novel approaches for compound screening in a more physiological
manner and allow screening of drug candidates in
target tissues. As has been stated ''.self-organizing tissues
with high functionality may be useful for drug screening.particularly
with patient-specific induced PSCs.''14
However, a limitation of natural hydrogels lies in their
isolation from animal sources, which may cause cell batch-tobatch
variations as well as the potential for pathogen contamination.
Consequently, there is a growing interest in
synthetic hydrogels, particularly those utilizing polyethylene
glycols (PEGs). Today, chemically defined synthetic hydrogels
are available, which are both biocompatible and devoid of
animal-derived materials or pathogens.16,17 The control of
stem cell differentiation involves spatial and mechanical cues,
as well as biochemical signals. Some cues can be imparted
by solid surface biomaterials designed to direct growth and
differentiation. Consequently, a diverse range of synthetic
hydrogel structures, including 3D matrices, scaffolds, and
microfluidic formats, are now being adapted for use in microtiter
plate formats. 3D matrices, notably, can be derived from
several PEG-based hydrogels with different porosities, pore
sizes, permeabilities, and mechanical characteristics, each of
which reflects a specific tissue ECM.18 These materials can be
further optimized to present oligopeptidic sequences tailored
for recognition by specific cellular adhesion molecules. Finally,
they can also be applied to microcarrier culture, which
utilizes beads derived from several porous polymers as 3D
support for anchorage-dependent cells.19,20
3D culture is, therefore, gaining acceptance into drug discovery,
particularly as primary cells or PSC-differentiated
cells are becoming available in the large reproducible
amounts required for screening. 3D culture is already having
an impact in the area of preclinical lead optimization, particularly
in screening compounds for metabolic liability or
cellular toxicity. This is perhaps unsurprising, since many 3D
coculture methods have been refined using liver cells for
compound evaluation.
3D CELL CULTURE LIMITATIONS
One major limitation of using 3D culture in drug screening
lies in the technical aspects that relate to assay protocols.5,6
These include the need to optimize and standardize procedures
for cell harvesting, cell lysis, production scale-up, as
well as control of pH and temperature to reduce well-to-well
and lot-to-lot variation. Since 3D culture can be more heterogeneous
than 2D culture, interpretation of data is more
challenging. In addition, the potential for compound nonspecificity
may be increased due to the more complex culture
conditions used, as well as physicochemical issues, including
compound access to the cells within the 3D complex.
Nonetheless, as occurred in the adoption of 2D cell culture
procedures in HTS/HCS, defined protocols-and novel instrumentation-are
now being developed for 3D culture that
could circumvent these limitations.
A second, more general, limitation of 3D culture is the lack
of organoid vascularization, consequent lack of oxygenation,
and removal of metabolic side products. In this respect, current
3D culture is inferior to 2D cell culture. However, the
hypoxic interior ofthe spheroid may actually better mimic the
hypoxic core of a tumor, as well as access of the novel compound
to cells in the interior. 3D cultured cells exhibit lower
sensitivity than those grown in 2D culture, although other
factors may be involved here, such as cell surface receptor
expression, cancer gene expression, and uniformity of cell
differentiation stages-all of which differ between cells from
2D or 3D cultures.5
More fundamental is the development of 3D cell culture
models for growing cells in a matrix that mimics the in vivo
ECM dynamically. Many 2D or 3D environments are static and
lack exposure to mechanical forces that influence tissue and
organ morphogenesis (including shear forces provided by
blood flow). Potentially, the next wave of3D cell cultures lies in
models that better mimic the microstructure of living organs,
particularly those using microfabrication technologies. In the
most advanced concepts, organ-on-a-chip systems dynamically
link multiple miniaturized organs through channels lined
by endothelial cells.21 It remains to be seen if such systems can
be utilized in HTS or whether they will play a more limited role
in preclinical lead ADME/toxicology evaluation.
CONCLUSION
The availability of techniques to generate large reproducible
batches of human primary cells is now impacting drug
discovery, notably in compound screening, but also lead optimization.
It is unclear if the use of3D culture will reduce the
compound attrition rate, although the cellular responses to
drug treatments in 3D are similar to what are seen in vivo.5 The
ª 2022 MARY ANN LIEBERT, INC. ASSAY and Drug Development Technologies 29
Assay and Drug Development Technologies

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