Lab Technology
In Vitro Tissue Models:
Working in the Third Dimension
By Emma Sceats
at Zyoxel Ltd
Figure 1: Threedimensional cell culture
using the TissueFlex®
system with 12
microbioreactors. Each
bioreactor is individually
supplied with culture
medium through a
multi-channel peristaltic
pump, and effluents are
collected downstream
for analysis
In vitro systems featuring three-dimensional cell cultures permit
more accurate prediction of drug toxicity and efficacy by enabling
cells to be tested in an environment that more closely mimics
the in vivo state.
The use of in vitro platforms in research presents
obvious ethical and cost advantages over in vivo
models. In vitro models can also offer important
scientific advantages; for example, the study of
biological mechanisms of action has been expedited by
these models because it is easier to measure the impact
of an experimental variable – such as a drug – on a
simple, well controlled system.
three-dimensional (3D) arrangements of cells.
Furthermore, in a 2D static cell culture the environment
is constantly changing as nutrients in the media are
depleted and metabolites accumulate. This is clearly
unrepresentative of the in vivo state where cells are
maintained in a chemostatic environment, courtesy of a
constant fresh supply of nutrients and the removal of
waste products via the circulatory system.
In the pharmaceutical industry, the advantages of in vitro
models have led to their widespread use in research and
permitted the discovery and development of new
and more efficacious drugs by enabling a better
understanding of disease pathology.
Isolated perfused organs and tissue slice models offer the
closest in vitro models of the in vivo state. These models
– which are commonly referred to as ‘natural tissue
models’ – preserve the in vivo cellular heterogeneity and
structural complexity, and ensure that important cell-cell
and cell-matrix interactions are maintained. However,
the high cost and short supply of organs, the difficulties
associated with maintaining organ/tissue viability ex vivo
and donor specific issues make these models unsuitable
for routine testing purposes.
3D VERSUS 2D
Traditionally, in vitro research has been conducted using
two-dimensional (2D) cell cultures. However, an
increasing number of researchers have questioned the
validity of studying cells in an environment that is so far
removed from the in vivo state. After all, organisms, and
the tissues and organs of which they are comprised, are
Three-dimensional cell culture (1) offers a practical
alternative to natural tissue models (see Figure 1). These
systems provide an environment in which one or more
Culture medium
(with defined dose
of chemicals)
Spent media for metabolic
functional and bioassays
Source: ZF Cui, University of Oxford
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cell types can be encouraged to form tissue-like
constructs, often using natural or synthetic scaffolds
such as collagen, alginate, polyethylene glycol hydrogels
and nanofibres to provide structural support to the
growing tissue. Scaffolds can also provide – through
modification or by virtue of their natural composition –
growth factors and other proteins to replicate the
biochemical cues offered by the extracellular matrix
(ECM) in native tissues. Though the most sophisticated
3D tissue models cannot replicate the complexity of
natural tissues or a whole organ, 3D models offer many
benefits over natural tissue models. Notably, 3D cell
culture models can provide a platform for high
throughput and systematic experimentation, reducing
the need for animals, and permitting a more
straightforward understanding of cause and effect in
drug safety and efficacy studies.
3D tissue models also offer numerous benefits over
simple 2D cell culture models. In recent studies, cell
viability, proliferation, differentiation, morphology, gene
and protein expression and function have been shown to
exhibit significant differences in 3D compared with 2D,
with 3D constructs more closely mirroring what is
observed in vivo. Undoubtedly, the cell-cell and cellmatrix interactions established in 3D play a significant
role in the function of both healthy and diseased tissues
and can affect the cellular response to drugs. The use of
perfusion in 3D cell culture affords the added benefit of
improved long term cell viability and tissue specific
function, enabling analysis of the culture over several
days or weeks.
This growing body of research is contributing to the
increased use of 3D tissue models to complement 2D cell
culture and animal model studies. This article will
highlight some of the more established 3D models and
explain the uses of these models in pharmaceutical
research and development.
Cardiac tissue models may also prove to be a
useful tool in regenerative medicine studies.
Cardiomyocytes are terminally differentiated in
adults and so cardiovascular diseases can
result in permanent damage to heart tissue
because there are no self-repair mechanisms.
Stem cell-derived cardiomyocytes may offer
new therapeutic opportunities in the treatment
of myocardial infarction and heart failure.
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CARDIAC TISSUE MODELS
In vitro cardiac tissue models have principally been
developed for the study of cardiac diseases and in the
prediction of drug cardiotoxicity (2).
Scaffold-based approaches and scaffold-free cultures have
been used to prepare models that structurally and
electrophysiologically resemble native cardiac tissue.
