Three-Dimensional Cell Cultures in Drug Discovery and Development

Abstract

The past decades have witnessed significant efforts toward the development of three-dimensional (3D) cell cultures as systems that better mimic in vivo physiology. Today, 3D cell cultures are emerging, not only as a new tool in early drug discovery but also as potential therapeutics to treat disease. In this review, we assess leading 3D cell culture technologies and their impact on drug discovery, including spheroids, organoids, scaffolds, hydrogels, organs-on-chips, and 3D bioprinting. We also discuss the implementation of these technologies in compound identification, screening, and development, ranging from disease modeling to assessment of efficacy and safety profiles.

Full text

https://doi.org/10.1177/2472555217696795
SLAS Discovery
1 –17
© 2017 Society for Laboratory
Automation and Screening
DOI: 10.1177/2472555217696795
journals.sagepub.com/home/jbx
Review
Introduction
Cell-based assays have been widely used in drug discovery
for several decades. Historically, two-dimensional (2D)
monolayer cells cultured on a variety of planar substrates
were the only practical option for cell-based screening and
have proven to be a convenient and effective means to dis-
cover drug candidate molecules. Nowadays, 2D cell models
can be used to effectively predict in vivo drug responses for
many targets and pathways and are still very useful in drug
discovery. However, it is evident that these 2D cultures suf-
fer disadvantages associated with the loss of tissue-specific
architecture, mechanical and biochemical cues, and cell-to-
cell and cell-to-matrix interactions,1,2 thus making them rela-
tively poor models to predict drug responses for certain
diseases such as cancer. For instance, compared with 2D cul-
ture, colon cancer HCT-116 cells in 3D culture have been
found to be more resistant to certain anticancer drugs such as
melphalan, fluorouracil, oxaliplatin, and irinotecan3; such che-
moresistance has been observed in vivo as well.4
The past decade has seen the accelerating implementa-
tion of 3D cell cultures in early drug discovery, principally
fueled by the need to continuously improve the productivity
of pharmaceutical research and development (R&D).5–7 The
use of 3D cell cultures, together with better cell models
such as stem cells and primary cells, would allow greater
predictability of efficacy and toxicity in humans before
drugs move into clinical trials,8,9 which, in turn, would
lower the attrition rate of new molecular medicines under
development. The 3D cell culture and co-culture models are
advantageous in that they not only enable drug safety and
efficacy assessment in a more in vivo–like context than tra-
ditional 2D cell cultures but also eliminate the species dif-
ferences (vs. animal models) that often impede interpretation
of the preclinical outcomes by allowing drug testing directly
in human systems.
In this review, we examine the new opportunities for the
application of 3D cell culture technologies in early drug dis-
covery, such as disease modeling, target identification and
validation, screening, and drug efficacy and safety assess-
ment. We also discuss emerging opportunities of 3D cell
cultures in drug development. Future directions and techni-
cal challenges for 3D cells-based drug discovery and devel-
opment are also discussed.
3D Cell Culture Technologies
Recent advances in cell biology, microfabrication tech-
niques, and tissue engineering have enabled the develop-
ment of a wide range of 3D cell culture technologies. These
include multicellular spheroids, organoids, scaffolds,
696795JBXXXX10.1177/2472555217696795SLAS DiscoveryFang and Eglen
research-article2017
1Biochemical Technologies, Corning Research and Development
Corporation, Corning Incorporated, Corning, NY, USA
2Corning Life Sciences, Corning Incorporated, Tewksbury, MA, USA
Received Sep 19, 2016, and in revised form Jan 28, 2017. Accepted for
publication Jan 30, 2017.
Corresponding Author:
Richard M. Eglen, Corning Life Sciences, Corning Incorporated, 836
North St., Building 300, Suite 3401, Tewksbury, MA 01876, USA.
Email: eglenrm@corning.com
Three-Dimensional Cell Cultures in Drug
Discovery and Development
Ye Fang1 and Richard M. Eglen2
Abstract
The past decades have witnessed significant efforts toward the development of three-dimensional (3D) cell cultures as
systems that better mimic in vivo physiology. Today, 3D cell cultures are emerging, not only as a new tool in early drug
discovery but also as potential therapeutics to treat disease. In this review, we assess leading 3D cell culture technologies
and their impact on drug discovery, including spheroids, organoids, scaffolds, hydrogels, organs-on-chips, and 3D bioprinting.
We also discuss the implementation of these technologies in compound identification, screening, and development, ranging
from disease modeling to assessment of efficacy and safety profiles.
Keywords
3D cell culture, 3D bioprinting, disease models, efficacy, organoids, organs-on-chips, safety, screening, multicellular
spheroid, toxicity

2 SLAS Discovery
hydrogels, organs-on-chips, and 3D bioprinting, each with
its own advantages and disadvantages (see Table 1 for a
summary). These 3D cultures, although different in princi-
ple and protocols, are used to restore the morphological,
functional, and microenvironmental features of human tis-
sues and organs. This section briefly describes the key fea-
tures of these technologies.
Spheroids
Multicellular spheroid cultures were initially developed by
Sutherland and coworkers in 1970 to recapitulate the func-
tional phenotype of human tumor cells and their responses
to radiotherapy.10,11 Since then, spheroid cultures have been
applied to many other types of cells, including stem cells,
hepatocytes, and neuronal cells (Table 1). Furthermore,
tumor spheroid monocultures or co-cultures with immune
or endothelial cells have been adapted to experimental can-
cer research and recently to oncology drug screening (see
below). The spheroid model compensates for many of the
deficiencies seen in monolayer cultures. For instance,
spheroids can develop gradients of oxygen, nutrients,
metabolites, and soluble signals, thus creating heteroge-
neous cell populations (e.g., hypoxic vs. normoxic, quies-
cent vs. replicating cells). In addition, spheroids have a
well-defined geometry and optimal physiological cell-cell
and cell-extracellular matrix (ECM) interactions. However,
there are several practical challenges associated with spher-
oid culture, including the development and maintenance of
spheroids of uniform size, the formation of spheroids from
a small seed number of cells, the precise control of specific
ratios of different cell types in spheroid when co-culture,
and the lack of reliable, simple, standardized, and high-
throughput compatible assays for drug screening using
spheroids.
There are four different approaches to enable spheroid cul-
tures. The first approach is to use low-adhesion plates to pro-
mote the self-aggregation of cells into spheroids12 (Fig. 1a).
These plates not only have an ultralow attachment surface
coating to minimize cell adherence but also possess a well-
defined geometry (e.g., round, tapered, or v-shaped bottom) to
drive and position a single spheroid within each well. The key
advantage of this approach is to form, propagate, and assay the
spheroids within the same plate, thus enabling high-throughput
screening (HTS) or high-content screening (HCS).
The second approach is to use hanging drop plates (HDPs)
to promote the formation of multicellular spheroids13 (Fig.
1b). When cells in media are dispensed into the top of an HDP
well, cells are segregated into the discrete media droplet
formed below the aperture of the HDP well bottom opening,
eventually forming spheroids. Similar to the low-adhesion
plates, the HDP can also be used for spheroid co-culture,
wherein multiple cell types are added either at the time of ini-
tial dispensing or sequentially. However, a clear caveat of this
approach is that spheroids are required to transfer from the
HDP to a second plate for assays.
The third approach is to use a bioreactor (e.g., spinner flask
or microgravity bioreactor) to drive cells to self-aggregate into
Table 1. Advantages and Disadvantages of Different 3D Cell Culture Techniques.
Technique Advantages Disadvantages
SpheroidsaEasy-to-use protocol
Scalable to different plate formats
Compliant with high-throughput screening (HTS)/high-content screening (HCS)
Co-culture ability
High reproducibility
Simplified architecture
Organoids Patient specific
In vivo–like complexity
In vivo–like architecture
Can be variable
Less amenable to HTS/HCS
Hard to reach in vivo maturity
Complication in assay
Lack vasculature
May lack key cell types
Scaffolds/hydrogels Applicable to microplates
Amenable to HTS/HCS
High reproducibility
Co-culture ability
Simplified architecture
Can be variable across lots
Organs-on-chips In vivo–like architecture
In vivo–like microenvironment, chemical, physical gradients
Lack vasculature
Difficult to be adapted to HTS
3D bioprinting Custom-made architecture
Chemical, physical gradients
High-throughput production
Co-culture ability
Lack vasculature
Challenges with cells/materials
Difficult to be adapted to HTS
Issues with tissue maturation
aDiscussion is limited to low-adhesion plates.

