COBIOT-1034; NO. OF PAGES 7
Available online at www.sciencedirect.com
Synthetic 3D multicellular systems for drug development
Markus Rimann and Ursula Graf-Hausner
Since the 1970s, the limitations of two dimensional (2D) cell
culture and the relevance of appropriate three dimensional (3D)
cell systems have become increasingly evident. Extensive
effort has thus been made to move cells from a flat world to a
3D environment. While 3D cell culture technologies are
meanwhile widely used in academia, 2D culture technologies
are still entrenched in the (pharmaceutical) industry for most
kind of cell-based efficacy and toxicology tests. However, 3D
cell culture technologies will certainly become more applicable
if biological relevance, reproducibility and high throughput can
be assured at acceptable costs. Most recent innovations and
developments clearly indicate that the transition from 2D to 3D
cell culture for industrial purposes, for example, drug
development is simply a question of time.
Address
Zurich University of Applied Sciences, Institute of Chemistry and
Biological Chemistry, Einsiedlerstr. 31, 8820 Wädenswil, Switzerland
Corresponding author: Graf-Hausner, Ursula ([email protected],
[email protected])
Current Opinion in Biotechnology 2012, 23:1–7
This review comes from a themed issue on
Tissue, cell and pathway engineering
Edited by Hal Alper and Wilfried Weber
0958-1669/$ – see front matter
# 2012 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2012.01.011
Introduction
During the past decades, a conversion from 2D to 3D cell
culture has been taking place, hoping to ensure higher
physiological relevance of experiments. The usage of 3D
cell culture systems is manifold and covers areas such as
regenerative medicine, basic research and drug development/screening. Recognising the inefficient translation
from basic research to industrial and clinical applications
the US National Institute of Health (NIH) established
the National Centre for Advancing Translational Sciences
(NCATS). The aim is to accelerate the development of
diagnostics, devices and therapeutics by: (1) Supporting
researchers to find bottlenecks in the therapeutic development pipeline, (2) circumventing bottlenecks with new
innovations and (3) testing innovations in industrial setup
[1]. Interdisciplinary research groups targeting 3D cell
culture have been formed world-wide. Considerable
effort is presently being made to firmly entrench 3D cell
culture also in the industrial environment. The formation
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of competence centres with the goal to combine basic and
applied research with industrial aspects of pharmaceutical, biotech-related and medical companies is crucial. A
non-exhaustive list of centres formed is published in
Mironov et al. [2].
This review aims to (1) shortly report on 3D culture
systems currently entering industrial R&D laboratories
and to (2) emphasize the advantages and potential, but
also challenges and hurdles of these novel developments.
Limitations of 2D culture
Cellular behaviour (proliferation, differentiation, metabolism) is strongly influenced by the microenvironment,
that is, the cellular surrounding [3–5]. It has been shown
that primary mouse mammary luminal epithelial cells
(MEC) cultured on 3D basement membrane matrix
exhibit extended proliferation time compared to 2D
cultures [5], which is essential to analyse effects of gene
deletion. Using a CreER system, Jeanes et al. provided
valid evidence that the deletion of b1 integrin expression
and subsequent protein loss leads to cell cycle defects in
primary cells [5]. Furthermore, 3D culture conditions can
better simulate in vivo metabolic activity (e.g. more
tumour-like lactate production of MCF-7 breast cancer
cells in chitosan-based scaffolds than in 2D culture) or
drug efficacy (e.g. more tumour-like tamoxifen-resistance
of MCF-7 cells in chitosan-based scaffolds than in 2D
culture, indicated by its 10 higher IC50 value) [6].
Regarding safety and efficacy testing during drug development, the metabolic processes of not only the target
cells, but also of neighbouring cells, introduced in 3D by
co-culture, need special consideration. Froeling et al.
recognised the importance of organotypic co-culture systems in pancreatic cancer modelling [7]. When seeding
pancreatic cancer cells on top of ECM gels with pancreatic stellate cells (stromal cells), the pancreatic cancer cells
developed apical-basal structures, which were visualized
with positive staining for phosphorylated ERM (Ezrin,
Radixin, Moesin) at the apical membrane. Furthermore,
Froeling et al. could show that Ezrin translocation from
the apical to the basal compartment led to cellular processes invading ECM gels being an early event of cancer
invasion.
