Assay and Drug Development Technologies - 20

RYAN ET AL.
pigmented cells can be clarified by incorporating tissue
clearing methods in the assay preparation work flow.54,55
As alluded to previously, one of the main issues with 2D
culture is the gap between in vitro and in vivo cellular modeling
and drug efficacy studies. A number of techniques now exist to
culture patient tumor tissue in either a tissue culture type system
such as organoids56-58 or using patient-derived xenograft (PDX)
murine models.59,60 This allows for the gene and protein characterization
of the patient's cancer and/or normal tissue, in addition
to the determination of the patient's cells response to a
number ofdrugs in either system.56,61 Again, 3D systems would
appear to be superior at evaluating drug efficacy, with breast
cancer as one such example.62 Importantly a number of these
systems are fully compatible with HTS and HCS and will lead to
improved personalized medicine strategies for patients. Some
groups have used multiple cell types in 3D model systems such
as the lung63 and breast.64 These models consisted of epithelial
cells, fibroblasts, and other stromal components resulting in
physiologically relevant microenvironments.63,64 Model systems
using multicellular types provide enhanced 3D models as more
unicellular 3D spheroids will vary in size and lack a vascular
system.13 ADMET (absorption, distribution, metabolism, elimination,
toxicity) studies also aid in the drug screening process
and help to ensure that only the most efficacious therapeutic
targets are investigated leading to a greater number ofsuccessful
compounds. These types ofstudieshelptocharacterizethe drugs
properties and also identifypossible drug-drug interactions.65,66
These models could be modified to use a wide variety ofcells
and/or patient tissues cultured in 3D systems,67,68 including
those cultured in our own colloidal suspension medium (CSM),
which will be discussed later. The latter is currently being utilized
for the optimization ofassays involving patient samples in
a range of cancer and normal cells.
Although traditional 3D culture tends to promote the culture
of single cell types, work is ongoing to develop this to encompass
multiple cell types, which could further mimic the
ECM and stromal compartment within tumors. These advances
will also lead to superior 3D models ofnormal cells such as the
kidney and liver, therefore enhancing the capability oftoxicity
testing in vitro. This would also benefit ADMET and PDX
models as normal models could be produced from corresponding
normal tissues derived from the patient, therefore
allowing an increased scope to investigate drug effects on seriously
ill patients. Support in the literature for 3D cell culture
highlights discrepancies observed between 2D and 3D culture
and in the majority of cases promotes 3D as the preferred
method.69-73 Some examples are described in Table 2. The idea
is that by simulating an in vitro environment, which encourages
cells to proliferate, aggregate, and differentiate as they do
in vivo, we may yield more biologically and clinically relevant
Table 2. Pharmacological and Genomic Comparisons Between 2D and 3D Cell Culture
Reference
2D
NA8 melanoma cells were cultured in 2D, 2D in the
presence of ECM (collagen) and 3D.62
Comparison of gap-junction protein expression in bladder
carcinoma cell lines HCV-29, RT4, J82 with different
urothelial differentiation.63
HCT-116 colorectal carcinoma cell line was treated with a
number of anticancer drugs.64
Cell-matrix interactions implicated in carcinogenesis were
examined using epithelial ovarian cancer cell lines
OV-MZ-6 and SKOV-3.65
The potential for human adipose-derived stromal cells to
undergo osteogenic differentiation both in vitro and
in vivo was characterized using a novel 3D system.66
Combined 2D and 3D similarity comparisons were used to
reveal possible new functions and/or side effects of
known bioactive compounds.67
Significant upregulation of five genes in the
presence of ECM compared to standard 2D culture.
Lower differentiation compared with 3D culture.
3D
Significant modulation in gene expression, with 106
genes upregulated and 73 downregulated compared
with standard 2D culture.
Greater differentiation compared with 2D culture.
All drugs highly active.
Less efficacy with gradual loss of activity.
20% survival rate after exposure to paclitaxel.
40%-60% survival rate after exposure to paclitaxel.
Decreased differentiation potential and ECM
production compared with 3D cultures.
Some similarities untraceable in relation to 3D.
Increased differentiation potential and ECM
production compared with 2D culture.
Identified new functions and side effects of the
known VEGFR inhibitor Vatalanib.
20 ASSAY and Drug Development Technologies
ª 2022 MARY ANN LIEBERT, INC.
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