Models employing animal embryonic cardiomyocytes or
human cardiomyocyte cell lines have proved useful for
understanding the underlying mechanisms of tissue
damage resulting from cardiac diseases. However, they
do not always accurately predict human responses and
have been of limited use in toxicological studies. Human
primary cardiomyocytes might offer more accurate
toxicological results but these primary tissues are
notoriously difficult to maintain in culture and their
limited supply makes them unsuitable for widespread use
in drug safety screening. To overcome these limitations of
supply and human relevance, 3D cardiac tissue models
based on human embryonic stem cell (hESC)-derived
cardiomyocytes are being developed. Cardiotoxicity is a
leading cause of approved drugs being removed from the
market, so it is anticipated that there will be continuing
significant effort towards the development of humanrelevant in vitro cardiac tissue models.
Cardiac tissue models may also prove to be a useful tool
in regenerative medicine studies. Cardiomyocytes are
terminally differentiated in adults and so cardiovascular
diseases can result in permanent damage to heart tissue
because there are no self-repair mechanisms. Stem cellderived cardiomyocytes may offer new therapeutic
opportunities in the treatment of myocardial infarction
and heart failure. However, the techniques for ensuring
that stem cells grow and differentiate in a controlled
manner still need rigorous preclinical testing and in vitro
3D tissue models are likely to prove useful in this regard.
LIVER TISSUE MODELS
The liver is the primary detoxification organ in the
human body. Studying the metabolism of drugs by the
liver is an important component of drug discovery
programmes. In vitro assays and animal models are
used prior to human clinical trials to predict the
potential toxicity of a drug, or the by-products of its
metabolism, and to learn how drug metabolism affects
bioavailability.
The performance of in vitro liver models is assessed by
the maintenance of cell viability, liver specific function
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and enzymatic activity for drug metabolism. The most
clinically relevant models employ primary human
hepatocytes; however cell lines are used more frequently
because of low primary cell functionality in 2D culture,
lack of availability and cost. Unfortunately, current in
vitro 2D cell-based assays are often poorly predictive of
drug hepatotoxicity. For example, these assays may
give false-positives, with the 2D cell cultures being less
able to withstand cytotoxic agents than 3D tissue,
meaning that potentially safe and efficacious
compounds may be removed unnecessarily from drug
development programmes.
Studies have shown that many of these limitations can be
overcome by using 3D hepatocyte cultures and such
models are becoming more accepted for liver toxicity and
drug metabolism assessment (3). Scaffold-free cellular
spheroids and scaffold-based liver tissue models have
been developed, and in perfused culture these systems
can maintain excellent primary cell viability for two to
three weeks. Further improvements in cell viability have
been achieved by using co-culture and sandwich culture
techniques, where hepatocytes are cultured with nonparenchymal cells such as epithelial cells and fibroblasts.
3D cell culture also enables liver specific functions –
including urea and albumin production and cytochrome
P450 enzyme activity, an important class of metabolic
enzymes – to be maintained for up to 10 days at close to
in vivo levels (see Figure 2). The effect of drugs on in
vitro liver tissue function can be a useful indicator of in
vivo hepatotoxicity and drug metabolism.
Epithelial tissue is the membranous tissue
that covers internal organs and other
internal and external surfaces of the body.
Human epithelial tissue models are some of
the most well developed 3D tissue models,
although they are sometimes described as
pseudo-3D models because they are built up
from multiple 2D layers of cells.
Numerous studies have shown that the
physiological relevance of epithelial tissue
www.iptonline.com
TUMOUR TISSUE MODELS
The development of in vitro tumour tissue models has
been driven by the need for better preclinical methods
for predicting the efficacy of anticancer drugs. The 2D
Figure 2: Human
hepatocyte (C3A) urea
and albumin levels in
TissueFlex® 3D perfused
culture compared with
2D static culture
conditions. Hepatocyte
function is improved in
3D perfused culture and
much closer to in vivo
function than in 2D
static culture
2.5
2D urea
3D perfused area
20
2.0
2D albumin
3D perfused area
15
1.5
10
1.0
5
0.5
0
0.0
0
Source: Zyoxel Ltd
2
4
6
8
Time (days)
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Albumin in medium µg/24h/106 live cells
EPITHELIAL TISSUE MODELS
Skin, corneal, oral and vaginal tissue models are now
commercially available and can be used in validated
toxicological assays. Skin tissue models are undoubtedly
the most widely used epithelial tissue models and they
are commonly used in testing drugs that are
administered topically (4). These models are also useful
in testing the penetration and sensitisation potential of
non-drug chemicals. This has been of significant
interest to the cosmetics industry which is increasingly
using skin tissue models as in vitro substitutes for the
murine local lymph node assay (LLNA), the goldstandard method for testing the safety and allergenic
properties of cosmetics.