Despite the clear disadvantages of 2D culture, it is still
commonly used in the pharmaceutical industry. As convincing example the transition from 2 to 3D has largely
been made in the cosmetic industry. The use of in vitro
human skin equivalents for compound testing is imperatively needed to replace animal testing (legally banned
from 2013). As 3D cell culture holds obvious advantages,
Current Opinion in Biotechnology 2012, 23:1–7
Please cite this article in press as: Rimann M, Graf-Hausner U. Synthetic 3D multicellular systems for drug development, Curr Opin Biotechnol (2012), doi:10.1016/j.copbio.2012.01.011
COBIOT-1034; NO. OF PAGES 7
2 Tissue, cell and pathway engineering
the pharmaceutical industry is interested in making this
transition once their major concerns (large-scale, automation, low costs, wide applicability and predictability)
will be fulfilled.
cell culture is a promising way to improve drug development processes and to save time. Using a better screening process following the ethical 3R principle for animal
experiments (reduce, replace, refine), and also reducing
costs related to drug development gives pharmaceutical
companies a competitive advantage.
Potential of 3D culture
3D cell culture systems allow simulating the composition
of extracellular matrix like using collagen or hyaluronic
acid based scaffolds [8,9], with selective incorporation of
signalling factors, adhesion factors, and proteins. Therefore, it is possible to design an environment that better
mirrors the situation in the body, enabling the cells to
behave in a physiologically relevant manner. 3D culture is
often more time-consuming than 2D culture, but different 3D systems enable rapid experimental manipulations
and testing different hypotheses with high physiological
relevance. So far, evaluation of new drug candidates has
mostly been performed by high-throughput 2D cellbased screening assays. The major pitfall was the failure
of chosen candidates during subsequent animal testing or
clinical trials, leading not only to high costs, but also to the
loss of precious time [10]. The failure of candidates can of
course have multiple reasons, for example, species differences in drug metabolism and poor predictability of 2D
test systems with non-physiological cell behaviour. 3D
Types of 3D culture systems
A large
market
free or
culture
variety of 3D culture systems is currently on the
(Table 1). They are classified as either scaffoldscaffold-based (natural or synthetic origin) 3D
systems (Figure 1).
Scaffold-free 3D culture
The generation of scaffold-free microtissue spheroids by
gravity-enforced self-assembly in hanging drops has been
used for decades. This technology has shown its versatility in vitro over the past few years [12]. Kelm et al.
produced microtissues in Terasaki plates by culturing cell
suspensions upside down [23]. They could show that
microtissues made from human aortic fibroblasts that
were coated 2 days later with human umbilical vein
endothelial cells produced a capillary network throughout
the microtissue without additives. This was achieved by
endogenous expression of vascular endothelial growth
Table 1
3D cell culture systems. This table shows a non-exhaustive list of commercially available 3D cell culture systems and specific features. It
is subdivided into scaffold-free, scaffold-based and degradable or non-degradable scaffolds. Furthermore, companies providing m-fluidic
devices are listed
3D cell culture system
Vendor
Scaffold material
Degradable
Format
Components of
animal origin
References
comments
Microtissues
NanoCulture Plate
InSphero AG
Scivax
No
No
–
–
96–384 well plate
96 well plate
No
No
[11,12]
[13]
3D-Insert PCL
3D Biotek
Polycaprolactone
Yes
96 well plate
No
Extracel Hydrogel Kit
Glycosan
Biosystems
Invitrogen
Hyaluronane,
Gelatine, PEG
Alginate
Yes
Scalable
Yes
Scalable
[15]
QGel
PEG
Yes
Scalable
Yes, chemically
defined
No, chemically
defined
No
Product:
Recently
launched
[14]
Advanced
BioMatrix
BD Biosciences
Collagen I
Yes
Scalable
Yes
[17]
Basement
Membrane
Mixture
Polyvinylalcohol,
PEG
Yes
Scalable
Yes
[18]
Yes
Scalable
No
Product
launch: 03.
2010
AlgiMatrix 3D
Culture System
QGel MT 3D Matrix,
degradable
PureCol
BD Matrigel Basement
Membrane Mix
[16]
3D Life PVA Hydrogel,
CD-Link
Cellendes
Alvetex
3D-Insert PS
QGel MT 3D Matrix,
non-degrad.