Fang and Eglen 3
spheroids under dynamic culture condition14 (Fig. 1c). This
approach permits large-scale production of spheroids.
However, this approach has disadvantages associated with flu-
idic flow-induced shear stress, as well as nonuniformity in size
of spheroids produced.
The fourth approach is to use micro-/nano-patterned sur-
faces as the scaffolds to control cell adhesion and migration,
thus enabling spheroid cultures15 (Fig. 1d). This approach
offers a wide range of nanoscale scaffolds imprinted onto a
flat substrate for the selection of appropriate patterns and
adhesive properties for a variety of cell types. Similar to
low-adhesion plates, these micropatterned plates have little
well-to-well and plate-to-plate variation, which make them
compliant with HTS. However, one caveat is that bubbles
may easily form during the culture, and pipetting often
damages the micropatterned surfaces.
Organoids
Organoids, also termed organ buds, represent a rapidly expand-
ing family of dish-based, 3D developing tissues that show real-
istic microanatomy.16–18 An organoid is “a collection of
organ-specific cell types that develops from stem cells or organ
progenitors and self-organizes through cell sorting and spa-
tially restricted lineage commitment in a manner similar to in
vivo.”16 Organoids are classified into tissue and stem cell
organoids, depending on how the organ buds are formed.19
Tissue organoids refer to stromal cell–free (or mesenchyme-
free) culture and mostly apply to epithelial cells because of
their intrinsic ability to self-organize into tissue-like structures.
Stem cell organoids are generated from either embryonic stem
cells (ECSs) or induced pluripotent stem cells (iPSCs) or pri-
mary stem cells such as neonatal tissue stem cells or tissue-
resident adult stem cells. To date, several in vitro organoids
have been established to resemble various tissues, including
functional organoids for thyroid,20 pancreas,21 liver,22,23
stomach,24,25 intestine,26 vascularized cardiac patch,27 cerebral
cortex,28 thymus,29 kidney,30,31 lung,32 and retina.33 Table 2
summarizes key features (e.g., cell types culture techniques
used, and organotypic features) of these organoids.
Numerous different approaches have been used to obtain
organoids (see Table 2 for specifics).34 The first approach is
to directly culture cells as a monolayer on a bed of feeder
cells or an ECM-coated surface, so the organoids are formed
after the cells differentiate. The second approach is to use a
mechanically supported culture to allow the further differ-
entiation of primary tissues. For example, human keratino-
cytes can further differentiate and self-assemble into a fully
stratified tissue when the supported culture is in contact
with an air-liquid interface over a period of weeks.35 The
third approach is to generate embryoid bodies on the low-
adhesion plates or through hanging drop culture, similar to
spheroid cultures. The fourth approach is to use serum-free
floating culture of embryoid body-like aggregates with
quick reaggregation in low-adhesion plates.
Organoids mimic some, but not all, of the structure and
function of real organs.16 First, all organoids lack vascula-
ture, which is essential to nutrient and waste transport.
Second, some organoids may lack key cell types found in
vivo. Third, some organoids replicate only the early stages
of organ development. For example, retinal organoids do
not have the outer segments, and photoreceptors fail to fully
mature to become light sensitive, whereas the cerebral
organoids fail to fully develop later features, such as corti-
cal plate layers.16 Technical challenges still remain to pro-
duce organoids with in vivo–like complexity, increasing
maturity, and screening-compatible reproducibility.
Scaffolds and Hydrogels
Scaffolds refer to synthetic 3D structures made of a large
variety of materials with different porosities, permeability,
Figure 1. Four different techniques used for spheroid cultures. (a) A well of low-adhesion plates that have a round bottom with
an ultralow cell attachment coating.12 (b) A droplet of hanging drop plate where cells are partitioned and self-organized into a
spheroid.13 (c) Suspension culture in bioreactor where cells become self-aggregated into spheroids.14 (d) A representative pillar of
micropatterned plates where the cells are enriched on the top of the pillar to form a spheroid.15