25
Urea in medium µg/24h/106 live cells
3D liver tissue models, derived from
hepatocyte cell lines, are now commercially
available for use in liver toxicity and
metabolism testing. However, further
development will be required to permit the
routine use of primary human hepatocytes or
stem cell derived hepatocytes in drug testing.
models is highly dependent on the components of the
extracellular matrix (ECM). The ECM is important for
controlling the stiffness of epithelial tissues and
resisting tension, and this is critical for preventing
damage to underlying tissues and organs. The ECM
also acts as a semi-permeable barrier, controlling the
transport of substances to underlying tissues and
organs. Models that most closely replicate epithelial
physiology, function and biochemistry tend to
incorporate natural ECM gels such as collagen or fibrin
or their synthetic mimics.
cell culture models that are employed in early preclinical studies tend to suggest that anticancer drugs
have a much higher efficacy than is subsequently
observed in vivo. Studies indicate that the 3D
architecture of tumours and the ECM – which provides
structural support and acts as a depot for growth
factors that regulate cell growth, proliferation and
differentiation – all contribute to the higher in vivo
resistance to anticancer drugs.
Tumour xenograph models, which involve the
transplantation of human tumour cells or cell lines into
immunocompromised animals, can overcome many of
the shortcomings of 2D cell culture by providing
systems that are more representative of a tumour in its
native state. However, these models are timeconsuming, expensive and technically challenging to
develop making them unsuitable for routine testing.
State of the art in vitro models aim to replicate the
structural, functional and mass transport properties of
tumours by culturing cells in 3D (5).
Multicellular tumour spheroid (MCTS) models, which
are small spheroids consisting of a mixture of tumour
cells and stromal cells, are useful 3D models because
they can reproduce many characteristics of avascular
tumour nodules and micrometastases of large solid
tumours, and they better replicate the barrier to drug
penetration which is presented by dense tumour tissues.
The principle limitations associated with MCTS
models are the absence of vasculature, which results in
lower nutrient supply to the tumour than would be
observed in vivo, and the requirement for relatively long
culture periods.
human tumour cell lines. Scaffold-based tumour
models demonstrate significantly higher drug resistance
than 2D cell culture, making them useful models for
secondary drug screening. Disadvantages of scaffoldbased models include the artificial barrier created by the
scaffold, which can inhibit efficient transport of large
biomolecules and drugs in the tumour.
In addition to their utility in drug efficacy screens, 3D
tumour models may prove useful in the development of
new anticancer drugs. Both MCTS and scaffold-based
models are known to better reflect in vivo gene and
protein expression patterns and signalling pathways,
making it possible to identify new cellular targets for
anticancer drugs.
CONCLUSION
In vitro 3D tissue models are increasingly being used to
complement 2D cell culture and animal model studies,
in an attempt to improve the predictive capabilities of
preclinical drug safety and efficacy testing. The
development of 3D tissue models with high cell
viability and functioning that more closely replicates
in vivo tissue behaviour will push these in vitro models
into the mainstream of pharmaceutical screening
programmes. The potential utility of 3D tissue
models in regenerative medicine and stem cell research
will also contribute to the more widespread use of
these models.
References
1.
Elliott NT and Yuan F, A review of threedimensional in vitro tissue models for drug
discovery and transport studies, J Pharma Sci n/a.
Scaffold-based cancer models involve the culture of
cells on natural ECM proteins, such as collagen and
Matrigel®, or synthetic polymer scaffolds. Scaffoldbased techniques offer high versatility and 3D tumour
models have now been developed for a variety of
doi: 10.1002/jps.22257
2.
(a) Franchini JL et al, Novel tissue engineered
tubular heart tissue for in vitro pharmaceutical
toxicity testing, Microsc Microanal 13: pp267-271,
2007. (b) Bursac N et al, Cardiac muscle tissue
engineering toward an in vitro model for
electrophysiological studies, Am J Physiol 277:
Emma Sceats is Business Development Manager at Zyoxel
Ltd (Oxford, UK), a company spun-out of the University of
Oxford to exploit TissueFlex®, a novel bioreactor technology for
three-dimensional cell culture. Prior to joining Zyoxel, Emma
was Licensing Manager at Isis Innovation, the University of
Oxford Technology Transfer Office, with a specialisation in
enabling technologies for pharmaceutical research and
development. She holds an MSci in Chemistry from the University of Bristol
(UK), an MS in Chemistry from the Massachusetts Institute of Technology (US)
and a DPhil in Chemistry from the University of Oxford (UK).
Email: [email protected]
pp433-444, 1999
3.
Khetani SR and Bhatia SN, Microscale culture
of human liver cells for drug development,
Nat Biotechnol 26 (1): pp120-126, 2008
4.
Carlson MW, et al, Three-dimensional tissue
models of normal and diseased skin, Curr Protoc
Cell Biol , 2008 Dec, Chapter 19, Unit 19.9, 2008
5.
Kim JB, Three-dimensional tissue culture models
in cancer biology, Semin Cancer Biol 15 (5):
pp365-377, 2005
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