Reinnervate
3D Biotek
QGel
Polystyrene
Polystyrene
PEG
No
No
No
12 well plate
96 well plate
Scalable
No
No
No
[19]
[20]
[16]
Iuvo Microchannel
5250
MiCA Microfluidic
plate
BellBrook Labs
–
–
–
–
Depends on
scaffold chosen
Depends on
scaffold chosen
[21]
CellASIC
192 well format
(SBS/ANSI formatted)
32 well format (SBS/
ANSI formatted)
Current Opinion in Biotechnology 2012, 23:1–7
[22]
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3D cell culture systems Rimann and Graf-Hausner 3
Figure 1
(A)
(B)
(C)
Current Opinion in Biotechnology
Organotypic tissue models and devices. (A) Scaffold-free gravity-enforced self-assembled tumour microtissue using InSphero’s technology, (B)
scaffold-based tumour tissue using 3D Life polyvinylalcohol Hydrogel (Cellendes), and (C) m-fluidic assay chip for 3D cell culture kindly provided by Dr.
Laurent Barbe (CSEM). Scale bar = 200 mm.
factor. The advantages of this technique are the precise
size control and the uniformity of the microtissues with
only one microtissue per drop. Furthermore, co-culture of
different cell types is possible and crosstalk is guaranteed
[23]. InSphero AG introduced a novel technology to
optimize the procedure and render microtissue production high throughput-compatible. The 96 well design
allows the automated production of microtissues by
applying cell suspensions with standard liquid handling
robots from top through a capillary to build drops. A
similar system developed by Tung et al. uses a 384 well
plate format that is also loaded from the top with cell
suspensions to form microtissues in hanging drops [11].
This small format is especially interesting because 384
well plates are the standard format for cell-based assays in
industry. Like the 96 well format developed by InSphero
AG, the plates used by Tung et al. contain liquid reservoirs to reduce evaporation of the small microtissuecontaining drop [11]. Another approach to generate
scaffold-free microtissues was employed by MarkovitzBishitz et al. They produced micrometre-sized honeycomb-shaped non-adhesive structures to generate one
microtissue in each unit of the honeycomb. The honeycomb structures are manufactured by deep reactive-ion
etching (DRIE), a technique to produce deep microchambers with no constraints on their diameters. This
system can be used in any well plate format and enables
analysis of growth parameter and apoptosis directly in the
honeycomb culture plates, owing to transparent bottom
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parts [24]. Because of the formation of many microtissues
per well, subpopulations such as cancer stem cells could
be detected from a disassembled tumour and even isolated for further analysis. Furthermore, Yoshii et al. produced nanoimprinted well plates with a non-adhesive
bottom in 96 well plates [13]. The square patterned
structures of 1 mm line depth and 2 mm line spacing
enabled microtissue production with Colon-26 and
HT-29 cell lines. Unlike microtissues produced in hanging drops and honeycomb structures, the microtissue size
in nanoimprinted well plates varies. This leads to limitations in its use as narrow microtissue size distribution may
be necessary, for example, for hypoxia studies. Nevertheless, Yoshii et al. detected hypoxic cores in microtissues by overexpression of hypoxia-inducible factor-1 and
accumulation of hypoxia indicator pimonidazole leading
to upregulation of, for example, vascular endothelial
growth factor A. On the basis of these innovations, scaffold-free 3D culture has become increasingly relevant for
industry, for example, in cancer research and screening of
novel anti-cancer drugs [25].
Scaffold-based 3D culture
Many companies provide scaffold-based 3D culture systems (Table 1). Generally, a cell-containing scaffold is
obtained by using either of two techniques: (1)
Cell-seeding on an acellular 3D matrix (e.g. 3D
Biotek, Alvetex and AlgiMatrix); (2) Dispersion of cells
in a liquid hydrogel, followed by polymerization
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COBIOT-1034; NO. OF PAGES 7
4 Tissue, cell and pathway engineering
(e.g. Matrigel, Cellendes, Glycosan Biosystems, QGel).
The most obvious way to grow cells in 3D is seeding cells
in a polystyrene scaffold being the same material as for
standard 2D culture. This approach has, for example,
been used most recently to investigate personalized drug
screening procedures. Caicedo-Carvajal et al. found
increased proliferation of lymphoma cells (HBL-2 cells,
a mantle cell lymphoma cell line) in 3D polystyrene
scaffolds with stromal dermal neonatal fibroblasts compared to 2D. The subsequent lymphoma cell isolation
contained less contaminating fibroblasts. Therefore, this
method is suitable for clinical applications to isolate and
expand cancer cells in vitro for medication screening [20].
Another typically used scaffold material is polyethylene
glycol (PEG) as it can easily be functionalized. A highly
flexible fully synthetic PEG-based scaffold has been
employed by Loessner et al., which consists of 8-arm
PEGs that are functionalized with or without integrinbinding sites (RGD) and matrix-metalloproteinase(MMP) sensitive sites [26]. By fine tuning the different
components, hydrogels with different stiffnesses, integrin-binding sites and degradation sites are obtained.