4 SLAS Discovery
surface chemistries, and mechanical characteristics designed
to mimic the microenvironment of specific tissues. Scaffolds
can be classified into biological and polymeric scaffolds.
Biological scaffolds mostly use naturally derived ECM
such as Matrigel and collagen to promote appropriate cell
attachment and reorganization into 3D structures. Compared
to synthetic scaffolds, Matrigel can provide a more physio-
logically relevant microenvironment of soluble growth fac-
tors, hormones, and other molecules with which cells
interact in an in vivo environment.36 Matrigel has been
widely used as the gold standard scaffold material to pro-
vide 3D cell cultures for a wide range of cell types. However,
the disadvantages associated with Matrigel are commonly
occurring lot-to-lot variability during manufacturing and
complexity in composition, which are often ill-defined,
making it difficult to determine exactly which signals are
promoting cell function. Other natural gels such as fibrin,
hyaluronic acid, chitosan, alginate, or silk fibrils have also
been used for 3D cell culture; however, these natural gels
have less versatility to promote 3D culture than Matrigel.
Table 2. Organoids and Their Origin, Culture Techniques, and Applications.
Organoid Origin Culture Technique Endpoints Ref.
Thyroid mESCs EB differentiation in hanging
drops
Functional thyroid organoid 20
Pancreas Mouse embryo pancreas
progenitor
Matrigel embedding Epithelial derivatives including
endocrine cells
21
Liver mLGR5+ SC Matrigel embedding Bile ducts and hepatocytes to
model alpha-1 antitrypsin
deficiency and Alagille
syndrome
22
Liver hPSCs Co-culture with HUVECs and
hMSCs on Matrigel after
monolayer differentiation
toward endoderm
Liver bud derivative 23
Stomach Adult SC/gastric glands
(m/h)
Matrigel embedding Adult SC + all stomach epithelial
derivatives, excluding parietal
cells, to model Helicobacter
pylori infection/gastric cancer
24, 25
Intestine hESCs/PSCs Spheroids embedded Matrigel
after monolayer
differentiation toward hindgut
Intestinal bud, epithelial and
mesenchymal derivatives
26
Vascularized cardiac patch hESCs High FCS Contractile muscle 27
Cerebral cortex m/hESCs EBs generated in low-adhesion
U-shaped plates
Embedded in Matrigel and
cultured in spinner flask
Cerebral cortex to model
microcephaly
28
Thymus Fibroblasts Reprograming induced by
FOXN1
All types of thymic epithelial
cells on transplantation
29
Kidney hESCs/PSCs Subculture in air-liquid
interface after differentiation
and dissociation
Nephrons associated with
a collecting duct network
surrounded by renal
interstitium and endothelial
cells
30
Kidney hPSCs Sandwiched between two layers
of Matrigel, differentiation
with GSK3β inhibitor
Proximal tubules, podocytes,
and endothelium
31
Lung mAdult SCs Matrigel co-culture with lung
endothelial cells
Epithelial derivatives +
mesenchymal derivatives
32
Retina hESCs SFEBq in low-adhesion
V-shaped plates with
Matrigel embedding day 2,
transfer to Petri dish day 12
Epithelial + retinal derivatives 33
EB, embryonic body; ESCs, embryonic stem cells; FCS, fetal calf serum; FOXN1, transcription factor forkhead box N1; HUVECs, human umbilical vein
endothelial cells; LGR5, leucine-rich repeat containing G protein–coupled receptor 5; m/h, mouse or human; MSCs, human mesenchymal stem cells;
PSCs, induced pluripotent stem cells; SCs, stem cells; SFEBq, serum-free floating culture of EB-like aggregates with quick reaggregation.

Fang and Eglen 5
Polymeric scaffolds use synthetic hydrogels or other bio-
compatible polymeric materials to generate the physical
supports for 3D cultures.37,38 The hydrogels used for 3D
culture include poly(ethylene glycol) (PEG), poly(vinyl
alcohol), and poly(2-hydroxy ethyl methacrylate).38
Furthermore, hydrogels can be made to be hydrolytically or
enzymatically biodegradable by incorporating poly(lactic
acid) units39 or enzyme cleavable peptide sequences40 into
the polymer network backbone. The biodegradability is
critical to applications in which cell utilization is a must,
such as tissue engineering and regenerative medicine.
Synthetic scaffolds have several clear advantages over
Matrigel or other natural gels for 3D cultures. First, the use
of synthetic materials can minimize the relatively poor
reproducibility of biological ECMs between batches and
the resulting lack of consistency between cultures, as they
are often simply processed and manufactured. Second,
these scaffolds allow for fine tuning of biochemical and
mechanical properties, so it is possible to optimize both
mechanical and chemical cues for 3D cell cultures. Third,
these hydrogels possess high water content, enabling trans-
port of oxygen, nutrients, waste, and soluble factors, all of
which are important to cell functions.41 However, these
hydrogels do not contain the endogenous factors but act
mainly as a template to regulate cell behavior. In addition,
these hydrogels pose challenges related to oxygen availabil-
ity, heterogeneities present in the synthetic cellular micro-
environment, and uneven distribution of soluble growth
factors within the matrix and complication in imaging and
cell analysis.38
The scaffold characteristics, along with the material
properties, can regulate cell adhesion, proliferation, activa-
tion, and differentiation.42,43 For instance, naive mesenchy-
mal stem cells (MSCs) were shown to specify lineage and
commit to phenotypes with extreme sensitivity to substrate
mechanical stiffness.44 MSCs were neurogenic on soft
matrices but myogenic on stiffer matrices that mimic mus-
cle and osteogenic on comparatively rigid matrices that
mimic collagenous bone. Upon treatment with soluble fac-
tors, MSCs were found to differentiate into the lineage
specified by matrix elasticity.
Scaffolds can be made using a variety of techniques,
such as 3D printing,45 particulate leaching,46 or electrospin-
ning.47 Alternative approaches include gas foaming, fiber
meshes/fiber bonding, phase separation, melt molding,
emulsion freeze drying, solution casting, or freeze drying
(reviewed in ref. 48). The types of scaffolds obtained were
dependent on the fabrication techniques. In general, partic-
ulate leaching or solvent casting can be used to produce
porous scaffolds, whereas electrospinning is useful for fab-
ricating fibrous scaffolds, and 3D printing can be used to
produce scaffolds with defined shapes and geometries. All
of these types of scaffolds have been realized for 3D cul-
ture. For instance, to overcome the progressive loss of
functionality of MSC expansion in 2D monolayer culture,
freshly isolated bone marrow nucleated cells were directly
cultured within 3D porous hydroxyapatite ceramic scaf-
folds in a perfusion-based bioreactor system.49 The stromal
tissues obtained were enzymatically treated to yield CD45-
MSCs, which gave rise to a 4.3-fold higher clonogenicity
and the superior differentiation capacity toward all typical
mesenchymal lineages, compared with the 2D expansion
culture. Of note, cells grown on fibrous scaffolds are often
not considered to truly represent 3D culture, as cells typi-
cally adhere and elongate along the fibers.50 In addition,
porous scaffolds have issues associated with their limited
diffusion properties, which make it difficult to fabricate
more complex tissues such as heart and liver.
Organs-on-Chips
An organ-on-a-chip refers to an artificial, miniature model
of a human organ on a microfluidic cell culture chip. The
chip is made with great precision using microfabrication
techniques such as soft lithography, photolithography, and
contact printing.51 The chip usually consists of a series of
well-defined structures, patterns, or scaffolds. Therefore,
the position, shape, function, and chemical and physical
microenvironments of the cells in culture can be controlled
with high spatiotemporal precision using microfluidics.52
Organs-on-chips are designed to reconstitute the structural,
microenvironmental, and functional complexity of living
human organs. However, most organs-on-chips are often
made to capture only the critical features of an organ type or
a disease model due to practical reason, so researchers can
reproduce clinically relevant disease phenotypes and phar-
macological responses.53,54 To date, a wide range of organs-
on-chips have been reported, including skin,55 lung,56–58
vasculature,59 heart,60,61 muscle,62 liver,63–65 intestine,66 and
several others (see below for specific applications of some
of these organ systems).
Organs-on-chips have been adapted to microplate for-
mats, the de facto footprint used in drug discovery. For
instance, a liver-on-a-chip that uses a bioreactor to foster
maintenance of 3D tissue cultures under constant perfusion
was developed in a multiwell plate format67 and used for
drug metabolism profiling and pharmacokinetic evalua-
tion.68 However, most organs-on-chips lack vasculature and
also are difficult to adapt to HTS.
Three-Dimensional Bioprinting
Three-dimensional bioprinting refers to the printing of
cells, biocompatible materials, and supporting components
into complex 3D living tissues with the desired cell/organ-
oid architecture, topology, and functionality using additive
manufacturing.69 Three-dimensional bioprinting usually
involves layer-by-layer positioning of biological materials,