Using this system, Loessner et al. could demonstrate that
clusters of human epithelial ovarian cancer cells (OVMZ-6 cell line) grew faster and were larger on RGDfunctionalized PEG, indicating that tumour progression
is integrin-dependent [26]. Such fully synthetic systems
with high chemical and mechanical flexibility offer
additional potential compared to animal-derived scaffold
systems such as Matrigel basement membrane matrix
(Table 1). Other efforts aim at miniaturization leading to
m-engineering and m-fluidics in combination with 3D
scaffolds (Figure 1). Huang et al. developed a m-fluidic
cell culture chip with 48 chambers, using an agarose
scaffold. This chip is perfused by integrated pneumatic
micropumps and is transparent to allow microscopical
analysis [27]. When culturing primary articular chondrocytes for 4 days in 3D agarose scaffolds under perfusion,
the cells produced more glycosaminoglycans compared
to static 3D cultures. These results emphasize the
importance of perfused 3D culture systems. Recent
reviews are summarizing latest inventions in m-fluidic
devices for drug development [28,29]. In conclusion,
current inventions may finally allow implementing scaffold-based 3D culture into routine activities of pharmaceutical industry.
Novel analysis for 3D culture
While results have been delivered to optimize automated
3D cell culture, little progress has been made with regard
to novel analysis methods that meet the industrial
requirements. Confocal laser scanning microscopes,
which are traditionally used for analysis, are slow. An
emerging technology called light-sheet based fluorescence microscopy (LSFM) decreases photobleaching/
phototoxicity because of the selective illumination of the
focal plane with a light-sheet obtained by a cylindrical
Current Opinion in Biotechnology 2012, 23:1–7
lens. The objective lens is placed perpendicular to the
light-sheet and the specimen is moved through the lightsheet. A plane resolution of 0.5 mm is achieved. This
technique holds great promise for the analysis of large
numbers of samples with simple preparation, fast recording speed, high resolution and multi-channel fluorescence
imaging [10]. However, so far this technology is not yet
adapted for a high throughput setting. Adanja et al. developed an automated cell tracking system to monitor the
migration of cells into a collagenous matrix based on timelapse phase-contrast microscopy [30]. This method
proved to be useful to monitor drug-induced effects on
migration of unlabeled cells without the need for cumbersome staining procedures or transfections. The
authors showed the anti-migratory effect of Cytochalsin
D on A549 colic tumour cells embedded in collagen I gels.
Analysing differential fluorescent samples in a high
throughput-manner is proposed by Chen et al. [31]. They
could discriminate differentially treated PEG-encapsulated cells (microtissues, size 500 mm) with large
particle Flow Cytometry and near infrared (NIR) scanning. Microtissues of photoencapsulated HepG2 cells
(hepatoma cell line) were exposed to toxic and non-toxic
concentrations of doxorubicin in combination with BCLXL gene silencing. To distinguish between the four
different treatments microtissues were pre-labeled
(during encapsulation) with a fluorescent dye and postlabeled (after encapsulation) with a NIR-tag. When analysing pooled microtissues they could show that BCL-XL
gene knockdown combined with high doxorubicin concentration is most effective in killing the cells [31].
Recent developments in mass spectrometry presented
by Li and Hummon show the usefulness of imaging mass
spectrometry to analyse spatial distributions of proteins in
3D cell culture [32]. They embedded spherical colon
carcinoma cell line HCT-116 microtissues in gelatine and
cut them into 10 mm slices. Slices were analysed with
MALDI-MS to generate ion intensity maps. With this
approach they could identify a specific signal only present
in the core region of the microtissue. In order to identify
the proteins, MALDI-MS results were combined with
data obtained by nanoflow liquid chromatography tandem
MS. The possibility to determine also unknown target
molecules is an advantage. But analysis of tissue slices,
which necessitate the sectioning of the organotypic culture, makes this approach cumbersome and not high
throughput-compatible. Innovative analytical tools are
urgently needed and will complement emerging 3D
culture techniques in industrial drug development.
Pitfalls and limitations of 3D culture
3D culture holds great promise for drug development.
However there are still many hurdles and unmet needs.