6 SLAS Discovery
biochemicals, and living cells. There are three approaches
used for bioprinting. The first one is biomimicry, which
employs biologically inspired engineering to replicate the
cellular and extracellular components of a tissue or organ
(e.g., human ears).70 The second approach is autonomous
self-assembly, which relies on the cells as the primary driver
of histogenesis to produce the desired biological microar-
chitecture and functional tissues.71 The third approach is to
fabricate and assemble mini-tissue building blocks, such as
a kidney nephron, into the larger construct by rational
design, self-assembly, or a combination of both.72,73
Three-dimensional bioprinting has been used to generate
functional tissues, such as multilayered skin, bone, vascular
grafts, tracheal splints, heart tissue, and cartilaginous struc-
tures, for transplantation applications.74 Furthermore, 3D bio-
printing has been used not only to create scaffolds for 3D cell
cultures but also to directly produce 3D-bioprinted tissue mod-
els for drug screening and profiling.75 Bioprinting has several
advantages, such as custom-made microarchitecture, high-
throughput capability, and co-culture ability. However, com-
pared with other 3D cell cultures, 3D bioprinting faces many
additional challenges associated with cell and material require-
ments as well as tissue maturation and functionality.69
3D Cell Cultures in Drug Discovery
Drug discovery is a long, complex process with growing dif-
ficulty. Three-dimensional cell cultures have been penetrating
into the early drug discovery process, starting from disease
modeling to target identification and validation, screening,
lead selection, efficacy, and safety assessment (Fig. 2). This
section discusses how to best implement different 3D cell cul-
ture technologies into different stages of drug discovery
process.
Disease Modeling
Drug discovery often starts with a disease or a clinical con-
dition without suitable medical products available.76 As
growing efforts have been directed toward unmet therapeu-
tic needs in recent years,77 disease modeling has become
increasingly important to the success of drug discovery pro-
grams. As they promise to bridge the gap between 2D cul-
ture and in vivo, a range of 3D cell cultures have been
applied to understand the mechanisms of different diseases.
In particular, 3D models have gained popularity in elucidat-
ing tumor biology, as standard 2D models are inadequate to
address questions regarding indolent disease, metastatic
colonization, dormancy, relapse, and the rapid evolution of
drug resistance.78
Three-dimensional cultures on ECM gels have provided
models to detect architecture transformation from preinva-
sive breast carcinoma to full malignancy induced by the
progressive loss of tissue architecture and aberrant signal-
ing79 or from nonmalignant breast epithelial cells to malig-
nant tumors induced by tuning stiffness of Matrigel/
Collagen I gels used in 3D culture.80 Recently, Drost et al.81
investigated the phenotypes of sequential cancer mutations
in cultured human intestinal stem cells by combining organ-
oid culture and CRISPR/Cas9 gene editing. Here, normal
human intestinal stem cells isolated from patients were
genetically edited using CRISPR/Cas-9 for the four most
commonly mutated colorectal cancer genes (APC, P53,
KRAS, and SMAD4), followed by culturing on Matrigel or
basal membrane extract–coated plates in medium contain-
ing the stem-cell-niche factors WNT, R-spondin, epidermal
growth factor, and noggin. Results showed that the epithe-
lial organoids obtained remained genetically and phenotyp-
ically stable for long periods of time, and xenotransplantation
of quadruple mutant organoids into mice resulted in tumors
with features of invasive carcinoma. Remarkably, the com-
bined loss of APC and P53 was found to be sufficient for the
appearance of extensive aneuploidy, a hallmark of tumor
progression.
Spheroid cultures have become useful for modeling the
tissue architecture, signaling, microenvironments, and inva-
sion and immune behaviors of cancer, as well as for study-
ing and expanding the cancer stem cells (CSCs).82 Human
Figure 2. How different three-
dimensional culture techniques
have been implemented in
different stages of drug discovery
and development processes.
Representative references for
each application are cited in
parentheses in the graph.

Fang and Eglen 7
cancer is known to harbor several heterogeneous subpopu-
lations of CSCs that play distinct roles in tumor initiation,
maintenance, and metastasis. For instance, in colon cancers,
there were three types of CSCs isolated from patients:
tumor-initiating cells that have limited or no self-renewal
capacity but are contributed to tumor formation only in pri-
mary mice, self-renewal CSCs that allow long-term tumor
growth, and rare delayed contributing CSCs that were
exclusively active in secondary or tertiary mice.83 Tumor
invasion and metastasis is a multistep cascade process. It
begins with local invasion of cancer cells through the ECM
and stromal cell layers, then intravasation into the lumina of
blood vessels. This is followed by transit through the lym-
phatic and hematogenous systems and arrest and extravasa-
tion out of the circulatory system, which leads to the
formation and growth of micrometastatic lesions into mac-
roscopic tumors at a distant site.84 Spheroids of cancer cell
lines have been used to investigate different aspects of the
cancer invasion process, including the invasion of cells in a
3D spheroid into the surrounding 3D ECM structure85,86 and
endothelial cell–tumor cell interactions.87 For instance, we
had developed a label-free, real-time, single-cell, and quan-
titative assay to monitor the invasion of cells in a spheroid
through a 3D Matrigel (Fig. 3). We found that epidermal
growth factor accelerates the invasion of the colon cancer
cell line HT-29, whereas vandetanib dose-dependently
inhibits the invasion.88 Vandetanib is a multitarget kinase
inhibitor that has been clinically approved for the treatment
of late-stage (metastatic) medullary thyroid cancer in adult
patients who are ineligible for surgery and also has potential
to treat non–small-cell lung cancer. Although the results
obtained using the label-free assay are largely expected as
vandetanib is known to inhibit vascular endothelial growth
factor receptor, this assay enables real-time quantification
of its effect on cancer invasion through the Matrigel, a capa-
bility that is otherwise difficult to obtain using conventional
endpoint assays. We further found that PTEN knockout
increased the invasion rate of HCT116 cells in spheroid
through 3D Matrigel, and PI3K inhibitors LY294002 and
wortmannin drastically reduced the invasiveness of the
Figure 3. A label-free, single-cell, real-time assay to measure the invasion of cells in a single spheroid through a three-dimensional
extracellular matrix (Matrigel). (a) Principle of the assay, which consists of four critical steps: coating the biosensor surface with
Matrigel; adding medium to the well; transferring a spheroid from an ultralow attachment, round-bottomed microplate and placing
it onto the top Matrigel surface; and monitoring the invasion of cells through the matrix and adhesion on the sensor surface in
real time. (b–d) The time series dynamic mass redistribution (DMR) images before and after a single spheroid was placed onto the
biosensor surface coated with 10 µL 0.1 mg/mL Matrigel: 0 min (b), 1 h (c), and 24 h (d). Spatial scale bar: 500 µm. Intensity scale bar:
–500 pm to 2000 pm. (e) A DMR image taken 24 h after a spheroid was placed on the top Matrigel surface. Scale bar: 500 µm. (f)
Representative pixelated real-time DMR signals for the black line indicated in (e). (g) The adhesion events versus cell types. (h) The
adhesion time to reach 200 pm under different conditions. For (e–h), coating was 0.2 mg/mL Matrigel. Data represent mean ± SD for
g (n = 3). ***p < 0.001. This figure is adapted from ref. 89 with permission.

8 SLAS Discovery
cells.89 This label-free imaging technique has revealed that
besides the accelerated invasion kinetics, PTEN knockout
expedites cell dissociation from the spheroidal structure and
adhesion onto the surfaces. This study also indicates that the
mechanisms governing cell invasion are sensitive to ECM
matrix density, and the invasion inhibitory sensitivity of
PI3K inhibitors is also sensitive to the PTEN expression
level.
Organoid cultures have also been applied to model can-
cer, besides a great number of other diseases including
developmental disorders, infectious diseases, and neuronal
degeneration.16 For example, several different intestinal
organoids were obtained and used for modeling a range of
diseases. Specifically, the human intestinal organoids
derived from the ESC line WA09 were used to examine gas-
trointestinal infection with rotavirus.90 The human intestinal
organoids generated using intact crypts from human intes-
tines were used to examine Cryptosporidium parvum infec-
tion.91 The organoids obtained by culturing CD44+CD24+
cells enriched for colorectal CSCs in the HT29 and SW1222
cell lines were used to study colon CSC biology.92 The
intestinal organoids obtained using murine primary intesti-
nal cells were used to study genetically reconstituted tumor-
igenesis (e.g., by knockdown adenomatous polyposis coli
[APC]),93 whereas the intestinal organoids cultured from
patient biopsies were used to study genetic disorders.94,95
Many genetic disorders that have been difficult or impos-
sible to model in animals can be modeled by using organoid
cultures of patient iPSCs or, alternatively, through the
introduction of patient mutations into human PSCs using
genome-editing technologies, such as CRISPR/Cas9. For
instance, the CRISPR-Cas9 genome-editing system was
used recently to introduce multiple recurrent mutations in
colon cancer patients into organoids derived from normal
human intestinal epithelium.96
Organs-on-chips are also useful for cancer modeling.
For instance, cultured human skin tissue has been success-
fully used as a surrogate for modeling melanoma cancer
growth.55 Here, when human melanoma cell lines were
incorporated, the cultured skin tissue recapitulated natural
features of melanocyte homeostasis and melanoma progres-
sion in human skin. They displayed the same characteristics
reflecting the original tumor stage (vertical and radial
growth phases and metastatic melanoma cells) in vivo.
Organs-on-chips have also been used to model other dis-
eases. For instance, a lung-on-a-chip was developed to
mimic breathing by stretching and compressing an artificial
alveolar-capillary barrier using a cyclic vacuum machine.
This was used to model pathogen infection and inflamma-
tory responses to air pollutants56 or the development and
progression of pulmonary edema induced by the toxicity of
interleukin-2.57 Recently, the airway-on-a-chip device lined
by living human bronchiolar epithelium from normal or
chronic obstructive pulmonary disease (COPD) patients
was connected to an instrument that “breathes” whole ciga-
rette smoke in and out of the chips to study smoke-induced
pathophysiology in vitro.58 This enables the detection of
smoke-induced ciliary micropathologies, COPD-specific
molecular signatures, and epithelial responses to smoke
generated by electronic cigarettes.
Target Identification and Validation
Target identification and validation is often the rate-limiting
step in preclinical drug discovery.97 Three-dimensional cul-
tures have the potential to discover novel mechanisms and
targets and to accelerate target identification and validation,
given that the gene expression patterns found in 3D models
are one step closer to in vivo, compared to 2D monolayer
models.98 For instance, gene expression analysis of meso-
thelioma cell lines cultured in spheroids had revealed the
underlying causes of chemoresistance in malignant pleural
mesothelioma.99 Here, the spheroids were found to acquire
increased chemoresistance compared with 2D monolayers.
A total of 209 genes were differentially expressed in com-
mon by the three mesothelioma cell lines in spheroids,
among which argininosuccinate synthase 1 (ASS1) was the
only consistently up-regulated gene in both 3D spheroids
and human tumors. siRNA knockdown of ASS1 signifi-
cantly sensitized mesothelioma spheroids to the proapop-
totic effects of bortezomib or cisplatin plus pemetrexed.
These results suggest that ASS1 may be a druggable target
to undermine mesothelioma multicellular resistance.
In another recent study, a microfluidic vasculature chip
was developed to model intravascular steps in metastasis.59
Here, the chip consisted of an upper intravascular compart-
ment and lower stromal chambers, separated by a semipo-
rous membrane lined with human microvascular endothelial
cells. Upon stimulation of microvascular endothelium from
the basal side, CXCL12 acted through the CXCR4 receptor
on endothelium to promote adhesion of circulating breast
cancer cells. This suggests that targeting CXCL12-CXCR4
signaling in endothelium may limit metastases in breast and
other cancers.
Screening for Hit Identification
Screening using cell-based assays has frequently been the
starting point for identifying hit compounds in the early
stage of drug discovery. In the past three decades or so,
target-based HTS has been dominating in the hit identifica-
tion process, given that HTS-compatible cellular assays
have simplicity, relatively low cost, and high efficiency.
However, in recent years, there has been a renaissance in
phenotypic screening, driven by three factors. First, con-
tinuous improvement in the productivity of pharmaceutical
R&D calls for innovative strategies for drug discovery.
Second, although target-based screens are more effective