Many novel 3D culture systems focus on a very specific
application [33,34]. By contrast, pharmaceutical industry
is searching for a universal standardized 3D culture system for drug development. While in academia the main
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3D cell culture systems Rimann and Graf-Hausner 5
goal is to create 3D systems with excellent biological
relevance, industrial application relies on efficient readout, automation and acceptable costs. Therefore, the
optimal screening tool for pharmaceutical industry has to
combine biological relevance, wide applicability/versatility, high throughput and scalability/automation at low
costs – a challenge that researchers in specialized
centres/networks are prepared to meet [2]. In the following
section specific drawbacks of 3D systems are summarized.
- Most of the existing systems fail to mimic the
biomechanical characteristics of tissue in vivo and thus
only represent a static condition [35].
- Animal-derived or human-derived scaffold materials
risk the potential transmission of diseases.
- For scaffold-based culture systems, reproducibility
between different batches is unsatisfactory, especially
if animal-derived components are used. In order to
circumvent the batch to batch variability of naturally
derived materials, many fully synthetic or chemically
defined scaffolds have been developed (Table 1).
- Commonly used fully synthetic scaffolds are often
PEG-based. PEG is cell-compatible but inert.
Embedded cells are not able to attach to the matrix
without modifications like RGD-sites covalently
attached to PEG hydrogels [36].
- Methods to gently and rapidly recover encapsulated
cells (e.g. for isolation of RNA or protein) are missing or
still need to be optimized, particularly in scaffold-based
systems. However, many different enzymatic and nonenzymatic reagents are meanwhile available to specifically digest the scaffold without harming the cells,
indicating that this may not be an issue in 3D culture
anymore in the nearby future. In addition for assays like
luminescent ATP content measurements recovery of
cells is obsolete because the reagent penetrates the
scaffold to produce a luminescent signal.
- Methods directly applying screening and bioprocessing
in 3D culture systems like imaging tools are scarce and
face scaffold-typical problems such as autofluorescence
of collagenous scaffold.
- Limitations of the scaffold-based 3D culture systems
are potential interactions of screening compounds with
the scaffold [37]. Scaffold absorption of compounds
strongly relies on the compounds properties (hydrophilic, hydrophobic) as shown by Nugraha et al. [37].
Therefore, it is important to compare different scaffoldbased systems for their absorption properties of
compounds or to switch to scaffold-free 3D culture
systems without additives.
In summary, the benefits of 3D cell culture systems in
terms of biological relevance especially for industrial
applications are obvious. For high throughput application
and analytical read-out, the different 3D systems need to
be adapted. It seems likely that there is not only one
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suitable model system for all applications. Solutions have
to be adapted from case to case.
Conclusions
Tremendous progress has been made to adjust 3D culture
techniques for industrial application. The state-of-the-art
technologies still however fail to provide standardized
and validated 3D tissue models with relevant read-out
that can be used in automated processes. In the past,
collaborations between material scientists and biologists
resulted in a huge variety of 3D cell culture systems, but
for a successful integration of 3D culture in industry,
automation and high throughput analysis is still a matter
of concern. Therefore, tremendous effort is currently
being made to develop novel analysis methods for 3D
culture, such as improved techniques for light sheetbased fluorescence microscopy and imaging mass spectrometry. This task is easier achieved by forming research
and application clusters such as the National Centre for
Advancing Translational Sciences (NCATS) established
by the NIH and other competence centres. They will
speed up well-directed research towards industrial applications and join forces of researchers and industrial partners. As effective implementation of 3D cell culture
systems in the industrial setting is a complex task, these
competence centres certainly need to be equally complex, that is, multidisciplinary and divers as shown in
Figure 2.
Figure 2
Tissue models
Automation
HTS
Costs
Competence
Centre
Relevance
Predictability
Analysis
Read-out
Current Opinion in Biotechnology
Competence centres: In order to successfully translate 3D cell and
tissue culture into industrial applications competence centres have to be
formed to combine biology and technology.
Current Opinion in Biotechnology 2012, 23:1–7
Please cite this article in press as: Rimann M, Graf-Hausner U. Synthetic 3D multicellular systems for drug development, Curr Opin Biotechnol (2012), doi:10.1016/j.copbio.2012.01.011
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6 Tissue, cell and pathway engineering
Acknowledgements
We thank Dr. Karin Wuertz for critical comments and fruitful discussions
about the manuscript. The competence centre ‘‘Tissue Engineering for
Drug Development’’ TEDD is provided with start-up financing by GebertRüf Foundation, Switzerland (GRS-040/10).
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Current Opinion in Biotechnology 2012, 23:1–7
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