Fang and Eglen 9
for discovering follow-on drugs for which molecular mode
of action is known, phenotypic screens are more productive
for discovering first-in-class drugs.100 Third, advances in
detection technologies have made it feasible to perform
phenotypic screens with high throughput as well as more
biologically relevant information relative to conventional
molecular assays.101–104
Incorporating 3D cell cultures with HTS processes is
still in infancy but shows promise in directly identifying
clinically relevant compounds, enabling effective transla-
tional research. Unfortunately, not all 3D cell culture mod-
els are compatible with HTS or HCS in a routine and
cost-effective manner. Among all 3D models under devel-
opment, spheroids cultured in the low-adhesion plates have
started gaining popularity in oncology drug screening
because of their easy-to-use protocols, high-density micro-
plate formats (e.g., 384-well and 1536-well), and compati-
bility with automation and multimode detection systems.
For instance, using glucose-deprived multicellular tumor
spheroids of colon cancer cell lines with inner hypoxia that
were cultured in 384-well low-adhesion plates, Senkowski
et al.105 screened 1600 compounds with documented clini-
cal history to identify five compounds that selectively target
the hypoxic cell population. All five compounds inhibited
mitochondrial respiration, suggesting that cancer cells in
low-glucose concentrations depend on oxidative phosphor-
ylation, instead of solely glycolysis. The antiprotozoal drug
nitazoxanide was found to activate the AMPK pathway and
down-regulate c-Myc, mTOR, and Wnt signaling at clini-
cally relevant concentrations. Combining nitazoxanide with
the cytotoxic drug irinotecan showed anticancer activity in
vivo. Similar results were obtained from the HCS of 1120
compounds against spheroids of the human breast cancer
cell line T47D.106 At the 2016 SLAS annual conference, Dr.
Timothy Spicer and his colleagues at The Scripps Research
Institute presented results using Corning nonadherent 1536-
well spheroid plates to screen the entire Scripps Drug
Discovery Library of more than 650,000 compounds in less
than 2 wk (personal communication).
Three-dimensional co-cultures of a cancer cell with
another cell type (e.g., an immune or fibroblast cell line)
have also been developed in high-throughput formats. For
instance, using a multilayered organotypic culture contain-
ing primary human fibroblasts, mesothelial cells, and ECM,
Kenny et al.107 performed a screen of 2420 pharmacologi-
cally active compounds. This organotypic culture was used
to reproduce the human ovarian cancer metastatic microen-
vironment. Subsequent validation in secondary in vitro and
in vivo assays confirmed two active compounds, β-escin
and tomatine, that prevented ovarian cancer adhesion, inva-
sion, and metastasis, leading to the improved survival in
mouse models. This study shows the power of complex 3D
models to improve the disease relevance of assays used for
drug screening.
Efficacy Profiling for Lead Identification
Following hit identification is lead identification. Once
identified in a screen, hits are first confirmed based on
dose-response curves using the same assay for screening
and orthogonal testing with different assay(s). Once con-
firmed, hits are further evaluated for synthetic tractability,
freedom to operate, drug-likeness, and possible toxicity,
metabolism, and stability-related risks. Medicinal chemis-
try optimization is the next step to generating lead candi-
date compounds with improved potency, reduced off-target
activities, and desired physicochemical and metabolic
properties. Critical to the entire process of lead identifica-
tion is to have cost-effective in vitro models that can more
reliably predict the efficacy, toxicity, and pharmacokinet-
ics of drug compounds in humans. Three-dimensional cell
culture models have a potential to play an important role
in lead identification and to reduce the use of animal test-
ing for preclinical studies.
Lacking in vivo efficacy is one of the key reasons why
some late-stage clinical trials fail.108 Three-dimensional cell
culture models have been shown to in some cases more accu-
rately evaluate drug efficacy than 2D models and may even
enable personalized approaches to identify the mechanisms
underlying disease and to screen and select the best drug(s)
for the patients.109–111 For instance, patient-derived spheroids
have been developed as a predictive test to identify the most
effective therapy for 120 patients with HER2-negative breast
cancer of all stages.112 Results showed that the tissue spher-
oid model reflected current guideline treatment recommen-
dations for HER2-negative breast cancer. Tissue spheroid
showed greater responses to anthracycline/docetaxel for hor-
mone receptor–negative samples, a higher response to fluo-
rouracil and anthracycline in high-grade tumors, and a higher
treatment efficacy to anthracycline treatment combined with
fluorouracil for smaller tumor size and negative lymph node
status. Recently, Tong et al. applied spheroids of three ovar-
ian cancer cell lines to investigate the differential oncolytic
efficacy among three different viruses: myxoma, double-
deleted vaccinia, and Maraba virus.113 They found that the
low-density lipoprotein receptor expression in ovarian cancer
spheroids is reduced, which in turn affects the binding and
entry of Maraba virus into cells.
Compared with spheroids, organs-on-chips provide a
viable strategy to further increase the complexity and physi-
ological relevance for reliable assessment of drug efficacy.
For instance, Aref et al.114 developed an organ-on-a-chip
consisting of lung cancer spheroids in a 3D matrix gel adja-
cent to an endothelialized microchannel to recapitulate
epithelial-mesenchymal transition during cancer progression.
Results showed that for the A549 cell model, there are both
qualitative and quantitative differences in drug response
between 2D monolayer cells and 3D spheroids. For instance,
for the TGF-βR inhibitor A83-01, the differences in

10 SLAS Discovery
effective dose between 2D and 3D culture were more than
three orders of magnitude (5 nM vs. 2.5 μM).
Toxicity Profiling for Lead Selection
Drug-induced toxicities in liver, heart, kidney, and brain
currently account for more than 70% of drug attrition and
withdrawal from the market.115 Adverse drug reactions are
often due to off-target interactions or excessive binding of
the drug molecule to toxicity-prone cells. Three-dimensional
cell culture models are powerful in assessing drug-induced
toxicity.
Organ buds of brain, liver, heart, and kidney can be used to
assess drug toxicity.16 Recently, a brain organoid was produced
by combining human ESC-derived neural progenitor cells,
endothelial cells, MSCs, and microglia/macrophage precur-
sors on chemically defined polyethylene glycol hydrogels.116
Machine learning was used to build a predictive model from
changes in global gene expression when being exposed to 60
training compounds (34 toxic and 26 nontoxic chemicals). The
model was then used to correctly classify 9 of 10 additional
chemicals in a blinded trial. Human liver organoids obtained
using HepaRG cell line, a terminally differentiated hepatic cell
line derived from a human hepatic progenitor cell line, have
already been shown to produce human-specific metabolites.117
This is particularly useful because human liver often metabo-
lizes drugs in a manner distinct from animal liver. Of note,
these HepaRG 3D organotypic cultures are more sensitive to
acetaminophen- or rosiglitazone-induced toxicity but less sen-
sitive to troglitazone-induced toxicity than the 2D cultures.
Kidney organ buds from human iPSC cells were found to dif-
ferentially apoptose in response to cisplatin, a nephrotoxicant,
showing such organoids represent powerful models of the
human organ for drug-induced nephrotoxicity.30
Three-dimensional liver cell spheroid cultures are also
valuable for investigating drug-induced liver injury, func-
tion, and diseases. An organotypic culture of the human
hepatoma HepaRG cell line were obtained using hanging
drop culture and was able to detect the potent toxicity of
acetaminophen.117 Human primary hepatocyte spheroids
obtained using the low-adhesion plates were found to be
phenotypically stable and retained morphology, viability,
and hepatocyte-specific functions for at least 5 wk, enabling
chronic toxicity assessment of drug molecules.118 The
chronic toxicity of fialuridine was detected after repeated
dosing in this spheroid model; this type of toxicity was
impossible to detect using 2D models. However, the pri-
mary hepatocyte spheroids also retain the interindividual
variability, which may limit the ability of such models for
large-scale screening. To this regard, unlimitedly renew-
able, primary-like hepatocytes, such as HepatoCells,
HepRG, or iPS-derived cells, may be good alternatives for
screening.
Organs-on-chips and other 3D cell culture models were
also used to evaluate drug-induced toxicity.104 Heart-on-a-
chip devices were useful for assessing drug-induced cardio-
toxicity.60,61 The lung-on-chip model developed by Huh
et al.57 consisted of channels lined by closely apposed layers
of human pulmonary epithelial and endothelial cells that
experience air and fluid flow, enabling the detection of drug
toxicity-induced pulmonary edema observed in human can-
cer patients treated with interleukin-2 at similar doses and
over the same time frame.57 This study also found that both
angiopoietin-1 and GSK2193874 (a transient receptor
potential vanilloid 4 ion channel inhibitor) were effective at
preventing the drug toxicity-induced pulmonary edema. A
3D bioprinted, cell-based soft robotic device that was pow-
ered by the actuation of an engineered mammalian skeletal
muscle strip was recently used to sense, process signals, and
produce force.62 The muscle strip was made by printing
mouse skeletal muscle myoblast cell line C2C12 in the
presence of hydrogels and other biological components.
Skeletal muscle as a contractile power source is the primary
generator of actuation in animals. This device can be used
to assess drug-induced myopathy.
Pharmacokinetics and Pharmacodynamics
Profiling for Lead Selection
Inadequate pharmacokinetics and pharmacodynamics is
also a key factor in why drugs fail. Three-dimensional cell
culture models, in particular, liver spheroids, liver organ-
oids, and body-on-chips, are useful to investigate the phar-
macokinetic profiles of drug molecules. Liver spheroids
and organoids have been used to study the metabolism of
drug molecules.116 Several versions of liver-on-a-chip sys-
tems were used to measure rates of metabolic drug clear-
ance, which were compared with literature-reported
values.63–65 The gut-on-a-chip using the Caco-2 cell layer
on a porous support to separate two chambers was used to
reproduce characteristic absorptive properties and the bar-
rier function of the human intestine, enabling drug absorp-
tion studies.66 Integrating multiple organ types into one
chip, termed as body-on-a-chip, can be powerful for com-
prehending the pharmacokinetics and pharmacodynamics
of drug molecules.119,120 However, developing screening-
compatible body-on-a-chip remains a challenging task, in
particular when one considers the known allometric scaling
issue.121
3D Cultures in Cell Therapy and
Tissue Engineering
Cell therapy and tissue engineering have started entering
the market. They not only offer new hope for patients with
injuries, end-stage organ failure, or other clinical issues but

Fang and Eglen 11
also will eventually transform our lives. However, it is
becoming clear that realizing the full potential of cell ther-
apy and tissue engineering requires advances in cell culture
technologies to meet the demand in quantity, quality, and
process robustness for commercialization and clinical trials.
Three-dimensional cell cultures offer not only a solution for
cell scale-up production but also a new form of therapeutics
for treating many different diseases.
Stem Cell Spheroids for Regenerative Medicine
Stem cells are widely used as a cell source for regenerative
medicine and cell therapy applications. However, conven-
tional 2D culture techniques, in combination with the cur-
rent best practice, may be ineffective to expand stem cells
for clinical applications. This is reflected by the fact that 2D
cultures are inadequate to reproduce the in vivo microenvi-
ronment of stem cells.122 In addition, clinical observations
show that the beneficial effects of stem cell–based thera-
peutics seen in initial small-scale clinical studies are often
not validated by large, randomized clinical trials.123,124 In
fact, MSCs often decrease their replicative ability, colony-
forming efficiency, and differentiation capabilities over
time when culturing and passaging in 2D adherent mono-
layer.125,126 In contrast, MSCs cultured in spheroids display
a morphology that is significantly different from 2D cul-
ture.127 The MSCs are spherical inside and elongated out-
side the spheroid, with an overall reduction of cytoskeletal
molecules, ECM, and size (~75% reduction in individual
cell volume),128 indicating distinct differentiation prefer-
ences among different lineages.129 Furthermore, compared
with 2D culture, MSCs cultured in spheroids have different
gene expression patterns, with up-regulation of many genes
that are associated with hypoxia, angiogenesis, inflamma-
tion, stress response, and redox signaling.130
Spheroid cultures have been reported to improve the
efficacy of MSC-based therapeutics. Compared with 2D
cultures, MSC spheroid cultures were found to result in sev-
eral additional beneficial effects, such as enhanced anti-
inflammatory and tissue regenerative and reparative effects,
as well as better posttransplant survival of MSCs.130
Furthermore, compared with 2D cultured cells, spheroids of
human adipose–derived MSCs produced higher levels of
ECM proteins, exhibited stronger antiapoptotic and antioxi-
dative capacities, and increased the paracrine secretion of
cytokines.131 When injected into the kidney of model rats
with ischemia reperfusion-induced acute kidney injury,
these MSC spheroids were more effective in protecting the
kidney against apoptosis, reducing tissue damage, promot-
ing vascularization, and ameliorating renal function com-
pared with 2D cultured cells.
Spheroid cultures have been used to enrich patient-
specific stem cells for disease treatment. For instance,
Henry et al.132 applied spheroid culture to enrich adult lung
stem cells for use in treating idiopathic pulmonary fibrosis
in mice. Here, in a suspension culture, the outgrowth cells
from healthy lung tissue explants were self-aggregated into
spheroids, which recapitulated the stem cell niche and
acquired mature lung epithelial phenotypes. The mice that
received these spheroids showed decreases in inflammation
and fibrosis.
Spheroid cultures have also been used to scale up stem
cell products for use in clinical trials. For instance, the man-
ufacturing process of pancreatic endoderm cells (PEC-01)
involves dynamic suspension spheroid culture and differen-
tiation.133 The PEC-01 is derived from CyT49 human ESCs
and is the cellular component of the VC-01 combination
product from ViaCytes for treating type 1 diabetes. PEC-01
matures after transplantation and functions to regulate
blood glucose.
Organoids for Transplantation
Organoids could provide a source of autologous tissue for
transplantation, as organoid research advances rapidly. For
instance, renal organoids derived from pluripotent stem
cells were successfully transplanted under the renal cap-
sules of adult mice.134 Here, the organoid reconstituted the
3D structures of the kidney in vivo, including glomeruli
with podocytes and renal tubules with proximal and distal
regions and clear lumina. Furthermore, the glomeruli were
efficiently vascularized upon transplantation, which is a
promising step toward kidney replacement strategy.
Although early in development, organoid-based replace-
ment may find applications in other diseases, such as retinal
organoids obtained from human ESCs for treating certain
types of retinal degeneration and blindness,16 intestinal
organoids for replacement of damaged colon after injury or
following removal of diseased tissue,135 and gene-corrected
organoids for replacement of damaged organs with gene
defect(s).16 For instance, the intestinal organoids obtained
from Lgr5+ adult colonic stem cells have been transplanted
into superficially damaged mouse colon.136 Results showed
that the transplanted donor cells readily integrated into the
mouse colon, covering the area that lacked epithelium as a
result of the introduced damage in recipient mice. Long-
term (>6 months) engraftment with transplantation of
organoids derived from a single Lgr5+ colon stem cell was
observed after extensive in vitro expansion. This study
shows the feasibility of colon stem-cell therapy based on
the in vitro expansion of a single adult colonic stem cell.
3D Bioprinted Tissues/Organs for
Transplantation
Advances in tissue engineering, cell biology, and materials
sciences have made 3D bioprinting possible to create
functioning tissues or organ grafts with their natural

12 SLAS Discovery
microenvironments and architectures from autologous cells
for transplantation applications. Although printing an intact
organ still remains elusive, 3D-bioprinted bladders, tracheal
grafts, bone, and cartilage have proven to be functional after
development and implantation in animals or humans.74
These printed organs can be used as assist organs or viable
replacements. For instance, Atala et al.137 engineered a
human bladder by isolating autologous bladder urothelial
and muscle cells from the bladder biopsy, expanding the
cells in vitro and seeding them to a biodegradable bladder-
shaped scaffold made of collagen or a composite of colla-
gen and polyglycolic acid. About 7 weeks after the biopsy,
the autologous engineered bladder constructs were used for
reconstruction and implanted either with or without an
omental wrap. A clinical trial on seven patients in need of a
cystoplasty showed that the engineered bladder tissues, cre-
ated with autologous cells seeded on the collagen-polygly-
colic acid scaffolds and wrapped in omentum after
implantation, were safe and effective to use in patients. In
another example, a microfluidic device with double-coaxial
laminar flow was used to fabricate meter-long core-shell
hydrogel microfibers encapsulating ECM proteins and pri-
mary pancreatic islet cells.138 After transplanting through a
microcatheter into the subrenal capsular space of diabetic
mice, the microfibers containing the islet cells normalized
blood glucose concentrations for about 2 wk.
More recent efforts were focused on the development of
3D-bioprinted tissues, such as livers and kidneys, with inte-
grated vasculature.139 Integral vascular structures are criti-
cal to the survival of the transplanted organs or tissues.
Either autologous vascular conduits from deceased donor or
synthetic vascular grafts have been used for anastomosing
the new organ to the recipient when necessary; however,
both come with disadvantages. Printing using spheroids of
human umbilical vein smooth muscle cells and human skin
fibroblasts, along with agarose rods, has resulted in single-
and double-layered vascular tubes with small diameters.140
Furthermore, printing branched vascular structures using
human umbilical vein endothelial cells, 10T1/2 cells,
human fibroblasts, or human embryonic kidney cells is also
feasible.141,142
Challenges, Limitations, and Future
Perspectives of 3D Cell Cultures
Many challenges remain for the widespread adoption of 3D
cell culture technologies in the drug discovery process. In
fact, there are very limited 3D screens done with large com-
pound libraries, although a multitude of 3D assays, mostly
based on high-resolution fluorescence imaging techniques,
have been validated for HTS/HCS in recent years.143,144
First, many 3D models such as organoids exhibit signifi-
cantly more complex morphology and function than 2D
cultured cells, thus leading to challenges in systematic
assessment. Furthermore, current 3D cultures are diverse in
terms of complexity, size, morphology, 3D architecture, and
protocols for assaying. This leads to challenges in standardiza-
tion with respect to culture and assay protocols, phenotypes,
and output data for analysis. To this regard, the development of
high-density microtiter plate–based spheroid-forming plates
(e.g., 1536-well low-adhesion spheroid plates) represents
an attractive solution to streamline 3D spheroid-based drug
screening. The 1536-well spheroid-forming plates also
make HTS economically affordable.
Second, lacking the understanding of the relevance of a
3D phenotype measured to the in vivo drug effects sought
also possesses challenges for 3D screening, as typical 3D
assay techniques measure a wide range of cellular pheno-
typic parameters (e.g., spheroid size or morphology,
hypoxic core). As the mainstay in 3D assays, high-content
imaging techniques could measure many different pheno-
types. However, identifying a clinically relevant phenotype
that is measurable in 3D models is critical to streamline and
expedite the screening process. For instance, using spher-
oids of invasive human prostate cancer cell line PC3 cul-
tured in the Matrigel matrix, Booij et al.144 developed a
phenotypic imaging assay to measure more than 800 pheno-
typic parameters. Multiparametric analysis identified sev-
eral phenotypes that enable the discrimination of selective
inhibitors for c-Met, or epidermal growth factor receptor, as
well as putative biselective inhibitors of both receptor tyro-
sine kinases. However, this small-scale screen clearly high-
lights the complexity of identifying specific phenotypes for
screening.
Third, assays using 3D cell models are far less developed
with respect to imaging, analysis, quantification, and automa-
tion compared with established 2D methods. Confocal micros-
copy is the standard imaging tool for assessing cellular function
within 3D cell models; however, it is certainly limited in
throughput. Improvements in imaging modality, data acquisi-
tion throughput, and analysis tools are necessary for the wide
adoption of 3D cell cultures for screening.
Fourth, the predictive values of 3D cell cultures for drug
efficacy and toxicity need to be further determined and vali-
dated by using existing human data.145 Although data had
shown that the efficacy and toxicity of many drug mole-
cules obtained using 3D models are different from 2D cul-
tures, only a small set of these data confirmed that the
efficacy and toxicity of drugs in 3D models are close to the
clinical data.23,112,114,117
Fifth, regulatory authorities have yet to accept data
obtained from 3D cell models, such as organoids or organs-
on-chips, as a surrogate for preclinical animal testing. Partly
related to this is that historically, the assessment of new
technologies has been exceptionally slow (10 to 15 years)145
but more importantly is that these models often do not cap-
ture the full complexity of human organ function, such as

Fang and Eglen 13
lacking vascularization.146 In addition, organoid technolo-
gies face a common issue related to maturation.16
Nonetheless, 3D cell cultures have a bright future in drug
discovery and development. Three-dimensional cell cul-
tures would have enormous potential to model development
and disease, as advanced cell models under development
may fully capture the in vivo functions of organs and tis-
sues. Furthermore, the development of screening-compati-
ble 3D cell cultures would transform the drug discovery
process, as it becomes possible to obtain early the physio-
logically relevant efficacy and toxicity data. In addition, the
optimization of 3D cell cultures for scaling-up cell produc-
tion would improve quality, quantity, and efficacy, thus
making cells as therapeutics a reality.
Conclusion
A wide range of 3D cell culture technologies have been devel-
oped to address the need for continuously improving the pro-
ductivity of pharmaceutical R&D. Three-dimensional cell
cultures hold great potential as a tool for drug discovery—
ranging from disease modeling to target identification to
screening to lead identification—and as a new type of thera-
peutics/replacement therapy that may transform our lives.
Future developments in screening readily available 3D cell
models and assays, preclinically validated 3D cell models
for animal replacement, and functional, safe, and trans-
plantable 3D cell models will no doubt bring them closer to
reaching these potentials.
Acknowledgments
The authors appreciate valuable and constructive suggestions
from anonymous reviewers.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, author-
ship, and/or publication of this article.
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